日本・ポーランド二国間国際共同セミナー「生殖生理学の現状と将来―配偶子形成から新生子誕生まで―」
JSPS Japan-Poland Joint Seminar “Cutting-edge Reproductive Physiology-from Gamete to Baby-”

Preface
Kiyoshi OKUDA
Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan
In the recent decades, our knowledge about the regulating mechanisms of many reproductive phenomena has
dramatically increased. The new technologies, such as in vitro fertilization, embryo manipulation and so on, havebeen developed based on the understandings of the endocrine, cellular and molecular physiology controlling central
nerve system, ovarian and uterine functions, gamete and embryo.
In the long history of the scientific cooperation between Poland-Japan, the most recent achievements seem toconcentrate on the ovarian and uterine physiology around luteolysis or early pregnancy. The research developments
in reproduction of both countries are conspicuous in all over the fields, resulting in increasing number of collaborations among the researchers, especially after the first joint seminar of Poland-Japan held in Krakow/Poland, 2005,
with the title “Cutting-edge Reproductive Physiology -Regulation of Ovarian Function-”. Since both countries
have huge potential to develop reproductive research, it has been planed to hold the second joint seminar for exchanging information between both countries. This second seminar “Cutting-edge Reproductive Physiology –from Gamete to Baby-” is expected to be a crucial event which contributes in promoting future collaboration among young
scientists of the both sides. The participants, especially the young scientists have been expected to exchange theadvanced knowledge in the field of reproduction through their research presentations and discussions.
Since Reproductive Physiology covers a variety of fields, the subtitle “From Gamete to Baby” has been added toinclude the wide research fields such as “central nerve system”, “follicular development”, “regulation of corpus
luteum function”, “implantation and maternal recognition”, and “parturition”. Furthermore, the reproductive technology should also be included in the session, because the final goal of our research is to apply to animal reproduction. In the present seminar, only the young top scientists of both countries in the above research fields have been
invited as speakers.
I hope and believe that this seminar will encourage future collaborations among researchers of both countries.

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JP1-1

Cumulus Cells Play an Important Role to Induce Meiotic
Resumption and Progression of Oocyte
Masayuki SHIMADA
Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan

In preovulatory follicles, oocytes are surrounded by numerous layers of cumulus cells in a known as the cumulus cell-oocyte
complex (COC). After stimulation of ovulation by the LH surge, the morphology of COC is dramatically changed as a hyaluronanrich matrix accumulates within cumulus cell layer, and the oocyte resumes meiosis by progressing to the metaphase II. Althoughboth changes induced by LH surge are essential for successful fertilization in vivo, the expression of LH receptors (Lhcgr) is notdetected in the oocyte and is minimal (negligible) in cumulus cells compared with granulosa cells. However, cumulus cells
express members of the EGF receptor family (ErbB family), prostaglandin receptors (EP2 and EP4) and cytokine family receptors that respond to specific ligands secreted by granulosa cells during the ovulation process. EGF-like factors, especially
amphiregulin, that are expressed in a cAMP-PKA-CREB dependent manner in granulosa cells, act on EGFR expressed on cumulus cells. The phosphorylated EGFR induces the RAS-cRAF-MEK-ERK1/2 pathway, which increases the expression of genes
involved in cumulus expansion and oocyte maturation. One of the target genes is Ptgs2 that encodes the rate-limiting enzyme inthe synthesis of PGE2. PGE2 stimulates cAMP production, which results in the maintenance of cAMP level at the maximum
level in cumulus cells to progress oocyte meiosis to the MII stage. Cytokine family, especially IL-6 secreted from granulosa cells
also acts on cumulus cells. When COCs were cultured with IL-6, the developmental competence after in vitro fertilization issignificantly improved as similar to that in in vivo matured oocytes. Thus, by these intermediary steps, the cumulus cells mediate
LH signaling from granulosa cells to induce oocyte maturation.

JP1-2

Susceptibility of Particular Sperm Cell Features toDamages of Long Term-storage of Liquid Semen in
Normospermic Boars•´
Wojciech NIZANSKI
and Agnieszka PARTYKA

Department of Reproduction and Clinic of Farm Animals at Faculty of Veterinary Medicine,
Wroclaw University of Environmental and Life Sciences, Poland.
The aim of this study was to analyze the dynamic changes of sperm cell characteristics in boar semen extended in long-term
extender, in order to detect susceptibility to damage of particular structures of spermatozoa during cooling and storage at 17 C for
240 h post collection. The study included ejaculates collected from seven boars of Polish Large White breed in five replicates(n=35 ejaculates). The sperm characteristics were assessed at 0, 48, 96, 168 and 240 hr post collection with the use of flow
cytometer and computer assisted sperm analyzer. The significant decrease of motility parameters was recorded during the wholetime of semen storage. There were observed the gradual increase of the percentage of live spermatozoa with inactive mitochondria. The use of combination of SYBR-14/PI stains did not reveal any significant changes in the percentage of live sperm cells up
to 168 h of semen storage. Significant (P<0.01) deterioration of acrosome integrity was observed beginning from 168 h ofincubation. The sperm chromatin structure assay (SCSA) showed the significant increase (P<0.01) in DNA fragmentation index(%DFI) after 48 h of semen incubation. Further increase of %DFI was observed during the rest of time of semen storage. In
conclusion, storage of boar semen extended in long-term diluent at 17 C for 48 h induced the decrease of the integrity of sperm
DNA and rapid decrease of population of motile cells as well as all kinetic parameters of sperm cells such as velocity andlinearity. It seems that DNA structure of boar sperm cells in liquid stored semen is more susceptible to damage than the integrityof spermatozoal membranes.

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JP1-3Isolation of Ovarian Components Essential for Oocyte
Growth In Vitro
Yuji HIRAO
Livestock and Forage Research Division, Tohoku Agricultural Research Center (TARC), National
Agriculture and Food Research Organization (NARO), Morioka, Japan

Mammalian ovaries contain a large number of oocytes that degenerate either before or at various stages of growth. Such
oocytes can be rescued under suitable culture conditions. Because offspring have been derived from in vitro grown murine andbovine oocytes, it is evident that oocytes can grow into “normal” ova in vitro. Therefore, some growing oocytes can fulfill their
original function during culture. However, how faithfully the follicle, which contains the oocyte, should mimic the original invivo conditions remains a question. Finding an answer to this question is essential for a better understanding of the regulation ofoocyte growth. In the culture system for murine and bovine oocytes used in this study, the oocyte–granulosa cell complexes, atapproximately the late mid-growth stage, spread on a substratum. However, no theca cells were involved. The structural simplicity of this type of a system gives it an advantage with regard to narrowing down the basic conditions required for the regulation ofoocyte growth. Among the factors playing crucial roles in the ovary, FSH stimulated follicular growth, but was not indispensable
to oocyte growth in vitro. Androstenedione was used to compensate for the absence of theca cells, and it promoted both follicular
growth and acquisition of meiotic competence of oocytes. Besides the biological factors, high concentrations of polyvinylpyrrolidone (molecular weight: 360000) improved the growth of bovine oocytes. In addition, some oocyte-mediated bidirectional
interactions between oocytes and granulosa cells have been observed. Most of the oocytes cultured in a group were viable after
long-term culture, suggesting that unlike the events in the ovary, there was no exhaustive follicle selection. Collectively, theoocytes and the associated granulosa cells can establish independent units capable of supporting oocyte growth in appropriately
modified culture media.

JP1-4

Impairments of Reproductive Tract Function in Cows
Under Influence of Environmental Pollutants/
M. H. WRÓBEL, J. MLYNARCZUKand J. KOTWICA

Institute of Animal Reproduction and Food Research, PAS, Olsztyn, Poland
The aim of the studies was to investigate the effect of xenobiotics on function of bovine reproductive system. As a treatments
we used the chloroorganic compounds - polychlorinated biphenyls (PCBs), pesticide DDT and its metabolite (DDE), which arerecognized as an persistent environmental pollutants. Uteri and oviducts were collected from cows on day 1–5 of estrous cycle,
while ovaries were obtained on day 8–12 after ovulation. Endometrial, myometrial, epithelial of oviduct, granulosa and luteal
cells, as well as myometrial strips were exposed (2–72 h) to environmental pollutants (0.1–100 ng/ml): PCBs mixture (Ar1248),
individual PCBs congeners (30, 77, 126 or 153), PCBs hydroxy-metabolites (30 OH, 50 OH), DDT or DDE. Next, (a) viabilityof cells, (b) expression of mRNA for genes involved in the synthesis of prostaglandin (PG) F2a (cyclooxygenase-2 - COX-2;
PGF2a synthase - PGFS) in myometrium, and oxytocin (OT) (neurophysin-I/oxytocin - NP-I/OT; peptidyl-glycine-a-amidating
mono-oxygenase - PGA) in ovarian cells, (c) secretion of PGF2a from uterine and oviductal cells and OT from ovarian cells or (d)force of myometrial strips contractions (basal and OT-stimulated), were measured. Some myometrial strips were also pre-incubated with indomethacin, which blocks COX-2 and therefore synthesis of prostaglandins. None of used xenobiotics affected(P>0.05) the viability of studied cells. However, they increased (P<0.05) both, mRNA expression of studied genes and thesecretion of OT and PGF2a secreted from the studied cells. PCBs, DDT and DDE increased (P<0.05) the basal and OT-stimulated
force of myometrial strips contractions. However, the this effect was reduced (P>0.05), when the synthesis of prostaglandins wasinhibited. It can be concluded that the synthesis and secretion of OT in ovary and PGF2a in uterus is a part of the mechanism bymeans of which used xenobiotics may affect the force of myometrial contractions in cattle.
Supported by grant N N311 006536− j85 −

JP1-5

Regulation Mechanisms of Gene Expression in Bovine
Embryos Derived from Somatic Cell Nuclear Transfer
Ken SAWAI
Faculty of Agriculture, Iwate University, Morioka, Japan

High rates of early embryonic, neonatal, or postnatal abnormalities have consistently been observed in bovine cloning. The
cause of such abnormalities might be correlated with abnormal epigenetic modifications and gene expression in somatic cell
nuclear transfer (NT-SC) embryos. We examined expression patterns of several genes involved in segregation of inner cell mass(ICM) and trophectoderm (TE) such as OCT-4, CDX2, NANOG, and FGF4, and also DNA methylation level in bovine NT-SC
embryos. In NT-SC embryos at the blastocyst (BC) stage, transcript levels of all genes except CDX2 were lower than that in vivo
embryos. DNA methylation level in BC embryos derived from NT-SC was higher, and the value remained unchanged in theembryo disc and significantly decreased in the TE. Dnmt-1 expression in BC embryos derived by NT-SC was significantly lower
than that in vivo embryos; thus, differences in the DNA methylation status may reflect transcript levels of Dnmt-1. In order toclarify reasons of epigenetic errors, we examined the effect of torichostatin A (TSA) on gene expression in bovine NT-SC embryos, and also analyzed Xist mRNA expression in NT-SC embryos. TSA-treated NT-SC embryos developed to BC stage depending on TSA concentrations with 50 nM TSA showing the highest rate compared with NT-SC embryos without TSA. Histone
H3 acetylation level in NT-SC embryos treated with TSA was higher than that in NT-SC embryos without TSA. Xist expression
level in bovine NT-SC embryos was higher in both sexes than that in IVF embryos. These results raise the possibility that
abnormalities in the cloned fetus and placenta are related to the aberrant expression of genes involved in segregation and differentiation process in the early developmental stage. Although aberrant gene expressions may be caused by DNA methylation andHistone modification, these statuses are corrected as a result of demethylation and retention of methylation as the embryo develops and differentiates and TSA treatment of NT-SC embryos.

JP1-6

Searching for Optimal Conditions and Time Point for the
Derivation of Bovine Embryonic Stem Cells (bESC):
Pluripotency Markers Gene Expression
Zofia E. MADEJA, Berenika PLUSA1 and Dorota LECHNIAK-CIESLAK
Poznan University of Life Sciences, Department of Genetics and Animal Breeding, Poznan,
Poland, 1 The University of Manchester, Faculty of Life Sciences, Manchester, UK

The identification of stem cell specific markers is critical to the establishment of stable ESC form various species. Despite
many efforts, self-renewing ESC have only been derived from mouse (Evans, 1981) human (Thomson, 1998) and rat embryos(Buehr, 2008). It is now evident that although there are some shared mechanism regulating pluripotency and self-renewal in the
ESC, mouse and human ESC differ in morphology, surface marker or gene expression patterns. This question has not been fully
addressed in bovine and the existing data is often contradictory. We aim to analyse the canonical mouse and human pluripotency
and cell lineage markers in bovine. ICM and TE were mechanically dissected from hatched blastocysts (9dpi) and subjected toquantitative gene expression analysis. The preliminary data indicates, that at least at the mRNA level, the expression of NANOG,
OCT4, KLF4, FN1 is higher in bovine ICM and CDX2 and ELF5 in TE, what corresponds to the mouse model. Elf5 was recently
indicated as crucial for the induction of differentiation of mouse ESC to the TE lineage in vitro. A higher level of GATA6, REX1,
KRT-18 expression in bovine TE probably reflects species specific differences. Mouse implantation begins around 4.5dpi andbovine around 18–20dpi. Although, Rex1 is one of the main factors distinguishing mouse ESC from the epiblast stem cells, its
transcript was discovered at a very low level in TE (Rogers,1991). GATA6 is mouse primitive endoderm marker. Its higher
expression in bovine TE may reflect the fact that unlike in the mouse, bovine TE is formed from the cells surrounding theblastocyst cavity. Immunofluorescent analysis localises Cdx2 predominantly in the trophoblast and Oct4 in the ICM of bovine
blastocysts. We are at a very early stage of deriving bESC, but we are able to obtain uniform colonies from ICMs and maintainthem in culture without morphological signs of differentiation. The project is financed by the Foundation for Polish Science(Programme Powroty/Homing; HOM/2009/6B) as a part of Fellowship awarded to Dr Z. Madeja.

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JP2-1

Kisspeptin: A Key Molecule Controlling the Activity of the
Hypothalamo-pituitary-gonadal Axis
Yoshihisa UENOYAMA, Satoshi OHKURA, Hiroko TSUKAMURA and
Kei-ichiro MAEDA
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

Gonadotropin-releasing hormone (GnRH) neurons located in the preoptic area (POA) are final common pathway to control
gonadotropin release, which is critical for steroidogenesis and gametogenesis by the gonads. GnRH neurons are modulated by
feedback actions of sex steroids and several environmental factors. The afferent inputs to the GnRH neurons in the hypothalamus
remain elusive. Kisspeptin neurons, a potent candidate for afferent inputs to the GnRH neurons, emerged from genetic linkage
analyses of the patients of hypogonadotropic hypogonadism. These studies showed that loss-of-function mutations in GPR54 gene,a kisspeptin receptor, result in the hypogonadotropic hypogonadism. Ever since then, researchers found potent stimulatory effects offull-length kisspeptin or its C-terminal decapeptide on gonadotropin release through GnRH release. The present paper focuses on
the role of the two populations of brain kisspeptin neurons in controlling two modes of GnRH/luteinizing hormone (LH) release in
mammalian species. Kisspeptin neurons were limitedly localized in the anteroventral periventricular nucleus (AVPV) and hypothalamic arcuate nucleus (ARC) of female rats, which are prospected regions controlling GnRH/LH surges and pulses, respectively. In
male rodents, kisspeptin neurons are not found in the AVPV. The sexual dimorphism of the AVPV kisspeptin neurons is wellconsistent with the sexual difference in LH surges in rodents. In addition, estrogen enhances AVPV kisspeptin expression in females.
Thus, AVPV kisspeptin neurons would be responsible for surge mode of GnRH/LH release. On the other hand, ARC kisspeptin
neurons are still unknown but are considered to control pulse generation. Neuronal activities synchronized with LH pulses arerecorded in goat hypothalamus, when the electrode is placed at the kisspeptin neuronal cluster in the ARC. Kisspeptin biology
provides a clue as to the mechanism controlling two modes of GnRH release in mammalian species.
This work was supported in part by the PROBRAIN of Japan.

JP2-2

Neuroendocrine Regulation of Seasonality in Sheep
Dorota ZIEBA, Malgorzata SZCZESNA and Beata KLOCEK-GORKA
Agricultural University in Krakow, Poland

In temperate latitudes, sheep are seasonal breeders whose reproductive activity is controlled mainly by photoperiod throughnocturnal secretion of melatonin. However, neither the target sites for its action in the brain nor the neuropeptide circuits engaged
by the melatonin signal are well defined. Intense research has been carried out to provide explanations for the relationships
between hormones related to the regulation of energy homeostasis, metabolism and reproduction. Leptin, ghrelin and orexin
form a close web of interrelationships in the regulation of food intake and energy balance. The aim of this study was to determine
whether day-length modulates the interactions between leptin, ghrelin, growth hormone (GH) and orexin in seasonally breeding
sheep. Ovariectomised, E2-implanted ewes (n=24) were utilised. Ewes were assigned randomly to one of six treatments andinfused into III-ventricle three times at 0, 1 and 2 hr beginning at sunset. Treatment consisted of: 1) control; 2) leptin; 3) ghrelin;4) orexin B; 5) leptin antagonist then ghrelin; and 6) leptin antagonist then orexin. Blood samples were collected at 15-minintervals for 6 h. Leptin increased (P<0.001) plasma concentrations of melatonin during short-day (SD) and decreased themduring long-day (LD) photoperiods. Ghrelin decreased (P<0.01) melatonin during SD and LD, and orexin increased melatonin(P<0.001) only during LD. Leptin attenuated (P<0.05) ghrelin concentrations compared to controls during SD. Plasma concentrations of GH and orexin were lower (P<0.05) after leptin infusions during LD and SD; however, ghrelin had the opposite effects(P<0.01) on orexin concentrations. Orexin increased ghrelin concentrations during LD. Ghrelin and orexin concentrations wereincreased (P<0.001) after leptin antagonist infusions. Our data provide evidence that secretion of leptin, ghrelin and orexin are
not only seasonally dependent, with relationships that are under photoperiodic regulation, but that leptin is the predominant factor
regulating ghrelin and orexin release in sheep.
This work was supported by grants from the Polish National Research Council (N N311 318436 MNiSzW and DS 3242/
KHOiK/2010).

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JP2-3 The Corpus Luteum Development Regulated by Angiogenic
Factors and Polymorphonuclear Neutrophils in the Cow
Koumei SHIRASUNA, Sineenard JIEMTAWEEBOON, Akane NITTA,
Takashi SHIMIZU and Akio MIYAMOTO
Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary
Medicine, Obihiro, Japan
Development of the corpus luteum (CL) in ruminants occurs rapidly and time-dependently within 1 week after ovulation. Thesechanges involve luteinization of steroidogenic cells and angiogenesis to establish luteal function (progesterone (P) secretion). The
CL produces angiogenic factors including basic fibroblast growth factor (FGF2) and vascular endothelial growth factor A (VEGFA).
FGF2 and VEGFA systems exists higher levels in the early CL during the estrous cycle. Both FGF2 and VEGFA stimulate P
secretion from the early CL and progresses proliferation of luteal and endothelial cells. To investigate the impact of FGF2 and
VEGFA, we injected the specific FGF2 and VEGFA antibodies directly into the CL in the cow. These treatments markedly suppressed the increase in CL volume and P synthesis (plasma P concentrations and mRNA expression of StAR) together with changes
in mRNA expression of FGF2 and VEGFA. Therefore, FGF2 and VEGFA promote the establishment of a new vascular network andluteal function during CL formation in the cow. CL formation resembles an inflammatory response due to the influx of immune cells.
Polymorphonuclear neutrophils (PMN) play a role in inflammation, and secrete chemoattractants to stimulate angiogenesis. We
hypothesized that PMNs infiltrate in the developing CL and may play a role in angiogenesis of the CL. Considerable amounts of
PMNs and the high level of interleukin-8 (IL-8, a neutrophil-chemoattractant) were observed during the early luteal phase. The
PMN migration in vitro was stimulated by the supernatant from the early CL, and this activity was inhibited by neutralizing with ananti-IL-8 antibody. Moreover, the supernatant of activated PMNs and IL-8 stimulated formation of capillary-like structures of luteal
endothelial cells. In conclusion, angiogenic factors (FGF2 and VEGFA) are essential for the development of the CL, and PMNs is apotential regulator of angiogenesis together with IL-8 in developing CL in the cow.

JP2-4

Regulation of Corpus Luteum Function by Factors
Associated with Control of Energy Homeostasis
Nina SMOLINSKA, Anna NITKIEWICZ, Anna MALESZKA,
Jadwiga PRZALA, Iwona BOGACKA and Tadeusz KAMINSKI
Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, OlsztynKortowo, Poland

Reproduction is connected with metabolic status in mammals. Recent studies have indicated that the specific hormones –leptin, orexin A, orexin B and adiponectin – create link between feed intake regulation and control of reproductive system. The
expression of leptin, orexins, adiponectin and their receptors mRNAs and proteins has been found in the human, rat and porcine(our studies) corpus luteum (CL). The expression of these hormones and their receptors depends on phase of the oestrous cycle.
These cycle-related changes in expression level seem to be associated with hormonal milieu of animals and imply and importantrole for ovarian steroids as key regulators of these hormonal systems. The identification of leptin, orexins, adiponectin and their
receptors mRNAs and proteins expression in CL supports the conclusion that these hormones may act as the autocrine or/andparacrine factors within the ovary, and that CL are target tissues for leptin, orexins and adiponectin, which might have direct
effects on their functions. Direct effects of leptin, orexins and adiponectin on regulation of CL function have been investigated innumerous studies. These reports indicate that the hormones regulate steroidogenesis and may play a role in the process ofluteinisation. In addition, leptin, orexins, adiponectin and their receptors mRNAs and proteins has been found in human, rat andporcine (our studies) hypothalamus and pituitary. Studies on the hypothalamic-pituitary axis have identified that these hormones
regulate the secretion of GnRH, LH and FSH, and may indirectly participate in the control of CL function. Taken together, theevidences were presented that supporting the idea that leptin, orexins and adiponectin are more than factors associated withenergy homeostasis only. Their also serve as the metabolic signals controlling directly and indirectly (through hypothalamopituitary hormones) functions of corpora lutea.
This research was supported by Ministry of Science and Higher Education (project No N N308 122134, 2008-2011).

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JP3-1

Establishment of In Vitro Assessment System for Conceptus
Attachment to the Uterine Epithelial Cells
Toshihiro SAKURAI, Hanako BAI, Toshihiro KONNO and
Kazuhiko IMAKAWA
Laboratory of Animal Breeding, Graduate School of Agricultural and Life Science, the Universityof Tokyo, JapanThe establishment of pregnancy requires appropriate communication between the developing conceptus and the uterine endometrium. If implantation failure is in fact the major cause of pre-implantation embryo loss, studies should be directed toward
an understanding of biochemical communication between the conceptus and mother. The implantation proceeds when the conceptus and uterine developments are synchronized through secretory factors from each tissue. IFNT plays a central role in the
crosstalk between the conceptus and mother toward its attachment to the uterine endometrium. Because transcription of IFNT
gene starts to decline when the conceptus attaches to uterine epithelial cells, IFNT would be an ideal maker for evaluation onwhether or not the attachment of trophoblast cells to uterine epithelial cell occurs. If the assessment system of attachment in vitro
were established, each of attachment processes could be dissected for thorough evaluation and more importantly for the study offactors identified and/or yet unidentified required for conceptus attachment to proceed. We, therefore, attempted to establish thein vitro coculture system with bovine trophoblast CT-1 cells and uterine epithelial cells, which possibly mimic the attachment
process in vivo. Results from the coculture experiments with CT-1 and endometrial cells revealed that both the uterine flush frompregnant animals and physical contact with endometrial cells were essential for the down-regulation of IFNT in CT-1 cells.
Although further study is required for understanding the implantation process, the coculture system developed may become
useful for the assessment of biologically active factors during the implantation process.

JP3-2 The Role of HOXA10 as a Marker of Endometrial
Receptivity
Agnieszka BLITEK and Adam J. ZIECIK
Department of Hormonal Action Mechanisms, Institute of Animal Reproduction and Food
Research of Polish Academy of Sciences, Olsztyn, Poland
The establishment of a uterine environment suitable for successful implantation and further pregnancy establishment is crucial
and remains under strict regulation by ovarian steroids and conceptus signals. Search for markers of uterine receptivity revealed
that homeobox A10 (HOXA10) gene expression is essential for uterus development and embryo implantation in humans, nonhuman primates and mice. Recently we examined the expression of HOXA10 in the pig uterus. The endometrial HOXA10 gene
expression was increased on day 15 of pregnancy when compared to day 15 of the estrous cycle. Moreover, higher HOXA10transcript level was detected during implantation than at the period of “maternal recognition” of pregnancy. Immunoreactive
HOXA10 protein was detected in luminal and glandular epithelium, but also in stromal cells and blood vessels of the endometrium.
Estradiol (E2) alone or in the presence of progesterone (P4) increased HOXA10 transcript level in cultured stromal cells, as wellas incubated endometrial explants. Moreover, HOXA10 protein was stimulated in endometrial explants by E2, P4 and bothsteroids added simultaneously. Administration of PMSG/hCG to induce estrus resulted in decreased expression of HOXA10 in
the endometrium on day 12 of gestation, but had no effect on HOXA10 transcript levels in conceptuses. The observed downregulation of HOXA10 was accompanied by reduced serum P4 content in gonadotropin-treated gilts. Additionally, conceptus
products stimulated HOXA10 and PTGS2 transcript levels in luminal epithelium in vitro. Summarizing, these results indicate that
HOXA10 expression in the pig uterus is closely related to implantation process and stimulated by both maternal and conceptusderived factors.

This research was supported by grant N311 04732/2777 from MHE and basic grant of PAS.

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Plasma Estrogen Concentration and Placental Regulation ofJP3-3 Estrogen Activity at Parturition in Cows Carrying a
Somatic Clone Fetus
Hiroki HIRAYAMA

Animal Biotechnology Group, Animal Research Center, Hokkaido Research Organization,
Shintoku, Japan
The production of viable offspring from nuclear transfer with somatic cells has been reported in a range of domestic species,
although the production efficiency is still low. Pre-partum abnormality such as prolonged gestation and a lack of readiness forbirth in the recipient cows is one of the factors in decreasing the production efficiency of clone cattle. We analyzed hormonal
concentrations during the pre-partum period and placental function regarding regulation of estrogen activity and steroidogenesis
in placentome at parturition. Placental estrogens have been suggested to play important roles in the parturition process. In clone
pregnancies, pre-partum rises in concentrations of plasma estrone (E1) and estradiol-17b (E2) in the recipient cows were subtle.
Estrone sulfate (E1S) is an inactive form of E1 catalyzed by estrogen sulfotransferase (SULT1E1). The plasma concentration of
E1S in clone pregnancies increased gradually from pre-initiation of parturition induction whereas control cows that received invivo-derived embryos showed a significant increase at parturition. In clone pregnancies, the ratio of E1/E1S was low during the
pre-partum period. Messenger RNA expression of SULT1E1 in the placentome at parturition was significantly higher in clone
pregnancies and was localized in binucleate cells. SULT1E1 mRNA abundance was negatively and positively correlated withconcentrations of maternal E1 and E1S at parturition respectively. Estrogens are synthesized by aromatase (CYP19) from androgens. Sulfoconjugated estrogens may also be precursors of the production of free estrogens, depending on placental estrogensulfatase (STS) activity. STS and CYP19 mRNA abundances were similar between clone and control pregnancies and showed
positive correlations with maternal E2 concentration. These results suggest that excess estrogen sulfoconjugation is the reason
for the perturbed low ratio of active to inactive estrogens and the resulting hormonal imbalance contributes to the lack of overt
signs of readiness for parturition in cows pregnant with clones.

JP3-4 The Role of Immunological Disturbances in the
Pathogenesis of Pre-eclampsia
D. DARMOCHWAL-KOLARZ 1, M. KLUDKA-STERNIK2,
J. TABARKIEWICZ2, B. KOLARZ2, J. ROLINSKI2 and J. OLESZCZUK2
12

Department of Obstetrics and Perinatology, Medical University of Lublin, Poland
Department of Clinical Immunology, Medical University of Lublin, Poland

It seems that abnormal activation of the immune system may play a role in the etiology of pre-eclampsia. Many authors havefound a number of changes in the innate and adaptive immune system which may contribute to the development of pre-eclampsia.
The aim of our study was to estimate the prevalence of CD3+CD4+ T lymphocytes producing IL-17, IL-2, IFN-g and IL-4, as wellas CD4+CD25+FoxP3+ T regulatory cells (Tregs) in peripheral blood of patients with pre-eclampsia and healthy women in thethird trimester of normal pregnancy. Furthermore, the purpose of our study was to assess the immunosuppressive activity of Treg
cells of pre-eclamptic patients in comparison with the controls. : The percentages of T CD3+CD4+ lymphocytes producing IL17A were significantly higher in pre-eclampsia when compared to healthy normotensive pregnant women in the third trimester ofnormal pregnancy (p<0.001). The population of CD4+CD25+FoxP3+ Treg cells was significantly lower in the study when compared to the control group (p<0.05). There were no changes in the proliferative response of CD3+CD4+CD25- T lymphocytes ofpre-eclamptic patients during in vitro assay without Treg cells and after the addition of autologous Tregs. In normal pregnancy
the proliferative response of CD3+CD4+CD25- T lymphocytes was significantly higher without Treg cells when compared to this
response after addition of autologous Tregs (p<0.05). The results obtained suggest the up-regulation of Th17 immune response in
pre-eclampsia. It seems that the decreased number and function of Treg cells may be responsible for the activation of inflammatory response in this disorder. The predominance of Th17 immunity can act through the modulation of Th1/Th2 immune response
in pre-eclampsia.

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Closing Remarks
Dariusz J. SKARZYNSKI
Department of Reproductive Immunology and Pathology, Institute of Animal Reproduction and Food Research ofPAS, Olsztyn, Poland
Studying the regulatory mechanisms of biology of gametes, ovarian function, early embryo development and implantation is important as a basic data for the establishment of new methods of the diagnostic and treatment forsterility in medical and veterinary sciences. In addition, it is essential for the fields of experimental zoology andanimal husbandry to develop new techniques of reproductive control, enable efficient animal production andbiodiversity.
Japanese and Polish researchers who are young pioneers in reproductive physiology, biotechnology and pathology
have met for four days on our joint Seminar and have discussed the present situation of reproductive biology in bothcountries. The seminar covered seven main topics of mutual interest and discussion: (1) Gametes biology and biotechnology of reproduction, (2) Embryo, (3) Central mechanisms of reproduction, (4) Ovarian function – including
follicular development, (5) Corpus luteum function, (6) Pregnancy recognition and implantation, (7) Pregnancy,
placenta, parturition. The main objective of the Seminar was to encourage the young scientists of both countries toestablish further long-lasting collaboration which should result in joint research projects. The new directions of the
future research were analyzed and defined and we can expect new development in field of reproductive biology andpathology. Finally we hope that the Seminar should enable us to make at least the research in our filed more public
and practical.
Here I want to particularly thank Dr. Ken SAWAI for their hard work in organizing the current meeting. Organizing an international meeting is not an easy job. I want to also thank all our speakers and all of you for participating in
the meeting. I believe that in the soon future we can further increase our collaboration. We hope that every participants will return home stimulated and with new research ideas and new friendships.

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