More than 99% of follicles undergo a degenerative process known as “atresia”, in mammalian ovaries, and only a few follicles ovulate during ovarian follicular development. We have investigated the molecular mechanism of selective follicular atresia in mammalian ovaries, and have reported that follicular selection dominantly depends on granulosa cell apoptosis. However, we have little knowledge of the molecular mechanisms that control apoptotic cell death in granulosa cells during follicle selection. To date, at least five cell death ligand-receptor systems [tumor necrosis factor (TNF)α and receptors, Fas (also called APO-1/CD95) ligand and receptors, TNF-related apoptosis-inducing ligand (TRAIL; also called APO-2) and receptors, APO-3 ligand and receptors, and PFG-5 ligand and receptors] have been reported in granulosa cells of porcine ovaries. Some cell death ligand-receptor systems have “decoy” receptors, which act as inhibitors of cell death ligand-induced apoptosis in granulosa cells. Moreover, we showed that the porcine granulosa cell is a type II apoptotic cell, which has the mitochondrion-dependent apoptosis-signaling pathway. Briefly, the cell death receptor-mediated apoptosis signaling pathway in granulosa cells has been suggested to be as follows. (1) A cell death ligand binds to the extracellular domain of a cell death receptor, which contains an intracellular death domain (DD). (2) The intracellular DD of the cell death receptor interacts with the DD of the adaptor protein (Fas-associated death domain: FADD) through a homophilic DD interaction. (3) FADD activates an initiator caspase (procaspase-8; also called FLICE), which is a bipartite molecule, containing an N-terminal death effector domain (DED) and a C-terminal DD. (4) Procaspase-8 begins auto-proteolytic cleavage and activation. (5) The auto-activated caspase-8 cleaves Bid protein. (6) The truncated Bid releases cytochrome cfrom mitochondrion. (7) Cytochrome c and ATP-dependent oligimerization of apoptotic protease-activating factor-1 (Apaf-1) allows recruitment of procaspase-9 into the apoptosome complex. Activation of procaspase-9 is mediated by means of a conformational change. (8) The activated caspase-9 cleaves downstream effector caspases (caspase-3). (9) Finally, apoptosis is induced. Recently, we found two intracellular inhibitor proteins [cellular FLICE-like inhibitory protein short form (cFLIP_S) and long form (cFLIP_L)], which were strongly expressed in granulosa cells, and they may act as anti-apoptotic/survival factors. Further in vivo and in vitro studies will elucidate the largely unknown molecular mechanisms, e. g. which cell death ligand-receptor system is the dominant factor controling the granulosa cell apoptosis of selective follicular atresia in mammalian ovaries. If we could elucidate the molecular mechanism of granulosa cell apoptosis (follicular selection), we could accurately diagnose the healthy ovulating follicles and precisely evaluate the oocyte quality. We hope that the mechanism will be clarified and lead to an integrated understanding of the regulation mechanism.

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Gene expressions and their interaction are complex and have not been definitely clarified in the placenta. To identify interactions of gene products previously not studied, we applied cDNA subtraction analyses to the placenta between days 12 and 16, days 12 and 14, days 14 and 16 of pregnancy. Among subtracted cDNAs cathepsin M, Q and R in PECs were specifically identified on days 14 and 16 pregnancy. All of these gene expressions exhibited a similar pattern to the mPL-II gene expression determined by northern blot and RT-PCR analyses. By means of in situ hybridization, these mRNAs were localized in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Double staining studies of cathepsin Q or cathepsin R mRNA by in situ hybridization followed by immunohistochemical staining of mPL-II in the same section revealed that signals for cathepsin Q and cathepsin R mRNAs were colocalized in mPL-II immunopositive trophoblast cells in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Possible association of cathepsins with mPL-II may play important roles in placental functions during the latter half of pregnancy in mice.

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The present study was conducted to clarify the difference in the color of the cytoplasm in immature follicular oocytes from prepubertal and adult minke whales. The four lipid contents (triglyceride, total cholesterol, phospholipids and non-esterified fatty acids) in vitrified immature oocytes from prepubertal and adult minke whales, and also in fresh and vitrified immature porcine oocytes, were measured. The lipid contents in vitrified-warmed minke whale oocytes were similarly high compared with those in vitrified-warmed porcine oocytes. In particular, the total cholesterol and phospholipid contents in the vitrified immature oocytes from prepubertal and adult minke whales were significantly (P<0.05) higher than those from prepubertal pigs. Furthermore, the distribution of lipid droplets in fresh and vitrified immature oocytes was observed in transmission electron microscopy. Lipid droplets in the prepubertal minke whale oocytes were distributed throughout the cytoplasm. In contrast, adult minke whales had larger lipid droplets which were distributed mainly in the central portion of the cytoplasm. The lipid droplets of immature oocytes from prepubertal pigs were larger than those in minke whale oocytes. These results indicated that the difference in the distribution of the cytoplasmic lipid droplets may result in the difference in the color tone of both prepubertal and adult whale oocyte cytoplasm.

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Blastocyst formation in a chemically defined medium (mR1ECM) of rat oocytes soon after sperm penetration is less frequent than in those undergoing male pronuclear formation. This inhibition is released by preculturing the oocytes for a few hours in modified Krebs-Ringer bicarbonate solution (mKRB). The present study examined the effects of phosphate (Pi), bovine serum albumin (BSA) and osmolarity during preculture of sperm penetrated rat oocytes on their development to blastocysts in mR1ECM in vitro. These are the major factors that differ between mR1ECM and mKRB. When oocytes collected at 0730-0800 h on the day following mating and freed from cumulus cells were precultured for 5 h in mKRB or Pi-free mKRB and then cultured for 127 h in mR1ECM, about 73-74% of oocytes developed to blastocysts. In both media, replacement of BSA with polyvinylalcohol (PVA) or osmolarity of 246 mOsM reduced blastocyst formation compared with media containing BSA or with osmolarity of 304 mOsM; blastocyst formation was greatly inhibited when oocytes were precultured in media with PVA and osmolarity of 246 mOsM. On the other hand, when precultured in mR1ECM or mR1ECM with osmolarity of 304 mOsM or BSA instead of PVA, fewer oocytes developed to blastocysts than those precultured in Pi-free mKRB and mR1ECM with osmolarity of 304 mOsM and BSA. These results indicate that both BSA and osmolarity, but not Pi, are essential factors during preculture of rat oocytes soon after sperm penetration for promoting their further development to blastocysts in a chemically defined medium.

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We previously found that bovine oocytes 90-99 μm in diameter in early antral follicles grew to nearly their final size in serum-free medium, with some of the oocytes acquiring the nuclear competence to reach the second metaphase. In the present study, we examined the competence of the fertilization and pre-implantational development of the oocytes grown in serum-free medium. Bovine early antral follicles, 0.4-0.7 mm in diameter, were collected mechanically using fine forceps, embedded in collagen gels, and cultured in serum-free medium for 16 days. Grown oocytes which were enclosed by granulosa cells and did not show disintegrated ooplasm were recovered as normal oocytes, were transferred to the maturation medium, and then inseminated with spermatozoa. Ten to 12 h after insemination, 28% (41/145) of the oocytes were penetrated by spermatozoa. Of the penetrated oocytes, 18 (12%) formed a female and a male pronuclei, and 10 (7%) had a female pronucleus and an enlarged sperm head. Among the abnormally penetrated oocytes (13/41), 10 were penetrated by multiple spermatozoa and 3 were penetrated by a spermatozoon at the first metaphase stage. Of the 106 inseminated oocytes grown under serum-free conditions, 8 oocytes had cleaved and developed to the 2-cell stage 48 h after insemination, and 3-4-cell embryos and 5-8-cell embryos were observed after 72-96 h. However, no embryo developed to the blastocyst stage within 8 days. These results indicate that bovine oocytes grown in serum-free medium can be fertilized, but acquire insufficient embryonic development competence under the employed culture conditions.

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To reveal the molecular regulation mechanism of selective follicular atresia in porcine ovaries, we isolated the porcine cDNA encoding cellular FLICE-like inhibitory protein (cFLIP), which inhibits death receptor-mediated apoptosis signal transduction. Two alternative splicing isoforms of cFLIP, porcine cellular FLIP-short form (pcFLIP_S, 642 bp and 214-aa) and -long form (pcFLIP_L, 1446 bp and 482-aa), were identified from a cDNA library prepared from follicular granulosa cells of pig ovaries. pcFLIP_S and pcFLIP_L indicated high identities with human and murine cFLIP, and both of them contain two tandem specific amino acid regions (death effector domain: DED) in their N-terminal, suggesting that pcFLIP_S and pcFLIP_L inhibit the death receptor-mediated apoptosis signal by binding to other pro-apoptotic factors mediated by DED. pcFLIP_S contains a short C-terminal region, while pcFLIP_L has a caspase-like domain in the C-terminal region. The reverse transcription-polymerase chain reaction analysis revealed that both pcFLIP_S and pcFLIP_L mRNAs were highly expressed in granulosa cells of healthy follicles, suggesting that these cFLIPs play important roles in the regulation mechanism of apoptosis in ovarian follicular granulosa cells. The present data will contribute to understanding of the physiological roles of cFLIPs in the apoptosis regulation in porcine tissues.

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The LH-producing cell line, LβT2, and non LH-producing cell line, αT3-1 cells, established from a pituitary tumor, were employed for cDNA subtraction cloning to identify genes with expression unique to LH producing cells. Several cDNAs that code for known as well as for many unidentified clones were discovered. Most clones were the spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1, the abnormality of which causes neurodegeneration and loss of cerebellar Purkinje cells. We examined whether the expression of SCA1 gene in LβT2 cells is related to hormone production. We also compared the expression of SCA1 with that in various other pituitary tumor derived cell lines, and confirmed the prominent expression of SCA1 in LβT2 cells. The effect of gonadal factor(s) for SCA1 gene expression was examined. The expression level in female rats was low and did not change during the estrus cycle, but increased significantly after ovariectomy and did not return to the normal level under low and high doses of estrogen. In the male pituitary SCA1 gene expression increased markedly after castration and was not decreased by estrogen or testosterone. The Ontogeny of SCA1 gene expression was investigated in porcine fetal and postnatal pituitaries and reveaed biphasic and sexually dimorphic expression. Transient expression of SCA1 gene was observed at fetal day 50 and 65 in males and day 40 in females, followed by a decline and increased expression before birth in both genders. Thus the expression of SCA1 gene is prominent in LH-producing cells and is not under direct control of gonadal factor(s) in both genders. In addition to the variable expression of SCA1 gene during the fetus period, the present results provide a novel aspect to the understanding of Boucher-Neuhauser syndrome (Ataxia Hypogonadism Choroidal Dystrophy).

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The Prnp (prion protein) gene, which encodes a soluble protein anchored to the cell surface by glycosylphosphatidylinositol (GPI), might be involved in cell-to-cell interaction. The expression of Prnp is strongly observed not only in the brain, but also in non-neuronal tissues. In order to examine the Prnp expression sites in mouse testes, we carried out Northern blot and in situ hybridization analyses. By Northern blot analysis, two kinds of Prnp transcripts (major band of 2.2 kb, and minor band of 1.1 kb) were detected in testes. The 2.2-kb transcript was observed in testes throughout the postnatal development, whereas the 1.1-kb transcript was observed in testes from 2 to 70 weeks old. In situ hybridization analysis showed that the positive signals for Prnp mRNAs were predominantly observed in spermatogenic cells, but not in somatic cells such as Sertoli cells, Leydig cells and peritubular myoid cells. The signals were observed moderately in spermatogonia, and strongly in spermatocytes and round spermatids, but not in elongate spermatids and spermatozoa. These results suggest that Prnp may be involved in germ cell differentiation during mammalian spermatogenesis.

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To elucidate synthesis, processing, and subcellular localization of mouse ADAM3 (cyritestin) during spermatogenesis and epididymal sperm transport, we carried out immunoblotting and immunohistochemical analysis of testicular germ cells, and epididymal and vas deferens sperm, using affinity-purified anti-ADAM3 antibody. ADAM3 was initially synthesized as a 110-kDa precursor in round spermatids, and the precursor was then processed into a 42-kDa mature protein during the sperm transport into and/or once in the epididymis. The mature ADAM3 was localized on the anterior part of capacitated sperm heads and was rapidly removed from the head region during the calcium ionophore A23187-induced acrosome reaction. These results demonstrate that the mature form of ADAM3 is involved in the binding of sperm to the egg zona pellucida, not in the membrane fusion between sperm and egg.

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To develop a reliable follicle culture system, mouse preantral follicles 150-200 μm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro.

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The effect of glucose supplementation at different times in in vitro culture on the developmental competence of in vitro produced (IVP) porcine embryos was examined. In Experiment 1, when IVP embryos were cultured in modified NCSU-37 supplemented with pyruvate and lactate (IVC-pyr/lac) for 0 h, 24 h, 48 h, 72 h, 96 h, or 118 h and subsequently in modified NCSU-37 supplemented with glucose (IVC-glu) until Day 6 (Day 0=day of in vitro fertilization), the rates of blastocyst formation were significantly higher in embryos cultured in IVC-pyr/lac for 24 or 48 h (24.4% and 23.0%, respectively) than in embryos cultured in IVC-pyr/lac for the whole culture period (14.5%). However, there were no significant differences between embryos obtained after the energy source replacement and embryos cultured in IVC-glu for the whole culture period on the rates (15.2%-24.4%, and 16.8% respectively). Replacement of pyruvate/lactate with glucose at 58 h of culture in Experiment 2 significantly enhanced the rate (31.3%) compared to those after replacement at 48 h, 53 h and 63 h of culture (20.6%, 20.8%, and 21.1%, respectively). In conclusion, replacement of pyruvate/lactate with glucose as the energy substrate was optimal at 58 h of culture for the development of porcine embryos to the blastocyst stage.

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The effect of developmental stage on the survival of bovine somatic cell nuclear-transferred blastocysts after freezing and thawing was evaluated. We also investigated how freezing affects nuclear-transferred (NT) embryos and in vitro fertilized (IVF) bovine embryos. Advanced-stage bovine NT blastocysts survived freezing better than early-stage NT blastocysts (86 vs 14%). The trend was similar with IVF embryos (87 vs 30%). At the stages tested, there was no significant difference in the survivability of NT and IVF embryos from advanced (86 vs 87%) or early-stage blastocysts (14 vs 30%). The average survival rate did not differ between NT and IVF bovine embryos (50 vs 51%). The higher survival rate of advanced-stage blastocysts compared to early-stage blastocysts in NT and IVF bovine embryos might be due to their higher cell number. In NT (128 ± 25 vs 53 ± 20) and IVF (128 ± 29 vs 75 ± 22) groups, advanced-stage blastocysts contained a significantly higher total cell number than early-stage blastocysts. There was no difference in total cell number between advanced-stage NT and IVF blastocysts (128 ± 25 vs 128 ± 29), however, early-stage NT and IVF blastocysts (53 ± 20 vs 75 ± 22) differed significantly.

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Gene silencing using small interfering RNA (siRNA) may be useful for functional analyses of unidentified genes expressed during cell differentiation. The present study was performed to evaluate RNA interference (RNAi) in porcine granulosa cells stimulated with bovine FSH, by using two fluorescence reporter genes: a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescence microscopy and analyzed by flow cytometry. Strong fluorescence was observed after introduction of both plasmids into cells. The intensity of green fluorescence generated by GFP was greatly suppressed by introduction of GFP-siRNA, showing an approximate 70% decrease in the ratio of green to red fluorescence. Consequently, we concluded that gene silencing by siRNA can be used to analyze the functions of genes of interest during differentiation of porcine granulosa cells.

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