国際生殖シンポジウム「生殖生物学の革命と展望：体外受精、胚性幹細胞、エピジェネティクスの展望」
International Meeting for Evolution of Reproductive Biology and Task of Frontiers: Trajectory and Prospects of IVF,
Stem Cell and Epigenetic Studies

IS-PS
Fertilization Studies and Assisted Fertilization in Mammals: Their
Development and Future
Ryuzo YANAGIMACHI
Institute for Biogenesis Research, University of Hawaii Medical School, Honolulu, Hawaii 96822 USA
Scientific studies of fertilization began when it was recognized that spermatozoa and eggs, large or small, aresingle cells (von Koelliker, 1840; Gegenbaur, 1861) and their fusion, in particular the fusion of their nuclei, is theessence of fertilization and the beginning of life of a new individual (Hertwig, 1876; Fol 1879). Early studies offertilization were done using invertebrates (such as sea urchins and annelids) because their gametes could be collected readily and fertilization and embryo development could be examined readily in a dish of sea water. Before
1960 mammalian fertilization studies were difficult. Collection of a large number of fully mature eggs at a time wasdifficult in many species, and no one knew what kinds of media they should use to handle gametes, zygotes andpreimplantation embryos. Thanks to many researchers’ endeavor, fertilization research in a variety of mammalian
species has become far easier. Discovery that mammalian spermatozoa, unlike sea urchin spermatozoa, require“capacitation” to become fertilization-competent (Chang, 1951; Austin, 1951, 1952) triggered active research of
mammalian fertilization. Today, papers dealing with mammalian fertilization published annually by far exceed thosedealing with fertilization in non-mammalian vertebrates and invertebrates combined.
Progress in structural, biochemical and molecular studies of mammalian fertilization during the last 50 years isimpressive (for reviews, see Austin, 1961; Schatten, 1994; Yanagimachi, 1994; Wassermann and Florman, 1997;
Moro and Okabe, 2011). Progress, however, never ends. Even a long-standing dogma that mammalian spermatozoa
undergo the physiological acrosome reaction after contact with the zona pellucida of eggs has been challenged thisyear by Jin et al. (2011) who demonstrated that most fertilizing (mouse) spermatozoa are acrosome-reacted alreadybefore they contact with zona surfaces. The concept that egg’s zona pellucida is the sole biological molecule thattriggers the physiological sperm acrosome reaction must be abandoned. Such a simple question as “how does
mammalian spermatozoon pass through the zona pellucida?” has not been settled (Bedford, 2008). Molecular mechanisms by which spermatozoa become competent to fuse with egg plasma membrane (Kiawah et al., 2008) are yet to
be clarified.
Among major technical breakthrough in the last century are: (1) cry preservation of gamete and embryos, (2) invitro maturation of eggs, (3) assisted fertilization such as IVF and ICSI, (4) sperm-sexing, (5) non-surgical embryotransfer, and (6) preimplantation genomic analysis of embryos.
Subjects of future investigations related, directly or indirectly, to fertilization and the improvement of animal andhuman reproduction include: (1) permanent preservation of spermatozoa at ambient temperature, (2) sex selection ofindividual spermatozoa, (3) mass-production of mature gametes in vitro, (4) selection of genomically normal gametes
prior to assisted fertilization, (5) conversion of diploid (somatic) cells to haploid (germ) cells, and (6) genomic
manipulation of gametes for the production of superior animals.

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IS1-1A Historical Perspective on IVF, In Vitro Embryo Culture and ET in
Japan
Yutaka TOYODA
Professor Emeritus, Obihiro University of Agriculture and Veterinary Medicine
In Japan, a pioneering research on mammalian in vitro fertilization (IVF) was performed by Jinshin Yamane (1889–1972)
during the early 1930s. He observed the extrusion of the second polar body in the rabbit eggs following ‘insemination’ andconcluded that the activation was caused by sperm penetration into the egg. Evidence of fertilization itself was not confirmed,
however. Gregory Pincus (1903–1967), another pioneer in the U.S.A., was confronted with similar difficulties. Unequivocal
cytological evidence of IVF was first obtained in 1954 by Charles Thibault and his colleagues in France, by using ‘uterine
spermatozoa’ recovered from mated females, after the discovery of capacitation by C.R.Austin and M.C.Chang in 1951. Chang(1959) then obtained live offspring by transferring the IVF rabbit embryos into pseudopregnant recipients, as the definitive proof
of normal developmental capability.
Since the first success in IVF-ET by Chang, the progress has been accelerated and the research fields expanded rapidly. Two
major technical advances are considered most critical; that is (1) induction of in vitro capacitation, and (2) improvement of
culture medium for preimplantation embryonic development.
The possibility of in vitro capacitation was first demonstrated by Yanagimachi and Chang (1963) who showed that hamster eggs
can be fertilized in vitro by epididymal spermatozoa almost as readily as by uterine spermatozoa. Inspired by this landmark paper,
young Japanese scientists challenged new subjects of IVF research in Chang’s lab at the Worcester Foundation in Shrewsbury,
U.S.A., in rapid succession during the next 15 years, resulting in a large amount of scientific contributions, including the first mouse
IVF by epididymal sperm (Iwamatsu & Chang, 1969), the first rat IVF by uterine sperm (Miyamoto & Chang, 1973), and the first
IVF-ET in the rat (Toyoda & Chang, 1974).
After coming back from Chang’s lab to Japan, A. Hanada led the national team to put the IVF-ET to practical use for cattle
production and finally succeeded in delivering a newborn calf in 1985, by non-surgical transfer of IVF bovine embryos derived
from in vitro matured eggs. Fortunately non-surgical ET in cattle had already been established in this country by pioneering workof T. Sugie (1965). Moreover, Oguri & Tsutsumi (1974) had successfully applied the Sugie’s procedure to equine blastocysts
developed in vivo, as the first non-surgical ET in the horse.
In parallel to the progress in laboratory and farm animals, basic as well as clinical aspects for human IVF were extensively
studied in several medical institutions in Japan, and the first IVF baby was born at Tohoku University Hospital, Sendai (M.Suzuki
et al, 1983). It was five years later than the birth of the world’s first IVF baby, Louise Brown, in Cambridge, U.K. (Steptoe &Edwards, 1978).
Current methodologies for in vitro culture of mammalian embryos are largely dependent on early studies on mouse preimplantation embryos, notably by J.D. Biggers, R.L. Brinster, W.K. Whitten and D.G. Whittingham. Defined culture media developedby them in the late 1960s to early 1970s have provided the bases of nearly all the IVF media developed thereafter, including thosefor humans. At about the same time, we could establish a repeatable procedure for mouse IVF (Toyoda, Yokoyama & Hosi, 1971)
and our medium (TYH) has become widely used, although it is limited to the IVF, not for embryo culture beyond the 2-cell stage.
Efforts to improve the IVF medium have been continuing to date and will be continued in the future. Every effort to minimize
the differences between in vivo and in vitro will provide an insight into the mechanisms of mammalian reproduction and development, thus leading to the refinement of assisted reproductive technologies (ART).

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IS1-2
Frontiers of IVF in Livestock Species: Factors Affecting Efficiency
Christopher G. GRUPEN
Faculty of Veterinary Science, the University of Sydney, Camden, NSW 2570, Australia
Embryo in vitro production (IVP) systems in sheep, cattle and pigs have been established for over twenty years, yet
the goals to fully understand the interaction of the gametes and achieve the highest fertilization rates possible remain
fundamental to researchers. The embryo IVP efficiency equation is simple; in vitro fertilization (IVF) of the best
quality oocytes with the best quality sperm under the optimal in vitro conditions will produce the best quality embryos.
Our understanding of oocyte maturation processes has progressed substantially over the past decade. It is now well
accepted that communication between the maturing oocyte and its associated cumulus cells is a complex two-way
interaction and that the intact follicle regulates meiotic progression at a level not yet achieved in vitro. Examinationof the properties of cumulus-oocyte complexes classified according to antral follicle diameter, long known to be anindicator of oocyte quality, is still providing useful insight into the molecular and cellular events taking place during
maturation. The development of embryo IVP systems in which oocytes are matured and fertilized individually, andthe resulting embryos are cultured individually, are anticipated to accelerate our understanding of how developmental
competence is acquired by the oocyte. Applying this knowledge in IVP embryo transfer programs may increase thenumber of transferrable embryos obtained per oocyte donor, which has remained static for many years.
For a multitude of reasons, sperm used in IVF systems of livestock species are almost always cryopreserved beforehand. Also, IVF incorporating sex-sorted sperm is regarded as an effective way to overcome the limitations of thesex-sorting technology, particularly in species that require large artificial insemination doses. Both the cryopreservationand sex-sorting processes expose sperm to stressors that reduce their fertility. The addition of seminal plasma or its
components to processing media provides some benefit; seminal plasma improved the post-thaw quality ofcryopreserved ram sperm and PSP-I/II spermadhesin reduced the capacitation-like changes in sex-sorted boar sperm.
However, negative effects of seminal plasma addition have also been reported, and inconsistencies between studies
highlight the biological variability of its composition and the differences in sperm membrane responsiveness. Proteomic
analysis is being increasingly used to identify proteins of seminal plasma and sperm membranes that are associatedwith sperm function and male fertility. Understanding the role of these proteins during in vivo and in vitro fertilization remains a key challenge.
The methods used to prepare and select sperm for IVF and the in vitro conditions used during the co-incubation of
sperm and oocytes also affect the efficiency of fertilization. While swim-up incubation and density gradient centrifugation are commonly implemented to select motile, viable sperm for IVF, the procedures often need to be refined
depending on the quality of the sperm sample or the type of processing the sperm have been subjected to. Some novel
approaches to selecting high quality sperm for IVF have potential to improve fertilization efficiencies. Finally,
limiting the gamete co-incubation period is an effective strategy for reducing the incidence of polyspermy in porcine
IVF, and may improve embryo development by decreasing oxidative damage associated with the production of reactive oxygen species. The continued refinement of IVF conditions to increase the rate of normal sperm penetration
will undoubtedly benefit from an increased understanding of the differences in sperm properties between species,
between males of the same species, and between ejaculates from the same male.

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IS1-3Puppies from Frozen Embryos
Hiroshi SUZUKI
Obihiro University of Agriculture and Veterinary Medicine
The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer and cryopreservation of
gametes have considerably contributed to the development of biomedical sciences in addition to improved breedingin domestic animals and infertility treatment in humans. However, ARTs used in canine species have strictly limited
utility when compared with other mammalian species including humans. Although successful somatic cell cloning
has been reported, artificial insemination by frozen semen is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind to date. Cryopreservation for mammalianoocytes and embryos has been used for several purposes such as to preserve the genes of elite livestock, increase theefficiency of animal breeding, preserve valuable genetically modified animals and endangered species, and use germ
cells effectively in ARTs in animals and humans. The first mammalian species to have embryos successfully
cryopreserved was the mouse in the early 1970s, and over the next three decades this has been extended to embryosof most species including humans. However, cryopreservation of canine embryos has not progressed far enough. I
describe here successful cryopreservation of embryos and subsequent embryo transfer in dogs. Embryos were collected from excised reproductive organs of Labrador Retrievers on day 2–10 after artificial insemination (on day 7–
15 after LH surge) and subsequently cryopreserved by ethylene glycol based vitrification solution with a cryotop
sheet. The embryos were examined under ultraviolet light using epifluorescent microscopy and the plasma membrane integrity of embryos was assessed with PI staining. The embryos that had blasotomeres negative for PI staining
accounted for more than 75% were assessed as normal. For embryo transfer, the embryos were not stained with the
PI. The localization of developing embryos in the reproductive tracts of Labrador Retrievers was clarified in thisstudy. Embryos at morula stage migrated from the oviduct into uterus on day 10 after the LH surge. This transport
might be completed within 24 hrs. By day 12 after the LH surge, all of developing embryos moved and localized inuterus. Embryos developed to morula by day 11–12 and to blastocyst by day 12–13 after LH surge, respectively. To
assess membrane integrity of the cryopreserved embryos, when vitrified-warmed embryos were stained with PI, theviability of those embryos at 4– to 16–cell stage, morula and blastocyst was 90–100%, 50% and 40%, respectively.
When vitirified-warmed canine embryos were non-surgically transferred with a cystoscope to recipients, normal pups
were delivered reproducibly. Namely, of nine embryo transfer experiments five recipients became pregnant and fourof them delivered seven pups in total. Percentages of development to newborn after the transfer of cryopreserved
canine embryos were 9.1%. The microsatellite genotypes clearly demonstrated that those delivered pus were derived
from donor embryos but not from the recipient animal. These results clearly indicate that cryopreserved canine
embryos are able to develop to term, even if developmental rates after the transfer were lower. The suitable combination of stages between transferring embryo and estrus of recipient might be when 8-cell to 16-cell stage of embryos
were transferred to recipients on day 8–9 post LH surge. The cryopreservation of embryo in canine species will makeavailable for transportation, storage of genetic materials, and elimination of vertically transmitting diseases. And thistechnique will contribute to improved breeding of companion and working dogs such as guide dog, drug detecting
dog or quarantine dog.

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IS1-4
Sexing Mammalian Sperm
George E. SEIDEL, JR.
Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523-1683,
USA
The only procedure that has proven efficacious for sexing sperm is flow cytometry/cell sorting while measuring
fluorescence of Hoechst 33342, which binds to DNA quantitatively. Since X-chromosome-bearing sperm of most
mammals have about 4% more DNA than Y-chromosome-bearing sperm, this difference is the basis for sexing sperm.
Refinements in sperm sorters enable sexing over 10 million sperm of each sex per hour per sorting nozzle with 90 %accuracy. This procedure for sexing sperm has been used for many mammalian species. However, sexing sperm onan every day, commercial basis is only being done with two species, humans and cattle, the latter at several million
insemination doses per year, mostly with dairy cattle. There have been many research applications of sexing sperm,
and some use has occurred in wildlife. Commercialization is just beginning with horses, deer, and sheep.
Sexed sperm usually are cryopreserved using standard procedures, and usually are used via artificial insemination,
but in vitro fertilization followed by culturing and then transferring resulting embryos is another option. The limited
number of sperm that that can be sorted per unit time economically has resulted in using about 2 million sexed sperm
per insemination dose for cattle rather than typical doses of unsexed sperm of 10 million or more sperm. With goodmanagement of cattle, fertility of this low dose sexed sperm product usually is 70 to 90% of fertility of controls of 10
million unsexed sperm per insemination dose. About one-third of this decreased fertility is due to using fewer sperm,
but the majority of the decrease is due to mechanical damage of the sperm during the sorting process. The exact cause
and nature of this damage is not known. Thus, pregnancy rates, even with 10 million sexed sperm per inseminationdose, are still low under most conditions.
Studies of thousands of calves produced via sexed sperm have not revealed any differences in incidences of abnormalities compared to control calves produced with unsexed sperm. While there are many applications for sexed
sperm, the main one in cattle is to produce more females for breeding purposes or sale, primarily by breeding heifers
to have heifer calves. An important human application is to avoid sex-linked genetic diseases by having female
offspring. Sperm sexing technology is protected by numerous patents, and licenses for use in animals are controlled
by the company Sexing Technologies, located in Navasota, Texas. For human applications, the intellectual property
is controlled by the company Genetics and IVF Institute, in Fairfax, Virginia. These companies have made hugeinvestments in developing and refining sperm sexing technology.

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IS1-5
From a Functional to a Molecular Assessment of Totipotency
Jean-Paul RENARD, Helene KIEFER, Helene JAMMES, Isabelle HUE,
Nathalie BEAUJEAN and Véronique DURANTHONINRA, UMR1198 Biologie du Développement et Reproduction, F-78352 Jouy-en-Josas, France
ENVA, F-94704 Maisons Alfort, France
Totipotency refers to the ability of a single cell to give rise to all of the differentiated cells that contribute to thedevelopment of an organism including its germline, and in mammals the extraembryonic tissues. Totipotency is atransitory nuclear state since restriction in term of cell developmental potentialities leads to pluripotency, a property
defined as the ability of a (single) cell to differentiate in vitro and/or in vivo into the three primitive embryonic layers
and to contribute to the germ line of the organism but in chimera experiments. The reprogramming of nuclear
functions to totipotency after fertilization is thus the first crucial event of development.
Reprogramming the nucleus of a somatic cell to totipotency can be obtained following the introduction of its
nucleus into an enucleated oocyte, a procedure known as cloning. In the lab, we are using cattle clones as a pertinentmodel to characterize totipotency, this for two main reasons: first, fertile adults can be at obtained from cattle cloned
embryos at a relatively high rate and second, the three-weeks long preimplantation period of development allows anin vivo embryo sampling which encompasses the initiation of gastrulation when the three embryonic lineages are
already differentiated.
Measuring the 5-methylcytosine levels of DNA from circulating leukocytes, we evidenced a high variability among
healthy adult clones, a result which we confirmed at the tissular level by hybridization of immunoprecipitated methylated DNA on a cattle tiling array (MeDIP-chip) dedicated to 21416 promoter regions (UMD3.1 genome assembly).
This provides support to the view that the dynamic process of reprogrammation to totipotency is intimately bound toa long lasting epigenetic adaptation of the genome. Ongoing statistical analysis should allow identifying the genomic
regions with a high epigenetic variability in clones
To characterize this epigenetic adaptation at earlier developmental stages, when the functional reprogramming tototipotency can by definition not be determined, we used donor nuclei with different full reprogramming abilities but
similar implantation rates to generate cloned embryos. We found that the intra-and inter-variability of gene expression between these donor nuclei is unexpectedly low even for the extra-embryonic tissues of late pre-implanting
embryos (D18). At that stage, the embryonic lineages are properly individualised revealing reprogramming topluripotency. This indicates that the subsequent gene deregulations associated to abnormal placentation, the mostfrequently reported reprogramming failure after nuclear transfer, have not yet occurred or are still not detectable atthe onset of embryo patterning.
Molecular reprogramming of somatic nuclear activities to pluripotency after nuclear transfer is thus apparently
very close to the reprogramming of gamete genomes after fertilisation, but their reprogramming to totipotency is not.
Totipotency needs obviously additional and still uncharacterised epigenetic requirements. By studying the nuclear
distribution of the heterochromatin protein CBX1 together with that of histone H3K9me3 and centromeric proteins
CENPA/B just prior to embryonic genome activation, we have identified a subtype of nuclear organisation withcondensed heterochromatin blocks that could correspond to poorly reprogrammed cloned embryos in comparison to
fertilised controls. However, these embryos could to some extend be converted to a normal (totipotent) type byincreasing the initial exposure of the donor chromatin to the oocyte cytoplasm.
In conclusion our data indicate that totipotency could be made accessible to a structural and molecular pre ante andnot only to a functional ex post analysis as it is still mostly the case today.
Supported by the European program SABRE, “Cutting Edge Genomics for Sustainable Animal Breeding”, ref CT2006-01625 and French National research Agency (ANR), “EpiGrani”, ref ANR- 09-GENN-012-02.

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IS1-6
Abnormality and the Restoration of In Vitro-produced Porcine
Embryos
Kazuhiro KIKUCHI1, Tamás SOMFAI2, Michiko NAKAI1 and Takashi NAGAI2
1Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki, 305-0901, Japan2

In vitro production (IVP) of embryos including in vitro maturation (IVM), fertilization (IVF) and embryo culture(IVC) is now an important procedure not only for basic study in reproductive biology but also for obtaining live
offspring as an application technology. The in vitro developmental competence or viability of porcine IVM-IVF
oocytes to the blastocyst stage was first confirmed about 20 years ago [Mattioli et al. 1989]. Since then, live-born
piglets have been obtained from IVM-IVF embryos after IVC to the 2- to 4-cell stages [Mattioli et al. 1989; Yoshida
et al. 1993; Funahashi et al. 1996; Funahashi & Day 1997] and to the blastocyst stage [Marchal et al. 2001; Kikuchi
et al. 2002].
IVP procedures to generate porcine embryos have been improved, but the systems have still problems resulting inpoor developmental ability, and low quantity and quality of the IVP embryos. This leads to the failure of pregnancy
or embryo loss even after their transfer to recipients. One of the major causes of this problem is abnormal ploidy of
IVP embryos causing by 1) fertilization of oocytes arrested at the immature stage [Kikuchi et al. 1999; Somfai et al.
2005] and 2) polyspermy during IVF [Somfai et al., 2008].
It is important to make every effort to achieve normality in IVP embryos after IVM and IVF. However, it isbasically impossible to prevent immature meiotic arrest or polyspermy in all cultured oocytes even using currently
established technologies. Another approach for obtaining a good result under these conditions is to select fertilized
oocytes or embryos that guarantee the developmental competence to piglets when they are transferred to recipient
females. It is generally accepted that only monospermic fertilized oocytes that have matured to the metaphase-IIstage can be guaranteed to show normal embryonic development. On the other hand, in comparison with sperm,
oocyte sources are limited, especially those from rare genetic resources, genetically modified and cloned animals. To
apply in vitro reproductive technologies in such animals, it is important to utilize as many oocytes as possible from alimited number of females. For this propose, assuming some ability to develop to term, abnormally generated embryos may be used to generate offspring.
Aneuploidy during oocyte maturation has been noticed as a cause of embryo abnormality in pigs [Lechniak et al.
2007]. Mammalian embryos with abnormal ploidy have been known for long years to be capable of surviving eveninto the post-implantation period [Piko & Bomsel-Helmreich 1960; Bomsel-Helmreich 1971; Han et al. 1999]. However, the details of the mechanism involved have remained unclear. In our previous study, some IVM-IVF oocyteswith abnormal ploidy after polyspermy found to be able to develop into diploid embryos [Somfai et al. 2008]. The
mechanism by which such polyploid oocytes after IVF develop to a normal state during embryo development is still
not well understood. Attempts to clarify this mechanism would give an increased efficacy in IVP system including
other species, and also basic knowledge for human reproductive medicine.
In the present symposium, as well as in the previous review article [Kikuchi et al., 2009], we summarize the status
and consequences of generating abnormal porcine IVP embryos obtained as a result of polyspermy and also by
fertilization before completion of meiotic maturation. We also discuss the possibility of restoration of the abnormallygenerated embryos for improvement of porcine IVP efficacy.-− j64 −

IS2-1
Critical Involvement of Aberrant X Chromosome Inactivation in the
Defective Postimplantation Development of Cloned Embryos
Atsuo OGURA
RIKEN BioResource Center
Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome in the reconstructed cloned embryos. Reprogramming errors appear to arise randomly, but
the presence of many SCNT-specific phenotypes (e.g.; placental abnormalities, obesity, immunodeficiency, and perinatal death) suggests that some reprogramming errors may also arise in specific genes or regions within the donor
genome. We sought to identify the upstream dysfunction leading to impaired development of clones by DNA microarray
analysis using single cloned embryos.
When we compared the global gene expression patterns of SCNT blastocysts with those of IVF embryos, genes on
the X chromosome were specifically downregulated. This phenomenon was sex- and genotype-independent because
the average X:autosome (X:A) expression ratio in the four types of cloned embryos (two males and two females) was
consistently lower than in the corresponding control 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, theXist expression level was significantly higher in male and female cloned embryos than in corresponding IVF embryos. RNA fluorescent in situ 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 normalizationof the expression levels of the majority of downregulated genes, not just on this chromosome but also on autosomes.
This resulted in a 10- to 12-fold increase in the average cloning efficiency (producing birth rates of 5–18%), whichclearly suggests that the ectopic expression of Xist impaired the development of SCNT embryos. Furthermore,transient downregulation of Xist by a RNA interference (RNAi) technique also lead to 10 times more survival of the
Xist-siRNA embryos compared with controls as early as day 5.5 and this high survival persisted until term. This
clearly indicates that SCNT-specific XCI dysregulation can be confined to a narrow stage before implantation andthat Xist knockout exerts its greatest effect in rescuing cloned embryos during this very short period.
A detailed analysis of gene expression patters of Xist-knockout cloned blastocysts suggested the presence of another Xist-independent mechanism that specifically inactivated the Magea and Xlr family of genes on the X chromosome. These comprise gene clusters overlapping with regions enriched for histone H3 lysine 9 dimethylation(H3K9me2), which are highly conserved in somatic cells. We infer that they must have been in a constitutively
repressed state, inherited from the donor genome. In contrast to the successful conversion of ectopic Xist expression
by specific RNAi, this H3K9me2-induced epigenetic aberration was not rescued by RNAi against the G9a or Glp
genes, which are responsible for the establishment of the H3K9me2 modification.
Thus, our studies provide the clear evidence that SCNT induces epigenetic errors specifically on Xa, which are
likely beyond the ability of putative ooplasmic factors to remedy. Contrary to the general assumption, our results
suggest that the fate of cloned embryos may be determined predominantly before implantation by their XCI status.

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IS2-3Difference in the DNA Methylation Status Arises after Segregation ofTrophoblast and Embryonic Cell Lineages
Momo NAKANISHI, Koji HAYAKAWA, Kunio SHIOTA and Satoshi TANAKAThe University of Tokyo, Tokyo, JapanThe mammalian genome contains tissue-dependent and differentially methylated regions (T-DMRs). Cell typespecific DNA methylation status of T-DMRs constitutes DNA methylation profile of the cell, which ought to bereconstructed as cells differentiate. The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages resulting in the formation of trophectoderm (TE) and inner cell mass (ICM) inblastocyst. Although lower level of global DNA methylation in TE compared to ICM has been suggested byimmunostaining, dynamics of DNA methylation profile during the TE/ICM differentiation has not been carefully
studied.
We aimed to identify T-DMRs between trophoblast and embryonic cell lineages and analyzed their DNA methylation status in pre- and post-implantation embryos. Oct4 and Nanog are differentially methylated between trophoblaststem (TS) and embryonic stem (ES) cell lines (TS-ES T-DMRs). These two loci are hyper-methylated in TS cells andhypo-methylated in ES cells. Elf5 is another known TS-ES T-DMR but exhibits the opposite methylation pattern. A
number of other TS-ES T-DMRs have been also found so far in our laboratory. Bisulfite sequencing analysis of 11
TS-ES T-DMRs revealed that the DNA methylation status of them in E6.5 extraembryonic ectoderm and epiblast
coincide with those in TS and ES cells, respectively, showing that the TS-ES T-DMRs are surely cell lineage-based TDMRs. We then collected TE and ICM cells separately from E3.5 blastocysts and subjected to bisulfite sequencing.
Interestingly, genomes of TE and ICM exhibited no difference in the methylation status of all T-DMRs examined.
They were in extremely hypomethylated status. Thus we identified valuable T-DMRs for delineation of trophoblast/embryonic cell lineages. DNA methylation analysis of these T-DMRs suggested that the DNA methylation profiles
specific to each cell lineage are established after the morphological delineation of trophoblast and embryonic celllineages. DNA methylation may thus contribute to stabilize or fix the cell fate and the differentiation capacity.

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IS2-4
Regulatory Pathways Controlling Uterine Spiral Artery Remodeling
Michael J. SOARES, Damayanti CHAKRABORTY, Stephen J. RENAUD, Kaiyu KUBOTA,
Pengli BU and M.A. KARIM RUMI
Institute for Reproductive Health and Regenerative Medicine, Department of Pathology and Laboratory Medicine,
University of Kansas Medical Center, Kansas City, Kansas, USA
The maternal-fetal interface is a dynamic site where uterine and placental structures cooperate to promote development of the fetus. The rat, mouse, and human each possess a hemochorial placenta. This type of placentation ischaracterized by erosion of the maternal uterine epithelium and vasculature permitting the direct flow of maternal
nutrients to trophoblast. Remodeling uterine vasculature is critical for the success of pregnancy. As gestation progresses,
uterine spiral arteries supplying the placenta are modified creating flaccid, low resistance blood vessels. These
vessels are the conduit required to meet the nutrient demands of the fetus. Defective hemochorial placentation,
including impairments in uterine spiral artery remodeling, leads to pregnancy related disorders (preeclampsia, intrauterine growth restriction, preterm birth. The extravillous/invasive trophoblast lineage is of fundamental importanceto development of the maternal-fetal interface and understanding diseases of placentation and pregnancy. Based onin vitro analyses several “candidate” signaling pathways regulating the extravillous/invasive trophoblast lineage havebeen identified; however, the significance of most of these pathways during in vivo placentation are unknown. Organization of rat and human placentation sites show significant conservation, especially regarding trophoblast-directed
remodeling of the uterine spiral arteries. Both species exhibit deep trophoblast invasion. We have established relevant in vitro and in vivo research methodologies using the rat as an animal model to investigate the extravillous/
invasive trophoblast lineage. Two pathways controlling the extravillous/invasive trophoblast lineage have been identified: i) hypoxia/hypoxia inducible factor and ii) phosphatidylinositol 3-kinase/AKT/FOSL1. We have establishedthe importance of these pathways using both in vitro and in vivo experimentation. Dissection of these pathways has
facilitated identification of fundamental regulators of the extravillous/invasive trophoblast lineage. Understanding
molecular mechanisms underlying development of the extravillous/invasive trophoblast lineage is key to identifying
developmentally sensitive events that are potentially susceptible to dysregulation; and represent opportunities todiscover and evaluate approaches for the early detection and treatment of diseases of placentation. (Supported by
NIH-HD20676)− j68 −

IS2-5Novel Proteins Affecting Cell Proliferation and Angiogenesis of
Bovine Placenta
Toru TAKAHASHI 1,3, Koichi USHIZAWA 1,2, Shobu SASAKI 3, Misa HOSOE 1, Keiichiro
KIZAKI 4 and Kazuyoshi HASHIZUME 4
1

National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-0901, Japan
Present address: National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan
3
The University of Tokyo, Kashiwa, Chiba 277-8583, Japan
4
Iwate University, Morioka, Iwate 020-8550, Japan
2

Placenta is temporal organ that plays a role as an interface for feto-maternal dialogue. A variety of molecules is produced andutilized for the manifestation of placental function. Ruminants form unique cotyledonous placenta characterized by a large
number of placentomes. Two separate types of trophoblasts, trophoblast mononucleate cells (MNCs) and trophoblast giant/binucleate cells (BNCs), play a principal role on the placental function in ruminants.
MNCs produce interferon tau, trophoblast kunitz domain proteins, pregnancy-associated glycoprotein-2 and other MNCspecific proteins. Recently authors have identified a novel intronless gene uniquely expressed in ruminant MNCs. Since open
reading frame of this gene was flanked by reversed Alu sequences, we concluded this gene was one of the Alu integrated
retrotransposon. This gene was composed of 22-a.a.-long signal peptide and 78-a.a.-long single Ly-6 domain, therefore we
named this gene “Secreted protein Of Ly-6 Domain 1” (SOLD1). SOLD1 mRNA is exclusively transcribed in MNCs, however
SOLD1 protein is localized in the interstitial tissue of placenta. This apparent paradox is attributable to the anchoring of SOLD1
to extracellular matrix through interaction with type I collagen. SOLD1 protein regulates gene expression in cotyledonary fibroblasts in vitro and appears to participate in the construction of ruminant placenta. Our results suggest that the target site ofcollagen-anchored SOLD1 is restricted to near the storage site and biological signal of SOLD1 is triggered by their liberation
during the degradation of extracellular matrices. Except in ruminants, neither placental expression nor intronless structure of Ly6 family gene, have not been reported. The uniqueness of SOLD1 gene implies that SOLD1 is specific for ruminants at least in
cattle, sheep and goats. Since it has been proposed that mammals have evolved by the insertion of exogenous elements such as
retrotransposons and retroviruses, retroposed SOLD1 might have impacts on ruminant implantation and placentation.
Ruminant placenta produces an array of proteins structurally and functionally similar to prolactin (PRL). Of these only theplacental lactogen is known as a “classical” member because of functional similarity to PRL. The rest is called as “non-classical”
member without lactogenic activity. Biological activities of non-classical members in ruminants remain unclear. The authors
have cloned novel ovine PRL paralogue, named ovine prolactin-related protein (oPRP1). oPRP1 had a typical PRP nucleotide
sequence similar to bPRP1 and was exclusively transcribed in BNCs as well as bPRP1. However translational frame shiftingaround the C-terminus region of oPRP1 resulted in the premature termination, translated oPRP1 was anticipated to have a shortpolypeptide sequence. Recent studies revealed that rat and human PRLs could be cleaved by cathepsin and resulting fragments
acquired a new biological activity quite different from that of parental molecules. Since biological activities of oPRP1 wasappeared to be comparable with that of bPRP1, the authors hypothesized that bPRP1 expressed proper biological activity following C-terminal cleavage by some enzymes. Short-form (23–28 kDa) bPRP1 fragments co-existed with intact molecules in bovine
cotyledonary conditioned medium (BCCM). Recombinant bPRP1 was cleaved by cathepsins and matrix metalloproteinases(MMPs) resulting in N-terminal fragments ranging 24–28 kDa. BCCM itself was capable of bPRP1 cleavage and contained
MMP activity. Cleaved bPRP1 stimulated the proliferation of bovine brain microvascular endothelial cells in vitro. Our results
suggest that bPRP1 is potential substrate for placental enzymes including cathepsins and MMPs, and resulted N-terminal fragments stimulate placental angiogenesis.
Collectively, ruminant placenta produces many kinds of molecules those act harmoniously, particularly placental proteinsthose responsible for tissue remodeling and angiogenesis play crucial roles for the maintenance of gestation and fetal development. (Supported by grants from the JSPS Japan, 20380159 and 22580327)− j69 −

IS2-6
Select Nutrients in the Uterine Lumen of Sheep and Pigs AffectConceptus Development
Fuller W. BAZER1,2, Jinyoung KIM2, Hakhyun KA3, Greg A. JOHNSON4 and Guoyao WU1
1

Departments of Animal Science and 4Veterinary Integrative Biosciences, Texas A&M University, USA
WCU Biomodulation, Seoul National University, Korea
3
Department of Biological Resources and Technology, Yonsei University, Korea
2Interferon tau (IFNT), the pregnancy recognition signal from trophectoderm (Tr) cells of ruminant conceptuses (embryo andits extra-embryonic membranes), abrogate the uterine luteolytic mechanism to ensure maintenance of functional corpora lutea for
production of progesterone (P4). Additionally, IFNT acts in concert with P4 to induce expression of genes for production ofhistotroph. Histotroph includes nutrients, such as glucose and arginine (Arg), that activate mechanistic mammalian target of
rapamycin (MTOR) nutrient sensing cell signaling to stimulate proliferation, migration, differentiation and translation of mRNAs
by conceptus Tr.
In sheep, Arg and glucose increase 16- and 12-fold, respectively in uterine histotroph between Days 10 and 15 of pregnancy
due to expression of transporters for glucose (SLC2A1 and SLC5A1) and Arg (SLC7A2B) in uterine luminal (LE) and superficial
glandular (sGE) epithelia induced by P4 and stimulated by IFNT. In vitro, 0.2 mM Arg and 4 mM glucose (physiologicalconcentrations in plasma) stimulate phosphatidylinositol 3-kinase (PI3K) cell signaling to increase 3-phosphoinositide-dependent protein kinase 1 (PDPK1), V-akt murine thymoma viral oncogene homolog 1 (AKT1), cAMP response element-binding
protein 1 (CREB1), glycogen synthase kinase 3-beta (GSK3B), MTORC1, eukaryotic translation initiation factor 4e-binding
protein 1 (EIF4EBP1) and ribosomal protein S6 kinase (RPS6KB1). Further, in Day 16 ovine conceptus explants cultures, Arg
increases GTP cyclohydrolase 1 (GCH1) mRNA while both Arg and glucose increase ornithine decarboxylase (ODC), nitricoxide synthase 2 (NOS2), and GCH1 proteins, and Arg increases IFNT. GCH1 is the rate-limiting enzyme for synthesis oftetrahydrobiopterin, an essential cofactor for all NOS isoforms. Arg can be metabolized to nitric oxide (NO), by NOS, and topolyamines, by ODC1, via arginase. Both NO donors (SNAP and DETA) and putrescine (polyamine) increase proliferation ofoTr cells, and their effects are partially inhibited by L-NAME and Nor-NOHA that inhibit NOS and arginase, respectively.
Secreted phosphoprotein 1 (SPP1) in ovine uterine histotroph increases migration, focal adhesion assembly, adhesion, and perhaps Tr cell proliferation.
In pigs, total recoverable glucose, Arg, Leu and Gln increase in histotroph with advancing days of the estrous cycle andpregnancy; the greatest increase being between Days 12 and 15 of pregnancy. The glucose transporter SLC2A1 mRNA is highly
expressed in uterine LE of cyclic and pregnant gilts, but less so in conceptus Tr. Expression of SLC2A4 mRNA is similar to
SLC2A1, but expression is lower. SLC2A2 mRNA is abundant in conceptuses from Days 12 to 85 of pregnancy. SLC5A1 mRNAis expressed specifically in uterine LE on Days 12 and 13 of pregnancy. SLC2A3 mRNA is expressed very weakly in pig
conceptus and uterine tissues. The most abundantly expressed amino acid transporters are those for glutamate and neutral aminoacids (SLC1A1, SLC1A4) and cationic amino acids (SLC7A1, SLC7A2, SLC7A7, SLC7A9). SLC7A3 mRNA is highly expressedin conceptuses from Days 25 through 85 of gestation. Treatment of porcine Tr cells with either Arg (0.4 mM) or Leu (0.4 mM)
increases phosphorylated RPS6K, RPS6 and EIF4EBP1 in the nucleus or cytoplasm, and proliferation of Tr cells by 8.2- (P<0.01)
and 11.5-fold (P<0.01), respectively. Glucose and fructose also stimulate proliferation of pig Tr cells 3.4- (P<0.01) and 5.2-fold(P<0.01), while Azaserine, an inhibitor of hexosamine biosynthesis inhibits their effects on Tr cell proliferation.
These results indicate mechanisms whereby select nutrients act differentially to affect translation of of mRNAs for cell signaling molecules that affect conceptus growth, development, and survival during the peri-implantation period of pregnancy.
This research was supported by USDA CSREES National Research Initiative Grants 2006-35203-17283 and 2011-6701520028 and, in part by World Class University Program (R31-10056) through the National Research Foundation of Korea fundedby the Ministry of Education, Science and Technology.

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IS2-7
Gene Expression in the Bovine Endometrium: The Effects of Altered
Progesterone Concentrations and the Presence of the Conceptus
Niamh FORDE and Pat LONERGAN
School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Ireland
In cattle, the majority of pregnancy loss can be attributed to early embryonic loss which occurs prior maternal
recognition of pregnancy on Day 16 (Day 0 = ovulation). During this time, two significant processes are required toestablish and maintain pregnancy. Firstly, the developing conceptus (embryo and associated extra embryonic membranes) must undergo significant morphological changes from a blastocyst enclosed in a zona pellucida to a rapidly
elongating conceptus. This elongation process is entirely dependent on maternally derived secretions (termed histotroph) from the uterine endometrium as, in sheep, ablation of uterine glands renders the uterus incapable of initiating
this elongation process. Secondly the uterine endometrium must prepare for attachment and implantation of theconceptus which involves carefully orchestrated spatio-temporal alterations in the transcriptomic profile of the endometrium. The two main modulators of the endometrial transcriptome and by corollary maternally derived secretions are the hormone progesterone (P4) and the pregnancy recognition signal interferon tau (IFNT).
The important role of P4 in the establishment and maintenance of pregnancy have been well documented. Elevatedconcentrations of circulating P4, either endogenous or by exogenous supplementation, in the immediate post-conception period are associated with advanced conceptus elongation, increased IFNT production and higher pregnancy
rates in both beef and dairy cattle. Conversely, low P4 concentrations have been implicated as a causative factor in
the low pregnancy rates observed in dairy cows with recent data from demonstrating that post partum dairy cows havelower circulating concentrations of P4 associated with an impaired ability of the oviduct/uterus to support embryo
development, compared to their dairy heifer counterparts. The mechanism through with P4 impacts on conceptus
development is mediated through effects on the endometrium, rather than a direct effect on the embryo itself. Indeed,transfer of in vitro cultured embryos on Day 7 into recipient heifers that have had their P4 concentrations manipulated
have resulted in altered conceptus elongation. Transcriptomic analysis of the uterine endometrium has shown thatmodulation of circulating P4 concentrations alters endometrial gene expression which is beneficial (when P4 is supplemented) or detrimental (when P4 is reduced) to the developing conceptus. These changes in the endometrium involve
alterations to genes that can contribute to histotroph composition with demonstrated consequences for conceptus
elongation. In addition to these P4-induced alterations in endometrial gene expression, P4 is required to downregulate its own nuclear receptor in uterine luminal epithelium (LE) and superficial glandular epithelium (sGE) toestablish uterine receptivity to implantation and to allow the expression of genes that encode for secreted proteins andtransporters that drive conceptus elongation to be switched on.
In addition to P4 altering endometrial gene expression, the pregnancy recognition signal IFNT also has a significant impact on the endometrial transcriptome. IFNT, a type 1 interferon, includes the expression of classical interferon stimulated genes, which are predominantly maintained in the deep glandular epithelium and stromal cells, forprotection of the developing semi-allograph conceptus from the maternal immune system. However, IFNT can also
stimulate the expression of non-classical genes in the LE and GE which contributes to histotroph composition anddrive conceptus elongation. In summary, the successful establishment of pregnancy in cattle requires a sequence of
key events to ensure appropriate maternally derived secretions, establish uterine receptivity to implantation as well as
an adequate endometrial response to IFNT production.

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IS2-8Development of the Endometrial Spheroids as a Model for
Implantation In Vitro
Nobuhiko YAMAUCHI1, Kazuyoshi HASHIZUME2 and Masa-aki HATTORI1
1

Department of Animal and Marine Bioresource Sciences, Kyushu University, Japan
Department of Veterinary Medicine, Iwate University, Japan2

The endometrium is one of the most complex tissues; it undergoes dynamic changes in response to the process of
implantation and the stages of pregnancy. An in vitro model may provide a tool for clarifying the complex implantation process. The spheroid has been utilized in cell biology research, because it appears to mimic the morphology andphysiology of living tissues and organs are unlikely to conventional monolayer culture. The cells in multicellularspheroids are in close contact with each other and have considered useful in the investigation of intercellular interactions. Recently, we have developed multicellular spheroids composed of endometrial cells derived from bovine or rat
endometrium. In this study, interaction of endometrial spheroids and embryos was analyzed in a co-cultured system
in order to develop a model to clarify the mechanism of implantation in vitro.
Bovine endometrial spheroids were hetero-spheroids, composed of epithelial and stromal cells, generated using Lascorbic acid phosphate magnesium salt n-hydrate which stimulated collagen synthesis in stromal cells. Histologicalobservation revealed that hetero-spheroids were covered by epithelial cells on the outer layer. The spheroids expressed receptor genes for steroid hormones (ERα, PR) and INFτ (INFR1, INFR2). Moreover, the spheroids expressed Mx1 as a response to INF. The spheroids were co-cultured with the trophoblast tissues collected from bovine
elongated embryos (5 mm) at d 18 of pregnancy. P4 (1 µM) and IFNτ (100 ng/ml) were added to the medium duringco-culture. At the starting of co-culture, spheroids and trophoblast tissues were partially contacted. The spheroids
gradually adhered to the trophoblast tissues, and most part of the spheroids were embedded in to the tissue at 7 daysof co-culture. Further we observed that vesicles were developed from the trophoblast tissue during the co-culture.
Expression of MMP-9 was significantly increased in co-cultures, compared to single culture of spheroids or trophoblast tissues (P<0.05).
Rat endometrial spheroids were homo-spheroids, composed of only endometrial stromal cells derived from d 5 ofpregnancy, generated using salmon aterocollagen (SAC) gels. Diameters of the spheroids were about 500 µm at d 5
after detachment from SAC gels. The results of gelatin zymography showed that both MMP-2 and -9 were produced
in monolayer culture. However, after the detachment of the cell sheet, the production of both MMPs decreased
immediately and could not be detected until 15 days after detachment. In vitro decidualization of the spheroids were
induced by arachidonic acid (AA) treatment. RT-PCR analysis showed that typical marker genes for the decidualization,
desmin and d/tPRP, were expressed in the spheroids after 2 days of AA treatment, but not in the control groups
without the treatment. When the spheroids were co-cultured with hatched blastocyst rat embryos, they started toadhere at 24 h and embryos embedded in to the spheroids at 96 h after the beginning of co-culture. Furthermore,
activities of MMP-2 and -9 were detected in the co-cultures, whose activities were not detected in single culture of thespheroids or the embryos.
The present study demonstrated that the endometrial spheroids express receptors for factors important in the process of implantation, such as steroid hormones and INFτ. Furthermore, the spheroids and the embryos adhered andinteracted with each other for the occurrence of accompanying tissue remodeling which indicated by MMP expression. These results indicate that the endometrial spheroids provide a new insight into the study of endometrial functions and implantation, as a model for implantation in vitro.

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IS2-9Endogenous Retroviruses: A Model System for Understanding
Physiological Adaptation to a Rapidly Evolving Ruminant Genome
Thomas E. SPENCER1 and Massimo A. PALMARINI2
1
2

Washington State University, Pullman, Washington, USA
University of Glasgow Veterinary School, Glasgow, Scotland, United Kingdom

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and are remnants of ancient exogenous retroviral infections of the host germline transmitted vertically from generation to generation. Sheepbetaretroviruses offer a unique model system to study the complex interaction between retroviruses and their host1.
The sheep genome contains 27 JSRV-related endogenous betaretroviruses (enJSRVs) related to the pathogenic Jaagsiekte sheep retrovirus (JSRV). The enJSRVs have been integrating in the host genome for the last 5-7 million years.
By using enJSRVs as genetic markers and morphological traits2, it was determined that sheep populations originatingfrom early domestication process in the Middle East (approximately 9,000 years ago) dispersed across Eurasia andAfrica via two separate migratory episodes. The later migration, involving sheep with improved production traits,
shaped the great majority of present-day breeds. The ability to differentiate genetically primitive sheep from more
modern breeds provides valuable insights into the history of sheep domestication.
The exogenous JSRV is a causative agent of a transmissible lung cancer in sheep. Interestingly, enJSRVs are able
to protect their host against JSRV infection by blocking different steps of the viral replication cycle. Sixteen of the 27
enJSRV loci contain an env gene with an intact open reading frame, and those env can block entry of the exogenous
JSRV via receptor interference. Further, two enJSRV proviruses, which entered the host genome within the last 3
million years (before and during speciation within the genus Ovis), acquired in two temporally distinct events a
defective Gag polyprotein resulting in a transdominant phenotype able to block late replication steps of JSRV andrelated exogenous retroviruses. Both transdominant proviruses became fixed in the host genome before or aroundsheep domestication. Indeed, endogenization and positive selection of ERVs acting as restriction factors is a mechanism used by the host to fight exogenous retroviral infections.
The enJSRVs are most abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus (embryo and associated extraembryonic membranes) trophectoderm, and in utero loss-of-function experiments
found the enJSRVs envelope (env) to be essential for conceptus elongation and trophoblast growth and development3.
Recently, 5 of the 27 enJSRV loci were found to contain a complete and intact genomic structure and are able toproduce viral particles in vitro. New evidence supports the idea that those intact enJSRV loci expressed in the uterine
endometrial epithelia are shed into the uterine lumen and then transduce the conceptus trophectoderm. Infection of
the conceptus with endometrial-derived enJSRVs is hypothesized to influence development and differentiation of thetrophectoderm.
Collectively, available evidence in sheep and other mammals strongly supports the ideas that ERVs coevolved with
their hosts for millions of years and were positively selected for biological roles in genome plasticity, protection of
the host against infection of related pathogenic and exogenous retroviruses, and a convergent physiological role inplacental morphogenesis and mammalian reproduction.
1

Arnaud et al., 2008. Cell Mol Life Sci 65: 3422.

2

Chessa et al., 2009. Science 324: 532.

3

Dunlap et al., 2006. Proc Natl Acad Sci 103: 14390.

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IS3-1DNA Methylation Profiling by Tissue-dependent and Differentially
Methylated Regions (T-DMRs) in Pluripotent Stem Cells: Insights
into Genome Organization
Shintaro YAGILaboratory of Cellular Biochemistry, Department of Animal Resource Sciences/ Veterinary Medical Sciences,
Graduate School of Agricultural and Life Sciences, The University of Tokyo
Every cell type and tissue has a distinctive DNA methylation profile (an entity of epigenome) that consists ofthousands of tissue-dependent and differentially methylated regions (T-DMRs) exhibiting differentially methylation
status in a tissue-dependent manner. T-DMRs have been identified at promoter regions of genes encoding key molecules for cell phenotypes, such as Pou5f1 and Nanog genes involved in pluripotency of embryonic stem cells.
Genome-wide DNA methylation profile, which was investigated using a microarray-based analysis (D-REAM), illustrates the tissue/ cell-type specific function by illuminating the key transcription factors and their target genes,suggesting DNA methylation profile is fundamentals of genome function.
Uniqueness of DNA methylation profile to a cell type allows us to distinguish the kind of cell type from the others
exhibiting similar phenotypes. For example, iPS cells shows distinct methylation profiles from the other pluripotent
stem cells established from embryo, and incomplete methylation is observed at T-DMRs located in somatic tissue
specific genes, which are methylated in ES cells.
When we focus the genomic features associated with T-DMRs, quite unique characteristics were associated with TDMRs identified in ES cells in compared with somatic tissues/ cells. T-DMRs hypomethylated in the both human andmouse pluripotent cells were significantly associated with CpG islands (CGIs), and these T-DMRs were predominantly existed in short interspersed elements (SINEs) rich/ long interspersed elements (LINEs) poor genomic loci,spreading continuously over hundreds kb to 1 Mb containing multiple hypomethylated T-DMRs, which constituted
epigenetic clusters. These epigenetic clusters were found within the gene clusters, however, are not always. By using
mouse genome full-tiling array, T-DMRs were identified not only in genic but also in intergenic regions, and correlations between genomic activities and enrichment of transposable elements were observed around T-DMRs
hypomethylated in the pluripotent cells. These genomic features around T-DMRs were conserved between humanand mouse, suggesting that epigenome has an impact on genomic organization.
Gene clusters, generated by multipletimes of gene duplication, often show species or lineage specific patterns.
Expansion of prolactin gene families had occurred in rodent genomes, and members in these gene families are expressed in tissue-specific manners. We identified T-DMRs at promoter regions of all members in prolactin genefamily in mouse, which exhibit hypomethylation in tissues expressing the corresponding genes. Of our interest, weobserved the correlation between genomic organization, DNA methylation patterns and probable ancestral genomic
sequences. Considering the effects of DNA methylation in C->T conversion occurred in mammalian genomes, DNA
methylation profile provides us insights into involvement of epigenetic system in genome organization.

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IS3-3
Exposure to a Maternal High Fat Milieu Alters the Hepatic
Epigenome in Mice
Yoshinori SEKI 1 and Maureen J CHARRON 1,2
1

Department of Biochemistry, 2 Ob/Gyn & Women’s Health and Medicine, Albert Einstein College of Medicine,Bronx, NY, USA

Type2 diabetes mellitus (T2DM) and metabolic syndrome are modern day plagues in industrialized and developing nations alike. The incidence of these diseases has reached epidemic proportions. Exposure to a sub-optimal
maternal environment such as, low protein diet, global caloric restriction, prenatal stress and high fat (HF) diet are
known to be associated with increased incidence of cardiovascular disease, T2DM and metabolic syndrome later inlife. However, the molecular basis of this enhanced susceptibility for metabolic disease is poorly understood.
Dysregulation of the epigenome may play an important role in this process. Pathologic changes in gene expression
observed prior to development of overt disease support this hypothesis. To address this hypothesis, we performed
DNA microarray and genome-wide DNA CpG methylation analyses to measure mRNA expression and DNA methylation patterns in liver of 5 wk old male mice exposed to either a control or HF diet in utero.
Exposure to a HF diet in utero resulted in decreased fetal body weight, but accelerated weight gain, increased
adiposity and large adipocytes early in postnatal life. Serum glucose was increased and adipokines including PAI-I
and adiponectin were pathologically modulated in HF offspring. Furthermore, this is associated with the early life
manifestation of impaired insulin and glucose tolerance. Exposure to a maternal HF milieu activated genes of immune response, inflammation and hepatic dysfunction. Conversely, genes of lipid metabolism and biogenesis were
down regulated especially in the cholesterol biosynthesis pathway. DNA methylation was assessed by the HELP
assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR). This analysis revealed 3360 differentially methylated loci, the majority of which (76%) were hypermethylated in HF liver. DNA hypermethylation was accompanied by increased mRNA expression of DNMT1 and 3B in fetal liver. DNMT activity was also increased in fetal but
not 5wk old liver. The distribution of DNA hypermethylation was similar in promoters, gene bodies and intergenic
regions (75%, 82% and 72%, respectively). Less hypermethylation was noted at CpG islands particularly thosepresent in promoters (51% hypermethylation). This pattern of promoter methylation is consistent with the results ofthe gene expression microarray whereby the majority of genes were upregulated in HF vs. Control liver. Interestingly, the distribution of hypermethylated loci on Chromosomes 4 and 18 displayed clustered patterns whereas theother chromosomes had a random distribution of hypermethylated loci. On chromosome 4, half of the hypermethylatedloci (170 out of 347) mapped to the atherosclerosis QTL and 80% of the hypermethylated loci (67 out of 138) mapped
to the Insulin dependent diabetes susceptibility 21 QTL on chromosome 18. Most of the hypermethylated genes inthese hot spots are associated with cardiovascular system development and function. These results suggest that
hypermethylation of loci in the QTLs on Chromosome 4 and 18 may be strongly involved in the increased susceptibility to metabolic syndrome seen in offspring exposed to a maternal HF diet. Of these, Rho Guanine Nucleotide
Exchange Factor 19 (ARHGEF19), which plays a key role in adipocyte differentiation, was hypermethylated in the
promoter region and this was associated with a 60% reduction in mRNA in HF exposed offspring. In summary,
exposure to a maternal HF diet is associated with: 1) alterations in expression of hepatic genes in pathways of inflammation, fibrosis and cholesterol, 2) CpG DNA hypermethylation throughout the genome with clusteredhypermethylation of loci in QTLs associated with atherosclerosis and diabetes, 3) promoter hypermethylation andreduced expression of ARHGEF19, a negative regulator of adipogenesis. Our data strongly suggests exposure tomaternal HF diet programs increased susceptibility to insulin resistance, obesity and metabolic disease, in part, throughincreased DNMT activity and alterations in hepatic DNA CpG methylation.

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IS3-4
Genome-wide DNA Methylation Profiling of Male and Female Mouse
Embryonic Stem Cell Differentiation Using Massively Parallel
Sequencing
Masako SUZUKI and John M. GREALLY
Center for Epigenomics, Department of Genetics, Albert Einstein College of Medicine
DNA methylation is a chemical modification of genomic DNA and occurs predominantly at the cytosine residue of CpG
dinucleotides (CpGs) in mammals. The majority of CpGs in the mammalian genome are methylated, with only a minority of
CpGs remaining unmethylated. CpG-dense regions are referred to as CpG islands, and are often located in close proximity togene promoters, where they are normally unmethylated. Aberrant DNA methylation patterns are implicated in the pathogenesisof an increasing number of diseases, particularly tumorigenesis. More specifically, the acquisition of DNA methylation within
the promoter regions of tumor suppressor genes is frequently observed in numerous tumor types. DNA methylation plays animportant role in transcriptional regulation. When a CpG island adjacent to a transcription start site (TSS) becomes methylated,the corresponding gene tends to be down-regulated. Interestingly, recent research showed that methylation occurring within gene
bodies is also correlated with gene expression, notably DNA hypermethylation in the gene body is associated with active transcription. This is opposite to the association in promoter regions. These findings indicate the importance of analyzing DNA
methylation patterns genome-wide.
It is well known that DNA methylation is essential for normal development. Each tissue and cell type has a unique DNA
methylation profile and the acquisition of the appropriate tissue-specific DNA methylation profile is important for having normal
function of tissues. Inner cell mass (ICM) derived embryonic stem (ES) cells are pluripotent and are known to be hypermethylated
cell lines when maintained in an undifferentiated state. Interestingly, this hypermethylation is only found in male (XY) ES cells,
whereas DNA methylation in female (XX) ES cells is globally reduced with hypomethylation reported for repetitive elements andseveral unique sequences such as parentally imprinted genes. However, little is known about the differences in genome-wide
DNA methylation profiles between XY and XX ES cells. We tested the patterns of DNA methylation during in vitro differentiation of both XX and XY ES cells to understand the epigenetic differences between male and female stem cells.
To test the genome-wide DNA methylation profile, we employed our novel protocol, HELP (HpaII tiny fragment Enrichment
by Ligation-mediated PCR)–Tagging. HELP-tagging is a massively parallel sequencing (MPS) based technique. MPS is aremarkable new technology that has provided the foundation for many new genome-wide assays, with several protocols developed to test DNA methylation using MPS. The HELP-tagging assay is a rapid and relatively low-cost method for the quantitative,
genome-wide assessment of cytosine methylation that can examine the methylation status of greater than 98% of CpG islands. The
HELP assay uses the methylation sensitive enzyme HpaII and its methylation insensitive isoschizomer MspI to interrogate themethylation status of the CpG within the context of the CCGG sequence recognized by these enzymes. Tagging is accomplishedthrough the ligation of adaptors within which resides the sequence for the type III restriction enzyme EcoP15I, which can generate 27 bp of flanking sequence. This tagging procedure facilitates the unambiguous identification and location of about 80% of
MspI sites. Since MspI sites distribute evenly in unique and repetitive elements, genic and non-genic, and CpG dense or CpG
depleted regions, HELP-tagging can assess DNA methylation status not only in promoters of genes or CpG islands, but also ingene bodies and CpG depleted regions. Using this technique, we confirmed the hypomethylated status in XX ES cells and we
found the DNA methylation level of XX ES cells to increase during differentiation, consistent with prior observations. Furthermore we found that not only parentally imprinted genes but also non-imprinted genes and intergenic regions were hypomethylated
in these female ES cells. In this talk, we are going to discuss the genome-wide DNA methylation profiling of male and female ES
cells and the changes observed during in vitro differentiation.

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IS3-5
Epigenetics of Human Reproduction and Developmental
Abnormality
Kenichiro HATA
Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Japan
Epigenetic gene regulation is essential in mammalian reproduction. Especially, it is well known that genomic
imprinting, an epigenetic phenomenon whereby genes are differentially expressed according to their parental origin,
is known to be crucial for placental development and fetal growth in mammals. Most imprinted loci contain adifferentially methylated region (DMR) harboring allelic DNA methylation inherited from the male or the female
gamete (germline DMR). Mutations/epimutations affecting DMRs cause rare imprinting disorders such as SilverRussell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS). Whether imprinted genes also play criticalroles in the more common abnormal pregnancy cases such as preeclampsia and intrauterine growth restriction (IUGR)
is an important question yet to be fully evaluated. With the aim of addressing this question, we established quantitative COBRA (Combined Bisulfite Restriction Analysis) assay conditions for 30 DMRs from 22 loci that include themajority of known human imprinted DMRs. Using this COBRA assay system, we initially measured DNA methylation levels at these 30 DMRs in twenty blood samples from healthy individuals and twenty placental tissues fromnormal pregnancies, and determined the extent of tissue specificity and individual varieties of methylation levels.
Subsequently, we have screened for epimutations at these DMRs in the placental tissues from approximately 100
IUGR cases. In two cases, among the DMRs examined, only H19- and IGF2-DMRs were found to be hypomethylatedcompared to the normal controls. Several other cases showed hyper- or hypo-methylation at the DMRs other thanthose in the H19/IGF2 locus, and are being further characterized. The genetic etiologies of IUGR are largely unknown and considered to be diverse. Our results suggest that placental epimutation at DMRs may account for thegrowth restriction phenotype of a subset of IUGR cases.
We also try to make whole genome DMR mapping with very rare human cases, a parthenogenetic mosaicism andan androgenetic mosaicism.

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IS3-6
Role for PAD Enzymes in Chromatin Structure, Gene Regulation,
and Disease
Scott COONROD
Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, USA
The peptidylarginine deiminase (PAD) family of enzymes post-translationally convert positively charged arginine
residues in substrate proteins to the neutral, non-standard residue citrulline. Over the last few years we have been
investigating the role of PADs in the regulation of chromatin structure and disease. We have found that, in neutrophils for example, induction of PAD activity by TNFa leads to global histone hypercitrullination and subsequent
release of chromatin from the nucleus into the extracellular space. This citrullinated chromatin appears to form thestructural “backbone” for Neutrophil Extracellular Traps (NETS) which represent part of the innate immune responseto stimuli such as pathogens and inflammation. More recently, we have investigated the role of PADs in gene regulation in the mammary epithelium. We have found that, in this context, PADs appear to primarily function as transcriptional co-regulators. For example, we found that EGF treatment of breast cancer cells induces citrullination of
the oncogene Elk1 by PAD4 at promoters such as c-fos, leading to histone acetylation and transcriptional activation.
On the other hand, we found that treatment of the same cell line with estrogen (E2) leads to PAD2-mediated activation of specific subsets of ER target genes. Interestingly, the mechanism of target gene activation here appears to be
via citrullination of histone H3, which in turn, induces promoter-specific chromatin decondensation. Regarding
possible links between PADs and breast cancer, we have found that PAD expression and activity is strongly upregulated
in models of early stage breast cancer. Importantly, we have been testing several new PAD-specific inhibitors andfound that these drugs block tumor progression in cell lines and also in mouse xenograft models of human breast
cancer. Taken together, these findings suggest that, in some cases, PAD activity appears to play a positive role indisease resolution, while in other instances, induction of PAD activity appears to promote disease progression.

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ISP1

Trophoblast-specific DNA Methylation Occurs after the
Segregation of Trophectoderm and Inner Cell Mass inMouse Periimplantation Embryo○ Momo NAKANISHI, Koji HAYAKAWA, Kunio SHIOTA and Satoshi TANAKAThe University of Tokyo

Cell type-specific DNA methylation status of tissue-dependent and differentially methylated regions (T-DMRs) constitutes DNA methylation profile of cell, which ought to be reconstructed as cells differentiate. The first cell differentiation in
the mammalian development separates the trophoblast and embryonic cell lineages resulting in the formation of trophectoderm (TE) and inner cell mass (ICM) in blastocyst. Although lower level of global DNA methylation in TE genome
compared to ICM has been suggested, dynamics of DNA methylation profile during the TE/ICM differentiation has notbeen elucidated. To address this issue, we first focused on the TS-ES T-DMRs that have been previously identified bycomparison of trophoblast stem (TS) and embryonic stem (ES) cells. Bisulfite sequencing analysis verified that these TSES T-DMRs are differentially methylated between trophoblast and embryonic cell lineages not only in the in vitro cell line
but also in tissues of embryonic day (E) 6.5 mouse embryos. We also found that the human genomic regions homologous
to mouse TS-ES T-DMRs are differentially methylated between human placental tissue and ES cells. Collectively, wedefined them as cell lineage-based T-DMRs between trophoblast and embryonic cell lineages (T-E T-DMRs). We thenexamined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, at all T-DMRs examined, both TE andICM were in nearly unmethylated status and exhibited no difference, suggesting that the DNA methylation profiles specificto each cell lineage are established after the morphological specification. The T-E T-DMRs identified will serve as valuable
epigenetic markers for study of trophoblast/embryonic cell lineages.

ISP2

ATP Content in Bovine Oocytes May not Determine theTiming of First Cleavage and Further Developmental
Competence after IVF○ Vibuntita CHANKITISAKUL1,2, Tamas SOMFAI1, Yasushi INABA1,
Mongkol TECHAKUMPHU2 and Takashi NAGAI1
1National Institute of Livestock and Grassland Science, National Agriculture and Food Research
Organization, 2Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary
Science, Chulalongkorn University, Thailand
In bovine oocytes early timing of the first mitotic cleavage after fertilization is known to be associated with high developmental competence to the blastocyst stage (Lonergan et al., 1999). Also, in matured bovine oocytes ATP levels have beenfound to correlate with developmental competence (Stojkovic et al., 2001). This study was conducted to test the hypotheses
that ATP level in bovine oocytes directly affects the timing of first cleavage and blastocyst development after IVF. In the firstexperiment early cleaving 2-cell stage embryos were selected 27 h after IVF of IVM oocytes (early cleaving group), and ATP
levels and subsequent in vitro development were compared with those cleaving between 27–30 h of IVF (late cleaving group).
In the second experiment oocytes were treated with 0.6 mg/ml L-carnitine during IVM to increase ATP levels and the effect of
this treatment on timing of first cleavage and in vitro development after IVF was assessed. In early cleaving group theblastocyst formation rate was significantly (P<0.05) higher than that in late cleaving group (50.3% vs. 37.6%, respectively);
however, ATP levels at the first cleavage did not differ significantly between the two groups (1.83 pmol/embryo and 1.62
pmol/embryo, respectively). L-carnitine increased ATP levels from 2.26 pmol to 3.16 pmol in MII stage oocytes (P<0.05),
however, timing of the first cleavage and blastocyst formation rates after IVF were not affected. The results suggest that ATPlevel in IVM bovine oocytes may not determine fundamentally their cleavage speed and competence to develop to the blastocyst stage after IVF. This study was supported by RGJ-PhD grant and by NARO (N36G2Gr1).
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Excessive Pre-treatment of Mouse Spermatozoa with DNA
Non-interacting Agents Causes Zygotic Chromosomal
Damage○ Hiroyuki WATANABE1, Hiroshi SUZUKI2, Yutaka FUKUI2 and Hiroyuki TATENO1
1Asahikawa Medical University, 2Obihiro University of Agriculture and Veterinary Medicine[Background] Much time and effort has been invested to improve in vitro production (IVP) of embryos, and techniquessuch as sperm pre-treatment have been useful for obtaining fertilized eggs in IVP. However, the genetic risks to the
produced embryos have yet to be fully elucidated. The aim of this study was to investigate the genetic safety of sperm pretreatment, which is commonly used in IVP, and to elucidate the relationship between zygotic chromosomal integrity andembryonic development using a mouse model. [Methods] Spermatozoa were treated with methyl-β-cyclodextrin (0.75mM, 90 min), lysolecithin (0.02%, 1 min), Triton X-100 (0.02%, 1 min) and dithiothreitol (DTT; 5 mM, 10–60 min),followed by microinjection into oocytes. Injected eggs were used to examine chromosomal integrity at 1-cell metaphase,and to monitor pre- and post-implantation development. [Results] Chromosomal integrity of spermatozoa was impaired byin vitro incubation and chemical antagonism. Particularly in the DTT 60-min group, severe chromosomal damage wasobserved. Despite this chromosomal damage, however, the resultant embryos frequently developed to the blastocyst stage.
Embryos in the DTT 60-min group had significantly lower developmental competence to live fetuses. These results indicate that sperm pre-treatment such as DTT for 60 min generates severe chromosomal damage in ICSI oocytes, and fetal
development is not in line with blastocyst formation. These findings provide evidence that paternal chromosomal integrity
in zygotes is fully predictive of the fate of embryos in IVP. Given these issues, an innovative method of sperm genetic
screening during the pre-fertilization period is urgently required.

ISP4

Epithelial-mesenchymal Transition of Trophectoderm Cells
during Embryo Implantation○ Toshihiro KONNO, Sachiko YAMAKOSHI, Rulan BAI, Takashi CHAEN,
Mahiro EGASHIRA, Nana NOMURA, Toshihiro SAKURAI and Kazuhiko IMAKAWA
Graduate School of Agricultural and Life Sciences, The University of Tokyo

At the early stage of embryo implantation to the maternal endometrium, trophoblast and uterine epithelium initiate theirfirst contact via apical cell membranes. This epithelial-epithelial adhesion is recognized as one of the paradoxes in cellbiology. In humans and rodents, trophoblast cells invade maternal decidua during placentation. For the acquisition of thisinvasion competence, it has been postulated that the trophoblast cells undergo an epithelial-mesenchymal transition (EMT).
It has also been expected that the EMT participate in the trophectoderm acquisition of adhesion competence during implantation. In this study, we investigated the hypothesis that stimuli from the receptive uterus induce EMT in thetrophectoderm.Expressions of epithelial and mesenchymal makers were immunohistochemically examined on tissue sections from gestation day 22 bovine uteri. Expression of genes implicated in EMT process was assessed by RT-PCR analyses of RNAs from days 17, 20 and 22 bovine conceptuses. To evaluate the effects of receptive uterus on the blastocyst,
pregnant mice were injected with estrogen (E2) receptor antagonist Tamoxifen (TAM, 10 µg/animal) on gestation day 2.5.
Blastocysts from TAM treated or intact mice were collected and subjected further analyses.Our observations revealed thatthe bovine trophectoderm modified its epithelial property during implantation process, including loss of cytokeratin, expression of vimentin, switch from E- to N-cadherin and upregulation of MMP2 and MMP9. Mouse E2 receptor inhibition
model indicated that the down-regulation of blastocyst E-cadherin is regulated by E2 primed receptive uterus. We also
observed that the expression of trophoblast stem cell marker Cdx2 was down-regulated during the implantation window.
Collectively, our observations indicated that the EMT of trophectoderm was regulated by E2 induced secretion from theuterus, and the stimuli from the receptive uterus might also induce trophoblast differentiation.
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ISP5

Identification of Endogenous Retroviruses Derived-gene
Possessing a Zinc Finger Like Sequence during Bovine
Peri-attachment Period○ Hanako BAI1, Toshihiro SAKURAI1, So NAKAGAWA2, Toshihiro KONNO1,
Takayuki MIYAZAWA3, Takashi GOJOBORI2 and Kazuhiko IMAKAWA1
1

Graduate school of Agricultural and Life Sciences, The University of Tokyo; 2National institute of
Genetics, 3Institute for Virus Research, Kyoto University[Objective] The endogenous retroviruses (ERVs) are thought to be derived from ancient viral infections of germ cells.
Previous studies suggest that ERV genes play an important role in the development of the placenta and the trophoblast celllineage in mammalian species. The exact evolutionary pathways and the extent to which ERVs function are still unclear.
For the bovine species, the genes functioning in placental development are unknown despite their importance to the cattle
industry. In this study, we analyze bovine ERV-like genes that are expressed during bovine placentation. [Materials andMethods] We collected bovine conceptuses of days 17, 20, and 22 (day 0 = day of estrus) from super-ovulated Japanese
black cattle, and sequenced the expressed RNAs using the SOLiD3 System. We then searched for bovine ERV-like genes
on the bovine genome by computational analysis and found candidates sequences. [Results]We used SOLiD3 System andcomputational analysis and found five ERV-like candidate sequences. Using PCR analysis, the expression of candidate
genes were examined and found three candidates sequence’s mRNA in days 17, 20, and 22 bovine conceptuses. In particular, expression of one candidate gene on Chromosome 7 (Chr7-539) increased on day 22. The motif search for Chr7-539
revealed that Chr7-539 possessed zinc finger like structure. These results suggest that this gene might function as a transcription factor.

ISP6

Effect of Naloxone, a Mu-opioid Receptor Antagonist, on
Oocyte Maturation and Embryonic Development in Pigs
○ Thanh Quang DANG-NGUYEN1,2, Nguyen VIET LINH2,3, Rosa MINOIA5,
Masahiro KANEDA1, Tamas SOMFAI1, Seiki HARAGUCHI1, Satoshi AKAGI1,
Kazuhiro KIKUCHI4, Michiko NAKAI4, Atsushi TAJIMA2 and Takashi NAGAI1
1National Institute of Livestock and Grassland Science, National Agriculture and Food Research
Organization 2University of Tsukuba, 3University of Tokyo, 4National Institute of Agrobiological
Sciences, 5University of Bari, Italy
Studies on horses, cattle and dogs have shown that when added to a maturation medium for oocytes naloxone (Nx), a muopioid receptor antagonist, reduces metaphase II (M-II) rate. However, at low concentration, Nx increase the rate of M-II
oocytes. The purposes of this study are to investigate the expression of mu-opiod receptor (MOR) gene in oocytes and embryos at different stages, to examine the effect of Nx on nuclear and cytoplasmic maturation of oocytes, and on embryonicdevelopment in pigs. Also the combined effect of cAMP and Nx on maturation of oocytes was studied. MOR gene wasexpressed in oocytes at germinal vesicle and M-II stages, as well as 1-, 4-cell embryos and blastocysts. At concentrationof 10-8M Nx increased the M-II rate (P<0.05), but at concentration of 10-4M it reduced (P<0.05) the rate. However, Nx
had no effects on fertilization status and embryonic development (P>0.05). The supplementation of Nx into culture medium did not improve the blastocyst formation rate and cell number in blastocysts (P>0.05). At concentration of 10-4M, Nxdecreased the blastocyst formation rate (P<0.05). The addition of both cAMP and Nx into the maturation medium improved
the M-II rate (P<0.05) compared with the addition of either cAMP or Nx individually. In conclusion, MOR gene was expressed in porcine oocytes and embryos during the maturation and developmental culture, respectively. Nx improved M-II rate
at low concentration, but reduced the rate at high concentration. However, Nx did not promote cytoplasmic maturation andimprove embryonic development. The addition of Nx and cAMP into maturation medium had a synergistic effect on oocyte
maturation.
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