In the series of studies, changes of expression and regulation of luteinizing hormone (LH) receptor in the ovary of domestic ruminants were examined. Furthermore, mechanisms of formation of follicular cysts in domestic ruminants, caused by stress and so on, were endocrinologically elucidated. Results of the studies provide the following conclusions. (1) The quantity of LH receptor in the bovine antral follicles increases rapidly in the latter stage of its development. (2) The quantity of LH receptor and its mRNA in the bovine and caprine corpus luteum increase during their developments. The increase of the receptor in the caprine luteal development is regulated by LH through the receptor mRNA level. (3) At least, three splice variants of LH receptor mRNA exist in the bovine luteal tissue and the variant receptors are expressed at different cellular sites according to its structure. (4) Intracellular consecutive cysteine residues of LH receptor are palmitoylated and thereby inhibit internalization of the receptor. (5) As a mechanism of the bovine follicular cyst caused by stress, it is suggested that increased secretions of progesterone and cortisol from the adrenal gland exert inhibitory effects on the hypothalamus and follicle, respectively, and subsequently LH and FSH surges are blocked, then finally ovulation is suppressed and the follicle becomes cystic.

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In order to clarify the functional relationship between thyroid, adrenal and gonadal hormones, hypothyroidism was induced by administration of thiuoracil in adult male and female rats, and the effects of hypothyroidism on the adrenal and the gonadal axes were investigated in the present study. 1. The functional relationship between thyroid and adrenal hormones: Adrenal weights and corticosterone were lowered, whereas the secretion of ACTH, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) increased in hypothyroid rats compared to euthyroid rats. These results indicate that hypothyroidism causes adrenal dysfunction directly and results in hypersecretion of CRH and AVP from the hypothalamus. 2. The functional relationship between thyroid and gonadal hormones: The pituitary response to LHRH was lowered, whereas the testicular response to hCG was not changed in hypothyroid rats. Hypothyroidism suppressed copulatory behavior in male rats. These results suggest that hypothyroidism probably causes dysfunction in gonadal axis at the hypothalamic-pituitary level in male rats. In adult female rats, hypothyoidism inhibited the follicular development accompanied estradiol secretion, whereas plasma concentrations of progesterone and prolactin (PRL) increased in hypothyroid female rats. Hypothyroidism significantly increased the pituitary content of vasoactive intestinal peptide (VIP) though it did not affect dopamine synthesis. These results suggest that hypothyroidism increases pituitary content of VIP and this increased level of VIP likely affects PRL secretion in a paracrine or autocrine manner. In female rats, inhibition of gonadal function in hypothyroid rats mediated by hyperprolactinemia in addition to hypersecretion of endogenous CRH.

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The establishment of in vitro embryo production (IVP) system in pigs enables us to generate viable embryos with a quality equal to that of in vivo derived embryos. This technology contributes not only to developments in reproductive physiology and agriculture but also to biotechnologies for producing cloned or genetically modified pigs. The birth of piglets from in vitro matured and fertilized embryos at the two- to 4-cell stage was first achieved about 10 years ago, but it was only quite recently that piglets were produced after the transfer of IVP blastocysts. This improvement to the blastocyst stage of the in vitro culture system after in vitro maturation and fertilization can be expected to play a part in the development of an advanced IVP system. Here, we discuss the developmental ability of porcine embryos produced by our improved IVP system and the utilization of this technique in the new biotechnology.

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My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.

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Tumor necrosis factor-α (TNFα) is a multifunctional cytokine that was first described as a tumoricidal factor produced by activated macrophages. Extensive research over the last two decades has suggested that TNFα has physiologically diverse actions in ovarian function in a variety of species. TNFα and its specific receptors are present in the ovaries of many species. Furthermore, TNFα plays multiple and probably important roles in corpus luteum (CL) function as well as ovarian cell function throughout the estrous cycle. This review focuses on recent studies documenting TNFα in ovarian follicles and CL in several mammals. In addition, possible roles of TNFα in ovarian function throughout the estrous cycle and in the gestation period are discussed.

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A number of endocrine and paracrine factors regulate the follicular growth and atresia, which are closely associated with granulosa cell survival and apoptosis. However, the molecular mechanisms underlying the intracellular events induced by these factors are poorly understood. Here, we describe the correlation of mitogen-activated protein kinase (MAPK) activities with granulosa cell survival and apoptosis, and the cellular functions of protein tyrosine phosphatases (PTPs) in these cells based on our recent data. MAPKs play key roles in various cellular responses because numerous extracellular stimuli are integrated into MAPKs. The protein phospho-Tyr level regulated by protein tyrosine kinases (PTKs) and PTPs is a major control mechanism for processes as diverse as cell survival, proliferation, differentiation, and metabolism. Although PTKs are critically involved in granulosa cell survival and proliferation, there are no reports indicating the roles of PTPs in the ovary except for ours. Information about MAPKs and PTPs in these cells will provide a basis for the understanding of the molecular mechanisms controlling the fate of follicles.

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Active contractile pattern of the oviduct occurs during the periovulatory period for the movement of the gamete/embryo, which is strictly regulated by endocrine and paracrine/autocrine factors. In this review, an involvement of tumor necrosis factor α (TNFα) in the regulation of cow oviductal contraction is discussed. Oviductal epithelial cells express TNFα ligand and it's both receptor types; high expression during the follicular and postovulatory stages, while low expression during luteal stage and thus, TNFα system in the cow oviduct is most active during the periovularoty period. The immune cells present in large numbers in the oviduct during the periovulatory period of the estrus cycle, and these cells are also considered as another potential source for the TNFα in the oviduct. Using in vitro models, TNFα clearly stimulated local production and release of contraction related substances such as prostaglandins (PGs), endothelin-1 (ET-1) and angiotensin II (Ang II). Since these substances have been shown to activate directly the oviductal contraction in vitro, TNFα appears to stimulate the oviductal contraction during the periovulatory period and contribute to create an optimal local environment suitable for gamete/embryo transport. In addition, the ability of embryo to act as a source of TNFα in the oviduct cannot be excluded. To support this idea, the embryo at 2-4 cells stages indeed express TNFα, so that the minute quantities of TNFα secreted by the embryo may further acts locally to enhance the production of PG, ET-1 and Ang II in the oviduct, which may result in an active oviductal contraction in the microenvironment around the embryo. This may ensure the embryo to migrate into the uterus at the optimal time.

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The aim of the present study was to compare three methods of estrus synchronization in ewes during the non-breeding season. Forty-two ewes were randomly grouped for three treatments with different intravaginal devices for 12 days: Group A) CIDR, Group B) Self-made P sponge, Group C) MAP (medroxyprogesterone acetate) cream sponge. Furthermore, all groups were divided into two treatments with (R) or without ram presence to examine the "ram effect". Blood was collected from all treated ewes, and progesterone (P₄), estradiol 17-β (E₂) and luteinizing hormone (LH) concentrations were measured by enzyme-immnoassay. All ewes showed estrus behavior between Day 0 to 3 after device removal, and the mean onset times of their estrus were 23.0, 33.0 and 21.0 h for Groups AR, BR and CR, respectively. On Day 5 as examined by laparoscopy, the ovulation rates (and number of ovulated ewes) were 1.45 (11/11), 1.25 (12/14) and 1.21 (14/14) for Groups A, B and C, respectively. In Group C, the time to LH surge was significantly (P<0.05) later (32.4 h) than those in Groups A (27.0 h) and B (25.5 h). Ram presence did not affect the number of ovulated ewes, ovulation rate or time to LH surge. The ram introduction group had significantly (P<0.05) lower E₂ concentrations during the period from 0 h to 36 h than the groups without ram presence. These results suggest that the self-made P sponge or MAP cream sponge was effective as well as CIDR, and ram introduction was not necessary, for induction of estrus and ovulation during the non-breeding season.

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The objective was to determine the effect of glucose supplementation on development (to the blastocyst stage) of in vitro matured (IVM) porcine oocytes that were either in vitro fertilized (IVF) or electrically activated (EA). Embryos were incubated for 46 or 58 h post insemination (hpi) in an NCSU37-based medium containing 0.17 mM sodium pyruvate and 2.73 mM sodium lactate (IVC-PyrLac), and then transferred to an NCSU37-based medium containing 5.55 mM glucose (IVC-Glu) and cultured until Days 6 (Day 0 = day of EA or IVF). The proportions of oocytes that had formed full blastocysts by Day 6 following transfer to IVC-glu at 46 hpi was 23.5 and 41.2% in the IVF and EA groups respectively; these were lower (P<0.001) than the proportions of oocytes that formed full blastocysts after transfer at 58 hpi (60.3 and 78.7%). However, there was no significant difference in total cell number (at Day 6) between embryos transferred at 46 vs 58 hpi. We inferred that in vitro-derived pig embryos can efficiently use glucose as an energy source starting at approximately 58 hpi; exposure to glucose at that time enhanced development to the blastocyst stage as well as blastocyst quality.

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The present study examined the effects of pre-treatment of pig oocytes with different concentrations (0-50 μM) of calcium ionophore A23187 (CaA) on their activation, development and penetration in vitro. Although untreated oocytes were not activated and did not cleave in culture, high proportions of treated oocytes did so and 20% of oocytes developed to the blastocyst stage when treated with 6.25 μM CaA for 2 min. However, these proportions were reduced in a concentration-dependent manner. When inseminated in vitro with 1 × 10⁶ spermatozoa/ml, the penetration rate of oocytes treated with 6.25 μM CaA was similar to that of untreated oocytes. However, fewer oocytes treated with 12.5 and 50 μM CaA were penetrated than untreated oocytes. On the other hand, the proportion of monospermy of oocytes treated with 6.25 μM CaA was higher than the values in oocytes not treated or treated with 50 μM CaA. The time required for zona dissolution of oocytes treated with 6.25 and 12.5 μM CaA was not different from that in untreated oocytes, but oocytes treated with 50μM CaA required a longer time than untreated oocytes, indicating that zona solubility by protease does not reflect penetrability of oocytes in vitro. When oocytes were inseminated with different concentrations (1-10 × 10⁶ cells/ml) of spermatozoa, the highest penetration rate was observed at 1 × 10⁶ cells/ml in untreated oocytes and a similar result was obtained in oocytes treated with 6.25 μM CaA. There was no difference in the rate of monospermy in untreated oocytes among different concentrations of spermatozoa, but in treated oocytes, higher proportions of monospermy were observed at 0.5-5 × 10⁶ than 10 × 10⁶ cells/ml. At 1 × 10⁶ cells/ml, the proportion of monospermy was higher in treated than untreated oocytes. These results suggest that pre-treatment of pig oocytes with 6.25 μM CaA, an appropriate concentration, inhibits polyspermic penetration in vitro when insemination occurs with spermatozoa at a concentration of 1 × 10⁶ cells/ml.

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Although the biological role for whey acidic protein (WAP) in milk has been suggested, its true function is not known. This paper describes evidence for WAP function in the cell-cycle progression of EpH4/K6 (EpH4), mammary epithelial cells in vitro. The forced expression of exogenous WAP significantly impaired the proliferation of EpH4 cells, whereas it did not affect that of NIH3T3 cells. Apoptosis was not enhanced in the EpH4 cells with stable expression of WAP (WAP-clonal EpH4 cells). The analyses of BrdU incorporation revealed that forced WAP expression significantly reduced incorporation of BrdU in WAP-clonal EpH4 cells compared with control cells transfected with empty plasmid. Among G1 cyclins, the level expression of cyclins D1 was significantly lower in the WAP-clonal EpH4 cells than in control cells. The inhibitory action of WAP on the proliferation of EpH4 cells was enhanced by the presence of extracellular matrix (ECM), but not by the presence of a single component comprising ECM. The cultured medium of WAP-clonal EpH4 cells inhibited the proliferation of control cells without WAP expression. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary epithelial cells through an autocrine/paracrine mechanism.

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γ-Tubulin, a member of the tubulin superfamily, is a peri-centriolar component which is considered to be essential for microtubule nucleation. The dynamics of γ-tubulin during mouse oocyte meiotic maturation, fertilization, and early cleavage as well as the co-localization of γ-tubulin and α-tubulin during the formation of the meiotic I spindle were studied by confocal microscopy. We found that γ-tubulin was evenly distributed in the germinal vesicle (GV) stage oocyte. After germinal vesicle breakdown (GVBD) γ-tubulin dots were localized in both the cytoplasm and the vicinity of the condensed chromosomes, and aligned at both poles of the meiotic spindle at prometaphase I and metaphase I. At anaphase I and telophase I, γ-tubulin was detected between the separating chromosomes, while it was absent in the midbody. At the MII stage, γ-tubulin was again accumulated at the spindle poles. α-Tubulin had a similar distribution pattern as γ-tubulin in the cytoplasm and radiated from γ-tubulin foci close to the chromosomes during the meiotic spindle formation. After fertilization, γ-tubulin was translocated from spindle poles to the area between separating chromatids and distributed around the pronuclei. It aggregated into some dots during the interphase, but was distributed on the mitotic spindle poles in early embryos. Our results suggest that γ-tubulin is essential for microtubule nucleation and spindle formation during mouse oocyte meiosis, fertilization, and early embryo cleavage.

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The effects of adding cysteamine, EGF, and glucose as an energy substrate under low oxygen tension during in vitro maturation (IVM) were examined to find ways of improving the individual in vitro production (IVP) system in individually cultured bovine oocytes. The basic medium was mSOFaa containing 1 mg/ml polyvinyl alcohol. Immature oocytes were individually cultured in an IVM medium with 10 ng/ml EGF, 100 μM cysteamine, or EGF plus cysteamine under 20% or 5% O₂. Cleavage and blastocyst rates were significantly higher (P<0.05) in IVM culture was under 20% O₂ than in culture under 5% O₂. Under 5% O₂, neither EGF nor cysteamine improved embryonic development. The proportion of matured oocytes was significantly higher (P<0.05) in the presence of 1.5 mM glucose under 20% O₂ (68.6%), and 5.5 mM (66.7%) and 10 mM (65.5%) glucose under 5% O₂. The presence of 5.5 mM glucose significantly (P<0.05) increased the maturation rate compared with the absence of glucose, irrespective of addition of EGF and cysteamine. The addition of cysteamine alone in the maturation medium significantly (P<0.05) increased the intracellular GSH concentration in the oocytes. Also, under 5% O₂ cysteamine and/or EGF significantly (P<0.05) improved the proportions of penetrated oocytes, cleavage and blastocyst formation, which were similar levels to those of oocytes matured under 20% O₂. After vitrification, the re-expanding and hatching rates of blastocysts derived from the individual IVP system containing cysteamine under 5% O₂ were significantly (P<0.05) higher than those of blastocysts derived from the individual IVP system without cysteamine under 5% O₂ and the group IVP system under 20% O₂. The present study showed that a high glucose level (5.5 or 10 mM) was optimal in IVM culture under low (5%) oxygen tension. The addition of EGF and/or cysteamine to the maturation medium had no positive effect on nuclear maturation, but improved fertilizability, developmental competence and cryoresistance following vitrification, probably due to increased GSH synthesis during the IVM process.

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Gastrin-releasing peptide (GRP) has been suggested as a novel regulatory peptide in the female reproductive tract but the presence of GRP and GRP mRNA in the non-neurogenic tissue of the cervix has not yet been clarified. In the present study, immunohistochemistry and in situ hybridization were used to reveal the distribution of GRP immunoreactivity and expression of GRP mRNA in the bovine cervix. The cervixes from 21 non-pregnant and 20 pregnant cows, and 6 fetuses were used in the study. In the fetus, adult non-pregnant and pregnant specimens, GRP and GRP mRNA were predominantly detected in the luminal epithelial cells of basal areas of peripheral regions of the cervix. Positive staining of GRP in the epithelial cells of the cervix was first detected in the CRL 37 cm of the fetus. During the estrous cycles, the staining intensity of GRP in the epithelial cells was stronger in the follicular phase than in the luteal phase. During the early gestational period, GRP immunoreactivity was detected at relatively similar intensity to the follicular phase. In situ hybridization results ascertained the expression of GRP mRNA in the superficial epithelial cells of the cervix of non-pregnant and pregnant cows. The results suggest that GRP may be important both in the development of the fetal cervix and secretory activity of the epithelial cells of the cervix.

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In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.

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The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.

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The object of the present study was to investigate the validation of the sperm quality analyzer (SQA) and the hypo-osmotic swelling (HOS) test with standard sperm analysis methods in frozen-thawed ram and minke whale spermatozoa. In rams, highly significant correlations were observed in the percentage of motile spermatozoa (P<0.01) and sperm concentration (P<0.01) between the standard and SQA methods. But, the percentage of morphologically normal spermatozoa did not significantly correlate between the standard and SQA methods. The percentages of swollen spermatozoa at 15 minutes by the HOS test were significantly correlated with the motility by the standard (P<0.05) and by the SQA (P<0.05) methods. For minke whale spermatozoa, the SVI (sperm viability index) values by the standard method were significantly (P<0.001) correlated with the sperm motility index (SMI) values by SQA. The percentage of motile spermatozoa was also significantly correlated (P<0.01) with the motility measured by SQA. Using different hypo-osmotic solutions and incubation times, the HOS test with 25, 100 and 150 mOsM did not show significant variations. Motility observed by the standard method and the percentage of swollen spermatozoa were significantly correlated (P<0.05). These results indicate that the SQA and HOS test can be utilized to assess the post-thawing motility of ram and minke whale spermatozoa, and that the SQA and HOS test values are significantly correlated in ram spermatozoa. However, sperm concentration and morphologically normal spermatozoa are not assessed accurately by SQA in minke whales.

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