title:„Czech‐Japan Joint Symposium for Animal Reproduction“

subtitle:„From Gametes to Stem Cells“

date:20th ‐ 21st September, 2010

venue:

Conference Centre AV CR, Chateau Liblice, Liblice 61, 277 32 Bysice,
Czech Republic2Organizers:
Institute of Animal Physiology and Genetics AS CR, v.v.i.
Rumburska 89, 277 21 Libechov
Czech Republic
http://www.iapg.cas.cz
Institute of Animal Science, v.v.i.
Pratelstvi 815, 104 00 Praha Uhrineves
Czech Republic
http://vuzv.cz
Veterinary Research Institute, v.v.i.
Hudcova 70, 621 00 Brno
Czech Republic
http://vri.cz

Financial support:
BARIA, s.r.o.
Jizni 393, 252 44 Psary, Czech Republic
http://baria.cz
BioTech a.s.
Sluzeb 4, 108 00 Praha 10, Czech Republic
http://biotech.cz
Czech Association for Analytical Cytology
Institute of Biophysics AS CR
Kralovopolska 135, 612 65 Brno, Czech Republic
http://csac.cz
Institute of Animal Science, v.v.i.
Pratelstvi 815, 104 00 Praha Uhrineves,
Czech Republic
http://vuzv.cz
Olympus Czech Group, s.r.o.
Evropska 176, 160 41 Praha 6, Czech Republic
http://olympus.cz
Schoeller Pharma Praha s.r.o.
Videnska 124, 148 00 Praha 4, Czech Republic
http://schoeller.cz
Sigma‐Aldrich, s.r.o.
Sokolovska 100/94, 186 00 Praha 8, Czech Republic
http://sigmaaldrich.com

3

4

Dear Participants,
Welcome to the Czech‐Japan Joint Symposium for Animal Reproduction under the title “Fromgametes to stem cells”.
Liblice castle, now the Conference Centre of the Czech Academy of Sciences, was traditional
venue for animal reproduction meetings. I presented my first slides with maturing pig oocytes justhere. So our symposium logically continues in this line. But why the castle silhouette on the poster isbellow “Fuji san”, asked me several friends? All basic knowledge in the field of physiology of
reproduction flows into an essential concept of embryonic stem cells. Nearly two decades ofconcentrated effort focused to the mouse and human embryonic stem cells clarified several
principles of mammalian development. However, only the pioneering studies coming from Kyoto
University showed a new way to dedifferentiation of somatic cells. A unique property of mammalianegg, first demonstrated by Dolly, has been now proved under in vitro conditions. Professor Shiny
Yamanaka’s results have brought our dreams about stem cell therapy much closer to the reality.
The most beautiful lake bellow Mount Fuji is Yamanaka ko and I wanted to bring this allegory on theposter. However, a lake is not graphically optimal, so Fujisan represents this unique Japaneseachievement. The last session of the conference will demonstrate that large animal models that
represent an important step on the way to regenerative medicine. I hope that numerous discussions
will bring a new insight in a position of reproductive biotechnology that is now at the interface
between agricultural and biomedical research.
From my unique position as the conference organizer, I want to follow the Japanese tradition
in respect to old people and to remind you one colleague building the solid background of animal
reproduction before us. In this year, Luboš Holý, the emeritus professor of the University of
Veterinary and Pharamaceutical Sciences in Brno will be octogenarian. Luboš Holý devoted his great
effort in development of all new biotechnology methods in cattle reproduction and paved the way
for practical application of cattle embryo transfer, embryo micromanipulation and cryopreservation.
First of all he found the clever implementation of embryo transfer for improvement of herd
production. I wish you, Luboš, good health and I wish us to listen to your unique voice either in wise
advices or in Moravian songs.
We will also respect the tradition of opening the conference by the key note lecture. I amvery pleased that Professor Takeo Kishimoto has taken this duty. The Czech Academy of Sciences
recognized his concentrated scientific effort in the field of biology of reproduction and Professor
Helena Illnerova will award him the Johan Gregor Mendel medal as a symbol of pioneering researchin biology.
The whole Organizing Committee wishes you a productive and inspiring meeting. Our greathope is that you will leave this beautiful castle with fresh ideas, new collaborative connections andnew friendships since a human factor plays very important role in our scientific effort.

Jan MOTLIKHead of the Laboratory of Cell Regeneration and Plasticity
Institute of Animal Physiology and Genetics AS CR, v.v.i.
Libechov, Czech Republic5

6

Programme:
Day 1st – 20th September, 2010
08:00 – 09:30

Registration

09:30 – 10:00

Opening the Syposium
Josef FULKA
Institute of Animal Science, v.v.i., Prague‐Uhrineves, Czech Republic

10:00 – 10:30

Key note lecture
Takeo KISHIMOTOTokyo Institute of Technology, Japan„The cell cycle control: Lessons from starfish oocytes“
10:30 – 11:00

Johan Gregor Mendel medal for Takeo Kishimoto
Jan MOTLIKInstitute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic

Helena ILLNEROVA
Academy of Science of the Czech Republic, Prague, Czech Republic

11:00 – 11:20

Coffe break

11:20 – 11:30

Opening topic 1: Oocytes
Chair‐men: Takashi MIYANO, Radek PROCHAZKA

11:30 – 11:55

Jibak LEE, Tatsuya HIRANO
Chromosome Dynamics Laboratory, RIKEN Advanced Science Institute, Wako, Japan„Regulation of chromosome dynamics by cohesins in mammalian meiosis“
11:55 – 12:20

Takashi MIYANO, Mohammad MONIRUZZAMAN
Graduate School af Agricultural Science, Kobe University, Kobe, Japan„Activation and inactivation of primordial oocytes“
12:20 – 12:45

Koji SUGIURA1,2, You‐Qiang SU1, John J. EPPIG1
1

The Jackson Laboratory, Bar Harbor, ME, USA.
Laboratory of Applied Genetics, Graduate School of Agricultural and Life Science, The
University of Tokyo, Tokyo, Japan
2„Oocyte control of granulosa cell function and development“
12:45 – 13:10

Yuji HIRAO, Kosuke IGA, Kenji NARUSE, Naoki TAKENOUCHINational Agricultural Research Center for Tohoku Region (NARCT), Morioka, Japan„In vitro growth of bovine oocyte‐granulosa cell complexes after vitrification“

13:10 – 14:10

Lunch break

14:10 – 14:35

Radek PROCHAZKA
Institute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic„Regulation of pig oocyte maturation by EGF‐like peptides“714:35 – 15:00

Sugako OGUSHI1, Yasuko KATO1, Akira NAKAMURA1, Mitinori SAITOU2
1

Laboratory for Germline Development, RIKEN Center for Developmental Biology, Kobe, Japan.
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University,
Kyoto, Japan.
2„Functional analysis of maternal nucleolus in mouse oocytes“
15:00 – 15:25

Petr SOLC
Institute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic„Aurora‐A regulates MTOCs (centrosome) biogenesis but it does not trigger
G2/M transition in mouse oocytes matured in vivo“
15:25 – 15:50

Martin ANGER
Institute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic„Age‐related aneuploidy in mammalian oocytes“
15:50 – 16:10

Coffe break

16:10 – 18:30

Poster session
Poster competition: Jan MOTLIK, Kazuhiro KIKUCHI

18:30 – 19:00

Free time

19:00 – 20:30

Galla dinner

20:30 – 21:00„Baldrián“, the folklore group from Pardubice, Czech Republic

21:00

Drinks8

Day 2nd – 21st September, 201008:30 – 08:40

Opening topic 2: Fertilization
Chair‐men: Jiri KANKA, Naomi KASHIWAZAKI

08:40 – 09:05

Junya ITO1,2, Rafael A. FISSORE3, Naomi KASHIWAZAKI1,2
Laboratory of Animal Reproduction,
1
School of Veterinary Medicine,2
Graduate School of Veterinary Science, Azabu University, Sagamihara, Japan.
3
Department of Veterinary & Animal Sciences, University of Massachusets, Amherst, USA„Factors associated with calcium oscillations during fertilization in mammals“
09:05 – 09:30

Konosuke OKADA
Faculty of Applied Life Science, Nippon Veterinary and Life Science University, Masashio, Japan„Vole sperm cryopreservation: assessmants of viability, sperm DNA integrity
and fertilizing capacity“
09:30 – 09:55

Tereza TORALOVA
Institute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic„The role of CENPF in bovine preimplantation development“
09:55 – 10:20

Helena FULKA
Institute of Animal Science, v.v.i., Prague‐Uhrineves, Czech Republic„From oocyte to embryo – remodeling the parental genomes“
10:20 – 10:40

Coffe break

10:40 – 10:50

Opening topic 3: Stem cells
Chair‐men: Ales HAMPL, Noboru MANABE

10:50 – 11:15

Ales HAMPL
Department of Biology, Medical Faculty of Masaryk University, Brno, Czech Republic„Human embryonic stem cells“
11:15 – 11:40

Zuzana KOLEDOVA
Department of Biology, Medical Fac of Palacky University, Olomouc, Czech Republic„Centrosomal decisions on self‐renewal in mouse embryonic stem cells“
11:40 – 12:05

Petr VODICKA
Institute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic„iPS cells in miniature pigs“
12:05 – 12:30

Jana JUHASOVA‐HLUCILOVA
Institute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic„Bone marrow‐derived mesenchymal stem for the regeneration of cartilage
and bone“
12:30 – 12:55

Helena SKALNIKOVA
Institute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic„Proteomics of neural stem cells isolated from miniature pigs“
13:00 – 14:00

Lunch break9

14:00 – 14:10

Opening topic 4: Genetics manipulation
Chair‐men: Josef FULKA, Takashi NAGAI

14:10 – 14:35

Atsuo OGURA, Kimiko INOUERIKEN BioResource Center, Tsukuba, Japan„Improvement of somatic cell nuclear transfer in mice: Genetic andepigenetic approaches“
14:35 – 15:00

Masahiro KANEDA, Tamas SOMFAI, Seiki HARAGUCHI, Satoshi AKAGI, Shinya
WATANABE, Takashi NAGAI
National Institute of Livestock and Grassland Science, National Agriculture and Food Research
Organization, Tsukuba, Japan„Normal and abnormal epigenetic changes in the development of cloned
cattle“
15:00 – 15:25

Noboru MANABE1, Ichiro ONOYAMA1, Junyou LI1, Yutaka SENDAI2, Yoshito
AOYAGI3
1

Animal Resource Science Center, The University of Tokyo, Kasama, Japan.
Central Research Institute for Feed and Livestock, Zen‐Noh, Tsukuba, Japan.
3
Embryo Transfer Center, Zen‐Noh, Kami‐Shihoro, Japan.
2„Characteristics of prion, bovine spongiform encephalopathy (BSE) pathogen,homo‐knockout cow“
15:25 – 15:50

Michiko NAKAI1, Kazuhiro KIKUCHI1, Junya ITO2,3, Naomi KASHIWAZAKI2,3,
Hiroyuki KANEKO1
1

Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Japan.
Laboratory of Animal Reproduction,
2
School of Veterinary Medicine,
3
Graduate School of Veterinary Medicine, Azabu University, Sagamihara, Japan.

„Xenotransplantation of gonadal tissue for utilization of genetic resources inpigs“
15:50 – 16:15

Stefan JUHASInstitute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic„Miniature pig as a large animal model of Huntington disease“
16:15 – 16:30

Closing of the Symposium

10Key‐note lecture, invited by Czech Republic

Cell Cycle Control: Lessons from Starfish Oocytes
Takeo KISHIMOTO
Laboratory of Cell and Developmental Biology, Tokyo Institute of Technology, Nagatsuta, Midoriku,
Yokohama 226‐8501, Japan

Cell cycle control is a molecular system that ensures faithful duplication of the cell, andhence underlies wide range of life science from basic biology to various diseases, particularly cancer
and genetic disorders. Cell cycle studies at the end of the last century have established a conceptthat in eukaryotic cells, the cell cycle control system is consisted of two major elements, the cell cycle
engine and the checkpoint. The cell cycle engine performs progression of the cell cycle throughvarious cyclin‐Cdk (cyclin‐dependent kinase) complexes, while the checkpoint is negative feedbacks
that ensure correct replication and segregation of chromosomes. Based on these accomplishments,
the next subject would be to elucidate how the cell cycle control system is actually regulated invarious living cells.
The pattern of the cell cycle in higher eukaryotes can be divided into two types, the somatic
cell mitosis and the germ cell meiosis. The meiotic cell cycle, which is specialized to bring about
haploidy through two consecutive M‐phases, generally arrests twice in animal oocytes: the primaryarrest at prophase of meiosis I, and the second arrest at a later particular stage of meiosis until
fertilization. Although the primary arrest is released by various extracellular stimuli (typically bymaturation‐inducing hormone), the release from the primary arrest, or “meiotic reinitiation”, isconsistently equivalent to G2/M‐phase transition in somatic cells. In contrast, the stage of the secondarrest varies with the species: typically, metaphase of meiosis I (meta‐I) in insects and ascidians,
metaphase of meiosis II (meta‐II) in most vertebrates, or G1 phase (pronuclear stage) after
completion of meiosis II in echinoderms. But the second arrest, or “post‐meiotic reinitiation arrest”,
always enables mature eggs to prevent parthenogenesis and to begin development as a zygote only
after fertilization. It is an essential question how each of these different meiotic arrests is executedand released, and whether there is a common molecular principle. Model organisms to analyze thesearrests are mouse, frog and echinoderm starfish. Here I would like to review signalling pathways thatlead to the regulation of these primary and second arrests in starfish oocytes.
The release from the primary arrest in starfish oocytes is induced by 1‐methyladenine (1‐MeAde), which was identified first in the animal kingdom as a physiological maturation‐inducing
hormone by Kanatani and colleagues in 1969. Since then, the starfish oocyte has been for more than
40 years a model system to analyze the signalling pathway that leads to meiotic reinitiation. 1‐
MeAde acts on the oocyte surface and eventually causes the activation of cyclin B‐Cdc2 kinase, theuniversal trigger of G2/M‐phase transition, with no requirement of new protein synthesis. In thispathway, a putative surface receptor for 1‐MeAde is coupled to a heterotrimeric Gi‐protein, and thereleased G is suggested to activate PI3 kinase to produce PIP3, thus providing an occasion in which
PDK1, PDK2 and Akt/PKB meet each other. As a result, the phosphorylated and activated Akt/PKB
directly phosphorylates and inhibits Myt1, and thus reverses the balance between opposing Cdc25
and Myt1 activities towards the activation of cyclin B‐Cdc2. Downstream of cyclin B‐Cdc2, mitotic
choreographer kinases, such as Plk1 (polo‐like kinase 1), Aurora, Gwl (Greatwall kinase) and MAPK,
are activated to execute meta‐I. I will update on our progress in the study of 1‐MeAde signallings.
Although this pathway might be unique to the starfish in some aspects, the starfish oocyte is the only
system in which outline of the complete pathway from the maturation‐inducing hormone to cyclin B‐
Cdc2 has been unravelled.
After meiotic reinitiation, starfish oocytes complete meiosis and then arrest at G1 phase
unless fertilization occurs. The second arrest at G1 phase in mature starfish eggs relies on the Mos‐
MAPK pathway. While degradation of Mos upon fertilization is necessary and sufficient to release the11

G1 arrest, downstream of MAPK two separate pathways are operating to maintain the G1 arrest. One
is a Rsk‐dependent pathway that leads to prevention of entry into S‐phase, through suppression ofloading onto chromatin of Cdc45 which is a major regulator of transition from pre‐RC (pre‐replicativecomplex) to pre‐IC (pre‐initiation complex). And the other is a Rsk‐independent pathway that leads
to prevention of entry into M‐phase, through suppression of synthesis of mitotic cyclins A and B.
Such a “dual‐lock” downstream of MAPK is necessary for G1 arrest due to lack in functional
checkpoint in eggs. So far, this is the first demonstration that the Mos‐MAPK cascade separates into
Rsk‐dependent and Rsk‐independent pathways, thereby arresting the cell cycle prior to fertilization.
Originally, the Mos‐MAPK pathway was first identified in unfertilized mature frog eggs as an essential
component of CSF (cytostatic factor) to arrest the cell cycle at meta‐II. However, the role of its corecomponent, the Mos‐MAPK pathway, has expanded for various second arrests other than meta‐IIarrest, particularly for G1 arrest in starfish and meta‐I arrest in sawfly. Based on these, we now
propose a conceptual extension in the definition of CSF. CSF can be considered as a common
cytostatic arrest factor, irrespective of the particular stage of arrest, in metazoan eggs which are not
fertilized after meiotic reinitiation. The core of CSF is universal Mos‐MAPK, downstream of which
there are variable elements. The rewiring downstream of Mos‐MAPK produces cytostatic arrest at aparticular stage, either meta‐I, meta‐II or G1.
In conclusion, there should be meiosis‐specific modulations in the signalling to cell cycle
regulators. The starfish oocyte system provides insights into both common and unique modulations,thus contributing to the studies of cell cycle control in general.

References
Kishimoto, T. (2003). Cell cycle control during meiotic maturation. Curr. Opin. Cell Biol. 15, 654‐663.
Nishiyama, T., Tachibana, K., and Kishimoto, T. (2010). Cytostatic arrest: Post‐ovulation arrest until
fertilization in metazoan oocytes. In “Oogenesis: The Universal Process” (ed. Verlhac, M.H.), Chap. 14,
pp. 357‐384, Wiley‐Blackwell, UK.

12

Lectures:
Topic 1: Oocytes
Jibak LEE, Tatsuo HIRANO
Regulation of chromosome dynamics by cohesins in mammalian meiosis

15

Takashi MIYANO, Mohammad MONIRUZZAMAN
Activation and inactivation of primordial oocytes

16

Koji SUGIURA, You‐Qiang SU, John J. EPPIG
Oocyte control of granulosa cell function and development

17

Yuji HIRAO, Kosuke IGA, Kenji NARUSE, Naoki TAKENOUCHI
In vitro growth of bovine oocyte‐granulosa cell complexes after vitrification

18

Radek PROCHAZKA
Regulation of pig oocyte maturation by EGF‐like peptides

20

Sugako OGUSHI, Yasuko KATO, Akira NAKAMURA, Mitinori SAITOU
Functional analysis of maternal nucleous in mouse oocytes

21

Petr SOLC, Vladimir BARAN, Gabriela PANENKOVA, Adela SASKOVA, Tereza BOHMOVA,
Alexandra MAYER, Richard M. SCHULTZ, Jan MOTLIK
Aurora‐A regulates MTOCs (centrosome) biogenesis but it does not trigger G2/M
transition in mouse oocytes matured in vivo

22

Martin ANGER
Age‐related aneuploidy in mammalian oocytes

23

13

14

Regulation of chromosome dynamics by cohesins in mammalian meiosis
Jibak LEE and Tatsuya HIRANO
Chromosome Dynamics Laboratory, RIKEN Advanced Science Institute, Wako, Japan
Meiosis is a sexual reproduction process, in which haploid gametes (sperms and eggs) areproduced from diploid germ cells (spermatocytes and oocytes). For the reduction of chromosome
number, two successive meiotic divisions (meiosis I and II) follow a single round of DNA replication.
Meiotic chromosomes display unique behaviors particularly in meiosis I: homologous chromosomes
pair, synapse, and recombine with their partners during prophase I. As a result, homologous
chromosomes are connected with their partners by inter‐sister arm cohesion distal to chiasmata,thereby leading to the formation of bivalent chromosomes by metaphase I. At the onset of anaphaseI, resolution of the inter‐sister arm cohesion causes separation of homologous chromosomes while
sister chromatids are kept attached at their centromeric regions. The sister chromatids eventually
separate from each other when the centromeric cohesion is resolved in meiosis II.
Central to the regulation of the program of meiotic chromosome dynamics are cohesins, aclass of multi‐subunit protein complexes that play a main role in sister chromatid cohesion. In thispresentation, we will introduce two topics about cohesins in mammalian meiosis. The first topic
concerns recent advance in our understanding of the mechanism underlying the unique fashion of
meiotic chromosome separation in mammals. This mechanism involves the meiosis‐specific cohesin
subunit REC8, a counterpart of RAD21, and its centromeric protector called shugoshin (SGO2). In
mice, REC8 is localized along the inter‐sister‐chromatid axes at metaphase I. At anaphase I, REC8 islost from chromosome arms, but remains at the centromeres (Lee et al., 2003). Disruption of
separase, which cleaves RAD21 in mitosis, causes nondisjunction of homologous chromosomes atanaphase I, suggesting that cleavage of REC8 by separase is needed for homologous chromosome
separation in meiosis (Kudo et al, 2006). On the other hand, REC8 at centromeric regions survives
throughout meiosis I and remains until the onset of anaphase II. The protection of centromeric REC8is achieved by SGO2, since depletion of SGO2 by siRNA in oocytes causes the loss of centromeric
REC8 and precocious separation of sister chromatids at anaphase I (Lee et al., 2008). These studies,
together with other reports in yeasts, reveal that the basic mechanism of sister chromatid cohesion,
protection and separation in meiosis is essentially conserved from yeasts to mammals. The second
topic is about an unpublished study concerning a new cohesin subunit that we have very recently
identified as meiosis‐specific one. Our results allow us to propose that multiple forms of cohesins
coordinate proper progression of chromosomal events in mammalian meiosis.

15

Activation and inactivation of primordial oocytes
Takashi MIYANO and Mohammad MONIRUZZAMAN
Graduate School of Agricultural Science, Kobe University, Kobe, Japan
Ovaries of newborn animals contain a huge number of non‐growing oocytes in primordial
follicles (primordial oocytes). Some of them are selected to enter the growth phase (1st selection)and reach to their final size, while others become quiescent in the primordial follicles and are served
as the future stockpile. After animals reach puberty, small number of oocytes in primordial follicles in
the quiescent pool are activated to start to grow (2nd selection) and then some of the fully‐grownoocytes resume meiosis by the stimulation of gonadotropins to mature to metaphase II. This
recruitment of primordial oocytes continues throughout the female reproductive life. The
mechanisms underlying the above two selections of primordial oocytes are not understood well. We
have conducted a series of experiments of pig oocyte growth using in vitro or xeno‐transplantation
systems, and found that primordial oocytes in prepubertal pigs (6‐month‐old) took a longer time toinitiate their growth comparing those in neonatal animals (10‐ to 20‐day‐old). We think that
primordial oocytes in adults are different from those in neonatal animals.
A transcription factor FOXO3 has been implicated as one of the molecules responsible for thedormancy of primordial oocytes in mouse ovaries, since Foxo3‐/‐ female mice exhibited a distinctive
ovarian phenotype of global follicular activation leading to oocyte death [1]. In prepubertal pigs,
FOXO3 was detected in almost all (94+/‐2%) primordial oocyte nuclei, and in neonatal pigs, 42+/‐7%
primordial oocytes were FOXO3 positive. Further, FOXO3‐knockdown primordial follicles from
prepubertal pigs by the specific siRNAs developed to the antral stage accompanied by oocyte growthin the xenografts faster than those in the control [2]. The results suggest that ovaries of neonatal pigs
contain a mixed population of quiescent and activated primordial oocytes, and almost all theprimordial oocytes are quiescent in prepubertal/adult pigs, and that a transient knockdown of the
FOXO3 activates the primordial oocytes to enter the growth phase.
Usually, all of the primordial oocytes do not start to grow at the same time in the mammalianovary. Mammals have a long reproductive life span and the ovary is required to maintain a pool of
non‐growing oocytes throughout the female reproductive life. Most of the primordial oocytes
become dormant by a FOXO3‐related mechanism to establish a non‐growing oocyte pool in the ovaryfor the future recruitment, and perhaps the oocytes that have escaped the mechanism enter thegrowth phase at the 1st selection. After puberty, the quiescent primordial oocytes are activated atthe 2nd selection by certain stimulatory factors that inhibit or overcome the FOXO3‐related
inhibitory mechanism.
References
1. Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA. Suppression of ovarian follicle
activation in mice by the transcription factor Foxo3a. Science. 2003; 301:215‐8.
2. Moniruzzaman M, Lee J, Zengyo M, Miyano T. Knockdown of FOXO3 induces primordial oocyte
activation in pigs. Reproduction. 2010;139: 337‐48.

16

Oocyte control of granulosa cell function and development
Koji SUGIURA1,2, You‐Qiang SU1 and John J. EPPIG1
1

The Jackson Laboratory, Bar Harbor, ME, USA. 2Laboratory of Applied Genetics, Graduate Schoolof Agricultural and Life Science, The University of Tokyo, Tokyo, Japan

Mouse oocytes play a critical role in regulating granulosa cell function and development bysecreting several growth factors, including bone morphogenetic protein 15 (BMP15), growthdifferentiation factor 9 (GDF9), and fibroblast growth factors. In addition to the oocyte signals,
estrogen signaling is required for normal development of granulosa cells; however, coordination ofthese signals during follicular development was ill‐defined.
Expansion of cumulus cells, which requires expression of Has2 mRNA, is a prerequisite
process for normal ovulation. Participation of oocyte‐derived factors and estrogen is required for
acquisition and/or maintenance of the cumulus expansion competence. Therefore, to investigate
coordination of oocyte and estrogen signaling during follicular development, effects of estradiol (E2)and oocytes on development and function of cumulus cells were assessed using cumulus expansion
as a marker.
Exogenous E2 maintained competence to undergo expansion and to express full levels of
Has2 mRNA by cumulus cell‐oocyte complexes cultured in vitro. In contrast, exogenous E2 could not
maintain full expansion competence of isolated cumulus cells unless co‐treated with denuded
oocytes or recombinant GDF9 and BMP15. Furthermore, exogenous E2 promoted less‐differentiatedgranulosa cells from preantral follicles to become competent to undergo expansion in vitro.
Moreover, transcripts encoding NRIP1, a suppressor of estrogen receptor signals, were decreased incumulus cells by oocytes or GDF9 in vitro.
Therefore, oocytes‐derived GDF9 and BMP15 coordinate with the E2 to control granulosa cellfunction and development, and oocyte suppression of Nrip1 expression in cumulus cells may be oneof the mechanisms of oocyte and E2 signal crosstalk. Supported by grant HD23839 from the Eunice
Kennedy Shriver NICHD.

17

In vitro growth of bovine oocyte‐granulosa cell complexes after vitrification
Yuji HIRAO, Kosuke IGA, Kenji NARUSE, Naoki TAKENOUCHINational Agricultural Research Center for Tohoku Region (NARCT), Morioka, Japan
Introduction
The in vitro growth of oocytes and the cryopreservation of the growing oocytes are both
important technologies to be fully developed in the near future. Then the combination of them will
expand significantly the availability of mature oocytes. Studies of oocyte growth in vitro, and its
application to the vitrificaiton of growing follicles, have been conducted largely with mice [1]; thistendency is probably due to the ease in handling and culturing mouse oocytes compared to thebovine oocytes. Cryopreservation of growing follicles in other species has been reported [2], but
usually the oocytes were not followed up until they acquire the competence to mature. In thepresent study, we tested the possibility of preservation of growing bovine oocytes by using a step‐
wise vitrification, followed by a 14‐day growth culture using a system developed for bovine oocytes[3].
Materials and Methods
Bovine oocyte‐cumulus/granulosa cell complexes, hereafter referred to as simply complexes,
were dissected out from early antral follicles (0.5‐0.8 mm in diameter) in a Hepes‐buffered modified
Minimum Essential Medium, and subjected to a ten‐step equilibration treatment as previously
reported for fully grown immature mouse oocytes by Aono et al [4]. The eventual equilibration
solution contained 3% (v/v) ethylene glycol and 3% (v/v) DMSO. The complexes were thentransferred to a vitrification solution consisting of 15% ethylene glycol, 15% DMSO and 0.5 M
sucrose, and were placed onto the membrane of Millicell plates with a minimum volume of thesolution. After storage in liquid nitrogen, the complexes were thawed and washed twice in themedium described below.
In vitro culture of the complexes after thawing was performed as follows: the complexes
were placed on Millicell culture inserts and cultured for 14 days in a modified TCM199 supplemented
with 2 mM hypoxanthine, 5% fetal bovine serum, and 4% (w/v) polyvinylpyrrolidone (molecularweight: 360 000) [3]. The cultures were housed for 14 days in an atmosphere of 5% CO2 at 38.5°C,
but the O2 tension was switched from 5% to 20% after the first 3 days of culture. The oocyte
diameter was measured on Day 1 (the day of thawing designated Day 0); this measurement was toscreen out the oocytes with any signs of degeneration and to culture only oocytes 92–106 µm in
diameter. On Day 14, the oocytes showing no signs of degeneration were subjected to final size
measurement and in vitro maturation for 23 h.
Results and Discussion
The overall efficiency of the oocyte (and complex) recovery after vitrification and thawing
was 88% (72/82), as assessed by its morphology on Day 1. The viable complexes adhered to thesubstratum and grew outward. About a week later a characteristic dome‐like structure of granulosa
cells was formed, each containing a normal looking oocyte‐cumulus cell complex.
The percentage of oocytes/complexes surviving through the 14‐day culture period was 89%(64/72). The mean oocyte diameter on Day 1 was 100.0 µm, and the final size was 116.4 µm. This is asignificant increase in size, but smaller than that attained by unvitrified control oocytes, whose mean
diameter had increased from 101.3 µm on Day 1 to 119.4 µm on Day 14. The percentages of theviable oocytes progressing to metaphase II with a polar body emission after the subsequent 23‐hculture were 52% (33/64) and 50% (19/38) for the vitrified group and unvitrified control group,
respectively.
Several lines of evidence have indicated that the appropriate bidirectional interactions
between oocytes and granulosa cells are essential for normal development of the oocytes. The
results of the present study, exhibiting the oocyte viability, growth, maturation, and dome‐like18

structure formation, suggest that the functional associations between the oocytes and granulosa
cells were successfully preserved, or recovered, after the vitrification and thawing.
Conclusion
The present study clearly indicates that the growing bovine oocytes, isolated from early
antral follicles, can acquire the meiotic competence during the 14‐day culture after the vitrificationand thawing process
References
1) dela Pena EC, Takahashi Y, Katagiri S, Atabay EC, and Nagano M (2002) Birth of pups after transfer
of mouse embryos derived from vitrified preantral follicles. Reproduction, 123: 593‐600.
2) Santos RR, Tharasanit T, Van Haeften T, Figueiredo JR, Silva JRV, Van den Hurk R (2007)
Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid‐
surface vitrification methods Cell Tissue Res, 327: 167‐176.
3) Hirao Y, Itoh T, Shimizu M, Iga K, Aoyagi K, Kobayashi M, Kacchi M, Hoshi H, Takenouchi N (2004) In
vitro growth and development of bovine oocyte‐granulosa cell complexes on the flat substratum:
effects of high polyvinylpyrrolidone concentration in culture medium. Biol Reprod, 70: 83‐91.
4) Aono N, Abe Y, Hara K, Sasada H, Sato E, Yoshida H (2005) Production of live offspring from mouse
germinal vesicle‐stage oocytes vitrified by a modified stepwise method, SWEID. Fertil Steril, 84
Suppl 2: 1078‐1082.

19

Regulation of pig oocyte maturation by EGF‐like peptides
Radek PROCHAZKA
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic,
277 21 Liběchov, Czech Republic

Recent data indicate that LH‐induced events in preovulatory follicles are mediated indirectly:
LH binds to its receptor on mural granulosa cells and induces expression of Areg, Btc and Ereg. By
autocrine and paracrine mechanisms, these EGF‐like peptides induce expression of prostaglandin‐
endoperoxidase synthase 2 (Ptgs2) in both mural granulosa and cumulus cells, leading to increased
production of prostaglandin E2 (PGE2). PGE2 binds to its receptor on cumulus cells activating MAPK
14 and consequently stimulating production of AREG and EREG in cumulus cells. These peptides thenbind on EGF receptor of cumulus cells and in the autocrine manner stimulate transcription of genes
involved in regulation of meiotic resumption and cumulus expansion.
The aim of this work was to assess FSH‐stimulated expression of EGF‐like peptides in cultured
cumulus‐oocyte complexes (COCs) and to find out an effect of the peptides on activation of kinases in
the COCs, cumulus expansion, oocyte maturation and acquisition of developmental competence invitro. FSH significantly stimulated expression of Areg and Ereg, but not Btc in cultured COCs.
Expression of Areg and Ereg reached maximum at 2 and 4 h after FSH addition, respectively, no
significant increase in Areg, Ereg and Btc expression was found in COCs beyond 4 h of culture. FSH
significantly stimulated in the COCs expression of expansion related genes (Ptgs2, Tfnaip6 and Has2)
at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also
stimulated a prompt increase in expression of the genes, the dynamics of the expression was
somewhat different for EREG‐induced expression of Tfnaip6 and Has2, that persisted high at 20 h ofculture. In contrast to FSH, the AREG and EREG did not stimulate in the COCs expression of Cyp11A1
and an increase in progesterone concentration in culture medium. The EGF‐like peptides stimulated
a prompt activation of MAPk 3/1 and AKT kinase, but not activation of PKA. AREG and EREG
stimulated maturation of the oocytes and expansion of the cumulus cells, although the percentage ofoocytes that had reached metaphase II was significantly lower when compared to FSH‐induced
maturation. Parthenogenetic development to blastocyst stage of oocytes matured with AREG and
EREG or with combination of FSH and the EGF‐like factors was better then the development ofoocytes matured with FSH only. This feature was well documented by comparison of the proportionsof blastocysts among the cleaving activated oocytes.
We conclude that the EGF‐like factors do not reproduce all events elicited in the cultured
COCs by gonadotropins (FSH) – they do not stimulate activation of PKA, in vitro production ofprogesterone by cumulus cells and are not as efficient as the FSH in stimulating meiosis resumption
and completion. However, the oocytes that complete maturation following the stimulation by AREG
and EREG possess higher developmental potential than the oocytes stimulated by FSH only. Optimal
conditions for maturation of pig COCs in vitro and subsequent parthenogenetic development werereached in medium supplemented by both FSH and the EGF‐like factors.
Supported by GAČR No. 523/08/0111 and NAZV 101A166.

20

Functional analysis of maternal nucleolus in mouse oocytes
Sugako OGUSHI1, Yasuko KATO1, Akira NAKAMURA1, Mitinori SAITOU2
1

Laboratory for Germline Development , RIKEN Center for Developmental Biology, Kobe, Japan.
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto,
Japan.

2

The nucleolus is the site of ribosomal RNA (rRNA) synthesis and processing and ribosomal
subunit assembly. During growth in the ovary, the oocyte possesses a fibrillo‐granular nucleolus withactive transcription and ribosome biogenesis and stockpiles the proteins and RNAs for early
embryonic development. When the oocyte reaches full size, indicating that it has acquired
developmental competence, all its transcription ceases, including the transcription of rRNA.
Apparently, its nucleolus no longer contains DNA at this point and shows a highly compacted, fibrillar
morphology. As the nucleolus is eliminated during spermiogenesis, the spermatozoa do not havenucleoli. After fertilization, this maternally derived, transcriptionally inactive fibrillar nucleolus
appears in both male and female pronuclei and in the embryonic nuclei of each interphase until the4‐ or 8‐cell stage; that is the time at which zygotic RNA polymerase I activity is fully established.
When we produced an embryo lacking this inactive nucleolus, the embryo failed to develop
past the first few cleavages, indicating the requirement of the oocyte nucleolus for preimplantation
development. However, the detail timing of this nucleolus requirement in early embryonicdevelopment and function of inactive nucleolus are not fully understood.
We reinjected the nucleolus into oocytes and zygotes without nucleoli at various time points
to examine the timing of the nucleolus requirement during meiosis and early embryonicdevelopment. When we put the nucleolus back into oocytes lacking a nucleolus at the germinal
vesicle (GV) stage and at second metaphase (MII), these oocytes were fertilized, formed pronuclei
with nucleoli and developed to full term. When the nucleolus was reinjected at the pronucleus (PN)
stage, most of the reconstructed zygotes cleaved and formed nuclei with nucleoli at the 2‐cell stage,
but the rate of blastocyst formation and the numbers of surviving pups were profoundly reduced.
Moreover, the zygotes without nucleoli showed a disorder of higher chromatin organization not onlyin the female pronucleus but also, interestingly, in the male pronucleus. Thus, the critical time point
when the nucleolus is required for progression of early embryonic development appears to be at the
point of the early step of pronucleus organization.
To find oocyte nucleolus function, we focused on one protein, Nucleoplasmin2 (Npm2).
Npm2 knockout mouse has defect in oocyte‐nucleolus formation and in fertility. We found that theoocyte nucleolus contains Npm2 proteins, and the expression level of this protein is quite high inoocytes. To better understand oocyte nucleolus function, we sought to identify Npm2 associating
proteins in nucleolus by yeast two hybrid screening, and found interacted proteins. Our progress inunderstanding the nucleolus function will be presented.

21

Aurora‐A regulates MTOCs (centrosome) biogenesis but it does not trigger G2/M
transition in mouse oocytes matured in vivo
Petr SOLC1*, Vladimir BARAN2, Gabriela PANENKOVA1, Adela SASKOVA1, Tereza BOHMOVA1,
Alexandra MAYER1, Richard M. SCHULTZ3 and Jan MOTLIK1
1

Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic,
Libechov, Czech Republic, * e‐mail: solc@iapg.cas.cz
2
Institute of Animal Physiology, Slovak Academy of Sciences, Soltesovej 4‐6, 040 01 Kosice, Slovakia
3
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104‐6018, USA
Aurora‐A kinase (AURKA) is an important mitotic kinase involved in G2/M transition,
centrosome separation and spindle formation in somatic cells. Almost completely all known
information about a role of AURKA in cell cycle regulation is derived from in vitro cultured humancancer cells. Using transgenic models we provide insight on the physiological role of AURKA during
meiotic maturation (meiotic cell cycle progression) in mice. By means of Cre‐lox system we havegenerated mice with expression of both wild‐type (wt) and kinase‐dead (kd) aurka transgenes
specifically only in ovarian oocytes.
Intrafollicularly, control oocytes are arrested at prophase I and they resume meiosis in vivo (G2/M
transition) after LH/hCG stimuli. The first AURKA activation occurs shortly after LH/hCG peak. The
initial activation of AURKA is CDK1 independent but full activation of AURKA requires CDC25B
mediated CDK1 activation. The control oocytes contain 1‐3 microtubule organizing centres (MTOCs;
centrosome equivalent) at prophase I. At the time of nuclear envelope break down (NEBD), a firstvisible marker of meiosis resumption, MTOCs number increases up to several dozen (MTOCs
multiplication). MTOCs multiplication requires full AURKA activation and in this way it is dependent
on CDC25B.
Although expression of wt aurka transgene leads to premature activation of AURKA at
prophase I oocytes they do not resume meiosis without LH/hCG stimuli. After wt aurka transgene
expression in mouse oocytes, MTOCs multiplication occurs prematurely at prophase I without NEBD
although both control and kd aurka mice exhibit usually 1‐3 MTOCs per oocytes. At metaphase I
stage both wt aurka and kd aurka oocytes have more gamma‐tubulin. Both wt aurka and kd aurkatransgenic mice have normal fertility during first 7 mouth of their life. It suggests that the AURKA
induced premature MTOCs multiplication does not interfere with developmental competence ofoocytes. In conclusion these data imply that AURKA activity is responsible for physiological MTOCs
multiplication and that AURKA regulates independently of its kinase activity the amount of gamma
tubulin. AURKA is not a trigger kinase for G2/M transition in mouse oocytes.
This study was supported by the Czech‐U.S. cooperation grant ME08030.

22Age‐related aneuploidy in mammalian oocytes
Martin ANGER
Institute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic

It has been shown that mammalian meiosis is associated with a high incidence of errors
leading to chromosomal aberrations, which are more prevalent in female gametes and seem toincrease with maternal age. Oocyte aneuploidy has severe consequences, including pregnancy loss orbirth defects, and is probably originated in two steps during the oocyte development. According tothis theory, first error, created early in meiosis, for example during recombination, might lead toaneuploidy because of a failure of checkpoint mechanisms controlling chromosome segregation after
resumption of meiosis. Since our knowledge of chromosome segregation in meiosis is coming
predominantly from studies on yeasts, any research aimed at the etiology of oocyte aneuploidy mustbe focused first on characterization of the basic molecular mechanisms controlling meiotic programin mammals.
In meiosis, the reduction of number of chromosomes is achieved by combining two nuclear
divisions without DNA replication in between. Successful segregation of chromosomes during thisprocess is crucially dependent on stepwise removal of cohesion between sister chromatids, mediatedby cohesin complex. In order to ensure segregation of homologous chromosomes in meiosis I, andsister chromatids in meiosis II, cohesion between chromosome arms is removed during first meiosis,
whereas centromeric cohesion is maintained until second meiosis. We were able to show that in
mammalian meiosis, removal of cohesin complex during meiosis I, is dependent on proteolytic
cleavage of its Rec8 subunit by Separase. Deleting Separase from the oocyte or replacing Rec8 with
its uncleavable version would disrupt chromosome segregation. It was also hypothesized that theerrors in chromosome segregation in female gametes might be caused by malfunction of Spindle
assembly checkpoint (SAC), which in somatic cells is essential for monitoring of the correct
attachment of the chromosomes to the spindle. We have shown that intact SAC is essential in mouse
oocytes and its absence causes premature anaphase onset accompanied by massive chromosome
segregation defects. We now focus on characterization and quantification of changes in oocyte
meiosis specifically reflecting maternal aging. Using techniques, which can be applied on a single cell,such as live cell imaging or kinase assays, we are able to reveal subtle changes introduced bymaternal aging into meiosis and to address which components of the meiotic cell cycle machinery are
more susceptible to emphasize errors accumulated early during meiosis.

23

24

Lectures:
Topic 2: Fertilization
Junya ITO, Rafael A. FISSORE, Naomi KASHIWAZAKI
Factors associated with calcium oscillations during fertilization in mammals

27

Konosuke OKADA
Vole sperm cryopreservation: assessments of viability, sperm DNA integrity and fertilizing
capacity28

Tereza TORALOVA, Andrej SUSOR, Lucie NEMCOVA, Katerina KEPOKOVA, Jiri KANKA
The role of CENPF in bovine preimplantation development

29

Helena FULKA
From oocyte to embryo – remodeling the parental genomes

30

25

26

Factors associated with calcium oscillations during fertilization in mammals
Junya ITO1,2, Rafael A. FISSORE3 and Naomi KASHIWAZAKI1,2
Laboratory of Animal Reproduction, 1School of Veterinary Medicine,
Graduate School of Veterinary Science, Azabu University, Sagamihara, Japan.
3
Department of Veterinary & Animal Sciences, University of Massachusetts, Amherst, USA
2

The activation of the egg is the first stage in the initiation of embryo development. It
comprises a series of events that unfold soon after interaction of the gametes and that end with thecompletion of meiosis and progression onto the mitotic cell cycles. In all species studied to date, egg
activation requires a fertilization‐associated increase in the intracellular concentration of calcium([Ca2+]i). In mammals, the fertilizing [Ca2+]i signal consist of periodicals rises, which are also referred
to as [Ca2+]i oscillations. It is well known that at least two molecules (oocytes: type 1 inositol 1,4,5‐
trisphosphate receptor [IP3R1], sperm: phospholipase C zeta [PLC ]) are involved in the regulation of
calcium oscillations.
In vertebrate oocytes, it has been reported that IP3R1 is expressed at both mRNA and protein
levels. IP3R1 in mammals, or its homologue in other species, is responsible for the majority of Ca2+
release during fertilization. Production of inositol 1,4,5‐trisphosphate (IP3), the IP3R1 ligand, isdetected during fertilization, and entails hydrolysis of phosphatidylinostitol (4,5)‐bisphosphate by theaction of a phospholipase C (PLC). We previously reported that during maturation in mouse oocytes
the IP3R1 becomes phosphorylated at an MPM‐2 epitope, and that this phosphorylation persists until
the fertilization‐associated [Ca2+]i responses cease. We also found a role for the ERK pathway, as
oocytes matured in the absence of ERK activity showed decreased IP3R1 MPM‐2 reactivity andoscillatory ability. From these results, it is suggested an interdependence between the M‐phasekinases active during maturation, IP3R1 MPM‐2 phosphorylation and [Ca2+]i release. We recently
examined which kinase is responsible for IP3R1 MPM‐2 phosphorylation and how the ERK pathway
contributes to this phosphorylation. We found that IP3R1 is a novel target for Polo‐like kinase 1(Plk1), an M‐phase kinase with broad functions during meiosis/mitosis and cytokinesis. Plk1
phosphorylates IP3R1 at an MPM‐2 epitope, and these proteins interact in an M‐phase preferential
manner. IP3R1 and Plk1 co‐localize on the meiotic spindle poles, and on other sites in the ooplasm,
and this overlap, as well as IP3R1 MPM‐2 phosphorylation is reduced in oocytes matured without ERK
activity; these data demonstrate a novel regulatory role of the ERK pathway on IP3R1 reorganization.
We therefore propose that IP3R1 phosphorylation by Plk1, and possibly by other M‐phase kinases,
makes possible the delivery of spatially and temporally regulated [Ca2+]i signals to ensure faithfulprogression/completion of meiosis/mitosis and cytokinesis.
In addition, we focused on molecular mechanism of another factor which was derived from
sperm, is a sperm‐specific PLC isoform, PLCζ. During mammalian fertilization, PLCζ, is reportedly
responsible for IP3 production. After the discovery in the mouse, PLCζ has been identified in not only
mammalian species (rats, pigs, cows, monkeys, humans) but also non‐mammalian species (medaka,
chickens, quails). Injection of PLCζ mRNA and protein induced Ca2+ oscillations in mouse oocytes. On
the other hand, it has been demonstrated that PLC ‐deficient sperm in humans could not induce Ca2+
oscillations. These results lead us to the possibility that PLC is a sole factor which is required for Ca2+
oscillation. In domestic animals, injection of PLC into oocytes clearly showed induction of Ca2+
oscillations in the pig and the cattle. During fertilization, releasing of PLCζ from sperm was rapidly(probably within a first hour) occurred after sperm‐oocyte fusion. By using fluorescence‐tagged PLCζ,it has been showed that PLCζ is concentrated to the pronucleus (PN) formed and Ca2+ oscillations
were terminated in most of the mouse eggs before initiation of the accumulation. However, contrary
to mice, PLCζ in other species is never concentrated to PN. This result strongly suggests that species‐
specific difference is existed and molecular mechanism of PLC in other species is required to beclarified. We recently succeeded in molecular cloning of PLCζ in the horse. Equine PLCζ showedmuch higher activity than those in other species have been reported up to date.
In this symposium, we will show recent results about IP3R1 and PLC and discuss how Ca2+
oscillation is regulated by these molecules.
27

Vole sperm cryopreservation: assessments of viability, sperm DNA integrity andfertilizing capacity
Konosuke OKADA
Faculty of Applied Life Science, Nippon Veterinary and Life Science University, Musashio, Japan
Recently, a number of animals/plants have been rapidly threatened and the number of
threatened animals is more increasing. With regard to mammalian species described previously (total
5490 species), 20.8% (1142 species) of them are threatened and 1.4% (78 species) had alreadybecome extinct according to Red List (the International Union for Conservation of Nature; IUCN,
2009). In addition, half of mammals listed are small species, including rodents. Except forexperimental animals, however, small species have attracted less notice than larger species for astudy of conservation‐technology, although smaller animals are thought to be more vulnerable toenvironmental changes. We hence think that even small animals the research and maintenance ofthem might indirectly contribute to conservation of biological diversity, since the loss of biologicaldiversity would lead serious disadvantage for human life in future.
Vole (Animal; Chordata; Vertebrate; Mammallia; Rodentia; Muridae; Arvicolinae; Microtus),
small animal, widely inhabit from the Frigid Zone to the Temperate Zone and total 64 strains havebeen discovered so far. Ten of sixty‐four strains have been classified into the endangered category in
Red List (IUCN, 2009); M. bavaricus M. oaxacensis M. umbrosus M. breweri, M. schelkovnikovi, M.
quasiater, M. cabricus, M. sachalinensis, M. guatemalensis, and M. kikuchi. In our laboratory, threestrains have been maintained; M. montebelli (Japanese grass vole), M. arvalis (common vole), andM. rossiaemeridionalis (southern vole).
Microtus has been expected as a model for metabolic disease in herbivorous animal, since
this species is also herbivorous and possesses a complex stomach that is similar to ruminant animals,
although field vole dose not ruminate. Moreover, the chromosome number differs with strains; forinstance, Japanese grass vole, common vole, and southern vole that are maintained in our laboratory
possess 30, 46, and 54 chromosomes, respectively, and the analysis of speciation has been also
performed utilizing this variation of chromosome number. On the other hand, there are only a few
reports involved with its reproduction, characteristic of gametes, and related reproductive
technologies.
Our final goal is to establish the methods of preservation and regeneration in this species as
an animal genetic resource. To attain this objective, we firstly attempted to cryopreserve
spermatozoa isolated from Japanese grass vole and common vole using a conventional protocol formouse sperm cryopreservation. After frozen‐thawed, the rate of vole sperm viability assessed bynigrosin‐eosin staining was similar to control mouse, although the viability of frozen‐thawed
spermatozoa was significantly lower than that of fresh and such decline was equally to control.
Secondly, an alkaline comet assay was used to evaluate whether vole sperm DNA integrity wasprotected after frozen‐thawed. In both Microtus species, the rate of sperm DNA with a comet tail(this means damaged DNA) significantly increased after cryopreservation. Moreover, the rate ofdamaged sperm DNA from Japanese grass vole was higher than a result from common vole. Finally,
we used the interspecies ICSI procedure with mouse oocytes to test the fertilizing capacity ofcryopreserved vole spermatozoa, because of unstable collection of vole oocytes matured in vivo.
Most of mouse oocytes successfully injected with a single vole spermatozoon resumed meiosis,
extruded the second polar body, and formed female and male pronuclei. Taken together, volespermatozoa can be successfully cryopreserved using a conventional protocol in mouse, although theeffect of freezing‐thawing on vole sperm DNA may differ with strains. In addition to above results,characteristic of female gametes in Microtus will be illustrated in this symposium.

28

The role of CENPF in bovine preimplantation development
Tereza TORALOVA, Andrej SUSOR, Lucie NEMCOVA, Katerina KEPKOVA and Jiri KANKA
Institute of Animal Physiology and Genetics AS CR, v.v.i., Liběchov
CENPF (centromeric protein F; mitosin) is a large multifunctional protein that plays a key role
during cell division. It is expressed and localized in cell‐cycle‐dependent manner with peak at theG2/M phase boundary and is degraded at the end of mitosis. CENPF is one of the earliest proteinsassociated with kinetochores and helps to form the correct kinetochore–microtubule interactions.
The embryonic CENPF mRNA expression is started during the major genome activation at late 8‐cellstage. At the beginning of mammalian embryonic development all mRNAs are of maternal origin, theembryonic genome is activated during an event called embryonic genome activation (EGA), whichtakes place at late 8‐cell stage in cattle. The genes that are activated at this stage are supposed to beimportant for the preimplantation development.
Identification of these genes is essential for understanding early mammalian embryogenesis. Up tonow, several genes activated during EGA were identified, but only a small portion of them has beenstudied more thoroughly to uncover their exact role during preimplantation development. In order
to investigate the importance of CENPF, we have silenced the CENPF mRNA in bovine zygotes by
CENPF dsRNA injection and monitored the developmental competence in the embryos.
We showed that the embryos are able to develop without any developmental impairment until the
8‐cell stage (EGA). However, the developmental competence rapidly decreased then, and only 28.1%
of 8‐cell embryos with silenced CENPF mRNA were able to develop to 16‐cell stage or beyond (72% incontrol groups).
In order to understand what enables the development until the 8‐cell stage we used atranslational inhibitor cycloheximide (CHX). The CHX treatment of embryos before EGA does notchanged the intensity of CENPF immunofluorescence staining. However, in CHX treated post‐EGA
embryos, the intensity of staining was markedly decreased. This suggests that in pre‐EGA embryos,
CENPF is not degraded at the end of cell cycle, while in post‐EGA embryos, the protein behaves in thesame manner as in somatic cells, i.e. is degraded at the end of cell cycle. The latter statement was
further confirmed by immunofluorescent analysis, which showed the somatic‐cell‐like localizationand expression of CENPF in post‐EGA embryos.
These results show that microinjection of CENPF dsRNA leads to mRNA and protein silencing
in preimplantation embryos. The inhibition of CENPF mRNA does not influence the development ofembryos until 8‐cell stage, however the development thereafter is considerably deteriorated and the
majority of embryos arrest before reaching the 16‐cell stage. Hence, it shows that the expression of
CENPF mRNA is necessary for proper preimplantation development.
Supported by GACR 523/09/1035 and GACR 204/09/H084

29

From oocyte to embryo ‐ remodeling the parental genomes.
Helena FULKA
Institute of Animal Science, Prague, Czech Republic
Gametogenesis and early embryonic development in mammals are characterized byextensive epigenetic remodeling. Although many issues concerning such processes remainunresolved, it is clear that a functional link exists between this remodeling, gamete quality and/or
developmental potential of embryos. Thus, major epigenetic remodeling events take place during
four main phases – gametogenesis, fertilization, early embryonic development up to the blastocyst
stage and during the first differentiation at the blastocyst stage. During male gametogenesis, most
histones are replaced by protamines and the sperm DNA is packaged to almost crystalline state. On
the other hand, the oocyte genome is organized by histones throughout the whole phase of
gametogenesis – the only clear difference when compared to somatic cells is the absence of somatic
type linker histones. The above described changes go hand in hand with transcriptional silencing inboth male and female gametes. After fertilization both parental genomes decondense and the sperm
protamines are replaced by histones stored in the oocyte cytoplasm. At this phase, the parental
genomes are epigenetically unequal: the maternal genome retains high levels of DNA methylationand shows the presence of repressive histone modifications whereas the paternal genome
undergoes active DNA demethylation and exhibits the lack of repressive histone modifications andthe presence of the histone variant H3.3. Thus, although both parental genomes share a common
environment (zygotic cytoplasm) clear epigenetic asymmetry between parental genomes can bedetected. During subsequent divisions the parental genomes are combined and become intermingled‐ the epigenetic asymmetry is probably largely lost and equalized. This period is characterized bypassive DNA demethylation (replication dependent DNA demethylation) and global loss of certain
repressive histone modifications. On the other hand, some genes (imprinted genes) or specialized
structures (X chromosome) escape this phase of epigenetic equalization. Finally, a third phase of
epigenetic remodeling during early embryogenesis takes place during the blastocyst formation when
the differentiation of the trophectoderm (TE) and of the inner cell mass (ICM) occurs. These twolineages differ in the mode of X chromosome inactivation (random in ICM vs. imprinted in TE), in thelevel of DNA methylation and in the presence of certain histone modifications. Although we cancurrently only speculate about the impact of individual remodeling steps, it is possible that these
event influence processes such as embryonic genome activation and thus successful embryonicdevelopment. Moreover, it is clear that the epigenetic remodeling and erasure of epigenetic marks
during early embryogenesis is necessary for epigenetic resetting leading to totipotency of the early
embryo as both gametes are highly specialized and terminally differentiated cells.

30

Lectures:
Topic 3: Stem cells
Ales HAMPL
Human embryonic stem cells

33

Zuzana KOLEDOVA, Leona RASKOVA KAFKOVA, Alwin KRAMER, Vladimir DIVOKY
Centrosomal decisions on self‐renewal in mouse embryonic stem cells

34

Petr VODICKA, Katerina VODICKOVA‐KEPKOVA, Jan MOTLIKiPS cells in miniature pigs

35

Jana JUHASOVA, Stefan JUHAS, Jiri KLIMA, Jan STRNADEL, Alois NECAS, Robert SRNEC,
Ladislav PLANKA, Evzen AMLER, Eva FILOVA, Josef JANCAR, Jan MOTLIKBone marrow‐derived mesenchymal stem for the regeneration of cartilage and bone36

Helena SKALNIKOVA, Petr VODICKA, Petr HALADA, Martin MARSALA, Jan MOTLIK, Hana
KOVAROVA
Proteomics of neural stem cells isolated from miniature pigs

37

31

32

Human embryonic stem cells suffer from abnormal metabolism of centrosomes
Zuzana HOLUBCOVA1, Pavel MATULA3, Miroslava SEDLACKOVA4, Vladimir VINARSKY1,2, Dasa
DOLEZALOVA1,2, Tomas BARTA1,2, Petr DVORAK1,2, and Ales HAMPL1,2
1

Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Department of Molecular Embryology, Institute of Experimental Medicine, v.v.i., Academy of
Sciences of the Czech Republic, Brno, Czech Republic
3
Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Brno, Czech
Republic4
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech
Republic2

When propagated in culture human embryonic stem cells (hESCs) tend to harbor alterations
to their karyotype, which may limit their prospective use in patients. Chromosomal instability ofsome malignancies is considered to be driven, at least in part, by centrosomal overamplifications
perturbing balanced chromosome segregation. Here we for the first time report very high frequencyof mitotic cells with supernumerary centrosomes in cultures of hESCs. This inadequate situation
features strictly undifferentiated state of hESCs and becomes progressively suppressed upon
prolonged propagation in culture. We demonstrate that improved attachment to culture substratum
as well as inhibition of key molecular regulators of centrosomal metabolism, CDK2 and Aurora A,
diminish the frequency of multicentrosomal mitoses. In other words, attenuated cell attachment
and deregulation of machinery controlling centrosome numbers both contribute to centrosomal
overamplification in hESCs. Still, linking the number of centrosomes to the ploidy indicated that not
only overduplication within a single cell cycle but also mitotic failure were involved in generation ofnumerical abnormalities of centrosomes in hESCs. Collectively, our data point to the supernumerary
centrosomes as to be a significant risk factor for cultured hESCs in terms of maintenance of integrityof their genome, and as such should be evaluated when new culture conditions are being
implemented.

Supported by: MSM0021622430, AV0Z50390512, AV0Z50390703, 1M0538

33

Centrosomal decisions on self‐renewal in mouse embryonic stem cells
Zuzana KOLEDOVA1, Leona RASKOVA KAFKOVA1, Alwin KRAMER2 and Vladimir DIVOKY1
1

Department of Biology, Faculty of Medicine, Palacky University, Hnevotinska 3, 775 15 Olomouc,
Clinical Cooperation Unit for Molecular Hematology/Oncology, German Cancer Research Center(DKFZ) and Department of Internal Medicine V, University of Heidelberg, Im Neuenheimer Feld 581,
69120 Heidelberg, Germany
2

Mouse embryonic stem cells (mESCs) have a unique cell cycle structure with a peculiarly
short G1 phase that lacks functional G1 checkpoint; i.e. mESCs do not stop in G1 phase after DNA
damage. In our study, we attempted to investigate causes of G1 checkpoint non‐functionality in
mESCs and the relevance of rapid G1 phase progression for pluripotency of mESCs.
In somatic cells, in response to DNA damage in G1 phase, G1 checkpoint pathways (theChk1/Chk2–CDC25A and p53–p21 pathways) are activated to stop cell cycle progression by
downregulating CDK2 activity. However, as our studies revealed, in mESCs CDK2 activity is refractoryto DNA‐damage. We found out that G1 checkpoint pathways are not functional in mESCs due tocentrosomal sequestration of Chk1/Chk2 proteins and, importantly, localization of a large proportionof CDK2 molecules to centrosomes. Although alternative G1 checkpoint pathways, including p53–p21
and GSK‐3β–CDC25A pathways, were activated in mESCs after DNA damage, they did not impinge on
CDK2 activity, suggesting that centrosomal localization shelters CDK2 from checkpoint mechanisms.
Furthermore, our studies furnished evidence on CDK2 activity in underpinning rapid G1 phaseprogression during unperturbed mESC cycles, driving escape from G1 phase in the conditions of DNA
damage, as well as maintenance of self‐renewal: Downregulation of CDK2 activity slowed down G1
phase progression in mESCs and, importantly, induced somatic cell‐like cell cycle, differentiation‐
associated changes in mESC morphology and expression of differentiation markers.
From our observations, we conclude that centrosomal CDK2 might have a crucial function inmaintenance of self‐renewal in mESC in the conditions of DNA damage and that centrosomes mightplay an important, mediatory role in the cell fate game in mESCs, providing an interface for crucial
cell cycle and cell fate decisions to take place.
This work was supported by grants NR/9508 (Ministry of Health), MSM 6198959205 and
2B06077 (Ministry of Education, Youth and Sport), Palacky University grant LF 2010 013, and by fundsof the Deutsche Krebshilfe (108560) and Deutsche Jose Carreras Leukämie Stiftung (DJCLS R 06/04).

34

iPS cells in miniature pigs
Petr VODICKA1,2, Katerina VODICKOVA‐KEPKOVA1,2, Jan MOTLIK1,2
1

Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov,Czech Republic2
Center for Cell Therapy and Tissue Repair, Prague, Czech Republic

Pigs and miniature pigs are steadily gaining importance as large animal models in the field of
regenerative medicine and in stem cell research. Several somatic stem cell populations, including
mesenchymal stem cells or neural stem cells were successfully isolated in pigs, but establishment ofproper embryonic stem (ES) cell line, fulfilling all classical ES cells landmarks, including germ‐line
transmission remains so far elusive. Porcine ES cells would be useful both for production of gene‐modified animals and for testing potential stem cell therapies. Discovery of induced pluripotency
makes preparation of pig induced pluripotent stem (iPS) cells an attractive alternative to ES cells. We
have taken advantage of our experience with the isolation and culture of fetal porcine neuralprogenitors (NPC) and used them as a starting population for preparation of minipig iPS cells. NPC
express Sox2, one of the factors required for reprogramming into pluripotent state. We havetransduced several porcine NPC lines with either the combination of all four “Yamanaca factors”(Oct4, Sox2, Klf4 and c‐Myc), 3 factor combination without Sox2 or with Oct4 only. Using all thesefactor combinations, we were able to derive cell clones displaying morphology similar to human ES
cells, but proliferating on feeders in LIF containing medium, similar to mouse ES cells. We havechosen one of the clones transduced with 3 factor combination (B8) for further characterization andthe cells displayed alkaline phosphatase positivity, expression of Nanog, endogenous Sox2 and Oct4.
B8 minipig iPS cells formed embryoid bodies when placed in a suspension culture, and further
differentiated when placed onto gelatin coated dishes. Detailed characterization of more cell lines iscurrently under way and we hope to use minipig iPS cells as model system for stem cell therapies.
Acknowledgements
This study was supported by the AS CR project M200450971, Centre for Cell Therapy and Tissue
Repair (1M0538), Grant Agency of the Academy of Sciences of the Czech Republic (grant No.
KJB400500801) and Institutional Research Concept IAPG No. AV0Z50450515.

35

Bone marrow‐derived mesenchymal stem for the regeneration of cartilage andboneJana JUHASOVA1, Stefan JUHAS1, Jiri KLIMA1, Jan STRNADEL1, Alois NECAS2, Robert SRNEC2, Ladislav
PLANKA3, Petr GAL3, Evzen AMLER4, Eva FILOVA4, Josef JANCAR5, Jan MOTLIK1
1

Institute of Animal Physiology and Genetics AS CR, Laboratory of Cell Regeneration and Plasticity,Liběchov, Czech Republic2
Department of Surgery and Orthopaedics, Small Animal Clinic, Faculty of Veterinary
Medicine,University of Veterinary and Pharmaceutical Sciences Brno, Czech republic
3
Clinic of Pediatric Surgery, Orthopedics and Traumatology, The Faculty Hospital Brno, Czech Republic
4
Institute of Experimental Medicine of the Academy of Sciences of the Czech Republic, Prague, Czech
Republic
5
Institute of Materials Chemistry, Faculty of Chemistry, Brno University of Technology, Czech Republic
Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into avariety of cell types (osteoblasts, chondrocytes, tenocytes and adipocytes). They are also the primarycell source for cartilage and bone formation and so may be optimal for cell‐seeded scaffolds and canbe used for bone tissue repair (Sumanasinghe et al., 2006). Several studies reported that porcine or
rabbit MSCs are a valuable model system for mesenchymal basic research and tissue engineering(Ringe et al., 2002; Zhou et al., 2010).
The aim of our research was to obtain essential information on biological properties ofmesenchymal stem cells combined with newly synthesized polymeric biomaterials and nanofibres invivo as well as in vitro. We were focused mainly on development of effective methods of controlled
differentiation of MSCs into chondrocytes, osteocytes and fibrocytes and on testing compatibility
between MSCs and above‐mentioned materials. Composites of biomaterials and MSCs‐derived cells
were implanted into appropriate sites of musculoskeletal system of optimal animal models(miniature pigs and rabbits) to test their effectiveness in the treatment of serious diseases of joints
and bones. Osteogenic and chondrogenic ability of porcine and rabbit MSCs was tested by various
evaluation methods in different cultivation conditions and systems.
We performed an experimental study on New Zealand's white rabbits and miniature pigs tofind differences in the results of treating the distal physeal femoral defect by the transplantation of
autologous or allogeneic mesenchymal stem cells (MSCs) combined with scaffolds (collagen I ‐chitosan). The results showed that the transplantation of both autogenous and allogeneic MSCs into
a defect of the growth plate appears as an effective method of surgical treatment of physeal
cartilage injury. The next study evaluated macroscopically, histologically and immunohistochemicallythe quality of newly formed tissue in iatrogenic defects of articular cartilage of the femur condyle inminiature pigs treated with the clinically used method of microfractures in comparison with thetransplantation of a combination of a composite scaffold with allogeneic mesenchymal stem cells(MSCs) or the composite scaffold alone. The newly formed cartilaginous tissue filling the defects ofarticular cartilage after transplantation of the scaffold with MSCs had in 60% of cases a
macroscopically smooth surface. In all lesions after the transplantation of the scaffold alone or after
the method of microfractures, erosions/fissures or osteophytes were found on the surface.
Our experiments are essential for development of safe and effective technology prior to initiation ofclinical studies or prior to potential clinical use of these biomaterials as a substitutes for irreversibly
damaged cells and tissues in people.
This work was supported by AV0Z50450515, 1M0538, and 2B06130
Ringe J et al. Cell Tissue Res. 2002 Mar;307(3):321‐7.
Sumanasinghe RD et al. Tissue Eng. 2006 Dec;12(12):3459‐65.
Zhou J et al. Biomaterials. 2010 Feb;31(6):1171‐9.

36Proteomics of neural stem cells isolated from miniature pigs
Helena SKALNIKOVA1, Petr VODICKA1, Petr HALADA2, Martin MARSALA3, Jan MOTLIK1, Hana
KOVAROVA1
1

Department of Reproductive and Developmental Biology, Institute of Animal Physiology andGenetics, Academy of Sciences of the Czech Republic, v.v.i., Libechov, Czech Republic
2
Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic
3
Department of Anesthesiology, University of California San Diego, CA, USA
The mechanisms that regulate the stem cell self‐renewal and differentiation are complex andremain mostly unknown. Understanding neurogenesis and neural cell differentiation presents a
unique challenge for the treatment of nervous system disorders.
To characterise molecular mechanisms governing neural stem cell differentiation we
combined the advantage of porcine fetal neural stem cells in vitro differentiation model andcomplementary proteomic analyses. To monitor changes in cellular proteins, we applied 2‐
dimensional gel electrophoresis for protein separation followed by mass spectrometry for protein
identification. We demonstrated that differentiation of neural cells was accompanied by changes inthe expression of proteins involved in DNA and RNA binding, mRNA processing and transport, stressresponses, iron storage and redox regulation. To access signalling proteins that are present in cells invery low copies, we utilised the KinexTM Antibody Microarrays. The neural stem cells exhibited highlevels of G‐protein coupled receptor kinase 2 (GRK2), several members of apoptotic FAS pathway andphosphorylations of αB‐crystallin at Serin 45. For the differentiated neural cells were typicalenhanced integrin, platellet‐derived growth factor and MAP kinase signalling and increased levels ofheme‐oxygenase 2. In‐depth studies using immunoblot and immunocytochemistry verified
quantitative changes and localisation of selected proteins in neural cells. In addition, expression ofpotential biomarkers identified in porcine model was studied in human postmitotic neurons (hNTcells) and during differentiation of human spinal cord stem cells (566RSC cells).
We anticipate that characterisation of protein changes related to stem cell differentiation
will help define suitable biomarkers that might be necessary for partially differentiated cells to
optimally progress to a fully differentiated stage of neurons and glial cells. These biomarkers might
be then applied in selection of cells optimal for transplantation treatment of neurological disorders.
This study was supported by the Ministry of Education, Youth and Sports (projects 1M0538 andME10044) and Institutional research concept IAPG No. AVOZ50450515.

37

38

Lectures:
Topic 4: Genetics manipulation
Atsuo OGURA, Kimiko INOUE
Improvement of somatic cell nuclear transfer in mice: Genetic and epigenetic approaches

41

Masahiro KANEDA, Tamas SOMFAI, Seiki HARAGUCHI, Satoshi AKAGI, Shinya WATANABE,
Takashi NAGAI
Normal and abnormal epigenetic changes in the development of cloned cattle

43

Noboru MANABE, Ichiro ONOYAMA, Junyou LI, Yutaka SENDAI, Yoshito AOYAGICharacteristics of prion, bovine spongiform encephalopathy (BSE) pathogen,homoknockout cow

44

Michiko NAKAI, Kazuhiro KIKUCHI, Junya ITO, Naomi KASHIWAZAKI, Hiroyuki KANEKO
Xenotransplantation of gonadal tissue for utilization of genetic resources in pigs

46

Stefan JUHAS, Marian HRUSKA‐PLOCHAN, Jana JUHASOVA, Petr VODICKA, Antonin
PAVLOK, Monika BAXA, Atsushi MIYANOHARA, Martin MARSALA, Jan MOTLIK
Miniature pig as a large animal model of Huntington disease

48

39

40

Improvement of somatic cell nuclear transfer in mice: Genetic and epigenetic
approaches
Atsuo OGURA and Kimiko INOUERIKEN BioResource Center, Tsukuba, Japan
Cloning mammals by somatic cell nuclear transfer (SCNT) has been successfully applied tomore than 16 animal species. However, it is highly inefficient because of erroneous reprogrammingof the donor genome, the nature of which has not been fully understood yet. Recently, with growing
evidence for epigenetic alterations in SCNT embryos, agents used in modifying chromatin structure
or DNA methylation status in donor cells or reconstructed embryos have attracted attention as
possible treatments to overcome their developmental deficiency. One of the most successful
measures is the treatment of the recipient mouse oocytes with histone deacetylase (HDAC)
inhibitors, such as trichostatin A (TSA) and scriptaid, which led to significant increase in the cloning
efficiency for this species. As histone acetylation is a central factor regulating chromatin accessibility,
HDAC inhibitors are thought to facilitate the accessibility of putative ooplasmic factors to thetransferred nuclear genome, thus enabling potent genomic reprogramming. Therefore, it is probablethat they exert their effects on correcting random reprogramming errors in a global rather than aspecific manner. It is known that there are many HDAC inhibitors and each has its specific inhibitory
effect on several classes of HDACs. Although TSA and scriptaid work for cloned mouse embryos very
well, we may find other HDAC inhibitors that can be universally used for improvement of
mammalian SCNT.
Besides the non‐specific reprogramming errors described above, the presence of many SCNT‐
specific phenotypes (e.g., placental abnormalities, obesity, immunodeficiency, and perinatal death)suggests that SCNT inevitably induces specific epigenetic errors in the donor genome. These might benon‐random and definable characters, perhaps caused by fundamental epigenetic differences
between the somatic and germ cell genomes. However, no evidence for this assumption has been
proposed. To define the epigenetic characters specific for SCNT, we have recently analyzed global
gene expression patterns in mouse embryos cloned from different donor cells under strictly
standardized conditions in our laboratory. When the relative expression levels of filtered genes incloned embryos taken from a 44k oligo DNA microarray were plotted on the 20 chromosomes(except for Y), genes on the X chromosome were specifically downregulated. This phenomenon wassex‐ and genotype‐independent because the average X:autosome (X:A) expression ratio in the fourtypes of cloned embryos (two males and two females) was consistently lower than in thecorresponding control embryos. Interestingly, treatment of reconstructed oocytes with TSA could notameliorate the X‐linked downregulation of cloned embryos. The chromosome‐wide gene
downregulation on the X chromosome in cloned embryos was reminiscent of X chromosome
inactivation (XCI). As XCI is established by Xist RNA coating in cis, we next examined whether Xist was
expressed excessively in cloned embryos. As expected, the Xist expression level was significantly
higher in male and female cloned embryos than in corresponding IVF embryos. RNA fluorescent insitu hybridization (RNA FISH) revealed ectopic expression of Xist from the active X chromosome (Xa)in cloned embryos of the both sexes. Interestingly, deletion of Xist on Xa resulted in normalization of
the expression levels of the majority of downregulated genes, not just on this chromosome but also
on autosomes. Embryo transfer experiments are now under way.
Reprogramming mechanisms exist in the ooplasm primarily to modify the epigenetic statusof gametes into that of zygotes. The SCNT technique somehow utilizes this mechanism to reprogram
the donor somatic cell genomes, which are epigenetically different from those of gametes. Probably,
they might reflect fundamental differences in the nature of germ cells and somatic cells. Thus, we
can classify the epigenetic errors in the cloned embryos broadly into two categories: one is random
and can be overcome to some extent by enhancing the so‐called genomic reprogramming (e.g., by
HDAC inhibitor treatment), while the other is more specific and probably beyond the ability of theputative reprogramming factors. We demonstrated that the latter can be overcome at least to someextent by correcting the aberrated gene expression pattern by a genetic modification. The ultimate
41

goal of cloning researchers is to increase the efficiency of mammalian SCNT to a practical level (e.g., >20% per embryos transferred), because it is undoubtedly a promising technique with many potential
applications in biological drug manufacture, regenerative medicine and agriculture. To this end, weneed to know the fundamental differences between somatic and germ cell genomes. We expect that
SCNT will become more practical in future, by specifically targeting nonrandom epigenetic errors
associated with SCNT.

42

Normal and abnormal epigenetic changes in the development of cloned cattle
Masahiro KANEDA, Tamas SOMFAI, Seiki HARAGUCHI, Satoshi AKAGI, Shinya WATANABE andTakashi NAGAI
National Institute of Livestock and Grassland Science, National Agriculture and Food Research
Organization, Tsukuba, Japan
Since the first cloned sheep Dolly was born in 1996, lots of cloned animals in many species
have been produced by somatic cell nuclear transfer (SCNT). However, the success rate of SCNT isstill very low, less than 5‐10 % in all species. The reason is not yet revealed, but it is suggested thatthe incomplete reprogramming during SCNT causes epigenetic errors and abnormal gene expression.
Actually, there are lots of reports showing abnormal DNA methylation, histone modifications andgene expression patterns in cloned animals that die before or after birth. However, once the cloned
animals were born and healthily grew up to adulthood, their fertility is normal and their offspring do
not show any defects. This can be explained by the presumption that the first reprogramming step
(SCNT) is insufficient and abnormal epigenetic changes cause abnormal gene expression, whereas thesecond reprogramming step (only in germ cells) is sufficient and epigenetic errors should be erased
during germ cell development. Consequently, spermatozoa or oocytes from cloned animals are
epigenetically normal and therefore, offspring from cloned animals should be healthy. To test thishypothesis in cloned cattle, we compared DNA methylation levels of unique genes and repetitiveelements in cloned embryos, cloned bull somatic cells and spermatozoa. We observed aberrant DNA
methylation patterns in cloned embryos, however, there was no difference of DNA methylation insomatic cells and spermatozoa between cloned and non‐cloned bulls. These results suggest theoccurrence of proper reprogramming during cloned bull spermatogenesis leading to the normality ofcloned cattle offspring.

43

Characteristics of prion, bovine spongiform encephalo‐ pathy (BSE) pathogen,homo‐knockout cow
Noboru MANABE1, Ichiro ONOYAMA1, Junyou LI1, Yutaka SENDAI2 and Yoshito AOYAGI3
1

Animal Resource Science Center, The University of Tokyo, Kasama, Japan.
Central Research Institute for Feed and Livestock, Zen‐Noh, Tsukuba, Japan.
3
Embryo Transfer Center, Zen‐Noh, Kami‐Shihoro, Japan.

2

Abstract
Prion (Pr) protein plays a pivotal role in the infection of bovine spongiform encephalopathy(BSE). The aim of our research was to make Pr gene homo knockout (KO) Japanese black cows and toreveal their characteristics. Firstly, hetero Pr gene was knockouted in embryonic fibroblastic cells.
Somatic cell nucleus with hetero Pr gene KO was transferred into oocyte. After activation and in vitro
culture, somatic cell nucleus of blastocyst cell was transferred into oocyte to make homo Pr gene KO
embryo. The homo Pr gene KO embryos were transferred in to the uterus. Detailed data of Pr
distribution in organs of wild‐type and somatic cell nuclear cloning Japanese black cows were
demonstrated as follows: Pr protein were shown in peripheral organs: 7 skeletal muscles, 5 areas ofintestinal tracts and 5 areas of tongue (5‐ to 31‐ fold less Pr protein than the brain where high levelsof that, 9.0‐11.0 µg/g, were noted). Histochemistry showed that Pr protein was expressed in nerve
cells not only in brain but also in the tongue, skeletal muscles and intestines, indicating that theseorgans may serve as potential sources of BSE infection. No positive reaction for Pr mRNA or for Pr
protein was noted in Pr gene homo KO cows, indicating that the KO cows have no possibility of BSE
infection. In conclusion, to produce the KO cattle is the only way to make safe bovine derived foods,
drugs and medical materials.
Keywords: Bovine spongiform encephalopathy (BSE), Japanese black cow, Prion (Pr) protein, Pr gene
homo knockout (KO)Introduction
Misfolding of the Pr protein is involved in the pathogenesis of BSE [1, 2], but the mechanismsof prion transmission remains unresolved. One emerging hypothesis advocates that theabovementioned conversion phenomenon occurs at the site where the infectious agent (abnormal Pr
protein) binds with cellular Pr protein. Abnormal Pr protein has the potential to accumulate inperipheral tissues as well as in specified‐risk materials (SRM). In Japan, SRM were immediately
removed at slaughterhouse. Abnormal Pr protein was recently detected in the muscles of sheep [3‐5]and humans infected with Creutzfeldt‐Jakob disease [6, 7]. To assess the risk of abnormal Pr protein
accumulation in peripheral tissues, it is important to understand the distribution and expression
levels of cellular Pr protein in these tissues. To date, although studies have been performed in sheep[8, 9] and humans [10], Pr protein levels in the peripheral tissues of cattle remain to be determined.
To perfectly take away the risk and hazard factor for BSE infection, moreover, we produced Pr gene
homo knockout (KO) Japanese black cows.
Materials and Methods
Production of Pr KO cows: The cows lacking prion protein were produced using modified
methods reported previously [11]. Briefly, PRNP ‐/‐ fetal fibroblastic cell lines were made. Then, weused somatic cell nuclear transfer (SCNT) technique to produce the cloned fetuses and calves. Eightwild‐type and seven PRNP‐/‐ KO cows were produced.
Biochemical and histochemical analyses: RT‐PCR and in situ hybridization for Pr mRNA and Western
blotting, ELISA, protein misfolding cyclic amplification (PMCA) procedure and immunohistochemistry
for Pr protein in PRNP+/+ and PRNP‐/‐ cows were performed as previously described [12].

44Results and Discussion
Distribution of Pr mRNA and protein in organs: In situ hbridization and immune‐
histochemistry showed that Pr mRNA and protein was expressed in nerve cells not only in brain but
also peripheral tissues (tongue, skeletal muscles and intestines). Such data raise the possibility of Pr
accumulation in these tissues, indicating that these organs may serve as potential sources of BSE
infection.
Pr mRNA and protein levels in organs: Western blotting, ELISA and PMCA analyses revealed that highlevels of Pr (9.0‐11.0 µg/g wet tissue) was demonstrated in brain of normal and SCNT cows but not in
Pr KO cows. Lower levels of Pr were shown in other organs [in decreasing order as follows: skeletal
muscles (longissimus, iliocostalis thoracis, splenius, femoris biceps, triceps brachii, longissimus
thoracis and omotransversarius muscles), intestinal tracts (ileum, jejunum, duodenum, colon andcecum) and tongue (apex, posterior, anterior and radix areas) (5.0 ‐ to 31.0‐fold less Pr than thebrain).
Conclusion: We success to produce Pr gene homo KO Japanese black cows, which lack Pr mRNA orprotein. The Pr KO cows have no possibility of abnormal Pr protein infection. Thus, to produce the KO
cattle is the only way to make safe bovine‐ derived foods (beef and milk), drugs and medical
materials and equipments.
Acknowledgements. This study was supported in part by Grant‐in‐Aid for Food Safety Assessment
from the Food Safety Commission of Japan, by Grant‐in‐Aid for Creative Scientific Research
13GS0008 from the Ministry of Education, Culture, Sports, Science and Technology, and by Grants‐in‐
Aid for Challenging Exploratory Research 18658105 and 21658092 and Scientific Research
B18380164, B22380148 and S16108003 from the Japan Society for the Promotion of Science.
References
1. Prusiner SB: Prions. Proc Natl Acad Sci USA 95: 13363‐13383, 1998.
2. Jackson GS and Clarke AR: Mammalian prion proteins. Current Opini Struct Biol 10: 69‐74, 2000.
3. Masujin K et al.: Prions in the peripheral nerves of bovine spongiform encephalopathy affected
cattle. J Gen Virol 88: 1850‐1858, 2007.
4. Sakudo A et al.: Recent advances in clarifying prion protein functions using knockout mice andderived cell lines. Rev Med Chem 87: 589‐601, 2006.
5. Andreoletti O et al.: Protease resistant form Pr accumulation in myocytes from sheep incubating
natural scrapie. Nat Med 10: 591‐593, 2004.
6. Glatzel M et al.: Extraneural pathologic prion protein in sporadic Creutzfeldt‐Jakob disease. N
Engl J Med 349: 1812‐1820, 2003.
7. Kovacs GG et al.: Creutzfeldt‐Jakob disease and inclusion body myositis: Abundant diseaseassociated prion protein in muscle. Ann Neurol 55: 121‐125, 2004.
8. Horiuchi M et al.: A cellular form of prion protein exists in many non‐neuronal tissues of sheep. J
Gen Virol 76: 2583‐2587, 1995.
9. Moudjou M et al.: Cellular prion protein status in sheep: Tissue‐specific biochemical signatures. J
Gen Virol 82: 2017‐2024, 2001.
10. Pammer J et al.: Cellular prion protein expressed by bovine squamous epithelia of skin and uppergastrointestinal tract. Lancet 354: 1702‐1703, 1999.
11. Richt JA et al.: Production of cattle lacking prion protein. Nat Biotechnol 25: 132‐138, 2006.
12. Ano Y, et al.: Incorporation of S‐amyloid protein through the ileal epithelium before and after
weaning: Model for orally transmitted amyloidoses. Microbiol Immunol 52: 429‐434, 2008.

45

Xenotransplantation of gonadal tissue for utilization of genetic resources in pigs
Michiko NAKAI1, Kazuhiro KIKUCHI1, Junya ITO2,3 , Naomi KASHIWAZAKI2,3 and Hiroyuki KANEKO1
1

Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Japan. Laboratoryof Animal Reproduction,
2
School of Veterinary Medicine,
3
Graduate School of Veterinary Medicine, Azabu University, Sagamihara, Japan

Abstract
In vitro fertilization of in vitro matured oocytes in pigs has become the most popular methodof studying gametogenesis and embryogenesis in this species. In addition, because of recent
advances in in vitro culture of those embryos, in vitro embryo production now enables us to generate
viable embryos as successfully as for in vivo‐derived embryos and with less cost and in less time. In
addition to those in vitro production technologies, more advanced technologies such as xenograftingof gonadal (testicular and ovarian) tissue into immunodeficient experimental animals have beendeveloped in recent years, to help conservation and utilization of gamete resources. We have alreadyshown fertilizing ability and developmental ability of porcine embryos from gametes grown in mice.
Here, we discuss the possibility of conservation and utilization of pig resources by xenografting tomice.
Testicular tissues xenografring
The possibility of spermatogenesis by the transplantation of germ cells into mouse testis has
been suggested in mice [1, 2] and pigs [5]. In these cases, germ cells, including spermatogonial stem
cells, were injected directly into the seminiferous tubules. Spermatozoa from the introduced cells
would be expected to be produced in the ejaculate. However, this technique requires special skills
for the injection and also accurate separation procedures to select the spermatozoa derived from theintroduced cells. Although germ cell transplantation into mice has promoted complete donor‐derived
spermatogenesis in rodents, it has not yet been applied in large domesticated animals such as cattle
and pigs, and also wild animals. Xenografting of testicular tissues can be performed into
immunodeficient mice ectopicaly, such as under the skin of the back [6]. They succeeded in the
production of mammalian sperm grown in nude mice. Several years later, successful embryo
production by using these sperm cells from xenografted testicular tissues has been reported but
limited to rhesus monkeys [7] and pigs [8, 13]. In our previous study [13], blastocysts could be
produced after intra‐cytoplasmic sperm injection (ICSI). Furthermore, we have also reported thesuccessful live piglet production when the embryos just after the ICSI using xenogeneic sperm weretransferred to the recipients [14]. Although the efficacy of piglet production remains low, the results
suggests clearly that oocytes injected with a sperm differentiated from the gonocytes within the
xenografts have the ability to develop to viable offspring in a large mammal. In combination withcryopreservation of testis tissue, this procedure will be one of useful methods for conservation ofmale genetic recourses.
Ovarian tissue xenografting
Primordial follicles act as stores of ovarian follicles and are potential resources of oocytes for
medical, agricultural and zoological purposes. It was reported that full‐term culture of primordial
follicles to the mature stage and birth of viable offspring after in vitro fertilization and embryotransfer have been achieved only in mice [3]. However, ovarian xenografting is a potential method of
maturing the oocytes in the primordial follicles (primordial oocytes) of large mammals. To date,ovarian tissues could be prepared from some species phylogenetically distant from mice (including
pigs [9, 10]), and then been xenografted into immunodeficient mice. To our knowledge, only ourprevious studies, in which neonatal pig ovarian tissues were xenografted, had proven that primordial
oocytes can develop in the host mice and acquire in vitro‐fertilizing ability [10, 12] and also that
viable embryos can be generated after in vitro fertilization [11]. We further reported that theacceleration of follicular growth from the primordial stage and also the developmental ability of46

oocytes can be improved by treating with gonadotrophins (eCG or FSH) to the host mice [11].
However, it is also suggested that those oocytes derived from primordial follicles, even after in vivo
growth and maturation, seem to have difficulty achieving cytoplasmic maturation. Transfer of oocyte
chromosomes to an enucleated cytoplast with full developmental ability seems to be one possible
method for improving the ability. Fusion of an ooplasmic fragment(s) prepared by ‘Centri‐Fusion’
method [4] to the oocytes obtained from xenografts may be the other possibility. In combination
with cryopreservation of ovarian tissue, this procedure will be one of useful methods forconservation of female genetic recourses.
Acknowledgements
This study was supported in part by a Grants‐in‐Aid for Scientific Research (22380153 to K. K.
and 21380715 to H.K.) from the Japanese Society for Promotion of Science.
References
1. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonialtransplantation. Proc. Nat. Acad. Sci. USA 1994;91: 11303‐11307.
2. Brinster RL, Zimmermann JW. Spermatogenesis following male germ‐cell transplantation. Proc.
Nat. Acad. Sci. USA 1994;91:11298‐11302.
3. Eppig JJ, O’Brien MJ. Development in vitro of mouse oocytes from primordial follicles. Biol
Reprod 1996; 54: 197‐207.
4. Fahrudin M, Kikuchi K, Karja NWK, Ozawa M, Somfai T, Ohnuma K, Noguchi J, Kaneko H, Nagai T.
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reconstructed by the fusion of cumulus cells and cytoplasts prepared by gradient centrifugation.
Cloning Stem Cells 2007;9:216‐228.
5. Honaramooz A, Megee SO, Dobrinski I. Germ cell transplantation in pigs. Biol. Reprod.
2002a;66:21‐28.
6. Honaramooz A, Snedaker A, Boiani M, Scholer H, Dobrinski I, Schlatt S. Sperm from neonatal
mammalian testes grafted in mice. Nature 2002b;418:778‐781.
7. Honaramooz A, Li MW, Penedo MC, Meyers S, Dobrinski I. Accelerated maturation of primate
testis by xenografting into mice. Biol. Reprod. 2004;70:1500‐1503.
8. Honaramooz A, Cui XS, Kim NH, Dobrinski I. Porcine embryos produced after intracytoplasmic
sperm injection using xenogeneic pig sperm from neonatal teitis tissue grafted in mice. Reprod
Fertil Dev 2008;20:802‐807.
9. Kagawa N, Sakurai Y, Miyano T, Manabe N. Effects of long‐term grafting on follicular growth inporcine ovarian cortical grafts xenoplanted to severe combined immunodeficient (SCID) mice. J.
Reprod. Dev. 2005;51:77‐85.
10. Kaneko H, Kikuchi K, Noguchi J, Hosoe M, Akita T. Maturation and fertilization of porcine oocytes
from primordial follicles by a combination of xenografting and in vitro culture. Biol. Reprod.
2003;69:1488‐1493.
11. Kaneko H, Kikuchi K, Noguchi J, Ozawa M, Ohnuma K, Maedomari N, Kashiwazaki N. Effects ofgonadotropin treatments on meiotic and developmental competence of oocytes in porcine
primordial follicles following xenografting to nude mice. Reproduction 2006;131:279‐288
12. Kikuchi K, Kaneko H, Nakai M, Noguchi J, Ozawa M, Ohnuma K, Kashiwazaki N. In vitro and in vivo
developmental ability of oocytes derived from porcine primordial follicles xenografted into nude
mice. J. Reprod. Dev. 2006;52:51‐57.
13. Nakai M, Kaneko H, Somfai Tamas, Maedomari N, Ozawa M, Noguchi J, Kashiwazaki N, Kikuchi K.
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Theriogenology 2009;72:2‐9.
14. Nakai M, Kaneko H, Somfai T, Maedomari N, Ozawa M, Noguchi J, Ito J, Kashiwazaki N, Kikuchi K.
Production of viable piglets for the first time using sperm derived from ectopic testicularxenografts. Reproduction 2010;139.331‐335.

47

Miniature pig as a large animal model of Huntington disease
Stefan JUHAS1, Marian HRUSKA‐PLOCHAN1, Jana JUHASOVA1, Petr VODICKA1, Antonin PAVLOK1,
Monika BAXA1, Atsushi MIYANOHARA3, Martin MARSALA2, Jan MOTLIK1
1

Institute of Animal Physiology and Genetics AS CR, Laboratory of cell regeneration and plasticity,Liběchov, Czech Republic2
Department of Anesthesiology, Neuroregeneration Laboratory, University of California, San Diego,
CA, USA
3
Department of Pediatrics, Vector Development Lab, University of California School of Medicine, San
Diego, CA, USA
Huntington Disease (HD) is the most common and well‐studied polyglutamine
neurodegenerative disorder and is caused by an expansion of cytosine‐adenine‐guanine (CAG)
repeats in the huntintin gene, which leads to neuronal loss in the striatum and cortex and to theappearance of neuronal intranuclear inclusions of mutant huntingtin. Huntingtin plays a role inprotein trafficking, vesicle transport, postsynaptic signaling, transcriptional regulation, and apoptosis.
Thus, a loss of function of normal protein and a toxic gain of function of the mutant huntingtin
contribute to the disruption of multiple intracellular pathways. Excitotoxicity, dopamine toxicity,
metabolic impairment, mitochondrial dysfunction, oxidative stress, apoptosis, and autophagy havebeen implicted in the progressive degeneration observed in HD. Nevertheless, despite the efforts ofmultidisciplinary scientific community, there is no cure for this devastating neurodegenerative
disorder (Gil and Rego, 2008).
Research in this field is primarily based on rodent models which are, however, insufficient inconversion of the basic research to the human clinic comparing with the increasingly restricted
research on primates. Looking for a suitable and publicly acceptable large animal model for study ofneurodegenerative diseases, we have chosen the pig (miniature pig of our own breed) which has
several advantages over the rodents and can serve as a primate substitute (the pig is genetically wellcharacterized and has a large gyrencephalic brain with a primate‐like neuroarchitecture that allows
conventional brain imaging). In addition to general advantages of miniature pigs, our own breed(cross of Minnesota, Göttingen, Cornwall and Large White breed) is fully sexually matured as early as
4 months after birth and it has very small frontal sinus comparing to Göttingen miniature pig whichallow us to perform more precise neurosurgical operations (Hruška, 2009).
Our effort was targeted on the HD transgenic minipig generation and on the HD acuteminipig generation. HD transgenic minipig will be invaluable in the long‐term follow‐up examinations
which could lead to better understanding of the neurodegeneration mechanisms in the HD brain. In
addition to the transgenic model which can vary in the extent of gene expression, brain regions
involved, neuropathology, and behaviors, we decided to develop a rapid‐onset porcine model of HD,
in which timed neuropathology and motor impairments would be predictable, robust, andexperimentally testable.
This work was supported by CHDI, 1M0538, AV0Z50450515 and EMBO short term fellowship ASTF
308.00‐2009
Gil JM, Rego AC. Eur J Neurosci. 2008 Jun; 27(11):2803‐20.
Hruška‐Plocháň M. Clinical Genetics 2009, Vol 76 Issue s1, p 112

48

Poster session
Veronika BENESOVA, Tereza TORALOVA, Katerina VODICKOVA, Petr VODICKA, Jiri KANKA
Expression and function of nucleophosmin/B23 during the preimplantation bovine
development

51

Tereza BOHMOVA, Alexandra MAYER, Petr SOLC, Vladimir BARAN, Jan MOTLIK
Polo‐like kinase 1 and Aurora A cooperate in spindle assembly during meiotic maturationof mouse oocytes

52

Than Quang DANK‐NGUYEN, Masahiro KANEDA, Tamas SOMFAI, Kazutsugu MATSUKAWA,
Satoshi AKAGI, Kazuhiro KIKUCHI, Michiko NAKAI, Bui Xuan NGUYEN, Atsushi TAJIMA, Yukio
KANAI, Takashi NAGAI
Development of twin blastocysts derived from single blastomeres of the 2‐cell stageembryos in pigs

53

Katsuyoshi FUJIWARA, Daisuke SANO, Yasunari SEITA, Tomo INOMATA, Junya ITO, Naomi
KASHIWAZAKI
Calcium‐free medium including ethylene glycol for oocyte vitrification improves survivaland embryonic development of vitrified rat oocytes

54

Seiki HARAGUCHI, Tamas SOMFAI, Thanh Q. DANG‐NGUYEN, Masahiro KANEDA, Shinya
WATANABE, Kazuhiro KIKUCHI, Tomoyuki TOKUNAGA, Masaya GESHI, Takashi NAGAI
Single blastomeres within mouse 4‐cell embryo expresses different amounts of mRNA55

M. HORNAK, M. JESETA, P. MUSILOVA, A. PAVLOK, M. KUBELKA, J. MOTLIK, M. ANGER, J.
RUBES
Analysis of chromosome segregation errors oocytes using comparative genomic
hybridization

56

Jaroslav KALOUS, Michal KUBELKA, Jan MOTLIKInhibition of Polo kinase 1 (Plk1) affects resumption of meiosis in porcine oocytes

57

Pavla KARABINOVA, Lucie LISKOVA, Michal KUBELKA
Role of selected kinases in CPEB‐dependent cytoplasmic polyadenylation

58

Hirohisa KYOGORU, Takashi MIYANO, Josef FULKA Jr.
Enucleolation of growing and full‐grown pig oocytes

59

M. MACHATKOVA, M. JESETA, P. HULINSKA, S. KAPLANOVA, L. NEMCOVA, J. KANKA
Characteristics of mitochondria in bovine oocytes related to their meiotic competence

60

Shogo MATOBA, Atsuo OGURA
Generation of functional mouse oocytes and spermatids from isolated primordial germ
cells following ectopic transplantation in adult animals

61

M. MOROVIC, O. ØSTRUP, F. STREJCEK, I. PETROVICOVA, A. LUCAS‐HAHN, E. LEMME, H.
NIEMANN, J. LAURINCIK, P. HYTTEL
Nuclear and nucleolar remodelling during the first cell cycle in bovine somatic cell nuclear
transfer and tetraploid embryos

6249

Eva NAGYOVA, Radek PROCHAZKA, Lucie NEMCOVA, Antonella CAMAIONI, Alzbeta
MLYNARCIKOVA, Sona SCSUKOVA, Antonietta SALUSTRI
Involvement of EGFR and Smad2/3 activation in FSH‐induced porcine oocytes‐cumulus
cell complex maturation

63

Yoshiaki NAKAMURA
Gametegenesis following transplantation of primordial germ cells in domestic fowls

64

Chino NAKAUCHI, Katsuyoshi FUJIWARA, Yasunari SEITA, Junya ITO, Naomi KASHIWAZAKI
Sperm preincubation for intracytoplasmic sperm injection (ICSI) improves developmental
ability of ICSI oocytes to the blastocyst stage in rats

65

R. RYBAR, V. KOPECKA, P. PRINOSILOVA, S. KUBICKOVA, J. RUBES
Fertile bull sperm aneuploidy and chromatin integrity in relationship to fertility

66

Nozomi SUZUKI, Junya ITO, Naomi KASHIWAZAKI
Isobutyl‐methylxanthine facilitates capacitation‐associated protein tyrosine
phosphorylation of frozen‐thawed C57BL/6J mouse sperm

67

Milan TOMANEK, Tomas KOTT, Ewa CHRONOVSKA, Eva KOTTOVA
Telomerase (pTERT) mRNA expression in the pig granulosa cells in vitro

68

Irena VACKOVA, Zora NOVAKOVA, Leona URBANKOVA, Jan MOTLIKIsolation, cultivation and characterization of porcine cell lines derived from blastocysts
produced in vitro and in vivo

69

Katerina VODICKOVA KEPKOVA, Petr VODICKA, Tereza TORALOVA, Miloslava
LOPATAROVA, Svatopluk CECH, Radovan DOLEZEL, Vitezslav HAVLICEK, Urban
BESENFELDER, Anna KUZMANY, Marc‐Andre SIRARD, Jozef LAURINCIK, Jiri KANKA
Comparative transcriptomic analysis of in vivo and in vitro produced bovine embryos
using a custom bovine embryo‐specific microarray

70

Mayo WATANABE, Junya ITO, Naomi KASHIWAZAKI
Comparison of media for culture of matured and fertilized rat oocytes

71

50

Expression and function of nucleophosmin/B23 during the preimplantation bovine
development
Veronika BENESOVA, Tereza TORALOVA, Katerina VODICKOVA, Petr VODICKA, Jiri KANKA
Institute of Animal Physiology and Genetics AS CR, v.v.i., Liběchov, Czech Republic
Identification of genes that are differentially expressed during bovine preimplantation
development is crucial for proper understanding of early mammalian embryogenesis. Genes that are
differently expressed during preimplantation development are supposed to be important for normal
preimplantation development. Nucleophosmin, the significant nucleolar protein, functions as atransporting protein between cytoplasm and nucleoli. It is concerned in duplication of centrosomes,
formation of ribosomes and functions as a chaperoning protein. Localization of this protein is cell
cycle dependent. It is not known how important is embryonic expression of nucleophosmin for
subsequent development.
At the beginning of preimplantation development embryo lacks functionally active nucleoli.
Ribosomes from maternal oocyte are used for protein synthesis. Nucleoli become structurally
recognizable towards the end of the third and fourth post‐fertilization cell cycle ‐ at the time whenthe embryonic genome activation begins. The localization of nucleolar proteins reflects in thedevelopment of the nucleoli. Nucleophosmin, one of these nucleolar proteins, may be involved in
shuttling other proteins and in the assembly of pre‐ribosomal particles.
Bovine zygotes were microinjected with nucleophosmin dsRNA (800ng/ul) 20h post fertilization. Twocontrol groups were established – the uninjected group and a group injected with GFP dsRNA. After
micronjection, embryos were cultivated in vitro and collected at specific developmental stages (late8‐cell stage, 16‐cell stage, morula, blastocyst). The number of embryos that reached eachdevelopmental stage was counted and the quality of each embryo was determined. The level of
nucleophosmin mRNA was measured by a real‐time RT‐PCR; the expression of protein wascharacterized using immunofluorescence.
We did not observe significant differences in development of control and experimentalgroups. The embryos with silenced nucleophosmin mRNA were able to develop until the blastocyst
stage. The localization of protein in injected and uninjected embryos did not seem to be different.
Our study showed that the maternal nucleophosmin protein is probably stored even after theactivation of the embryonal genome and it is sufficient for embryonal development at least until
blastocyst stage.
In conclusion, maternal nucleophosmin protein seems to substitute embryonal protein in thepreimplantation development with no crucial defects on the embryo.
This study was supported by by a grant GAUK 43‐251133 and GA CR 523/09/1035.

51

Polo‐like kinase 1 and Aurora A cooperate in spindle assembly during meiotic
maturation of mouse oocytes
Tereza BOHMOVA1, Alexandra MAYER1, Petr SOLC1, Vladimir BARAN2 and Jan MOTLIK1
1

Institute of Animal Physiology and Genetics; Academy of Sciences of the Czech Republic; Libechov,Czech Republic;2
Institute of Animal Physiology; Slovak Academy of Sciences; Kosice, Slovakia

Polo‐like kinase 1 (Plk1) and Aurora A (Aurka) are Ser/Thr kinases specifically activated at
G2/M transition and involved in centrosome maturation and spindle assembly. Aurora A is known tobe the primary activator of Plk1 early in mitosis: in association with Bora cofactor it directly
phosphorylates Plk1 promoting mitotic entry. In later stages activated Plk1 mediates Bora
degradation and phosphorylates Tpx2. This enables the switch to Tpx2 –Aurka association which
triggers conformational changes of Aurka, stimulating its autophosphorylation and association withmicrotubules.
Loss of function of either Aurka or Plk1 during mitosis results in spindle abnormalities andmisaligned metaphases. However, the difference is that Aurora A disruption does not lead to SAC
activation and some cells enter mitosis with defects, whereas Plk1 disruption induces metaphasearrest and cell apoptosis.
In our studies we used BI2536 Plk1 inhibitor and MLN8054 Aurka inhibitor to elucidate theroles and interplay of these two kinases during meiotic maturation of mouse oocytes.
BI2536 treatment induced GVBD delay and consequently MI arrest as referred before on
somatic cells. Plk1 inhibition led to premature MTOC amplification in GV and their breakdown after
GVBD. In MI, MTOCs disappeared completely and gamma tubulin was not detected. Although inprinciple bipolar spindle formed, we observed some spindle defects and abnormal chromosome
congression.
Aurora A inhibition did not reduce the resumption of meiosis as markedly as Plk1 inhibition.
Neither the combination of two inhibitors increased the GVBD delay induced by Plk1 inhibition only.
However, the inhibition of Aurka in MI induced mislocalization of gamma tubulin and chromosomes.
Furthermore, when both kinases were inhibited, no spindle was polymerized.
We also revealed that Plk1 inhibition blocked Aurka activation in maturing oocytes.
Moreover, it prevented phosphorylation of Aurka substrate, Tacc3 protein which is involved inmeiotic MTOCs regulation. Our results suggest similar roles of Plk1 in mitosis and meiosis. Plk1probably participates in resumption of meiosis, process analogous to recovery from G2/M checkpointin mitotic cells, where Plk1 plays an important role. It also regulates spindle assembly and inhibitionof its function leads to MI arrest of oocytes. According to our data Plk1 and Aurka cooperate inmeiotic spindle assembly, however, their functions seem to be different. In contrast to mitosis, Plk1
mediated AURKA activation may occur from the beginning of meiotic maturation.
Paper referred:
Aurora kinase A controls meiosis I progression in mouse oocytes. Saskova A, Solc P, Baran V, Kubelka
M, Schultz RM, Motlik J. Cell Cycle. 2008 Aug;7(15):2368‐76. Epub 2008 May 29.

52

Development of twin blastocysts derived from single blastomeres of the 2‐cell stageembryos in pigs
Thanh Quang DANG‐NGUYEN1,2, Masahiro KANEDA2, Tamás SOMFAI2, Kazutsugu MATSUKAWA3,
Satoshi AKAGI2, Kazuhiro KIKUCHI4, Michiko NAKAI4, Bui Xuan NGUYEN5, Atsushi TAJIMA1, Yukio
KANAI1, and Takashi NAGAI2
1

University of Tsukuba, Tsukuba, Japan.
National Institute of Livestock and Grassland Science, National Agriculture and Food Research
Organization, Tsukuba, Japan.
3
Kochi University, Kochi Japan,
4
National Institute of Agrobiological Sciences, Tsukuba, Japan.
5Vietnamese Academy of Science and Technology, Hanoi, Vietnam.

2

In the present study, the developmental competence of intact and zona‐free porcine 2‐cellembryos and single blastomeres obtained from 2‐cell embryos (IN, ZF and SB groups, respectively),
and the quality of twin blastocysts derived from the sister blastomeres of 2‐cell embryos wascompared. Expression of transcripts related to developmental competence in blastocysts derived
from IN, ZF and SB groups was also assessed. There was no difference in blastocyst formation rates(of the total embryos cultured) among three groups. The blastocyst yield of SB group reached 90.2%of the original number of 2‐cell embryos from which the blastomeres had been separated, andsignificantly higher than those of IN and ZF groups. However, the total cell number in SB blastocysts(24.7 ± 1.7) was significantly lower than those in IN and ZF blastocysts (36.2 ± 2.5 and 35.0 ± 2.7,
respectively). The percentages of the sister blastomere pairs both developed to blastocysts (36.6 ±
5.3%) or both degenerated (46.3 ± 10.3%) were significantly higher than that of the pairs in which
one developed to blastocyst while the other degenerated (17.1 ± 7.8%). In the pairs both developed
to blastocysts one blastocyst had a significantly higher cell number (31.6 ± 2.9) than that of the otherone (19.1 ± 1.9). The expression of transcripts associated with cellular organization, TUBA1 and
TUBB, was significantly reduced in SB blastocysts compared with IN and ZF blastocysts. However, theexpression of transcripts associated with response to stresses (HSPE1, HSPD1, and HSPCA) andcatabolism of glucose (ENO1, COX6C, COX7B, NDUFA4, NDUFA13, UCRC, and UQCRFS1) in blastocysts
was not different among 3 groups. In conclusion, blastomere separation at 2‐cell stage remarkably
increased the blastocyst yield obtained from IVP embryos and might be an approach forconservation of rare pig breeds, in which the issue of lacking embryos for transfer is necessary to
overcome.

53

Calcium‐free medium including ethylene glycol for oocyte vitrification improves
survival and embryonic development of vitrified rat oocytes
Katsuyoshi FUJIWARA1, Daisuke SANO1, Yasunari SEITA1, Tomo INOMATA1,2, Junya ITO1,2, Naomi
KASHIWAZAKI1,2
Laboratory of Animal Reproduction,
Graduate School of Veterinary Science,2
School of Veterinary Medicine, Azabu University, Sagamihara, Japan1

Successful cryopreservation of unfertilized oocytes is one of the most valuable technologies
for efficient production of animals. Even though the laboratory rats are important experimental
animals and have been used for various research fields, information about cryopreservation of theoocytes is very limited. Since we recently reported successful vitrification of rat embryos at the
pronuclear stage by minimum volume cooling procedure (Cryotop), the objective of the present
study was to improve the vitrification protocol for unfertilized rat oocytes. Cumulus oocyte
complexes were collected from the oviducts and cumulus cells were removed. To determine theoptimal equilibration time, the oocytes were equilibrated in 7.5% ethylene glycol (EG) + 7.5%
dimethyl sulfoxide (DMSO) + 20% fetal calf serum (FCS) for 1, 4, 7 or 10 min and then 15% EG + 15%
DMSO + 0.5 M sucrose + 20% FCS for 1 min before being plunged into liquid nitrogen on Cryotops
when room temperature around at 24 C. Oocytes exposed to equilibration medium for 4 min showed
higher survival and cleavage rates after artificial activation. The survival and cleavage rates of vitrified
oocytes after activation were 98.3 and 78.4%, respectively. Oocytes vitrified in calcium‐free medium
including EG showed high survival rates after warming (79.4 ± 4.3%). After artificial activation, thecleavage and blastocyst formation rates of the oocytes were also high (72.8 ± 4.0 and 23.1 ± 4.2%,
respectively). The inhibitory effect of calcium and cryoprotectants (EG vs DMSO) in vitrification
medium on cortical granule exocytosis (CGE) was examined. In most of the oocytes vitrified incalcium‐free medium, CGE was strongly suppressed independent of cryoprotectants. The zona
penetration rate of vitrified/warmed oocytes was improved by using calcium‐free medium including
EG after in vitro fertilization. Thus, our data suggest that calcium‐free medium including EG improvesurvival, embryonic development and zona penetration of vitrified rat oocytes by spermatozoa.

54

Single blastomeres within mouse 4‐cell embryo expresses different amounts
of mRNA
Seiki HARAGUCHI1, Tamas SOMFAI1, Thanh Q. DANG‐NGUYEN1, Masahiro KANEDA1, Shinya
WATANABE1, Kazuhiro KIKUCHI2, Tomoyuki TOKUNAGA2, Masaya GESHI1, Takashi NAGAI1
1National Institute of Livestock & Grassland Science, National Agriculture and Food Research
Organization, Tsukuba, Japan.
2
Department of Animal Science, National Institute of Agrobiological Sciences, Tsukuba, Japan.
The aim of this study was to detect the expression patterns and levels of mRNAs in single
blastomeres from the same embryo. In mammalian preimplantation embryos, the study ofquantitative gene expression has been difficult because of the limited amount of starting material.
Although semi‐quantitative measurements of mRNA levels can be performed using several ways,
these are not applicable when working with single blastomere. Here, we show the comparative
quantitative analysis of mRNA from single blastomeres of mouse 4‐cell embryos. Four‐cell embryos
were mechanically separated into single blastomeres and subjected to RT‐PCR with a Gfp cRNA as anexternal control. After optimal number of cycles, in which only the Gfp signal could be detected, PCR
products were applied for Southern blotting analysis using a specific internal probe. The large
variation from cell to cell in the expression of mRNA levels was observed for all genes of interest; ß‐actin, Gapdh, CyclinB, Cateninß, Psen1, Oct4, and Nanog. These results clearly show that different
amount of mRNAs are expressed in single blastomere within a mouse 4‐cell embryo, implying thathousekeeping genes such as ß‐actin and Gapdh are not suitable for endogenous control. Moreover,in order to know whether there is a correlation between the expression pattern and the location of
blastomeres, each blastomere was marked with a dye and examined. However, the signal of mRNAs
was randomly detected, showing no correlation with the cell allocation. The biological meaning of
the phenomenon that blastomeres express high/low amounts of mRNA will be discussed.

55

Analysis of chromosome segregation errors in porcine oocytes using comparative
genomic hybridization
M. HORNAK1, M. JESETA1, P. MUSILOVA1, A. PAVLOK2, M. KUBELKA2, J. MOTLIK2, M. ANGER1,2, J.
RUBES1
1

Veterinary Research Institute, Brno, Czech Republic
Institute Animal Physiology and Genetics, Libechov, Czech Republic2

Although numerical chromosome errors are known to be prevalent and are likely to be aconsiderable factor influencing the mortality of early embryos and implantation failure, in farmanimals, data about the frequency and nature of errors is rather limited.
Studies concerning chromosome errors in gametes and embryos of animals utilized so far
FISH technique or karyotyping of chromosome spreads. However, internal drawbacks of the above
mentioned methods are significant ‐ FISH is capable of analysis of only a few chromosomes andkaryotyping provides reliable data only on ploidy errors due to poor chromosome quality.
Aim of our work was to implement reliable technique in order to investigate the wholechromosome set of single cells, therefore suitable for aneuploidy study of gametes or blastomeres.
We accomplished this task using methods of Whole Genome Amplification (WGA) and Comparative
Genomic Hybridization (CGH). WGA‐CGH is capable of detecting not only abnormalities of all
chromosomes in the entire chromosome set but also determines chromosome breakages. On theother hand, WGA‐CGH is not able to detect ploidy errors and balanced structural abnormalities.
In our subsequent study, we focused on porcine oocyte aneuploidy analysis. Using WGA‐CGH
approach we would like to provide new insight into oocyte aneuploidy and establish the frequency ofchromosome errors in the examined group of oocytes.

This study was supported by Grants GACR 523/09/0743 and GAAV IAA501620801.

56

Inhibition of Polo kinase 1 (Plk1) affects resumption of meiosis in porcine oocytes.
Jaroslav KALOUS, Michal KUBELKA and Jan MOTLIKInstitute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumburská
89, 277 21 Libechov, Czech Republic
The aim of the study was to investigate the implication of Plk1 in meiosis resumption duringin vitro maturation of porcine oocytes. When oocytes were treated with 1.5 μM of Plk1 inhibitor
BI2536, phosphorylation of Plk1 at its activation site Thr210 decreased and MPF activity was
inhibited as well. Moreover, resumption of meiosis was substantially suppressed as only 11% of
oocytes underwent GVBD during 24 h of treatment. However, this inhibition was fully reversible as
92% of oocytes passed GVBD and majority of them reached metaphase II ‐stage when BI2536‐
treatment was followed by culture in control conditions. In the case when oocytes were subjected toBI2536 only after 8h pre‐culture in control conditions the incidence of GVBD increased up to 62%.
These results indicate that Plk1 plays an important role in meiosis resumption, nevertheless, theimportance of Plk1 is decreasing when chromatin condensation and other events leading to GVBD
are already initiated.

57

Role of selected kinases in CPEB‐dependent cytoplasmic polyadenylation
Pavla KARABINOVA1,2, Lucie LISKOVA1 and Michal KUBELKA1
1Institute of Animal Physiology and Genetics, AS CR, v.v.i. Liběchov
2
Institute of Chemical Technology, Prague

Cytoplasmic polyadenylation of specific mRNAs is a mechanism of translational regulation
important for oocyte maturation and further development. This mechanism is mainly controlled byphosphorylation of cytoplasmic polyadenylation element binding protein (CPEB).
We focused on determination of kinases involved in CPEB phosphorylation and consequently the
CPEB‐dependent polyadenylation during mammalian oocyte cell cycle. Therefore we cultivatedporcine oocytes in the presence of specific kinase inhibitors (roscovitine, KN‐93, ZM447439, PD98059
and MLN8054). We employed poly(A)‐test PCR to monitor the effect of inhibitors on poly(A)‐tailextension of cyclin B1 and mos mRNAs as the markers of CPEB‐dependent cytoplasmic
polyadenylation. In addition, the effect of inhibitors on CPEB phosphorylation was observed by 2D–
PAGE western blot.
Our results show that CPEB is modified already in the GV‐stage porcine oocytes with intense
phosphorylation at the time of GVBD. This late phosphorylation, assumed responsible for CPEB
degradation in metaphase I, is sensitive to roscovitine implying it is mediated by CDK1 in porcine
oocytes. Furthermore, the inhibition of CDK and MAPK pathways from the beginning of maturation
affected the cyclin B1 mRNA polyadenylation. Thus these pathways may play a role upstream theregulatory cascade leading to the CPEB‐mediated polyadenylation. The CaMKII and Aurora kinase
inhibitors (KN‐93, ZM447439 and MLN8054) affected later process of maturation indicating differentrole of these kinases in meiotic maturation of porcine oocytes.
Financial support from specific university research (MSMT no. 21/2010), Institutional
Research Programme (IAPG No. AV OZ 50450515) and Czech Science Foundation GACR 204/09/H084
and P502/10/0944.

58

Enucleolation of growing and full‐grown pig oocytes
Hirohisa KYOGOKU1,2, Takashi MIYANO1 and Josef FULKA Jr.2
1

Graduate School of Agricultural Science, Kobe University, Kobe, Japan. 2Institute of Animal Science,
Prague, Czech Republic.

Recent research has shown that the maternal nucleolus is essential for embryonicdevelopment in mammals. The morphology of the nucleolus in growing oocytes differs from that infull‐grown oocytes. We examined the ability of nucleoli from growing oocytes to substitute fornucleoli of full‐grown oocytes in terms of supporting embryonic development in this study(Experiment 1). We also examined the effect of growing oocyte nucleoli on the maturation of full‐grown oocytes (Experiment 2). Pig growing oocytes (around 100 µm in diameter) and full‐grownoocytes (120 µm) were collected from small (0.6–1.0 mm) and large antral follicles (4–5 mm),
respectively. Nucleoli were aspirated from full‐grown and growing oocytes by micromanipulation.
Since the chromatins of growing oocytes were aspirated with the nucleolus during the enucleolation
process, growing oocytes were treated with actinomycin D before enucleolation. The nucleoli
collected from growing oocytes were transferred to the enucleolated full‐grown GV oocytes(Experiment 2) or enucleolated and matured full‐grown oocytes (Experiment 1). In the Experiment 1,
enucleolated full‐grown oocytes matured to metaphase II (MII). After being activated by electro‐
stimulation, nucleoli were formed in pronuclei of sham‐operated oocytes, and in the enucleolated
oocytes that had been injected with nucleoli from either full‐grown or growing oocytes. However,enucleolated oocytes did not form any nucleoli in the pronuclei. No enucleolated oocytes developed
to blastocysts, whereas enucleolated oocytes injected with nucleoli from full‐grown oocytes (15%) or
growing oocytes (18%) developed to blastocysts. In the Experiment 2, enucleolated growing oocytes
underwent germinal vehicle breakdown (45% vs. control 12%). On the other hand, enucleolated full‐grown oocytes which had been injected with growing oocyte nucleoli stopped maturation (MII: 21%
vs. control MII: 91%). These results suggest that the growing oocyte nucleolus inhibits oocyte
maturation, although it can substitute for the full‐grown oocyte nucleolus for early embryonicdevelopment.

59

Characteristics of mitochondria in bovine oocytes related
to their meiotic competence
M. MACHATKOVA1, M. JESETA1, P. HULINSKA1, S. KAPLANOVA1, L. NEMCOVA2, J. KANKA2
1

2

Veterinary Research Institute, Brno, Czech Republic
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov,Czech Republic

It is generally accepted that meiotic competence of oocytes increases with increasing follicle
size but it can be impaired by physiologic atresia at any time of follicular development. The present
study was designed to characterize mitochondria in oocytes that were recovered from different sizedfollicles and showed different signs of atresia. Cyclic cows, examined for ovarian status, were used as
oocyte donors. Oocytes recovered separately from medium (MF) or small follicles (SF) werecategorized on the basis of morphology of both cumulus and ooplasm as healthy, light‐atretic andmid‐atretic oocytes. Heavily atretic oocytes were excluded from the study. A half of the oocytes ofeach category was matured for 24 hours according to a standard protocol. The immature (GV) or
mature (MII) oocytes were denuded from cumulus cells, stained with MitoTracker Orange and fixed.
The morphology of active mitochondria (diffused, granulated or clustered), their distribution(peripheral or central) were evaluated by confocal microscopy. Expression of the mtDNA replication
related gene, mitochondrial transcription factor A (TFAM), was detected by real‐time RT‐PCR. The
results were analyzed by ANOVA.
Regardless of oocyte atresia level, a significantly higher (P < 0.05) percentage of GV oocyteswith granulated mitochondria or MII oocytes with clustered mitochondria was found for MF than for
SF oocytes (55.3 or 39.4% vs. 34.1 or 19.6%). Similarly, a significantly higher percentage of oocyteswith central distribution of mitochondria was determined for MF than for SF‐GV oocytes. The
difference in percentage of oocytes with centrally distributed mitochondria was not found between
MF and SF‐MII oocytes. Contrary to mitochondrial morphology and distribution, significantly higher(P < 0.05) level of TFAM transcript was detected for SF compared with MF‐GV oocytes. Thisdifference remained valid only for mid‐atretic SF‐MII oocytes.
In MF GV and MII oocytes, a significantly higher (P < 0.05) percentage of those with
granulated mitochondria and those with clustered mitochondria respectively, was detected inhealthy than in light‐atretic and mid‐atretic oocytes (73.3 vs 44.4 and 38.7%, and 60.5 vs 35.5 and8.0%). In contrast to MF oocytes, a significantly higher (P < 0.05) percentage of SF oocytes with
granulated and clustered mitochondria respectively, was found for light‐atretic than for healthy andmid‐atretic oocytes at GV (53.8 vs. 32.9 and 20.4%) and MII stage (36.6 vs. 17.2 and 2.9%).
At GV stage, a significantly higher (P < 0.05) percentage of oocytes with peripherally
distributed mitochondria was observed in healthy and light‐atretic, as compared with mid‐atretic MF
oocytes (36.6 and 20.0 vs. 4.5%). But on the other hand, a significantly higher (P < 0.05) percentageof oocytes with peripherally distributed mitochondria was determined in light‐atretic and mid‐atretic
in comparison with healthy SF oocytes (62.5 and 63.2 vs. 37.8%). Very high percentages of oocyteswith centrally distributed mitochondria, without differences among healthy, light‐atretic and mid‐atretic oocytes, were detected in both MF and SF oocytes at MII stage.
It can be concluded that bovine oocytes with different meiotic competence differ inproportions of those with diffused, granulated or clustered mitochondria. Healthy oocytes from themedium follicles and light‐atretic oocytes from the small follicles seem to be more competent interms of mitochondrial granula and cluster formation before and after maturation, respectively. The
morphology of mitochondria can characterize the cytoplasmic maturity of oocytes better thanmitochondrial distribution or quantification of TFAM.
This study was supported by Grants QI 91A018 and 0002716202 of the Ministry of Agriculture of the
Czech Republic.

60

Generation of Functional Mouse Oocytes and Spermatids from Isolated Primordial
Germ Cells Following Ectopic Transplantation in Adult Animals
Shogo MATOBA1, Atsuo OGURA1,2,3
1

RIKEN BioResource Center, Tsukuba, Japan,
Graduate School of Life and Environmental Science, University of Tsukuba, Tsukuba, Japan.
3
The Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo Japan.
2

Primordial germ cells (PGCs) are undifferentiated germ cells in developing fetuses. As theygive rise to definitive oocytes and spermatozoa that contribute to new life in the next generation,
their development must be under strict control regarding genetic and epigenetic aspects. However,
we do not know to what extent their development depends on the specific milieu. In this study, wetransplanted mouse PGCs isolated from male and female gonads at 12.5 days post coitum under thekidney capsule of adult mice, together with gonadal somatic cells. The transplanted cells constructed
testis‐like and ovary‐like tissues, respectively, under the kidney capsule within 4 weeks. Normal‐
looking round spermatids and fully grown germinal vesicle (GV) oocytes developed within thesetissues. Ectopic spermatogenesis continued thereafter, while oogenesis consisted of only a single
wave. The injection of these round spermatids directly into mature in vivo‐derived oocytes led to thebirth at term of normal pups. PGC‐derived GV oocytes were isolated, induced to mature in vitro, andinjected with normal spermatozoa. The injected oocytes were successfully fertilized and developed
into normal pups. Our findings demonstrate the remarkable flexibility of PGC development, which
can proceed up to functional gametes under a spatially and temporally non‐innate condition. Thistransplantation system may provide a unique technical basis for the induction of the development ofearly germ cells of exogenous origins, such as those from embryonic stem cells.

61

Nuclear and nucleolar remodelling during the first cell cycle in bovine somatic cell
nuclear transfer and tetraploid embryos
M. MOROVIC1, O. ØSTRUP2 , F. STREJCEK1 , I. PETROVICOVA1, A. LUCAS‐HAHN3, E. LEMME2, H.
NIEMANN3, J. LAURINCIK1, P. HYTTEL2
1

Constantin the Philosopher University, Nitra, Slovak Republic
2
University of Copenhagen, Copenhagen, Denmark3
Institute for Animal Breeding, FLI, Mariensee, Germany

Keywords: SCNT, tetraploid embryos, bovine embryo, reprogramming, UBF, fibrillarin
Cloning by the transfer of somatic nuclei into unfertilized eggs requires a dramatic
remodeling of chromosomal architecture. These radical changes are unambiguously related withwide range reprogramming of somatic cell genome. Even highly differentiated somatic nuclei can bededifferentiated in egg cytoplasm to acquire the totipotency essential for supporting normal
development to reproductive adulthood. In spite of this reprogramming process, somatic cell nuclear
transfer does not always result in ontogeny in mammals, and development is often associated withvarious abnormalities and embryo loss with a high frequency.
The study was aimed on evaluating influence of ooplasm on introduced somatic genome intetraploid (TP) embryos compared with somatic cell nuclear transfer (SCNT) embryos during the firstcell cycle. Following 20‐22 h of IVM, couplets of MII cytoplasts (SCNT), MII oocytes (TP) and starved
bovine fibroblasts were, after 2 h co‐culture, electrically fused, chemically activated by 5 µM
ionomycin for 5 min, followed by 3‐4 h incubation in 2 mM 6‐DMAP, and fixed at 4 and 12 hours post
activation (hpa). The SCNT and TP embryos were processed for immunofluorescence localization of
the nucleolar proteins: upstream binding factor (UBF) and fibrillarin at 4 and 12 hpa.. Embryos were
examined on confocal laser scanning microscope (Zeiss).
At 4 and 12 hpa, UBF was formed in discrete clusters surrounded by shell‐like structurelabeled by fibrillarin in SCNT embryos. This kind of localization of nucleolar proteins is distinguishedby nucleolus with large fibrillar centers (FC) segregated from the dense fibrillar component (DFC). At
4 and 12 hpa, all of the TP embryos displayed localization of fibrillarin to large spherical bodies.
During the first 4 hpa only 29.4 % of TP embryos was labelled for UBF. At 12 hpa, UBF displayed theincreased pattern of labelling in TP embryos (more than 65%).
Discrete localization of UBF in analysed SCNT and TP embryos can be explained by residual
association of this protein with rDNA characteristic for somatic cells during mitosis. This hypothesis isenhanced by partial recovery of nucleolus in SCNT embryos.
Acknowledgments
The work was supported by Marie Curie Intra‐European Fellowships (contract MRTN‐CT‐2006‐
035468, Clonet), VEGA 1/4323/07, VEGA 1/0057/08, CGA VI/7/2008, DFG and Alexander von
Humboldt fellowship.

62

Involvement of EGFR and Smad2/3 activation in FSH‐induced porcine oocytes‐
cumulus cell complex maturation
Eva NAGYOVA1, Radek PROCHAZKA1, Lucie NEMCOVA1, Antonella CAMAIONI2, Alzbeta
MLYNARCIKOVA3, Sona SCSUKOVA3, Antonietta SALUSTRI2
1

Institute of Animal Physiology and Genetics AS CR, Libechov, Czech Republic,
Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, Italy,
3
Institute of Experimental Endocrinology, Bratislava, Slovakia

2

It has been shown that the activation of both EGF receptor and SMAD2 and SMAD3 in mouse
cumulus cells is essential for their preovulatory maturation. In the present study we investigatedwhether these changes are also required for the final differentiation of pig cumulus cells. Porcine
oocyte‐cumulus complexes (OCCs) were stimulated in vitro with FSH in the absence or in thepresence of the following inhibitors: AG1478 (an EGFR tyrosine kinase inhibitor), SB431542 (a specificinhibitor of SMAD2 and SMAD3 activation) and SIS3 (a specific inhibitor of SMAD3 activation). We
found that EGFR inhibitor AG1478 completely blocked FSH‐induced cumulus expansion by preventing
hyaluronic acid production and its incorporation within the extracellular matrix. In addition, in thepresence of AG1478, most of FSH‐stimulated OCCs were in germinal vesicle stage (GV) and did not
mature. Conversely, AG1478 did not affect the stimulation of progesterone synthesis by FSH. In
agreement, EGF was unable to stimulate progesterone synthesis by OCCs cultured in vitro.
The SB431542, that inhibits the activation of SMAD2 and SMAD3, only partially inhibited cumulus
expansion by slightly decreasing both total production and matrix accumulation of hyaluronic acid.
Surprisingly, SIS, that inhibits only SMAD3, produced an inhibitory effect higher than SB431542. A
divergent effect of the two inhibitors was observed on steroid synthesis: FSH‐induced progesterone
synthesis increased in the presence of SB431542 and decreased in the presence of SIS. Bothinhibitors did not affect the induction of oocyte meiotic resumption promoted by FSH. These results
suggest that in pig, like in mice, FSH stimulates cumulus cells to produce EGF that acting in an
autocrine way induces the formation of the expanded matrix and the signaling for oocyte meiotic
resumption. The results also suggest that SMAD2 and SMAD3 activation is required for optimal
stimulation of hyaluronic acid and proteins involved in the organization of this polymer in the pig
OCC matrix. Finally, FSH stimulation of progesterone synthesis seems to be independent from EGF,
while it appears to be counteracted by SMAD2, but not SMAD3 activation, thereby suggesting thatspecific SMAD2 signaling pathway is probably indispensable to prevent luteinization of cumulus cells.
Supported by grant GACR 523/08/0111 and VEGA 2/0153/08.

63Gametegenesis following transplantation of primordial germ cells in domestic fowls
Yoshiaki NAKAMURA1
1

Interdisciplinary Graduate School of Science and Technology, Shinshu University, Nagano, Japan.
#
Research fellow of the Japan Society for the Promotion of Science

In avian species, unlike in mammal, techniques for cryopreservation of ova or nuclear
transfer are not available because of the large size of yolk structures. Therefore, primordial germ
cells (PGCs), the precursors of gametes, have been attracted attention as one of the powerfulmaterials for reproductive biology in chicken.
The first priority is clarification of the timing and migration pathway of chicken PGCs. PGCs
were scattered in the central zone of the area pellucida of blastoderm just after the eggs were laid.
Following the formation of primitive streak, PGCs moved anteriorly to the edge of the extra‐
embryonic region, so called that germinal crescent region. After enter of the developing vascularsystem, PGCs circulate temporarily in bloodstream and then migrate to the future gonadal region.
The unique mode of chicken PGCs migration during early development enables to combine
the cryogenic preservation of PGCs from early chicken embryos with the conservation of live animals.
PGCs were collected from the blood of early embryos, and cryopreserved in liquid nitrogen for over 6
months until transfer. Manipulated embryos were cultured until hatching; fertility tests indicatedthat they had normal reproductive abilities. Frozen‐thawed PGCs were transferred into thebloodstream of recipient embryos. Viable offspring derived‐from the frozen‐thawed PGCs wereobtained by crossing germline chimeras with manipulated chicken (efficiency was 12.6 %).
To obtain only donor‐derived offspring from germline chimeras, a novel technique for almost
complete replacement of the recipient germline with donor germ cells has been developed.
Following application of busulfan solublized in a sustained‐release emulsion (100 μg/50 μl) into theyolk of fertile eggs before incubation, nearly total depletion of the endogenous PGCs was achieved(0.6 % of control value). This was applied for preparing partially sterilized embryos to serve as
recipients for the transfer of donor PGCs. Immunohistochemical analysis showed that the proportionof donor PGCs in busulfan‐treated embryos was significantly higher than in controls (98.6% vs. 6.4%).
Genetic cross‐test analysis revealed that the germline transmission rate in busulfan‐treated chickens
was significantly higher than in controls (99.5% vs. 6.0%). Of 11 chimeras, 7 produced only donor‐
derived progenies, suggesting that these produced only donor‐derived gametes in the recipient’s
gonads.
These studies provide powerful tools for studying germline differentiation, for generatingtransgenic individuals, and for conserving genetic resources in avian species.

64

Sperm preincubation for intracytoplasmic sperm injection (ICSI) improvesdevelopmental ability of ICSI oocytes to the blastocyst stage in rats
Chino NAKAUCHI1, Katsuyoshi FUJIWARA1, Yasunari SEITA1,2, Junya ITO1,2, Naomi KASHIWAZAKI1,2
Laboratory of Animal Reproduction,
Graduate School of Veterinary Science,2
School of Veterinary Medicine, Azabu University, Sagamihara, Japan
1

Although in vitro fertilization (IVF) enables lots of oocytes to be fertilized at the same time,
IVF protocol using cryopreserved sperm has been very recently developed in rats. Intracytoplasmic
sperm injection (ICSI) is an alternative way to produce fertilized oocytes derived from cryopreserved
sperm. However, contrary to mice, it is known that rat ICSI‐oocytes show low developmental ability.
We previously reported that removal of acrosomal membrane from sperm head improvesdevelopment of ICSI‐oocytes in rats. Therefore, we next examined whether injection of capacitation‐induced sperm was effective in improving developmental ability of rat ICSI‐oocytes.
The cumulus‐oocyte complexes (COCs) were collected from the oviducts and used for IVF.
After insemination for IVF, the rest of sperm was collected and cryopreserved into liquid nitrogenusing a cryotube. The thawed sperm was separated into heads and tails by sonication and theninjected into the oocytes (IVF‐ICSI group). On the other hand, a part of sperm was incubated without
COCs. After collection and freezing‐thawing, the sperm was used for ICSI (Pre‐ICSI group). As control
group, fresh sperm was immediately used for ICSI. In control group, the proportion of 2 pronuclei
formation, the 2 cell stage and the blastocyst stage was 82.6%, 48.8% and 20.2%, respectively. In IVF‐ICSI group, these proportions were 82.1%, 66.0% and 34.7%, respectively. In Pre‐ICSI group, these
proportions were 91.0%, 65.8% and 42.4% , respectively. The rate of the blastocyst stage in Pre‐ICSI
group was tended to be higher than that in control group (p<0.1).
In conclusion, we demonstrated that the pre‐incubated sperm was effective in improving
developmental ability of rat ICSI‐oocytes, which suggests that induction of capacitation may be also
important for ICSI. Theses information will be useful for efficient production of rats via ICSI using
cryopreserved spermatozoa.

65

Fertile bull sperm aneuploidy and chromatin integrity in relationship to fertility
R. RYBAR, V. KOPECKA, P. PRINOSILOVA, S. KUBICKOVA, J. RUBES
Veterinary Research Institute, 621 00 Brno, Czech RepublicThis work was supported by the Grant Agency of the Czech Republic (523/07/P077) and by
the Grant Agency of the Ministry of Agriculture (1B44018).
Aneuploidy is associated with spontaneous abortions, perinatal mortality, mental retardation
and with embryonic and foetal mortality. Most of these abnormalities originate as a result of meiosis
errors during gametogenesis. The main purpose of the study was to analyse frequency ofaneuploidies of sex chromosomes and chromosome 6 by three‐colour FISH in 47 young bulls,
candidates for artificial insemination program with cryopreserved semen and to investigate theinfluence of aneuploidies and disturbed sperm chromatin integrity on non‐return rates, thefrequencies of abortions, perinatal mortality and stillbirths.
The average frequencies of sperm with disomy for chromosomes X, Y, XY and 6 were 0.032%,
0.005%, 0.003% and 0.039%, respectively. The incidence of XX66, YY66 and XY66 diploidy was
0.017%, 0.006% and 0.015%, respectively. Frequencies of meiotic II errors were significantly higher
than meiotic I errors (P<0.01). More X bearing sperm than Y bearing sperm were detected (5151 vs.
5022; P<0.01). Sperm chromatin damage expressed by DNA fragmentation index (DFI) was 5.3±3.7
and percentage of cells with defective chromatin condensation (HDS) was 1.4±0.8. No correlation
was found between sperm aneuploidy and basic sperm analysis. The relationship was found between
non‐return rate and total aneuploidy (r=‐0.310; P=0.036). Significant correlation was found between
sex disomy, total aneuploidy (disomy of chromosomes 6, X, Y and XY sperm and diploidy) andstillbirths (r=0.390; P=0.013; and r=0.331; P=0.037). Chromosome 6 disomy correlated with perinatal
mortality (r=0.317; P=0.047). HDS correlated significantly with total aneuploidy (r=0.449; P=0.002).
Our study indicated that aneuploidy frequencies in young fertile bull sperm are relatively low.
Nevertheless, there exists a variability in aneuploidy frequencies amongst bulls, which appears to beable to have an influence on the fertility of these animals.

66

Isobutyl‐methylxanthine facilitates capacitation‐associated protein tyrosine
phosphorylation of frozen‐thawed C57BL/6J mouse sperm
Nozomi SUZUKI1, Junya ITO1,2, Naomi KASHIWAZAKI1,2
Laboratory of Animal Reproduction, 1Graduate School of Veterinary Science, 2School of Veterinary
Medicine, Azabu University, Sagamihara, Japan
In vitro fertilization (IVF) using cryopreservation of mouse sperm is routinely applied for
efficient production of animals. Since lots of transgenic and gene‐targeting mice have been
generated, IVF using cryopreserved sperm will become more important. However, it is well known
that cryopreservation of mouse sperm in C57BL/6J strain which is commonly used for production oftransgenic and gene‐targeting mice, decreases the fertility markedly. We recently reported thattreatment of isobutyl‐methylxanthine (IBMX) dramatically improved capacitation‐associated protein
tyrosine phosphorylation of frozen/thawed rat sperm, succeeded in IVF for the first time using
cryopreserved sperm in rats. Therefore, in this study, it was examined whether IBMX treatment also
improved capacitation‐associated protein tyrosine phosphorylation of frozen/thawed mouse spermin C57BL/6J strain. Cauda epididymal sperm was collected from ICR and C57BL/6J strains andcultured in TYH medium up to 3 h. Frozen C57BL/6J sperm was thawed in TYH medium
supplemented with or without IBMX (0, 100, 200, 400, 800 M) up to 3 h. At every 1 h, sperm
samples were collected and used for detection of capacitation‐associated protein tyrosine
phosphorylation by western blotting. In both ICR and C57BL/6J fresh sperm, capacitation‐associatedprotein tyrosine phosphorylation was induced in a time‐dependent manner. On the other hand,capacitation‐associated protein tyrosine phosphorylation in frozen/thawed C57BL/6J sperm without
IBMX treatment was completely inhibited even at 3 h. However, IBMX treatment dramatically
improved capacitation‐associated protein tyrosine phosphorylation of frozen‐thawed C57BL/6J
sperm in a dose‐dependent manner. Taken together, our results suggest that IBMX treatment iseffective for improvement of capacitation‐associated protein tyrosine phosphorylation in frozen‐
thawed C57BL/6J mouse sperm. Now we are trying to clarify whether this IBMX treatment can also
improve fertility of oocytes fertilized in vitro using cryopreserved sperm in C57BL/6J strain.

67

Telomerase (pTERT) mRNA expression in the pig granulosa cells in vitro
Milan TOMANEK1, Tomas KOTT2, Ewa CHRONOVSKA1 and Eva KOTTOVA2
1

Institute of Animal Science, Department of Biology of Reproduction, Prague 10 – Uhříněves, Czech
Republic; tomanek.milan@vuzv.cz
2
Institute of Animal Science, Department of Molecular Genetics, Prague 10 – Uhříněves, Czech
Republic

Cell proliferation in the ovary is critical to the ovarian functions of producing and releasing
germ cells (oocytes) and hormones for reproduction. During the development of oocytes,
folliculogenesis is accompanied by significant proliferation of granulosa cells, which is tightly
controlled in a temporal and spatial manner.
Telomerase plays an essential role in cell viability and has been defined as a key factor for the
regulation of cell proliferation and senescence. Under various physiological and pathologicalconditions the dynamics of telomerase expression and activity in proliferating, regenerating ordifferentiating tissue is regulated by cellular microenvironment and growth factors (Bayne and Liu,
2005). We described telomerase enzymatic activity in pig granulosa cells in vitro and we suggested
that its activity and function might be differentially regulated in small and large developing ovarianfollicles (Tomanek et al., 2008). The aim of presented work was to develop a new RT‐PCR method of
pig telomerase (pTERT) mRNA quantification and its use for the study of pTERT mRNA expression in
the pig follicular granulosa cells in vitro.
Granulosa cells (GC) were isolated from small (SF‐GC) and large (LF‐GC) follicles and cultured
for 96 h in vitro in DMEM/F12 medium supplemented with 2% FBS, ITS, gentamycin, testosterone(10‐8M), EGF (10 ng/ml) and pFSH (50 ng/ml). Telomerase activity was assayed with the modified
protocol based on the TRAPEZE® Telomerase Detection Kit, (Chemicon). For RT‐PCR, the total RNA
was isolated from granulosa cells with the use of Total RNA Chemistry method and 6100 Nucleic Acid
PrepStation (Applied Biosystems). Primers and TagMan MGB probes for pTERT were designed with
the Primer Expres ver. 3 Program. The mRNA was reverse transcribed into cDNA with the HighCapacity cDNA Archive Kit and RT‐PCR was performed on the ABI 7500 Fast Real‐Time PCR System(Applied Biosystems).
Freshly isolated SF‐GC expressed higher levels of pTERT mRNA than LF‐GC (3.363 vs. 1.834
RU) as well as higher levels of telomerase activity (112.11 vs. 71,20 TPG/prot.). During in vitro
culture, pTERT mRNA levels in SF‐GC increased in control and FSH treated cells (5.815 and 6.405 RU)
and slightly decreased in EGF treated cells (2.723 RU). On the other hand, LF‐GC expressedsignificantly lower pTERT mRNA levels (1.112, 0.78 and 0.95 for control, EGF and FSH ) as well as thelevels of telomerase activity (8.32, 9.66 and 11.12 TPG/mg prot. for control, EGF and FSH,
respectively).
In conclusion, we suggest that telomerase expression and activity in pig follicular granulosa
cells is related to their proliferative potential and function in developing ovarian follicles and pTERTmRNA expression and telomerase activity decline in highly differentiated, luteinized granulosa cells.
Supported by MZE CR project 0002701402
References
Bayne and Liu 2005. Mol.Cell.Endocrinol. 240: 11‐22
Tomanek et al. 2008. Anim. Reprod. Sci. 104:284‐298

68Isolation, cultivation and characterization of porcine cell lines derived fromblastocysts produced in vitro and in vivo
Irena VACKOVA1,3, Zora NOVAKOVA1,3, Leona URBANKOVA1 and Jan MOTLIK2,3
1

Institute of Animal Production, Pratelstvi 815, CS‐104 01 Prague, Czech Republic.
Institute of Animal Physiology and Genetics, Czech Academy of Science, CS‐277 21 Libechov, Czech
Republic.
3
Center for Cell Therapy and Tissue Repair, Prague, Czech Republic

2

The pig represents an interesting animal model for several branches of human medicine
including stem cell therapies, however, the full‐functional methodics for derivation of embryonicstem cells from porcine blastocysts has not been exactly established yet. The present study was
designed to compare the potential to generate porcine embryonic stem (ES) cell lines fromblastocysts produced under in vivo and in vitro conditions.
We found that significant proportion of inner cell masses (ICMs) from in vivo obtained
blastocysts attached to the feeder layer and started to grow in a monolayer. In particular,
proliferating cells formed three types of colonies – i/ tightly packed colonies of small cells resemblinghuman ES cells – ES‐like, ii/ flattened colonies of polyhedral shaped, weakly packed cells – epithelial‐
like, and iii/ big polyhedral shaped cells revealing large intracellular area with abundant lipid dropletsin the cytoplasm – trophectodermal cells. Immediately after attaching, ES‐like cells revealed markersof pluripotency. However, during cultivation cells derived from in vivo‐produced blastocysts (age 8‐
11 days) spontaneously differentiated into neural‐like or beating cells. In contrast to in vivo‐produced
blastocysts, the proportion of attached in vitro obtained blastocysts was low, moreover, proliferating
cells revealed mainly trophectodermal or epithelial‐like phenotype.
In summary, our results suggest that in vivo produced blastocysts represent more suitablematerial for derivation of porcine cell lines with various phenotype including ES‐like cells.

69Comparative transcriptomic analysis of in vivo and in vitro produced bovine
embryos using a custom bovine embryo‐specific microarray
Katerina VODICKOVA KEPKOVA1,2, Petr VODICKA1, Tereza TORALOVA1, Miloslava LOPATAROVA2,
Svatopluk CECH2, Radovan DOLEZEL2, Vitezslav HAVLICEK3, Urban BESENFELDER3, Anna KUZMANY3,
Marc‐Andre SIRARD4, Jozef LAURINCIK5, Jiri KANKA1
1

Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumburská
89, 277 21 Libechov, Czech Republic.
2
Veterinary and Pharmaceutical University, Faculty of Veterinary Medicine, Palackeho 1‐3, 612 42
Brno, Czech Republic
3
Reproduction centre ‐ Wieselburg, University of Veterinary Medicine, Vienna, Austria
4
Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université
Laval, Sainte‐Foy, Québec, Canada G1K 7P4.
5Constantine the Philosopher University, Faculty of Natural Sciences, Trieda A. Hlinku, SK‐949 74
Nitra, Slovak Republic
Pre‐implantation embryos derived by in vitro fertilization differ in their developmental
potential from embryos obtained in vivo. In order to characterize changes in gene expression profiles
caused by in vitro culture environment, we employed microarray constructed from bovine embryo‐specific cDNAs (BlueChip, Université Laval, Québec). The analysis revealed changes in level of 133
transcripts between in vitro derived and in vivo derived 4‐cell stage embryos and 96 transcripts weredifferentially expressed between 8‐cell stage in vitro and in vivo embryos. The expression profiles of
10 transcripts (BUB3, Fibrillarin, Nopp140, PCAF, Sf3a, GPBP, GABPα, TAF7, Cul1 and CNOT4) werestudied in detail by quantitative RT‐PCR during whole in vitro culture period (oocyte ‐ hatchedblastocyst). Protein level and localization of Cul1 and Fibrillarin in GV and MII oocytes and 2‐cell –morula stage embryos was also studied by immunofluorescence. Several identified genes can bepotentially useful as markers of embryo quality in future attempts to optimize in vitro culture
conditions.
Acknowledgements
This study was supported by the GA CR project No. 523/06/1226, GACR project No.204/09/H084,
MSM grant 6215712403, CLONET project MRTN‐CT‐2006‐035468, DFG and VEGA 1/0012/10 andInstitutional Research Concept IAPG No. AV0Z50450515.

70

Comparison of media for culture of matured and fertilized rat oocytes
Mayo WATANABE1, Junya ITO1,2, Naomi KASHIWAZAKI1,2
Laboratory of Animal Reproduction,
Graduate School of Veterinary Science,2
School of Veterinary Medicine, Azabu University, Sagamihara, Japan
1

Recently, embryonic stem cells have been successfully established in rats. Therefore, it
seems that in vitro manipulation and culture of rat oocytes/embryos become more important.
Although rat oocytes/embryos can be cultured in vitro, developmental ability of theoocytes/embryos is generally low compared to mice regardless in vitro manipulation. This result
suggests that development of more optimal medium is required for in vitro manipulation and cultureof rat oocytes/embryos. The objective of the present study was to clarify the factors involved indevelopmental ability of rat oocytes/embryos. Since it is known that rat oocytes are spontaneously
activated in vitro and this spontaneous activation is involved in the decrease of developmental abilityof the oocytes, we compared the development of oocytes cultured in different media which areusually used for in vitro culture of mouse and rat oocytes/embryos. Cumulus‐oocyte complexes werecollected from the oviducts in superovulated female rats and then cumulus cells were removed. The
oocytes were cultured in different media (R1ECM, mR1ECM, KSOMaa, TYH or CZB). Extrusion of
second polar body was investigated as indicator of spontaneous activation. At the result, theproportion of the extrusion in oocytes cultured in KSOMaa and TYH was lower than those in othermedia. When calcium was removed from KSOMaa, the extrusion was further suppressed. To clarify
the in vitro developmental ability of fertilized oocytes cultured in different media, in vivo fertilized
oocytes were collected from the oviducts from mated rats and then cultured in different media(R1ECM, mR1ECM, KSOMaa, TYH). As for in vitro development of fertilized oocytes to the 2 cell stage,
the proportion of the oocytes cultured in KSOMaa was higher than those in other media. However,
only fertilized oocytes cultured in R1ECM could be developed to blastocysts. In conclusion, our result
suggests that calcium‐free KSOMaa seems to be more optimal media for in vitro manipulation of rat
oocytes. Although further improvements will be required, KSOMaa may be also effective for in vitro
culture of fertilized oocytes in rats.

71

72

Mailing list:
ANGER Martin

anger@iapg.cas.cz

BARNETOVA Irena

barnetova.irena@vuzv.cz

BENESOVA Veronika

vercik.b@seznam.cz

BOHMOVA Terezabohmova@iapg.cas.cz

DANG‐NGUYEN Thanh Quang

adipy36@hotmail.com

FUJIWARA Katsuyoshi

ma0922@azabu‐u.ac.jp

FULKA Helena

fulka.josef@vuzv.cz

FULKA Joseffulka.josef@vuzv.cz

HARAGUCHI Seiki

sharaguchi@affrc.go.jp

HIRAO Yuji

yujih@affrc.go.jp

HORNAK Miroslav

hornak@vri.cz

HULINSKA Pavlina

hulinska@vri.cz

ITO Junyaitoj@azabu‐u.ac.jp

JESETA Michal

jeseta@vri.cz

KALOUS Jaroslav

Kalous@iapg.cas.cz

KANEDA Masahiro

mkaneda@affrc.go.jp

KANKA Jiri

kanka@iapg.cas.cz

KARABINOVA Pavla

karabinova@iapg.cas.cz

KASHIWAZAKI Naomi

nkashi@azabu‐u.ac.jp

KIKUCHI Kazuhiro

kiku@affrc.go.jp

KISHIMOTO Takeo

tkishimo@bio.titech.ac.jp

KOLEDOVA Zuzana

zkoledova@gmail.com

KUBELKA Michal

kubelka@iapg.cas.cz

KUCEROVA CHRPOVA Veronika

kucerova‐chrpova@iapg.cas.cz

KYOGORU Hirohisa

097a209a@stu.kobe‐u.ac.jp

LALOVA Helena

lalovah@vri.cz

LEE Jibak

leej@riken.jp

MACHATKOVA Mariemachatkova@vri.cz

MAN Petr

pman@biomed.cas.cz

MANABE Noboru

amanabe@mail.ecc.u‐tokyo.ac.jp

MATOBA Shogo

matoba@rtc.riken.jp

MAYER Alexander

mayer@iapg.cas.cz

MIYANO Takashi

miyano@kobe‐u.ac.jp73

MOROVIC Martin

mmorovic@ukf.sk

MOSKO Tibor

mosko.tibor@vuzv.cz

MOTLIK Jan

motlik@iapg.cas.cz

NAGAI Takashi

taku@affrc.go.jp

NAGYOVA Evanagyova@iapg.cas.cz

NAKAGAWA Shoma

shoma‐tira@hotmail.co.jp

NAKAI Michikonakai3@affrc.go.jp

NAKAMURA Yoshiaki

ynsu@affrc.go.jp

NAKAUCHI Chino

ma1015@azabu‐u.ac.jp

NEMCOVA Lucie

nemcova@iapg.cas.cz

NOVAK Petrpnovak@biomed.cas.cz

NOVAKOVA Zora

zorkanov@gmail.com

OGURA Atsuo

ogura@rtc.riken.go.jp

OGUSHI Sugako

ogushi@cdb.riken.jp

OKADA Konosuke

okada@nvlu.ac.jp

POMPACH Petr

pompach@biomed.cas.cz

PROCHAZKA Radek

prochazka@iapg.cas.cz

REZNIKOVA Jaroslava

reznikova@iapg.cas.cz

RUBES Jiri

hornak@vri.cz

RYBAR Roman

hornak@vri.cz

SKALNIKOVA Helenaskalnikova@iapg.cas.cz

SOLC Petr

solc@iapg.cas.cz

SUGIURA Koji

aks@mail.ecc.u‐tokyo.ac.jp

SUZUKI Nozomi

ma1010@azabu‐u.ac.jp

TOMANEK Milan

tomanek.milan@vuzv.cz

TORALOVA Tereza

moravcova@iapg.cas.cz

VACKOVA Irena

ivackova@seznam.cz

VODICKA Petr

vodicka@iapg.cas.cz

VODICKOVA KEPKOVA Katerina

kepkova@iapg.cas.cz

WATANABE Mayoma1027@azabu‐u.ac.jp

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Content
Organizers ……..………………………………………………………………………………………………………………………………………… 3
Financial support ………..…………………………………………………………………………………………………………………………… 3
Introduction …..……………………………………………………………………………………………………………………………………….. 5
Programme (day 1st) ….…………………………………………………………………………………………………………………………… 7
Programme (day 2nd) …………………………………………………………………………………………………………………………….. 9
Takeo KISHIMOTO (Key note lecture)
Cell cycle control: Lessons from starfish oocytes …………………………………………………………………………………….. 11
Lectures – topic 1: Oocytes …………………………………………………………………………………………………………………… 13
Jibak LEE, Tatsua HIRANO
Regulation of chromosome dynamics by cohesins in mammalian meiosis ……………………………………………… 15
Takashi MIYANO, Mohammad MONIRUZZAMAN
Activation and inactivation of primordial oocytes …………………………………………………………………………………. 16
Koji SUGIURA, You‐Qiang SU, John J. EPPIG
Oocyte control of granulosa cell function and development ………………………………………………………………….. 17
Yuji HIRAO, Kosuke IGA, Kenji NARUSE, Naoki TAKENOUCHI
In vitro growth of bovine oocyte‐granulosa cell complexes after vitrification ………………………………………… 18
Radek PROCHAZKA
Regulation of pig oocyte maturation by EGF‐like peptides …………………………………………………………………….. 20
Sugako OGUSHI, Yasuko KATO, Akira NAKAMURA, Mitinori SAITOU
Functional analysis of maternal nucleous in mouse oocytes ………………………………………………………………….. 21
Petr SOLC, Vladimir BARAN, Gabriela PANENKOVA, Adela SASKOVA, Tereza BOHMOVA,
Alexandra MAYER, Richard M. SCHULTZ, Jan MOTLIK
Aurora‐A regulates MTOCs (centrosome) biogenesis but it does not trigger G2/M
Transition in mouse oocytes matured in vivo …………………………………………………………………………………………. 22
Martin ANGER
Age‐related aneuploidy in mammalian oocytes …………………………………………………………………………………….. 23
Lectures – topic 2: Fertilization …………………………………………………………………………………………………………….. 25
Junya ITO, Rafael A. FISSORE, Naomi KISHIWAZAKI
Factors associated with calcium oscillations during fertilization in mammals ………………………………………… 27
Konosuke OKADA
Vole sperm cryopreservation: assessments of viability, sperm DNA integrity
and fertilizing capacity …………………………………………………………………………………………………………………………… 28
Tereza TORALOVA, Andrej SUSOR, Lucie NEMCOVA, Katerina KEPKOVA, Jiri KANKA
The role of CENPF in bovine preimplantation development ……………………………………………………………………. 29
Helena FULKA
From oocyte to embryo‐ remodeling the parental genomes …………………………………………………………………… 30
Lectures – topic 3: Stem cells ………………………………………………………………………………………………………………… 31
Ales HAMPL
Human embryonic stem cells …………………………………………………………………………………………………………………. 33

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Zuzana KOLEDOVA, Leona RASKOVA KAFKOVA, Alwin KRAMER, Vladimir DIVOKY
Centrosomal decisions on self‐renewal in mouse embryonic stem cells …………………………………………………… 34
Petr VODICKA, Katerina VODICKOVA‐KEPKOVA, Jan MOTLIKiPS cells in miniature pigs ……………………………………………………………………………………………………………………….. 35
Jana JUHASOVA, Stefan JUHAS, Jiri KLIMA, Jan STRNADEL, Alois NECAS, Robert SRNEC,
Ladislav PLANKA, Evzen AMLER, Eva FILOVA, Josef JANCAR, Jan MOTLIKBone marrow‐derived mesenchymal stem for the regeneration of cartilage and bone ……………………………. 36
Helena SKALNIKOVA, Petr VODICKA, Petr HALADA, Martin MARSALA, Jan MOTLIK,
Hana KOVAROVA
Proteomics of neural stem cells isolated from miniature pigs ………………………………………………………………….. 37
Lectures – topic 4: Genetics manipulation ……………………………………………………………………………………………… 39
Atsuo OGURA, Kimiko INOUE
Improvement of somatic cell nuclear transfer in mice: Genetic and epigenetic approaches …………………….. 41
Masahiro KANEDA, Tamas SOMFAI, Seiki HARAGUCHI, Satoshi AKAGI, Shinya WATANABE,
Takashi NAGAI
Normal and abnormal epigenetic changes in the development of cloned cattle ……………………………………… 43
Noboru MANABE, Ichiro ONOYAMA, Junyou LI, Yutaka SENDAI, Yoshito AOYAGICharacteristics of prion, bovine spongiform encephalopathy (BSE) pathogen,homoknockout cow …………………………………………………………………………………………………………………………………. 44
Michiko NAKAI, Kazuhiro KIKUCHI, Junya ITO, Naomi KASHIWAZAKI, Hiroyuki KANEKO
Xenotransplantation of gonadal tissue for utilization of genetic resources in pigs …………………………………… 46
Stefan JUHAS, Marian HRUSKA‐PLOCHAN, Jana JUHASOVA, Petr VODICKA, Antonin PAVLOK,
Monika BAXA, Atsushi MIYANOHARA, Martin MARSALA, Jan MOTLIK
Miniature pig as a large animal model of Huntington disease ………………………………………………………………… 48
Poster session ………………………………………………………………………………………………………………………………………… 49
Veronika BENESOVA, Tereza TORALOVA, Katerina VODICKOVA, Petr VODICKA, Jiri KANKA
Expression and function of nucleophosmin/B23 during the preimplantation
bovine development ……………………………………………………………………………………………………………………………….. 51
Tereza BOHMOVA, Alexandra MAYER, Petr SOLC, Vladimir BARAN, Jan MOTLIK
Polo‐like kinase 1 and Aurora A cooperate in spindle assembly during meiotic
maturation of mouse oocytes …………………………………………………………………………………………………………………. 52
Than Quang DANK‐NGUYEN, Masahiro KANEDA, Tamas SOMFAI, Kazutsugu MATSUKAWA,
Satoshi AKAGI, Kazuhiro KIKUCHI, Michiko NAKAI, Bui Xuan NGUYEN, Atsushi TAJIMA,
Yukio KANAI, Takashi NAGAI
Development of twin blastocysts derived from single blastomeres of the 2‐cell stageembryos in pigs ………………………………………………………………………………………………………………………………………. 53
Katsuyoshi FUJIWARA, Daisuke SANO, Yasunari SEITA, Tomo INOMATA, Junya ITO,
Naomi KASHIWAZAKI
Calcium‐free medium including ethylene glycol for oocyte vitrification improves
survival and embryonic development of vitrified rat oocytes ………………………………………………………………….. 54
Seiki HARAGUCHI, Tamas SOMFAI, Thanh Q. DANK‐NGUYEN, Masahiro KANEDA, Shinya WATANABE,
Kazuhiro KIKUCHI, Tomoyuki TOKUNAGA, Masaya GESHI, Takashi NAGAI
Single blastomeres within mouse 4‐cell embryo expresses different amounts of mRNA ………………………….. 5576

M. HORNAK, M. JESETA, P. PUSILOVA, A. PAVLOK, M. KUBELKA, J. MOTLIK, M. ANGER, J. RUBES
Analysis of chromosome segregation errors oocytes using comparative genomic hybridization ……………… 56
Jaroslav KALOUS, Michal KUBELKA, Jan MOTLIKInhibition of Polo kinase 1 (Plk1) affects resumption of meiosis in porcine oocytes …………………………………. 57
Pavla KARABINOVA, Lucie LISKOVA, Michal KUBELKA
Role of selected kinases in CPEB‐dependent cytoplasmic polyadenylation ………………………………………………. 58
Hirohisa KYOGORU, Takashi MIYANO, Josef FULKA Jr.
Enucleolation of growing and full‐grow pig oocytes ………………………………………………………………………………… 59
M. MACHATKOVA, M. JESETA, P. HULINSKA, S. KAPLANOVA, L. NEMCOVA, J. KANKA
Characteristics of mitochondria in bovine oocytes related to their meiotic competence …………………………. 60
Shogo MATOBA, Atsuo OGURA
Generation of functional mouse oocytes and spermatids from isolated primordial germ
cells following ectopic transplantation in adult animals ………………………………………………………………………….. 61
M. MOROVIC, O. ØSTRUP, F. STREJCEK, I. PETROVICOVA, A. LUCAS‐HAHN, E. LEMME,
H. NIEMANN, J. LAURINCIK, P. HYTTEL
Nuclear and nucleolar remodeling during the first cell cycle in bovine somatic cell nuclear
transfer and tetraploid embryos ……………………………………………………………………………………………………………… 62
Eva NAGYOVA, Radek PROCHAZKA, Lucie NEMCOVA, Antonella CAMAIONI, Alzbeta MLYNARCIKOVA,
Sona SCSUKOVA, Antonietta SALUSTRI
Involvement of EGFR and Smad2/3 activation in FSH‐induced porcine oocytes‐cumulus
cell complex maturation …………………………………………………………………………………………………………………………. 63
Yoshiaki NAKAMURA
Gametegenesis following transplantation of primordial germ cells in domestic fowls …………………………….. 64
Chino NAKAUCHI, Katsuyoshi FUJIWARA, Yasunari SEITA, Junya ITO, Naomi KASHIWAZAKI
Sperm preincubation for intracyzoplasmic sperm injection (ICSI) improves developmental
ability of ICSI oocytes to the blastocyst stage in rats ……………………………………………………………………………….. 65
R. RYBAR, V. KOPECKA, P. PRINOSILOVA, S. KUBICKOVA, J. RUBES
Fertile bull sperm aneuploidy and chromatin integrity in relationship to fertility …………………………………….. 66
Nozomi SUZUKI, Junya ITO, Naomi KASHIWAZAKI
Isobutyl‐methylxanthine facilitates capacitation‐associated protein tyrosine
phosphorylation of frozen‐thawed C57BL/6J mouse sperm …………………………………………………………………….. 67
Milan TOMANEK, Tomas KOTT, Ewa CHRONOVSKA, Eva KOTTOVA
Telomerase (pTERT) mRNA expression in the pig granulosa cells in vitro ………………………………………………… 68
Irena VACKOVA, Zora NOVAKOVA, Leona URBANKOVA, Jan MOTLIKIsolation, cultivation and characterization of porcine cell lines derived from blastocysts
produced in vitro and in vivo …………………………………………………………………………………………………………………… 69
Katerina VODICKOVA KEPKOVA, Petr VODICKA, Tereza TORALOVA, Miloslava LOPATAROVA,
Svatopluk CECH, Radovan DOLEZEL, Vitezslav HAVLICEK, Urban BESENFELDER, Anna KUZMANY,
Marc‐Andre SIRARD, Jozef LAURINCIK, Jiri KANKA
Comparative transcriptomic analysis of in vivo and in vitro produced bovine embryos
using a custom bovine embryo‐specific microarray …………………………………………………………………………………. 70
Mayo WATANABE, Junya ITO, Naomi KASHIWAZAKI
Comparison of media for culture of matured and fertilized rat oocytes …………………………………………………… 71

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Published by the Institute of Animal Physiology and Genetics AS CR, v.v.i., Libechov, Czech Republic
2010
Editorial Address:
Institute of Animal Physiology and Genetics AS CR, v.v.i.
Rumburska 89
277 21 Libechov
Czech RepublicTel.: +420 315 639 511, +420 315 639 505
Fax: +420 315 639 510
E‐mail: uzfg@iapg.cas.cz, hladka@iapg.cas.cz
http://www.iapg.cas.cz

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