A3

Abstracts of the presentations selected for 2007
JSAR Outstanding Presentation Award

Metastin may Induce Apoptosis of Luteal Cells via Gonadotropin
Releasing Hormone in the Corpus Luteum of RatsTomohiro YONEZAWA, Shiro KURUSU and Mitsumori KAWAMINAMI
Laboratory of Veterinary Physiology, Kitasato University
ABSTRACT. Corpora lutea formed during the estrous cycle regress without significant production ofprogesterone in rats. Once uterine cervical stimulation is given on proestrus (P), twice daily prolactin(PRL) surges are induced and the corpus luteum becomes functional. We have already reported that
gonadotropin releasing hormone (GnRH) and annexin 5 (AX5, a biomarker of GnRH signal) expressed in
corpus luteum and local GnRH is involved in the luteal regression. Recently, it has been reported thatmetastin, the ligand of GPR54, induces GnRH release in the hypothalamus. We investigated, in thisstudy, a putative functional relationship among luteal metastin, GnRH, AX5 and luteolysis. Cycling
Wistar-Imamichi rats were used. Corpora lutea were harvested at 1000 h and 1700 h of each day of
estrous cycle. Expression levels of mRNA were measured by real-time PCR respectively. All mRNAlevels of metastin, GnRH and AX5 were significantly high from the afternoon of D2 to the afternoon of P.
When PRL (10 IU/head) was administrated twice daily from estrus, the expression of metastin, GnRH
and AX5 mRNA were inhibited at 1700 h of D2. When metastin was administrated into the ovarianbursa using an osmotic mini pump (10–6 M/µl/h) from five days after uterine cervical stimulation, themRNA levels of metastin, GnRH, AX5, FasL and Fas were significantly augmented after two days.
TUNEL (TdT-dUTP nick end labeling) analysis showed apoptotic cells in the corpora lutea. Corpora
lutea harvested at 1000 h of D2 were cultured in the medium containing metastin with/without Cetrorelix(GnRH antagonist) under the condition of 40% O2 and 5% CO2. Metastin (10–7 M) stimulated theexpression of luteal metastin, GnRH, AX5, FasL and Fas mRNA levels during 6 h incubation, while
simultaneous treatment of Cetrorelix (10–7 M) with metastin blocked the augmentation of metastin,
GnRH, AX5, FasL and Fas mRNA expression. In addition, estrogen (10–10 to 10 –8 M) given for 6 hincreased metastin and GnRH mRNA levels in a dose-dependent manner. These results suggest thatluteal metastin is synthesized from the afternoon of D2 to P and it stimulates GnRH expression. Thesechange may result in the apoptosis of luteal cells. PRL is suggested to make corpus luteum functional, atleast partly, by inhibiting the expression of metastin, GnRH and AX5. It could be possible to postulatethat estrogen produced by a crop of new follicles could trigger a sequence of events from metastin toluteolysis.

A4

Abstracts of the presentations selected for 2007
JSAR Outstanding Presentation Award

A Unique Role of Maternal Nucleolus in Early MammalianDevelopment
Sugako OGUSHI1, 3, 4), Takashi MIYANO2), Mitinori SAITOU4) and
Josef FULKA Jr.3)
1)Graduate School of Science and Technology and 2)Faculty of Agriculture, Kobe

University, Kobe 657-8501, Japan, 3)Institute of Animal Science, Prague 104 01, Czech
Republic and 4)Laboratory for Mammalian Germ Cell Biology, RIKEN Kobe Institute,
Kobe 650-0047, Japan
ABSTRACT. Fertilization is a dynamic process of the transition from two highly specialized cell—theoocyte and spermatozoon—into the totipotent zygote. Paternal and maternal contributions to the zygote
are not only introducing a half set of the nuclear DNA for the next generation. They introduced acomplementary arsenal of structures and molecules necessary for fertilization, and make up for eachother after fusion to be functional zygotes. In mammals, mitochondria originate exclusively from theoocyte, whereas the centriole in mammals, except rodents, is of paternal origin. Another organelle thatcan be found only in the oocyte is the nucleolus. The nucleoli in fully grown oocytes are compact andtranscriptionally inactive, and chromatin, ribosomal RNAs and proteins are undetectable level inside of
the nucleoli. It is not known how or to what extent this nucleolar material contributes to the formation of
nucleoli in newly formed zygotes and early developing embryos. Considering that the oocytes have theability to mature, to be fertilized and then to develop as embryos, we may suppose that their nucleoli (ornucleolar material) play certain roles in maturation, fertilization, and development. Thus, we remove thenucleoli from the pig and mouse GV-oocytes microsurgicaly (enucleolation), and cultured in vitro. The
oocytes without nucleoli (enucleolated oocytes, ENL) progressed maturation as similar time course as
control oocytes and form the pronuclei (PNs) after activation. There was, however, no nucleoli inside ofboth male and female PNs. Re-injection of the oocyte nucleolus into ENL oocytes restored the formationof nucleoli in both pronuclei. In contrast, injection of the nuclei of somatic cells containing nucleoli into
ENL oocytes was unable to rescue the nucleolus reformation in pronuclei of zygotes derived from ENL
oocytes. ENL oocytes after maturation and activation were cultured up to the blastocysts, and theyhardly progressed to the blastocyst stage in both pig and mouse. Our results suggest that the oocyte
nucleolus provides the molecules to pronuclei in zygotes and even to pseudo-pronuclei formed after
somatic cell nuclear transfer to assemble their nucleoli. Moreover, the oocyte original nucleolar material
is absolutely essential for successful early embryonic development.

A5

Abstracts of the presentations selected for 2007
JSAR Outstanding Presentation Award

The Role of Arcuate Metastin Neurons in Regulation of Pulsatile
GnRH/LH Secretion in the Female Rat
Yoko INAMOTO, Maki MAEDA, Tamami HOMMA, Shunji YAMADA,
Yoshihisa UENOYAMA, Satoshi OHKURA, Kei-ichiro MAEDA and
Hiroko TSUKAMURA
Laboratory of Reproductive Science, Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya 464-8601, Japan
ABSTRACT. Pulsatile secretion of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH)
has a critical role in stimulating follicular development and steroidogenesis to maintain normal
reproductive functions in mammals. Metastin, a peptide coded by KiSS-1 gene, was found as a natural
ligand for GPR54, a G protein-coupled receptor. We have been focusing on the metastin-GPR54 signaling
in the brain, because accumulating evidence suggests that the signaling in the brain plays a critical rolein the regulation of reproduction in several mammalian species including human, monkey and rodents.
In the present study, we report that prolonged neonatal estrogen treatment abolishes pulsatile LH secretionand metastin neurons in the hypothalamus of female rats. Wistar-Imamichi strain female rats weretreated with daily injections of estradiol 3-benzoate (EB) or peanut oil for 10 days after birth. The vaginal
smears in the animals were daily examined after vaginal opening. The ovaries were removed andweighed when the rats reached 11–13 weeks of age. The animals were bled for 3 h at 6-min intervals todetermine the pulsatile LH release. The number of the metastin-immunoreactive cells was determined in
the hypothalamic arcuate nucleus (ARC) in the neonatally EB- or vehicle-treated ovariectomized (OVX)
rats. Neonatally EB-treated female rats showed persistent vaginal diestrus, while the vehicle-treatedcontrols showed regular 4-day estrous cycles after the puberty. Ovary weight in EB-treated rats was
significantly lower than vehicle-treated controls. Pulsatile LH secretion was profoundly inhibited in
OVX rats with neonatal EB treatment, resulting in significantly lower mean LH levels and frequency andamplitude of LH pulses compared with those in neonatally vehicle-treated controls. Few metastinimmunoreactive cells were found in the ARC in the neonatally EB-treated OVX rats, whereas manymetastin- immunoreactive cells were present in the ARC of the vehicle-treated controls, and the numberof the cells in the EB-treated animals was significantly lower than those in controls. The present results
indicate that prolonged neonatal EB treatment reduces the number of ARC metastin neurons with inhibited
GnRH/LH pulses and ovarian follicular development. Considering the stimulatory effect of metastin on
GnRH/LH release, the ARC metastin neurons may play a role in generating pulsatile GnRH/LH releaseto maintain the ovarian function in female rats.

A6

Abstracts of the presentations selected for 2007
JSAR Outstanding Presentation Award

Analysis of the Determinants of Meiotic and Developmental
Competence in Mouse Oocytes
Azusa INOUE, Masao NAGATA and Fugaku AOKI
Department of Integrated Biosciences, Graduate School of Frontier Sciences,
University of Tokyo, Chiba 277-8562, Japan
ABSTRACT. Mammals have two types of full-grown oocytes: those with germinal vesicles (GVs) inwhich the chromatin is condensed and surrounds the nucleolus (SN type), and those in which thechromatin is less condensed and does not surround the nucleolus (NSN type). Although SN oocytes
possess higher meiotic and developmental competence than NSN oocytes, the factors underlying thisdifference are unknown. To determine whether cytoplasmic or nuclear factors are involved, the GVs ofmurine SN and NSN oocytes were exchanged by nuclear transfer and the nucleus/cytoplasm of eachreconstructed oocyte was classified as follows: SN/SN, NSN/SN, SN/NSN, or NSN/NSN. After
reconstruction, the meiotic maturation and preimplantation development of the oocytes were analyzed.
Few mature SN/NSN and NSN/NSN oocytes were observed (20–26%). In contrast, 88% of the NSN/SN
oocytes matured; however, they rarely developed to the blastocyst stage after fertilization (4%), whereas
most of the SN/SN oocytes matured (84%) and reached the blastocyst stage (83%). These results suggestthat the differences in meiotic and developmental competence between SN and NSN oocytes are
determined by factors in the cytoplasm and nucleus, respectively.

A7

Abstracts of the presentations selected for 2007
JSAR Outstanding Presentation Award

A Novel Gonad-Specific Gene DD2-2 Involved in Early Zygotic
Gene Activation (ZGA) in Mouse Pre-Implantation Embryos
Seung-Wook SHIN, Toshiki MATSUOKA, Mikiko TOKORO, Manabu SATO,
Tomoko AMANO, Kazuhiro SAEKI, Yoshihiko HOSOI, Akira IRITANI and
Kazuya MATSUMOTO
Department of Genetic Engineering, Kinki University, Nishimitani 930, Kinokawa 6496493, Japan
ABSTRACT. ZGA take place at the G2 phase of the 1-cell stage in the mouse. However, its molecularmechanism has not been fully elucidated yet. Thus, we have investigated molecular functions of many
gene clusters, i.e., DD clones obtained by Differential-Display analysis between the ovulated eggs at the
MII stage and the 1-cell stage embryos at the G2 phase. This way, we have identified a novel gene (DD22 gene), whose function was unknown and further whose DNA sequence did not match any known
transcripts in the Gene Bank. Here, we report the expression profile and knockdown analyses of theDD2-2 gene in the mouse pre-implantation embryos. We have detected specific expression of DD2-2
gene in mouse gonads (testis and ovary) carrying out RT-PCR and Western blot analyses of mouse main
tissues (the brain, lung, liver, heart, spleen, kidney, testis, ovary, and skeletal muscle). In pre-implantationembryos, DD2-2 gene showed the unique expression profile: maternal DD2-2 transcripts were detectedin oocytes at GV and MII stages, whereas embryonic DD2-2 transcripts in early 2-cell embryos wereexpressed approximately 3-fold more than that of oocytes at GV and MII stages; however, the expression
dramatically decreased at the later stages and could not be detected in the stages of morulae andblastocysts. These results indicated that DD2-2 gene was specifically expressed during ZGA. Western
blot analyses with polyclonal antibody against specific peptides of DD2-2 amino acids showed that theexpression profile of DD2-2 protein was in parallel to that of DD2-2 transcript, implying that the expressionof DD2-2 gene was not regulated under translational control. Furthermore, we have performed
immunofluorescence cytochemical staining of DD2-2 in pre-implantation embryos using the polyclonal
antibody. The results obtained showed that DD2-2 localized in both the nuclei and cytoplasms ofoocytes or of embryos from GV stage to 2-cell stage at 36 hpi (hours post insemination). Further, toinvestigate DD2-2 involvement in the development of pre-implantation embryos, we have examined
knockdown effects of DD2-2 gene expression using antisense technology (pβ-act/antisense DD2-2/IRES/
EGFP) on the development of mouse pre-implantation embryo. The injected antisense expression vector
in EGFP-positive 1-cell embryos significantly inhibited their development to the 2-cell and 4-cell embryos
as compared with EGFP-positive embryos injected with a negative control expression vector (pβ-act/luc+/
IRES/EGFP)(54% (71/131) vs 72% (94/130), p<0.05). Further, in parallel to these phenomena, the amountof DD2-2 protein synthesized in EGFP-positive 1-cell embryos or 2-cell embryos which had been injectedwith antisense expression vector significantly decreased at 24 hpi by 52% when compared with that of
EGFP-positive 2-cell embryos injected with the negative control expression vector. Thus, these results
suggest that DD2-2 could be specifically involved in the transition from maternal gene activation toembryonic gene activation in mouse early pre-implantation embryos.

A8

Abstracts of the presentations selected for 2007
JSAR Outstanding Presentation Award

Purification of Fish Spermatogonia Using Flow Cytometer
Kazuyoshi KISE, Misako MIWA and Goro YOSHIZAKI
Department of Marine Biosciences, Tokyo University of Marine Science and Technology,
Tokyo 108-8477, Japan
ABSTRACT. The sorted population of spermatogonia is a powerful tool to study molecular events
regarding gametogenesis and surrogate broodstock technology by germ cell transplantation. We previously
succeeded to produce trout offspring from triploid salmon parents by germ cell transplantation. In thisexperiment, testicular cell suspension containing spermatogonia, together with large quantity of gonadal
somatic cells and meiotic germ cell, was transplanted. Therefore, in order to improve transplantation
efficiency, we purified spermatogonia showing the green fluorescence from pvasa-Gfp transgenic rainbow
trout using fluorescence-dependent flow cytometer. However, the resulting offspring, derived fromtransgenic spermatogonia became also transgenic individuals. This fact is not favorable for aquacultural
applications nor conservation usages. In this study, we purified fish spermatogonia by a flow cytometerusing forward and side light-scatter from non-transgenic individuals. First, to characterize size andgranularity of A-type spermatogonia we analyzed their forward light-scatter (FS) and side light-scatter(SS) using immature pvasa-Gfp transgenic rainbow trout possessing testes that contain only A-typed
spermatogonia. FS property is an indicator of relative cell size and SS property is determined by cell
shape and granularity. As a result, we found GFP-positive cell population (A-type spermatogonia)
showed high FS and low SS values. Second, cell fraction showing high FS and low SS values was sorted
from testicular cell suspension prepared from non-transgenic trout having only A-type spermatogonia.
The resulting cell population was applied to RT-PCR analyses with primer sets for germ cell marker(vasa) and somatic cell marker (Gsdf). The population only expressed vasa but not Gsdf. Further, in orderto check the purified cells have an ability to migrate and to be incorporated into the recipient’s genitalridges, the sorted cells labeled with PKH26 were transplanted into the peritoneal cavity of rainbow trout
embryos. The transplantation rate and the number of donor cells which were incorporated into therecipient’s genital ridges were 82 ± 7.0% and 8.4 ± 1.5 cells/fish, respectively, in case the sorted cells wereused. On the other hand, they were 29 ± 4.0% and 3.6 ± 0.6 cells/fish in case unsorted testicular cellsuspension was transplanted. Further, the same sorting condition could also be applied to fishes otherthan rainbow trout, such as masu salmon, Japanese char and nibe croaker. Thus, we succeeded to purify
A-type spermatogonia, which can be transplanted into the recipient embryos, from non-transgenic fishes.

A9

Abstracts of the presentations selected for 2007
JSAR Outstanding Presentation Award

ISG-15 Gene Expression Patterns Derived from Peripheral Blood
Leukocytes during the Pre- and Post-breeding Period in Dairy
CattleYuko HIROSAWA1), Takeshi OSAWA1), Norio YAMAGISHI1), Takumi FUKUDA1),
Michiko UCHIZA2), Nanae TAMEOKA2) and Yoshiaki IZAIKE1)
1)Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Morioka

020-8550, 2)National Livestock Breeding Center Iwate Station, Morioka 020-0123,
Japan
ABSTRACT. Early non-pregnancy diagnosis is essential to improve reproductive performance in dairy
cattle. Although mobile equipment of ultrasonography has ability to diagnose pregnant cows from 25days of pregnancy, it is difficult to diagnose them less than that stage. In recent years, however, various
interferon-stimulated genes (ISGs) have been shown to express in the endometrium in response to thesecretion of interferon at the time of pregnancy recognition in the ruminant. Increase in the expression of
ISG-15, one of the ISGs, in peripheral blood leukocytes (PBL) of pregnant cows has also been reported.
To clarify whether monitoring survivability of the embryos aged at less than 23 days is possible or not,we compared mRNA expression patterns of ISG-15 in PBL before and after breeding between pregnant
and non-pregnant animals. A total of 13 animals, comprising of 12 Holstein and Jersey cows and one
Holstein heifer, were used. All the animals were either inseminated on day 0 (day of estrus) or day 1, orserved as a recipient for non-surgical embryo transfer on day 7 or 8. Blood was taken on day 0 and every
other day from days 12 to 22. Additional blood samples were collected from two of the pregnant
animals every other day from days 22 to 30 and day 50. All the samples were collected from thecoccygeal vein into vacuum EDTA-2K tubes. PBL was separated from 1 ml of whole blood within six
hours after the collection, and was stored at –80 C until total RNA extraction. cDNA was synthesizedbased on the extracted total RNA, and the mRNA expression of ISG-15 in PBL was examined by RT-PCR.
Pregnancy was confirmed by ultrasonography either day 24, 25 or 26. An ISG-15 specific PCR productwas observed as a clear 87-bp band in the samples collected from days 18 to 22 or from days 20 to 22 inseven out of the 13 animals. All of these seven were confirmed to be pregnant and the other six animals
were confirmed to be non-pregnant by ultrasonography. The clear band was also shown in the samples
collected from days 24 to 30 in the two pregnant animals. In contrast, the clear band was not detectedconsistently in the samples collected from days 0 to 16 in all the 13 animals, and no band was observed
on day 50 in the two pregnant animals. In conclusion, the results of our study suggest that it is possible
to monitor the survivability of bovine embryo by examining the expression of ISG-15 mRNA in PBL
taken serially from days 18 to 22, and that a clear 87-bp band is not detected before day 15 and after day
50.