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Gonadotropins and Cytokines Affect Luteal Function through Control of Apoptosis in Human Luteinized Granulosa Cells

Department of Obstetrics and Gynecology, Nagoya City University Medical School, Nagoya 467-8601, Japan

Address all correspondence and requests for reprints to: Dr. Hirokazu Matsubara, Department of Obstetrics and Gynecology, Nagoya City University Medical School, Kawasumi 1, Mizuho-Cho, Mizuho-ku, Nagoya, Japan 467-8601.

The luteal phase in the normal human menstrual cycle is known to be about 14 days. The physiological mechanisms that regulate the corpus luteum remain to be clarified, although apoptosis is reported to be involved. This study was undertaken to investigate the regulation of luteal function by gonadotropins, cytokines, and PGs, concentrating attention on the incidence of apoptosis and its molecular mechanisms in cultured human luteinized granulosa cells collected at oocyte pick-up from patients undergoing in vitro fertilization and embryo transfer. Clusters of granulosa cells were pipetted in 0.1% hyaluronidase in phosphate-buffered saline. After cell separation by centrifugation using Ficoll-Paque, 1 x 10⁴ viable cells/mL in RPMI 1640 medium with 10% FCS were used for experimentation. Substances added were FSH (100 ng/mL), hCG (100 ng/mL), LH (100 ng/mL), interleukin-1ß (IL-1ß; 10 ng/mL), transforming growth factor-ß1 (TGFß1; 10 ng/mL), macrophage colony-stimulating factor (M-CSF; 10 ng/mL), tumor necrosis factor- (TNF; 10 ng/mL), and PGF₂ (10 ng/mL). After 24-h culture at 37 C under 5% CO₂ and air, cells were fixed with 4% neutral buffered formalin and stained with Hoechst 33258. Apoptotic bodies were counted under a fluorescence microscope, and immunostaining was performed using anti-Fas, Fas ligand, Bcl-2, Bax, and p53 antibodies. Incidences of apoptotic bodies in the group without substance addition were 0.7 ± 0.2% (0 h), 5.9 ± 0.6% (24 h), and 7.9 ± 1.2% (48 h); spontaneous increase was significant at the latter time points. Defining the incidence at 24 h as 100%, values after treatment were: FSH, 57%; LH, 84%; hCG, 44%; IL-1ß, 76%; TGFß1, 52%; M-CSF, 50%; TNF, 177%; and PGF₂, 147%. Significant suppression was observed with FSH, hCG, TGFß1, and M-CSF (P < 0.01). On the other hand, significant induction occurred with TNF and PGF₂ (P < 0.01). On immunostaining, the incidence of stained cells with anti-Fas, Fas ligand, Bax, and p53 antibody was increased after 24-h incubation without addition. This was reduced by hCG, TGFß1, and M-CSF. No stained cells were observed with anti-Bcl-2 antibody before or after incubation. In conclusion, our results suggest that both gonadotropins (FSH and hCG) and cytokines (TGFß1 and M-CSF) may be involved in the support of luteal function via suppression of apoptosis, and that TNF and PGF₂ may contribute to ovarian dysfunction and/or luteal regression via its induction in human luteinized granulosa cells. Our results also suggest that Fas, Fas ligand, p53, and Bax may play roles in this apoptosis controlled by hCG, TGFß1, and M-CSF.

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