Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes.
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It was previously shown that fully grown ovarian germinal vesicle (GV) oocytes of adult mice exhibit several nuclear configurations that differ essentially by the presence or absence of a ring of condensed chromatin around the nucleolus. These configurations have been termed, respectively, SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus). Work from our and other laboratories has revealed ultrastructural and functional differences between these two configurations. The aims of the present study were 1) to analyze the equilibrium between the SN and the NSN population as a function of the age of the mice and the time after hCG-induced ovulation and 2) to study the polymerase I (pol I)- and polymerase II (pol II)-dependent transcription in both types of oocytes through the detection of bromouridine incorporated into nascent RNA. We show 1) that ovarian GV oocytes exhibiting the SN-type configuration can be found as soon as 17 days after birth in the C57/CBA mouse strain and 2) that the SN:NSN ratio of ovarian GV oocytes is very low just after hCG-induced ovulation and then increases progressively with the time after ovulation. Furthermore, we demonstrate that the SN configuration correlates strictly with the arrest of both pol I- and pol II-dependent transcription in mice at any age. Finally, we show that ribosomal genes are located at the outer periphery of the nucleolus in the NSN configuration and that pol I-dependent perinucleolar transcription sites correspond to specific ultrastructural features of the nucleolus. Altogether, these results provide clear-cut criteria delineating transcriptionally active GV oocytes from those that are inactive, and confirm that the SN-type configuration is mostly present in preovulatory oocytes. (+info)
Endocrine biomarkers of early fetal loss in cynomolgus macaques (Macaca fascicularis) following exposure to dioxin.
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This study examines the endocrine alterations associated with early fetal loss (EFL) induced by an environmental toxin, TCDD (2,3,7, 8-tetrachlorodibenzo-p-dioxin), in the cynomolgus macaque, a well-documented reproductive/developmental model for humans. Females were administered single doses of 1, 2, and 4 microgram/kg TCDD (n = 4 per dose group) on gestational day (GD) 12. Urinary estrogen metabolites (estrone conjugates) were monitored to establish the day of ovulation, and serum hormones (estradiol, progesterone, chorionic gonadotropin, relaxin) were measured to assess ovarian and placental endocrine status before and after treatment. EFL occurred between GDs 22 and 32 in 10 of the 12 animals treated with TCDD. The primary endocrine alterations associated with TCDD treatment were significant decreases in serum estradiol and bioactive chorionic gonadotropin concentrations (p < 0.02). Less pronounced decreases in serum progesterone (p = 0.10) and relaxin (p < 0.08) also followed TCDD treatment. In contrast, immunoreactive chorionic gonadotropin concentrations were not reduced by TCDD exposure at any level, indicating that TCDD targets specific components of the chorionic gonadotropin synthesis machinery within the trophoblast to alter the functional capacity of the hormone. These data demonstrate the value of endocrine biomarkers in identifying a toxic exposure to primate pregnancy many days before direct signs of reproductive toxicity were apparent. The increased EFL that occurred after exposure to TCDD might reflect a toxic response initially mediated via endocrine imbalance, leading to placental insufficiency, compromised embryonic circulation, and subsequent EFL. (+info)
Molecular mechanisms of thyroid hormone-stimulated steroidogenesis in mouse leydig tumor cells. Involvement of the steroidogenic acute regulatory (StAR) protein.
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Using a mouse Leydig tumor cell line, we explored the mechanisms involved in thyroid hormone-induced steroidogenic acute regulatory (StAR) protein gene expression, and steroidogenesis. Triiodothyronine (T3) induced a approximately 3.6-fold increase in the steady-state level of StAR mRNA which paralleled with those of the acute steroid response ( approximately 4.0-fold), as monitored by quantitative reverse transcriptase-polymerase chain reaction assay and progesterone production, respectively. The T3-stimulated progesterone production was effectively inhibited by actinomycin-D or cycloheximide, indicating the requirement of on-going mRNA and protein synthesis. T3 displayed the highest affinity of [125I]iodo-T3 binding and was most potent in stimulating StAR mRNA expression. In accordance, T3 significantly increased testosterone production in primary cultures of adult mouse Leydig cells. The T3 and human chorionic gonadotropin (hCG) effects on StAR expression were similar in magnitude and additive. Cells expressing steroidogenic factor 1 (SF-1) showed marginal elevation of StAR expression, but coordinately increased T3-induced StAR mRNA expression and progesterone levels. In contrast, overexpression of DAX-1 markedly diminished the SF-1 mRNA expression, and concomitantly abolished T3-mediated responses. Noteworthy, T3 augmented the SF-1 mRNA expression while inhibition of the latter by DAX-1 strongly impaired T3 action. Northern hybridization analysis revealed four StAR transcripts which increased 3-6-fold following T3 stimulation. These observations clearly identified a regulatory cascade of thyroid hormone-stimulated StAR expression and steroidogenesis that provides novel insight into the importance of a thyroid-gonadal connection in the hormonal control of Leydig cell steroidogenesis. (+info)
Precocious estrus and reproductive ability induced by PG 600 in prepuberal gilts.
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A total of 29 SPF Large White prepuberal gilts (mean age 152 days at treatment) were examined for estrous and ovulatory responses after PG 600 treatment. After treatment, 85.2% of the gilts showed standing estrus within 6 days. Whereas the treatment-to-estrus interval and duration were 3.7 and 1.9 days respectively. As ovulation occurred on Day 5 to 6, appropriate timing of artificial insemination would be about 4 days after treatment. Fertility of gilts revealed to be excellent, giving rise to a high percentage of normal embryos, 85.3%. Meanwhile, development and growth of fetuses were mostly normal. Other reproductive performances recorded were: mean litter size 6.8; mean birth weight 1.26 kg; weaning-to-return estrus interval 5 to 8 days. In conclusion, PG 600 was found to be useful in inducing fertile estrus in prepuberal gilts, a result which will be of interest for commercial pig farms. (+info)
Utero-ovarian interaction in the regulation of reproductive function.
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The physiological regulation of fertile reproductive cycle in mammals depends on interactions between hypothalamus-pituitary, ovarian and uterine stimuli. Over the past 20 years, much has been learned about the interrelation between the affluent and effluent lymph and vascular drainage in and around both ovarian and uterine tissues. An essential feature in the regulation of the fertile cycle is the functional status of the ovary, particularly the corpus luteum. During the time of implantation and the early pregnancy, an active corpus luteum is essential. As human chorionic gonadotrophin (HCG) is important in the maintenance of the corpus luteum, we investigated if it was produced by the cyclic endometrium. Immunohistochemical and in-situ hybridization reactions were performed but neither identified the presence of HCG during the proliferative phase. Positive staining and beta-human chorionic gonadotrophin (beta-HCG) mRNA were observed during the secretory phase in the glandular cells of the endometrium. The results were confirmed by Western blotting of secretory phase endometrium extracts and assessment of the functional secretory capacity of primary endometrial cultures. Polymerase chain reaction (PCR) investigations showed a positive result in the secretory phase. We postulate that, based on the very close morphological interrelation between the uterus and the ovary, the beta-HCG of the endometrium is the primary factor for the maintenance of the corpus luteum and early pregnancy. (+info)
D-Aspartate stimulation of testosterone synthesis in rat Leydig cells.
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D-Aspartate increases human chorionic gonadotropin-induced testosterone production in purified rat Leydig cells. L-Aspartate, D-,L-glutamate or D-,L-asparagine could not substitute for D-aspartate and this effect was independent of glutamate receptor activation. Testosterone production was enhanced only in cells cultured with D-aspartate for more than 3 h. The increased production of testosterone was well correlated with the amounts of D-aspartate incorporated into the Leydig cells, and L-cysteine sulfinic acid, an inhibitor of D-aspartate uptake, suppressed both testosterone production and intracellular D-aspartate levels. D-Aspartate therefore is presumably taken up into cells to increase steroidogenesis. Intracellular D-aspartate probably acts on cholesterol translocation into the inner mitochondrial membrane, the rate-limiting process in steroidogenesis. (+info)
Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity.
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This study was undertaken to determine the modulation of uterine function by chorionic gonadotrophin (CG) in a nonhuman primate. Infusion of recombinant human CG (hCG) between days 6 and 10 post ovulation initiated the endoreplication of the uterine surface epithelium to form distinct epithelial plaques. These plaque cells stained intensely for cytokeratin and the proliferating cell nuclear antigen. The stromal fibroblasts below the epithelial plaques stained positively for alpha-smooth muscle actin (alphaSMA). Expression of alphaSMA is associated with the initiation of decidualization in the baboon endometrium. Synthesis of the glandular secretory protein glycodelin, as assessed by Western blot analysis, was markedly up-regulated by hCG, and this increase was confirmed by immunocytochemistry, Northern blot analysis, and reverse transcriptase-PCR. To determine whether hCG directly modulated these uterine responses, we treated ovariectomized baboons sequentially with estradiol and progesterone to mimic the hormonal profile of the normal menstrual cycle. Infusion of hCG into the oviduct of steroid-hormone-treated ovariectomized baboons induced the expression of alphaSMA in the stromal cells and glycodelin in the glandular epithelium. The epithelial plaque reaction, however, was not readily evident. These studies demonstrate a physiological effect of CG on the uterine endometrium in vivo and suggest that the primate blastocyst signal, like the blastocyst signals of other species, modulates the uterine environment prior to implantation. (+info)
CD9 is involved in invasion of human trophoblast-like choriocarcinoma cell line, BeWo cells.
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The CD9 molecule is expressed on human extravillous trophoblasts, which invade the endometrium during implantation and placentation. To elucidate the role of CD9 in trophoblastic function, we investigated the expression of CD9 protein and mRNA in BeWo cells, a human trophoblast-like choriocarcinoma cell line, using immunohistochemistry, Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). When BeWo cells were cultured with anti-CD9 monoclonal antibodies (mAb), their invasion through the extracellular matrices was significantly enhanced in a dose-dependent manner. Cell proliferation and human chorionic gonadotrophin production were unaffected. On the other hand, culture in the presence of mAb against integrins alpha3, alpha5 and beta1, which partially block the interaction with the extracellular matrices, inhibited BeWo cell invasion. Anti-CD9 monoclonal antibody had a stimulatory effect on BeWo cell invasion in the presence of anti-integrin alpha3 antibody. In contrast, it had no effect in the presence of mAb against integrins alpha5 and beta1, which were also highly expressed on BeWo cells. These findings suggest that CD9 has a function connected with the invasive properties of BeWo cells, which is partially mediated by integrin alpha5beta1. This may relate to the involvement of CD9 in trophoblastic invasion. (+info)