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This report studied the identification and sequence of a full-length cDNA for the bovine BCL2 antiapoptotic family member, BCL2-related protein A1 (BCL2A1), and its localized and quantitative expression in the placenta to clarify the regulatory mechanism of trophoblast cell proliferation and differentiation during implantation and placental development. We cloned a full-length bovine BCL2A1cDNA with 725 nucleotides and an open-reading frame corresponding to a protein of 175 amino acids. The predicted amino acid sequence shared 78% homology with human BCL2A1. All BCL2 homology domains (BH1, BH2, BH3, and BH4) in bovine BCL2A1 were conserved as well as in other mammalian BCL2A1. In the placentomes, in situ hybridization demonstrated that the BCL2A1was limited in binucleate cells expressing various pregnancy-specific molecules like placental lactogen. BCL2-associated X protein (BAX) was also expressed in binucleate cells. Quantitative real-time RT-PCR detection exhibited a high-level expression of BCL2A1in the conceptus at Day 21 of gestation, and it was expressed and increased in the extraembryonic membrane, cotyledon, and intercotyledon from implantation to term. BAXexpression intensity increased with progression of gestation and remained elevated in postpartum. Caspase-3 protein (CASP3) and mRNA (CASP3) were detected from late gestation to postpartum in placenta as well as in the results of TUNEL detection. We believe that the apoptosis of binucleate cells may be regulated by the balance of the BCL2A1 and BAX. BCL2A1genes produced a BCL2A1 protein in the mammalian cell-expression system. This molecule is a new candidate for antiapoptotic maintenance of the binucleate cells that support placental functions throughout gestation in bovine.

Using cDNA microarray methodology, we have shown previously that transcripts of progranulin gene (Grn,also known as acrogranin), a recently identified autocrine growth factor, were upregulated in mouse blastocysts adhered to the filter membrane in an in vitro-culture system. In the present study, we investigated the expression and effects of progranulin on blastocyst hatching, adhesion, and embryo outgrowth during the peri-implantation period in the mouse. During this period, substantial amounts of GrnmRNA were present in both inner cell mass (ICM) and trophectoderm. Progranulin was localized exclusively to the surface of the trophectoderm in early and pre- and postadhesion blastocysts as well as in trophoblast cells and ICM of outgrowth embryos, being secreted as a single, 88-kDa form into the surrounding medium. NIH3T3 cells that had been transfected with a progranulin expression construct secreted the 88-kDa form of the protein, from which a 68-kDa form could be generated by deglycosylation. In vitro treatment of blastocysts with recombinant progranulin promoted blastocyst hatching, adhesion, and outgrowth, whereas rabbit anti-mouse progranulin immunoglobulin G reduced the incidence of blastocyst hatching, adhesion, and outgrowth. Studies of bromodeoxyuridine incorporation and immunodissection of the ICM revealed that progranulin was effective on the trophectoderm but not on the ICM. These results indicate that progranulin is an important factor for the processes of blastocyst hatching, adhesion, and outgrowth, and they suggest that the effects of progranulin on blastocyst adhesion and outgrowth may have been triggered by the previous action of progranulin to induce hatching of the blastocysts.

Our objective was to develop a method of endowing oocytes from porcine primordial follicles with full maturation and fertilizing ability as a model for ovarian xenografting of large mammals. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the capsules of both kidneys of ovariectomized athymic mice. The host mice were treated with 5 IU of equine chorionic gonadotropin (eCG) for 10 days (eCG-10), 30 days (eCG-30), or 60 days (eCG-60) after detection of cornified epithelial cells in their vaginal smears. Cumulus-oocyte complexes, ovarian grafts, and blood samples were obtained 48 h after eCG treatment. Forty-five to 70 days after grafting, the host mice in all groups for the first time showed vaginal cornification, accompanied by the formation of a small number of antral follicles in the grafts. However, we recovered large numbers of full-sized oocytes only from mice in the eCG-60 group; the numbers of full-sized oocytes in the other groups were low. Peripheral levels of total inhibin were highest in the eCG-60 group; this supports our finding that the most enhanced growth of antral follicles occurred in this eCG-60 group. Of 573 oocytes obtained from the eCG-60 group, 98 (17%) were at the metaphase II stage after in vitro culture for maturation. Moreover, 55% of matured oocytes with the first polar body (n = 20) were fertilized in vitro. These results clearly demonstrate that fertilization of oocytes from porcine primordial follicles is achievable by a combination of xenografting and in vitro culture.

We have developed a method to monitor noninvasively, quantitatively, and in real-time transcription in living preimplantation mouse embryos by measuring expression of a short half-life form of enhanced green fluoresecent protein (EGFP) following microinjection of a plasmid-borne EGFP reporter gene. A standard curve was established by injecting known amounts of recombinant green fluorescent protein, and transcriptional activity was then determined by interpolating the amount of fluoresence in the DNA-injected embryos. This approach permitted multiple measurements in single embryos with no significant detrimental effect on embryonic development as long as light exposure was brief (<30 sec) and no more than two measurements were made each day. This method should facilitate analysis of the regulation of gene expression in preimplantation embryos; in particular, during the maternal-to-zygotic transition, and in other species in which limited numbers of embryos are available.

Polyspermy is one of the unresolved problems that exist regarding pig oocytes matured and inseminated in vitro. Quantitative study of the changes in the cortical granule (CG) population in oocytes is essential for understanding the mechanism of how oocytes block polyspermic penetration and for developing the optimum conditions for in vitro maturation (IVM) and in vitro fertilization (IVF). The present study was conducted to quantify the CG distribution in pig oocytes during IVM and IVF by using fluorescein isothiocyanate-labeled peanut agglutinin with laser confocal microscopy. The results indicate that CGs are distributed in the cortex cytoplasm of oocytes at the germinal vesicle (GV) stage with a mean number of 33.8 ± 7.3 CGs/100 µm2of cortex. As nuclear maturation proceeded to metaphase I and metaphase II, CGs migrated to the cortex and formed a continuous monolayer under the oolemma. No distinct CG-free domain was observed in oocytes during maturation. The migration of CGs to the cortex continued during maturation, with an increased CG density after the GV stage. All oocytes penetrated by spermatozoa were activated and released CGs from ooplasm with an average residual number of 3.5 ± 4.6 CGs/100 FLm2 of cortex at 18 h after insemination. Complete CG exocytosis was observed in 45% of oocytes. Calcium ionophore did not induce oocyte nuclear activation, but CGs were released from oocytes with an average of 7.1 ± 4.5 CGs/100 µm2of cortex still present when examined 18 h after treatment. An electrical pulse induced 89% of nuclear activation in matured oocytes, and CG exocytosis was observed only in nuclearactivated oocytes with an average residual number of 6.4 ± 9.4 CGs/100 µm2of cortex. Complete CG exocytosis was induced by ionophore and electrical pulse in 10% and 25% of the oocytes, respectively. These results indicate that CGs migrate to the cortex in pig oocytes during IVM and that the matured oocytes obtained under these maturation conditions possess the ability to release CGs upon sperm penetration, ionophore treatment, and electrical pulse. However, a functional block to polyspermic penetration in oocytes after CG exocytosis was not fully established in these studies. The present methods and results provide the approach for further investigation of the reasons for polyspermy in pig oocytes matured and inseminated in vitro.

Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) play critical roles for folliculogenesis, follicular development and ovulation in mammals. The two factors are thought to have synergistic effects in granulosa cell proliferation and to regulate the cumulus cell function in the periovulatory period. In this study, we investigated the roles of BMP15 and GDF9 on bovine oocyte maturation in vitro using their antibodies. Bovine cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries, and matured in medium199 supplemented 5% FCS, Estradiol (1mg/ml) and FSH (0.002AU/ml) with anti-bovine BMP15 antibody, anti-bovine GDF9 antibody or 0.5% normal rabbit serum. After in vitro maturation, some of COCs were denuded to observe the nuclear stages by aceto-orcein staining or to observe the distribution of cortical granules (CGs) as index of cytoplasmic maturation. For staining of the CGs, oocytes were stained with FITC-labeled Lens curinalis agglutinin. Distributional patterns of the CGs were classified into three types: type I, CGs distributed in clusters; type II, CGs dispersed; and partly clustered and type III, all CGs dispersed. The others of COCs were fertilized in vitro and cultured with IVD101 medium (Research Institute of Functional Peptide, Japan) to observe the polyspermy and blastocyst formation. After maturation culture, the percentages of oocytes at metaphase II treated with anti-BMP15 antibody, anti-GDF9 antibody or normal rabbit serum were not significantly different (71.1%, 72.8% and 71.8%, respectively). The percentages of type I of CGs distribution in oocytes treated with anti-BMP15 antibody were significantly higher than those in oocytes treated with normal rabbit serum (p<0.01), and the percentages of type III of CGs distribution in oocytes treated with anti-BMP15 antibody were significantly lower than that in oocytes treated with normal rabbit serum (p<0.01). The percentages of polyspermy in oocytes cultured with anti-BMP15 antibody, anti-GDF9 antibody or normal rabbit serum were were not significantly different (13.3%, 5.4% and 7.8%, respectively). And a significantly (p<0.05) lower population of oocytes cultured with anti-BMP15 antibody developed to blastocyst stage than those treated with anti-GDF9 antibody and normal rabbit serum. The present results show that BMP15 has a crucial role for the cytoplasmic maturation of oocytes and affects subsequent developmental competence of embryos in cows.

Endogenous retrovirus (ERV) envelope (env) genes are involved in the differentiation of trophoblastic cells in humans and mice. However, there is limited information about their roles in ruminant trophoblastic cells. The purpose of this study is to explore the possible roles of ERV elements in the binucleation of bovine trophoblastic cells using three in vitrobovine trophoblastic (BT) cell lines, BT-1, BT-C and BT-K. On-Matrigel culture was used to induce binucleate cells (BNC) in the BT cell lines. During induction of BNC culture, the expression profiles of ERV elements (bERVE-A, bERVE-B, BERV-K1 envand BERV-K2 env) were examined by a quantitative RT-PCR (qRT-PCR) and immunocytochemistry, and BNC specific genes (bCSH1, bPRP-1and bPAG1) were examined for the confirmation of binucleation. The binucleation was found in BT-1 and BT-C but not in BT-K during on-Matrigel culture, i.e., the BNC specific genes were confirmed more than 10-times by qRT-PCR. The increment of bPRP-1 expression cell numbers was found in BT-C immunocytochemically. The expression intensities of bERVE-Aand BERV-K1 envwere increased in BT-1 and BT-C but not in BT-K during on-Matrigel culturing, and also the cell number of BERV-K1 Env protein expressed was increased in BT-C. The expression levels of these ERV elements in BT cells were consistent with those of BNC specific genes during on-Matrigel culturing (P< 0.01). These results suggest that bERVE-Aand BERV-K1 envare closely involved in the expression of BNC specific genes. The on-Matrigel culture system using BT cell lines supplies a convenient in vitrotool for studying bovine trophoblastic cell lineages. This study was supported by JSPS (Kiban-kenkyu B 23380162 and Bilateral Program).

Trophoblastic cell line is a useful tool to explore the placental functions and feto-maternal interaction in vitro. We raised thirteen bovine trophoblastic cell lines from in vitro fertilized ova. These cells expressed trophoblast cell specific genes and their expression patterns were different and depend on the cell lines, respectively. In the present study, we examined comprehensive genes expression profiles to confirm the characteristics of individual cell lines. Four different cell lines were used for microarray analysis: BT-1 (230 and 330 passages), BT-C (70 passages) which mainly expressed interferon-tau (IFNT) in previous study, BT-J (55 passages) which expressed IFNT with less placental lactogen (CSH1) and BT-K (41 passages) which expressed prolactin-related protein 1 (PRP1) with less IFNT. Some in vivo embryos were collected from Holstein cows on Day 20 of gestation and only trophoblastic tissue was separately collected into TRIzol and kept -80 C until analysis. Total RNA was extracted from cultured cells and embryonic trophoblast tissues and they were subjected to microarray and quantitative RT-PCR. Gene expression data were analyzed by GeneSpring GX 10 (Agilent). Although a custom microarray involves about 15,000 genes, the comparison between 230 and 330 passages in BT-1 showed only 16 genes different and they included no trophoblast-specific genes. Global genes expression analysis of BT-C showed that about 1,000 genes were difference from those of BT-1 (passage 330), and similar expression difference was found in BT-J cell. However over 4,000 genes were significant difference between BT-K and BT-1 (330). The gene expression profiles in new BT cells were similar to those in BT-1 confines to trophoblast-specific genes. However new cells still kept the characters of in vivo trophoblast cells, trophoblastic tissues from embryos on Day 20 of gestation had wider range of associated genes. All cell lines including BT-1 have been maintained some signal regulatory genes like OCT3/4, CDX2, but less of STAT3, GATA4, SOX2 and NANOG. Day 20 embryonic trophoblast tissues expressed various TGFB super-family genes including INHA, INHBA, FST, SMAD2, and both BT-J and BT-K carried out the features of in vivo embryonic trophoblast tissues. The trophoblastic cell lines and in vivo embryonic trophoblast tissues expressed BMP4, BMP15 and BMP receptors with lower intensity. These gene expression profiles suggest that BT cell lines maintain a specific characteristic of embryonic trophoblast tissues during peri-implantation. A series of BT cell lines will provide a useful tool and open a wedge of new insight for implantation and trophoblast cell lineage. This study was supported by a Grant-in-Aid from JSPS (Kiban-Kenkyu B 20380159).(poster)

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a transmembrane glycoprotein located on the surface of normal and pathological cells. EMMPRIN stimulates the production of several matrix metalloproteinases (MMPs) through direct cell-cell interaction and/or as a paracarine manner. Apart from its MMPs inducing ability, it also facilitates cellular differentiation, developmental process and female fertility. Regarding the expression and role of EMMPRIN in the bovine endometrium is still obscure. Therefore, the aim of this study was to 1) analyze the expression and cellular localization of EMMPRIN along with MMPs during estrous cycle and early gestation and; 2) regulation of MMPs by direct cell-cell and cell-matrix interaction using cultured cells and collagen type-I matrix. EMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at day 35 of gestation than the cyclic endometrium. The fetal membrane at day 35 of gestation also expressed EMMPRIN mRNA but the intensity of expression was slightly lower than that of the endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium. On the other hand, the intense band migrated approximately 51 and 32 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively; whereas, a 32 kDa band was detected weakly in the stromal cells. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on day 19 conceptus. At day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on day 30 of gestation, slightly increased expression was found at the site of placentation. In the endometrium, expression of EMMPRIN mRNA was limited in the stromal compartment. Expression of MMP-2 and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at day 19 of gestation however it was also expressed at the site of placentation at day 30 of gestation as observed for EMMPRIN. Therefore, we have expected that EMMPRIN from epithelium may stimulate stromal MMP-14 and MMP-2 expression in the bovine endometrium; and regulate the endometrial functions during early gestation. Our in vitro study showed that collagen type-1 significantly stimulated the expression of EMMPRIN, MMP-2 and MMP-14 by both the stromal and epithelial cells. Furthermore, direct cell-cell interaction of epithelial and fibroblastic cells significantly stimulated both MMP-2 and MMP-14 mRNA expression. Taken together, expression of EMMPRIN on feto-maternal interfaces may have crucial role in adhesion and fusion of embryo to luminal epithelium by directly itself through physiological tissues remodeling and developmental process, and/or stimulating MMPs to compensate endometrial functions. Therefore, this study for the first time suggests that EMMPRIN is an important glycoprotein and its differential expression regulates endometrial remodeling during early gestation in cows. Supported by a grant from the Research Project for Utilizing Advanced Technologies (05-1770) from the Ministry of Agriculture, Forestry, and Fisheries of Japan and grants (Kiban-kenkyu C 19580335; Kiban-kenkyu B 20380159) from the Ministry of Education, Culture, Sport, Science, and Technology of Japan.(poster)

The differentiation of the trophoblast lineage in mammals is recognized from the appearance of phenotypic cells distinct in morphology and function. Bovine trophoblastic binucleate cells (BNCs) are large cells with two nuclei, differentiate from the mononuclear trophoblastic cells (MNCs) at the beginning of implantation and throughout pregnancy, and represent about 20% of trophoblastic cells. The BNCs produce hormones such as placental lactogens (CSH1), prolactin-related proteins (PRPs) and pregnancy-associated glycoproteins (PAGs), and play an important role in fetal development and maternal adaptation to pregnancy. Although BNCs are known to produce these specific molecules, the characteristics and differentiation mechanisms remain to be investigated. Therefore, we try to isolate the BNCs and MNCs from bovine placenta, and characterize the both cell types. The placentomes were collected from cows which were ranged from middle to late gestation. In brief, fetal cotyledonary tissue was separated manually from placentomes, washed and cut into small pieces. The pieces of tissue were treated with collagenase, and the digested tissue was filtered through stainless steel meshes to remove undigested tissue. The filtrate was washed with the medium and layered on discontinuous Percoll gradients with isopycnic densities at 1.03–1.07 g/ml, and centrifuged. The interface between 1.04 and 1.06 g/ml were collected, and the cell fractions were incubated with Hoechist 33342 and propidium iodide. Further cell fractionation was performed using high performance cell-sorting system EPICS ALTRA MultiCOMP. The nuclear phase, cell size and the inner complexity of the cells were used as separation indicator. In flow cytometric analysis, the analyzed trophoblastic cells exhibit approximately 80% diploid and 20% tetraploid (or hexaploid) cells, and two different sizes of tetraploid cells in the fraction were observed. These cells were sorted and the mRNA expression of trophoblastic cell specific molecules were analyzed by quantitative RT-PCR. The mRNA of CSH1, PRP1and PAG1were highly expressed in tetraploid and hexaploid cells compared with diploid cells. However, the transcriptional factors including CDX2, ETS2and POU5F1mRNA levels were not different in each cell types. The present results imply that BNCs and multinucleated throphoblastic cells were highly contained in the tetraploid and hexaploid cell fractions. These results further suggest that the methodology using high performance cell-sorting system following discontinuous density gradients centrifugation in the present study may supply an appropriate technique for isolation of BNCs from bovine placenta, and allow the investigation of trophoblastic cell differentiation and function. Supported by a Research Project for Utilizing Advanced Technologies (05-1770) grant from the MAFF of Japan; grants (Kiban-kenkyu B 20380159, Kiban-kenkyu C 19580335) from Ministry of Education, Culture, Sport, Science.(poster)

Trophoblast cells derive from the outer layer of a blastocyst and are the first differentiated cell from fertilized embryo. They take crucial roles for implantation and placentation in interface between embryonic membrane and maternal endometrium. In ruminant, trophoblast has two cell linages; mononucleate and binucleate. Both cells express and produce various hormones and cytokines as endocrine cells, respectively. However some trophoblastic cell lines in bovine like BT-1, which we previously established as one of bovine trophoblastic cell lines, key factors for trophoblastic lineages are unknown and there are no effective procedures for establishing cell lines yet. To explore the effective methods for raising trophoblastic cell lines, we examined culture procedures and the effects of one of transforming growth factor beta super-family, bone morphogenetic protein 4 (BMP4), on the differentiation of embryonic cells for trophoblast. In vitro fertilized ova were culture in CR1aa containing 5% calf serum with recombinant BMP4 (0, 1, 10, 100 ng/ml) for 7 days, then cultured in TCM-199 medium containing 20% FCS with recombinant BMP same as above for 6 days. The embryos were transferred into 24 wells culture dishes and cultured in containing 20 % FCS DMEM/F12 continuously. Spread cells were subcultured into T-25 tissue culture flask. The mRNA was extracted from cultured cells and used for gene expression analysis by quantitative RT-PCR (Q-PCR). Most of embryos attached on the 24 wells dish and about 80% of them spread. Over 10 proliferation doubling level (PDL) passage, only 3% embryos attained to cell lines in control culture, however, about 10% of embryos grew cell lines in BMP4 groups. We picked out some cell lines and examined their characteristics at passed over 30 PDL stage. They still have been keeping bovine trophoblast cell features: Binucleate-like cell was found in the monolayer which shaped cobble-stone like to epithelial cells. Doubling time was about 7 days and this period was same as that in BT-1 cell. In this new bovine trophoblastic cell lines, they contain at least three different characteristic cell lines. All these three lines expressed placental lactogen (CSH1) and bovine prolactin-related protein 1 (bPRP1), but they showed different expressions in interferon-tau (IFNT); namely first one expressed CSH1 and IFNT, which are very similar features as that in BT-1. Second one mainly expressed IFNT. Third one mainly expressed CSH1 not IFNT. Although all cell lines expressed same transcription factors; ETS2, CDX2 and POU5F1, similar to the BT-1, one line expressed less ASCL2. BMP4 may be an inductive factor for trophoblastic cell lineages and stimulate the establishment of trophoblast cell lines in bovine effectively. These different features cell lines will serve as a useful in vitro tool for the analysis of the molecular and/or cellular mechanisms in bovine trophoblast cells differentiation and placentation. Supported by a Research Project for Utilizing Advanced Technologies (05-1770) grant from the MAFF of Japan; grants (Kiban-kenkyu B 20380159, Hoga-kenkyu 19658101) from Ministry of Education, Culture, Sport, Science.(poster)

Environmental pollutants are potentially hazardous for endocrine system. Particularly some estrogenic compounds have been reported to be endocrine disruptors in many species. In cows, since circulating estrogen levels are considerably lower than those in rodents and primates, it is plausible that cows are potentially sensitive and have a fragile homeostatic base against exogenous endocrine disruptors. To evaluate the effects of endocrine disruptors in cows, toxicogenomics platform needs to be established as a powerful tool for genome-wide research. In the present study we established bovine oligonucleotide array and evaluated the use of trophoblast cell line (BT-1) for studying the effects of endocrine disruption in cows. Sixty-mer nucleotides probes for customized microarray were designed and synthesized on the glass slide. Array fabrication was carried out by the Agilent Technologies. The annotated bovine oligonucleotide array represented 10263 sequences. Of which, 4466 genes were known bovine genes, 5697 unknown sequences were possible candidates for novel bovine genes, and 100 internal references. BT-1 cells, generated from the trophectoderm of bovine blastocyst, were maintained over 300 passages upon establishment. BT-1 cells were cultured in collagen-coated flasks that contained Dulbecco fs modified eagle medium/Ham's F-12 medium supplemented with 20% fetal bovine serum and antibiotics in an atmosphere of 5% CO2 in air at 37C. The cells were grown up to 70%-confluent and further cultured in the presence of estradiol 17-β (E2), diethylstilbestrol (DES), nonylphenol (NON), octylphenol (OCT) (10-7M each) or ethanol (vehicle 0.1%) for 48h. Total RNA was isolated and used for microarray analysis. The labeling, hybridization, scanning and data analysis were carried out by using two samples (tested chemical versus vehicle) labeled with different fluorochromes. Samples were reciprocally labeled for dye-swap normalization. Genes that showed either more than 2-fold (test/vehicle) induction or repression were selected for further analyses. The expression levels of only 137, 11, 5 and 4 genes were changed more than 2-fold following treatment with E2, DES, NON and OCT, respectively. The expression levels of representative genes were confirmed in BT-1 cells by real-time PCR and spatial localization of the gene in utero-placental tissues was examined by in situ hybridization. Results in the present study show that genome-wide analysis with customized bovine oligonucleotide array is capable of measuring the alteration of gene expression profiles in BT-1 cells following exposure to endocrine disruptors. In the present study, the numbers of genes responding to endocrine disruptors were relatively fewer than those of previous reports analyzed with in vivo tissues. Thus reduced numbers of responding gene may be due to the homogeneous cell lineage of BT-1 cells. (Supported by grants in aid RPUAT-05-1770 from MAFF, NIAS-05-201 from NIAS and HC-2261 from NIAES)

Fish sperm are immotile in the seminal plasma and become activated on ejaculation. Sperm of many freshwater and marine teleosts are quiescent at the osmolality of seminal plasma (isotonic). Their motility is initiated in the hypotonic freshwater and hypertonic seawater, respectively. On the other hand, the euryhaline teleosts can acclimate and perform reproduction in both conditions. In order to get deep insight into the regulatory mechanism of fish sperm motility, we have investigated the sperm motility of euryhaline teleosts, such as tilapia and medaka. Sperm of medaka kept in fresh water are motile in fresh water to isotonic water but not in seawater. When the fish is acclimated in seawater, the motility in hypertonic water was observed. Addition of Ca2+improved as well as that observed in tilapia sperm. Demembranation-reactivation experiments also revealed that Ca2+was an intracellular key factor for motility activation. Neither cAMP nor cGMP accomplished the motility activation. We also demonstrated that CO2is the candidate for an inhibitory factor for sperm motility in the sperm duct. Extra-cellular CO2inhibited sperm motility, but extra-cellular HCO3-did not. Reactivation of the demembranated sperm was inhibited by low pH and HCO3-. In addition, inhibition of sperm motility did not occur in the presence of the inhibitor of carbonic anhydrase, ETZ. These facts suggested that the influx of extracellular CO2through plasma membrane causes the increase in the concentration of HCO3-and H+ from CO2by carbonic anhydrase to inhibit the sperm motility. Above 5.6 mM CO2was enough to inhibit motility. These results suggest that the increase of Ca2+and the depletion of CO2together activate the motility on ejaculation. Studying sperm of medaka will lead to comprehension of the adaptation mechanism to wide range of osmolality at sperm cell level.

In monovular species including cattle, usually one follicle is selected and continues growing to become dominant during the follicular wave. Although the dominant follicle acquires ovulatory capacity, if it fails to ovulate, the follicle undergoes atresia and new follicular wave emerges. It is well documented that hormonal milieu plays critical role in the fate of the dominant follicle. In addition, it seems that the growth and atresia of dominant follicle is regulated by specific changes of numerous genes expressed in follicular cells. In this study, we tried to identify genes differentially expressed during growth and atresia of dominant follicle using microarray analysis. Paired ovaries were obtained from five Japanese-black cows and five follicles (≥8 mm in diameter) were dissected. Total RNA was extracted from individual follicles and used for microarray analysis. Sixty-mer nucleotides probes for customized microarray were synthesized on the glass slide. Array fabrication was carried out by the Agilent Technologies. The annotated bovine oligonucleotide array represented 10263 sequences. Of which, 4466 genes were known bovine genes, 5697 unknown sequences were possible candidates for novel bovine genes, and 100 internal references. The expression levels of 78 genes were found to be enhanced exceeding twenty-fold among the follicles. Follicles were classified into two groups (Group 1 and Group 2) by hierarchical cluster analysis. Group 1 follicles were characterized by higher expression of 46 genes including FSHR, CYP19 and inhibin α subunit. Whereas other 32 genes including IGFBP5, TIMP1 and uPA, were predominately expressed in Group 2 follicles. Therefore, follicles in Group 1 and 2 were considered to be growing and atretic follicles, respectively. Expression profiles of representative 19 genes (9 genes highly expressed in Group 1 and 10 genes highly expressed in Group 2) were confirmed by using quantitative RT-PCR. In summary, we analyzed genome-wide transcriptome profiles of dominant follicles in cows by using customized oligonucleotide array. Results in the present study suggested that gene expression profiles of dominant follicles were classified into 2 groups representing the status of follicular development. Expression of stage-specific genes in dominant follicles may be closely associated with its growth or atresia. Several genes identified in this study will provide intriguing candidates for the determinant of follicular growth. This study was supported by JSPS Research Fellowships for Young Scientists.

Nuclear reprogramming by somatic cell nuclear transfer (SCNT) to derive patient-specific embryonic stem cells (ESCs), which is known as therapeutic cloning, remains a very promising medical therapy for many currently incurable human diseases. Normally, SCNT is conducted using fresh unfertilized or fertilized oocytes. A major obstacle for therapeutic cloning to be applied in humans is that donation of fresh human oocytes by volunteer is very limited and controversial, Utilization of leftover cryopreserved oocytes donated with informed consents by infertile couples undergoing in vitro fertilization (IVF) treatment is possible and has minimal ethic concerns. Recent clinical reports showed that cryopreserved oocytes could be successfully used for human fertility treatment. However, it is not clear whether frozen oocytes can be successfully used for cell reprogramming to generate ESCs. In this study, we tested the validity and efficiency of deriving ESCs from cryopreserved oocytes in a mouse model via parthenogenetic activation and SCNT using skin fibroblast cells. Our study showed that after thawing, 97.3 % (579/595) of cryopreserved oocytes were viable as judged by cell membrane integrity. The rates of blastocyst development of the frozen/thawed oocytes were 78.5% (117/149) following parthenogentic activation and 56.3% (108/192) following SCNT. Moreover, we successfully generated 7 ESC lines (7/106; 6.6%) from parthenogenetic blastocysts, and 25 SCNT-ESC lines (25/101; 24.5%) from cloned blastocysts. We selected five cell lines for various ESC in vitro characterization to confirm thier pluripotent status. The derived cell lines expressed genes and antigens characteristic of pluripotent ESC and produced full-term pups upon tetraploid embryo complementation. Overall, our results showed that cryopreserved oocytes could be successfully used for ESC generation alone by parthenogenetic activation or by SCNT reprogramming. Our results present the intriguing implication that unused, leftover cryopreserved oocytes may serve as a source of human oocytes for generating patient-specific ESC lines. Research supported by funding to XCT and XY and NSC 96-2321-B-002-033 to LY.

In general, placentomes develop only on the caruncle and not on other areas in the bovine endometrium, but aberrant development of placentomes has been found in rare cases. To evaluate the meaning of subplacentomes function and developing mechanism, expressed genes in subplacentomes that are usually small in sizes and irregular shapes were examined with microarray. The subplacentomes were collected from three different cows which were ranged from approximately days 100 to 250 of gestation, mainly determined the pregnancy days by the size of craniocaudal axis. Normal size placentomes and subplacentomes were collected and extracted RNA for microarray analysis using a custom-made bovine oligo DNA microarray which involved 11,000 oligo DNA based on bovine utero-placental and liver cDNA sequences. Some endometrial tissues including subplacentomes and normal size placentome were fixed in 10% formaldehyde solution, embedded in paraffin wax and made sections for histological and in situ hybridization analysis. Subplacentomes were smaller but different in sizes with irregular shapes with 0.5 × 1.0–2.0 cm, however histological examinations showed very similar structures of fetal and maternal components; just smaller of septa and trophoblast areas. There were well-developed trophoblast cells, like mononucleate and binucleate cells. The binucleate cells expressed their specific proteins like placental lactogen, Pregnancy-associated glycoproteins (PAGs) and Prolactin-related proteins (PRPs). Microarray showed about 200 genes expressed higher in subplacentomes than those in normal size placentomes. To analyze the difference among the normal size placentomes, intact endometrial tissues and subplacentomal tissues, expression rate between the caruncular (CAR) and cotyledonary (COT) tissues in normal size and in subplacentomes (sCAR, sCOT) were examined, respectively. There were no remarkable changes in specific genes in binucleate cells, like PAGs and PRPs on day 100 of gestation. Some matrix degradation enzymes like MMP-1, -3 and -9 were rather high but MT1-MMP and TIMP-3 were lower in CAR and COT. These data suggest that sCAR and sCOT have similar structures and functions as those in normal size placentomes. Supported by Kiban-kenkyu B (17380172) from JSPS and a grant of Animal Remodeling Project from NIAS. (poster)