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

Abstract

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-1beta (IL-1beta; 10 ng/mL), transforming growth factor-beta1 (TGFbeta1; 10 ng/mL), macrophage colony-stimulating factor (MCSF; 10 ng/mL), tumor necrosis factor-alpha (TNFalpha; 10 ng/mL), and PGF2alpha (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-1beta, 76%; TGFbeta1, 52%; M-CSF, 50%; TNFalpha, 177%; and PGF2alpha, 147%. Significant suppression was observed with FSH, hCG, TGFbeta1, and M-CSF (P < 0.01). On the other hand, significant induction occurred with TNFalpha and PGF2alpha (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, TGFbeta1, 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 (TGFbeta1 and M-CSF) may be involved in the support of luteal function via suppression of apoptosis, and that TNFalpha and PGF2alpha 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, TGFbeta1, and M-CSF.

Effect of apoptosis and reactive oxygen species production in human granulosa cells on oocyte fertilization and blastocyst development

“…Other known apoptosis inductive agents are androgens and GnRH [17,18]. On the other hand, the agents that have antiatretogenic effects are estrogens, FSH, LH and HCG [17,19]. The women in our study were stimulated with recombinant FSH that is supposed to posses an antiatretogenic effect Springer and GnRH antagonists.…”

TGF-β superfamily members and ovarian follicle development

“…It has also been demonstrated that prolactin can stimulate TGF-b2 expression in rat luteal macrophages (Matsuyama et al 1990). Human granulosa-lutein cells treated with TGFb1 showed a significant reduction in apoptosis (Matsubara et al 2000). Therefore, it appears that TGF-b isoforms in concert with prolactin have the potential to support the CL by enhancing progesterone production and suppressing apoptosis.…”

ATP-induced apoptosis of human granulosa luteal cells cultured in vitro

“…An immunohistochemical study showed that human granulosa cells express hCG receptors (46). It is also well established that when gonadotropins, such as LH, FSH, and hCG, added to human granulosa luteal cells in culture affect luteal function through control of apoptosis (47). The role of hCG in luteal rescue has been attributed to up-regulation of a tissue inhibitor of metalloproteinase by human granulosa luteal cells (48) or down-regulation of P 2Y receptor message in human granulosa luteal cells (49).…”