Rat ovarian genes induced by the treatment of immature rats with pregnant mare serum gonadotropin (PMSG) were isolated by a subtraction cloning method. Amongst them was obtained a probable rat homologue of steroidogenic acute regulatory protein (StAR), which has been recently identified as a protein that is an acute regulator of the rate limiting transfer of cholesterol from the outer to the inner mitochondrial membrane. Structure of rat StAR was determined by nucleotide sequence analysis. Northern blot analysis revealed that StAR mRNA levels were rapidly and strongly increased by PMSG/hCG. In situ hybridization revealed that the expression of StAR mRNA was strongly induced by PMSG in theca interna cells as well as in corpora lutea. These findings indicate that expression of StAR mRNA is restricted to and induced in the ovarian steroidogenic cell types where cholesterol is used as a substrate for synthesis of steroid hormones. To study the mechanisms of regulation of StAR in rat granulosa cells, we used granulosa cells obtained from diethylstilbestrol-treated immature rats. Northern blot analysis revealed two major transcripts of about 3.6 kb and 1.6 kb of rat StAR mRNA. Rat StAR mRNA had strongly increased within 2 h due to the treatment of FSH or 8-Br-cAMP in this culture, a parallel increase of transcripts of both sizes was observed. Compared to the control, StAR mRNA levels increased in a dose-dependent manner in the presence of increasing concentrations of FSH (1-100 ng/ml) and 8-Br-cAMP (0.25-5 mM). Although co-treatment of rat granulosa cells with FSH and TGF-β did not change FSH-induced StAR mRNA levels, these levels in granulosa cells were markedly increased by pretreatment with TGF-β before being acutely (2 h) stimulated with an effective dose of FSH. The stimulatory effect of TGF-β was concentration-dependent (1-30 ng/ml).

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Androgen, largely through its aromatized estrogen, plays an important role in sexual differentiation of the rat brain during the perinatal period. To demonstrate the regulation of estrogen receptors α (ERα), and β (ER β), androgen receptor (AR), and aromatase (AROM) gene expression, the mRNA levels of these mRNAs were determined in the hypothalamus of male and female rats at 2, 6, and 10 days of age using the competitive RT-PCR method. ERα mRNA level did not differ between males and females, except that gene expression on day 6 was lower in males than in females. There were no significant differences in gene expressions of ERβ and AR in the hypothalamus between males and females throughout the neonatal period. AROM mRNA levels were higher on males for all 3 days of determination. Subcutaneous estrogen treatment at 2 days of age decreased ERα mRNA levels, while it increased ERβ, AR and AROM gene expressions. Our results suggest that sexual differences and steroid-dependent regulation of these genes might be important in the sexual differentiation of the rat brain.

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To investigate the effects of vitamin E on the in vitro development of mouse embryos from 1-cell to blastocysts, embryos were cultured in two media, Brinster's BMOCIII and BMOCIII supplemented with 100 μM vitamin E (BMOCIIIvitE). The incorporation and oxidation of glucose were also compared between the embryos cultured in these media. Both the number of embryos that developed to the 1-cell stage and to the blastocyst stage were much larger for BMOCIIIvitE than BMOCIII. No significant differences in the incorporation of ¹⁴C-glucose or in the oxidation of ¹⁴C-glucose were found between the 2-cell embryos incubated in BMOCIIIvitE and BMOCIII. On the other hand, the incorporation and oxidation rates of ¹⁴C-glucose were significantly higher (p<0.05) in the embryos cultured in BMOCIIIvitE compared with those in BMOCIII at the blastocyst stage. These findings indicate that vitamin E has a beneficial effect on embryo development in mice, perhaps through protecting the cells from oxygen radicals in vitro.

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Parthenogenetically activated mouse oocytes, induced by 7% ethanol for 5 min at room temperature then treated with a low (0.5 μg/ml) or a high (5 μg/ml) concentration of cytochalasin B (CCB), developed into different types of eggs in vitro. Pronuclei formations were observed 6 h after the commencement of ethanol treatment, and 81% of the eggs showed an immediate cleavage with a pronucleus in each blastomere at 0.5 μg/ml CCB, while at a higher concentration of 5 μg/ml CCB, 90% of the eggs showed two-pronuclei in a single cell. Without CCB treatment, single-pronuclei with extruded second polar bodies were mainly seen in the majority (63%) of the eggs. Observations of both actin filaments and nuclei by confocal laser scanning microscopy showed that no spindle rotation prior to extrusion of the second polar body resulted in immediate cleavage in the presence of a low concentration of CCB. Therefore the mechanism of spindle rotation strongly depends on actin filaments.

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We have previously demonstrated that parthenogenetic activation of mouse oocytes can be induced by exposure to 100 μM progesterone for 6 h, and activated eggs developed to the blastocyst stage. Here we examined the pre- and post-implantation development of these parthenogenones in mice, and whether they could be rescued and develop to term by forming aggregation chimeras with in vivo-derived embryos or triploids. When these parthenogenetic embryos were diploidized by 5 μg/ml cytochalasin B, 23% of them developed to the blastocyst stage, and the implantation rate was 51% after transfer to recipients. Live parthenogenetic fetuses were also found on day 10.5 of gestation, but parthenogenones could not survive beyond day 10.5 of gestation. To rescue these lethal parthenogenetic embryos, aggregation chimeras of parthenogenones and in vivo-derived embryos were made at the 4-cell stage. Sixty six percent of chimeric embryos which developed to single blastocysts were transferred to 8 recipients. Two of them delivered a total of nine live pups, and one of them was chimeric. In the present study we confirmed that parthenogenetic mouse embryos, even when activated by progesterone, developed to the mid-gestation stage, and could be rescued by forming chimeras consisted of parthenogenetic and diploid embryos. On the other hand, aggregation chimeras derived from parthenogenetic and triploid embryos were lethal after implantation.

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Active angiogenesis occurs during early luteal development. Angiogenesis in the corpus luteum (CL) is regulated by various growth factors in many species. The purpose of the present study was to determine the effect of hepatocyte growth factor (HGF) on the proliferation of the microvascular endothelial cells derived from developing bovine CL. The expression of HGF and HGF receptor (c-met) mRNAs in cultured bovine endothelial cells was also observed by reverse transcription-polymerase chain reaction analysis. The cells were exposed to HGF (50 ng/ml) for 1, 2, 4, and 6 days. HGF significantly increased the total DNA in endothelial cells at all exposure times (P<0.05). When the endothelial cells were exposed to HGF (1-50 ng/ml) for 4 days, total DNA was increased in a dose-dependent manner (P<0.05). Moreover, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) also increased the total DNA. However, when the endothelial cells were simultaneously exposed to HGF (50 ng/ml) with bFGF (50 ng/ml) or VEGF (50 ng/ml), the effect of HGF was not augmented. Furthermore, the mRNA expressions of both HGF and c-met were determined in the endothelial cells. The overall results suggest that HGF is involved in luteal angiogenesis by stimulating proliferation of endothelial cells in cattle.

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The present study was undertaken to examine the effect of methyl-β-cyclodextrin (MBCD) and fertilization promoting peptide (FPP; pGlu-Glu-ProNH₂) on the capacitation and acrosome reaction of boar spermatozoa in a protein-free medium. After washing with a protein-free medium, ejaculated spermatozoa (resuspended at 1 × 10⁶ cells/ml) were cultured in 2 ml of modified Medium-199 containing polyvinylalcohol (mM199-PVA) supplemented or not supplemented with MBCD and/or FPP for 2 h in an atmosphere of 5% CO₂ in air at 39 C. Effects of MBCD and FPP on boar spermatozoa were determined by chlortetracycline fluorescence assessment. When spermatozoa were cultured in various MBCD concentrations, capacitation was induced in a concentration-dependent manner, but spontaneous acrosome reaction was not affected. When the effect of FPP in mM199-PVA was determined, sperm capacitation was induced and spontaneous acrosome reaction was inhibited in 100 nM FPP, regardless of the absence or presence of BSA. In the last experiment, spermatozoa were cultured in mM199-PVA supplemented with various concentrations of MBCD and/or FPP. An accelerative effect of MBCD on sperm capacitation was observed in a concentration-dependent manner even in the presence of FPP, whereas 1-2 mM of MBCD neutralized the stimulatory effect of FPP on capacitation. Furthermore, FPP inhibited spontaneous acrosome reaction even in the presence of MBCD. These results demonstrate that MBCD and PFF can induce capacitation of boar spermatozoa in a protein-free medium and that FPP alone also inhibits spontaneous acrosome reaction.

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Uterine quiescence and activity during pregnancy are regulated by local generation and action of prostaglandins. In this study, we measured the activity of phospholipase A₂, that is responsible for release of their precursor arachidonic acid, in the rat uterus and cervix throughout gestation and at parturition. Phospholipase A₂ activities of both uterine and cervical cytosol were relatively low until day 15 of pregnancy, showed highest values from days 21 to 23 (the delivering day) of pregnancy, and declined by day 2 postpartum. Prostaglandin F_2a levels in both tissues showed similar fluctuations with greater amplitudes to those of phospholipase A₂ activity during gestation. The enhanced enzyme activity on the day of delivery was mostly suppressed by the specific inhibitor for cytosolic phospholipase A₂. Immunohistochemical examination revealed cytosolic phospholipase A₂ localization in uterine endometrial epithelial cells, longitudial myometrium, and cervical connective tissue. Obtained data show the presence of cytosolic phospholipase A₂ in rat uterine myometrium and cervix and its activity was regulated during pregnancy. They also suggest that the enhanced activity of this enzyme may contribute to muscle contraction and cervical dilatation at parturition through local prostaglandin synthesis.

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The present study was undertaken to develop a novel, practical and simple procedure for enzyme immunoassay of plasma estrone sulfate (E₁S) in cows. Diluted plasma was applied directly to wells without extraction and incubated with anti-estrone antibody and horseradish peroxidase-labeled estrone. The sensitivity of the assay was estimated as 40 pg/ml. The intra-assay and inter-assay coefficients of variation were 7.8-9.6% and 7.2-14.4%, respectively. When 2, 5, 10 and 20 ng of E₁S were added to 1 ml of plasma, the recovery rates ranged between 84.6 and 98.5%. Only 3 hr were needed to complete an assay to measure E₁S concentration. Plasma concentrations of E₁S were measured in six crossbred cows after the first trimester of pregnancy. A dramatic increase in the plasma concentrations of E₁S was observed from days 240 to 284 of gestation (7.40-13.00 ng/ml to 18.00-36.00 ng/ml). These results suggest that the present direct enzyme immunoassay is a practical and suitable method for measuring the plasma concentration of E₁S.

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The present study was undertaken to determine whether exposure of fertile eggs to an estrogenic chemical (diethylstilbestrol, DES) affects the structures of the testes and epididymis in matured Japanese quail. Fertile eggs were separated into 3 groups and immersed in test solutions prepared by dissolving DES in 95 % ethanol at concentrations of 0, 5 mg or 50 mg/100 ml (Groups C, L and H, respectively). The birds were sacrificed 100 days after hatching, and their testes, epididymis and cloacal glands were examined by histology and immunocytochemistry for androgen receptors. The structure of the seminiferous tubules in Group C birds was well organized with numerous sperm attached to the epithelium. Although the structure of seminiferous tubules in Group L did not show significant differences from those of Group C, the epithelium of the seminiferous tubules was thinner and the population of sperm attached to the epithelium was greatly reduced in Group H. The epididymis of Group C birds was well developed, consisting of the rete testis, proximal and distal efferent ductules and epididymal duct. In birds hatched from DES treated eggs, the epididymis was less well developed and had fewer tubules compared with Group C. However, immunoreactive androgen receptors were found in the epithelial cells of tubules in the epididymis of Groups C, L and H. The sizes of the cloacal glands were significantly smaller in Groups L and H when compared with Group C. These results suggest that DES exposure of fertile eggs causes structural and functional disruption of testes and epididymis, which may be one of the significant endpoints for endocrine disruptive effects in birds.

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In order to understand the molecular mechanisms responsible for hormone production by the anterior pituitary gland, information about novel molecules and their functions is required. We subjected proteins extracted from porcine pituitary crude nuclei to column chromatography and obtained a protein, Pit-G, with a molecular mass of about 85 kDa. An antibody against Pit-G was raised in a rabbit, and Western blot analysis using this antibody showed that Pit-G was present exclusively in the anterior pituitary gland, double immunocytochemical staining of which revealed that Pit-G-positive cells were localized only in growth hormone (GH)-producing cells. The result also indicated the presence of Pit-G in the cytoplasm. Interestingly, Pit-G was present in a subpopulation of GH-producing cells. We searched the literature for references describing molecular masses and localization of pituitary proteins, but found no information about proteins resembling Pit-G, which suggests that Pit-G is an 85 kDa pituitary protein that plays a specific role in some GH-producing cells.

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The expressions of the Smad family, which transmit transforming growth factor-beta (TGF-β) superfamily in cytoplasm, have been shown in previous studies, but the biological functions of the Smad in spermatogenesis remain to be elucidated. In the present study we examine the testicular localization of Smad2 and Smad3 involved in the intracellular signal transduction of activin and transforming growth factor-beta (TGF-β) under the influence of long and short photoperiods in Syrian hamsters. Immunohistochemistry detected both Smads in Leydig cells and the cytoplasm of pachytene spermatocytes in the active testis exposed to a long photoperiod. In the regressed testis exposed to a short photoperiod, both Smads proteins were detected in Leydig cells and accumulated in the nucleus of pachytene spermatocytes. Northern blots revealed that Plasminogen Activator Inhibitor-1 (PAI-1) mRNA, an index of the transduction of TGF-β and Smads, was expressed at high levels in the photoperiod-induced regressed testis. Therefore, these Smads might participate in the signal transductions of TGF-β in the pachytene spermatocytes of photoperiod-induced testis.

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