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M-A Hattori, E Yoshino, Y Shinohara, R Horiuchi, and I Kojima


It is well known that epidermal growth factor (EGF) induces down-regulation of LH receptors and desensitization to gonadotrophin stimulation in gonadal cells, including granulosa cells. In a previous study we showed that EGF receptor levels in rat granulosa cells were increased up to fourfold after 96 h of culture with human GH in the presence of FSH, and the present study has evaluated the action of EGF on these cells. The induced EGF receptors were identical in size to the pre-existing receptors as assessed by affinity labelling with 125I-EGF. After 48 h in culture, various amounts of EGF (0·5–10 ng) were added and the cells were cultured for a further 48 h. The addition of EGF caused down-regulation of LH receptors in cells expressing high levels of EGF receptors. However, this down-regulation was less than that in control cells. After the cells were washed, cAMP synthesis in response to human chorionic gonadotrophin (hCG) increased by two to three times the control value and this increase was closely correlated with an increase in EGF receptor content. However, stimulation with cholera toxin or forskolin showed no such augmentation, indicating that it may not be due to quantitative alterations in G proteins and their effector systems. Induction of EGF potentiation required long-term exposure to EGF, for at least more than 24 h. In addition, progesterone synthesis was sensitive to stimulation with lower doses of hCG. These findings indicate that the activation of hGH-induced EGF receptors may potentiate gonadotrophin action in granulosa cells.

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MA Hattori, N Nishida, K Takesue, Y Kato, and N Fujihara

The present study was designed to evaluate the regulation of nitric oxide (NO) synthesis in porcine oocytes during follicular development. Cumulus-oocyte complexes were obtained by aspirating the small follicles of immature porcine ovaries and cultured at 39 degrees C for 24-72 h with FSH in a serum-free medium. The oocyte-surrounding cumulus cells markedly proliferated and expressed LH receptor mRNA in response to FSH. The endothelial type of NO synthase (eNOS) (130 kDa) was detected in the oocyte, but not in the proliferated cumulus cells, by immunoblotting. The amount of oocyte eNOS did not significantly alter during culture, but measurement of nitrite and nitrate revealed FSH suppression of NO synthesis by approximately 50%. NO-releasing agents were added to the cultures to examine the effect of NO on the growth of cumulus cells. NO-releasing agents showed inhibitory effects on proliferation of the cumulus cells and expression of LH receptor mRNA. Thus, synthesis of eNOS-derived NO is suppressed in the porcine oocyte during development with no change in the enzyme amount, and it is suggested that it has an inhibitory function in the growth of cumulus cells.

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K Takesue, MA Hattori, N Nishida, Y Kato, and N Fujihara

The present study was designed to investigate nitric oxide (NO) synthesis and the expression of endothelial NO synthase (eNOS) gene in cultured porcine granulosa cells. Granulosa cells prepared from small follicles (1-4 mm diameter) were cultured in plastic dishes coated with fibronectin in chemically defined medium, and matured after 48 h of stimulation with FSH. The concentrations of nitrite and nitrate remained relatively constant until 42 h of stimulation, after which they increased significantly up to twofold at 48 h. NO synthesis was accompanied by an increase in cGMP. Gene expression for eNOS was studied by RT-PCR, and a PCR product of the expected size amplified. eNOS mRNA was expressed in the presence of FSH, but not in the absence of FSH. Although eNOS mRNA was not expressed in the initial period, it was expressed after 12 h of stimulation with FSH, and remained at a relatively constant level until 48 h. Expression of eNOS mRNA preceded expression of LH receptor mRNA, which showed a maximal level at 24 h of stimulation. These observations suggest that eNOS expression is not related to a rapid synthesis of NO in developing granulosa cells, and that the activation of NO synthesis is rigidly regulated in the initial period of development.