This Article Full Text Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matsubara, H. Articles by Suzumori, K. Search for Related Content PubMed PubMed Citation Articles by Matsubara, H. Articles by Suzumori, K.

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.

This article has been cited by other articles:

				R. E. Dickinson, M. Myers, and W. C. Duncan Novel Regulated Expression of the SLIT/ROBO Pathway in the Ovary: Possible Role during Luteolysis in Women Endocrinology, October 1, 2008; 149(10): 5024 - 5034. [Abstract] [Full Text] [PDF]

				I.-S. Hong, A. P. Cheung, and P. C. K. Leung Gonadotropin-Releasing Hormones I and II Induce Apoptosis in Human Granulosa Cells J. Clin. Endocrinol. Metab., August 1, 2008; 93(8): 3179 - 3185. [Abstract] [Full Text] [PDF]

				M. Tsai-Turton, B. T. Luong, Y. Tan, and U. Luderer Cyclophosphamide-Induced Apoptosis in COV434 Human Granulosa Cells Involves Oxidative Stress and Glutathione Depletion Toxicol. Sci., July 1, 2007; 98(1): 216 - 230. [Abstract] [Full Text] [PDF]

				P. G Knight and C. Glister TGF-{beta} superfamily members and ovarian follicle development. Reproduction, August 1, 2006; 132(2): 191 - 206. [Abstract] [Full Text] [PDF]

				K. A Slot, M. Voorendt, M. de Boer-Brouwer, H. H van Vugt, and K. J Teerds Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary J. Endocrinol., February 1, 2006; 188(2): 179 - 192. [Abstract] [Full Text] [PDF]

				W. V. Ingman, R. L. Robker, K. Woittiez, and S. A. Robertson Null Mutation in Transforming Growth Factor {beta}1 Disrupts Ovarian Function and Causes Oocyte Incompetence and Early Embryo Arrest Endocrinology, February 1, 2006; 147(2): 835 - 845. [Abstract] [Full Text] [PDF]

				Y. Kumazawa, K. Kawamura, T. Sato, N. Sato, Y. Konishi, Y. Shimizu, J. Fukuda, H. Kodoma, and T. Tanaka HCG up-regulates survivin mRNA in human granulosa cells Mol. Hum. Reprod., March 1, 2005; 11(3): 161 - 166. [Abstract] [Full Text] [PDF]

				R. Sasson, E. Rimon, A. Dantes, T. Cohen, V. Shinder, A. Land-Bracha, and A. Amsterdam Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases Mol. Hum. Reprod., May 1, 2004; 10(5): 299 - 311. [Abstract] [Full Text] [PDF]

				M. Zygmunt, F. Herr, S. Keller-Schoenwetter, K. Kunzi-Rapp, K. Munstedt, C. V. Rao, U. Lang, and K. T. Preissner Characterization of Human Chorionic Gonadotropin as a Novel Angiogenic Factor J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5290 - 5296. [Abstract] [Full Text] [PDF]

				J. K. Pru, I. R. Hendry, J. S. Davis, and B. R. Rueda Soluble Fas Ligand Activates the Sphingomyelin Pathway and Induces Apoptosis in Luteal Steroidogenic Cells Independently of Stress-Activated p38MAPK Endocrinology, November 1, 2002; 143(11): 4350 - 4357. [Abstract] [Full Text] [PDF]

				B. Pan, K. Sengoku, K. Goishi, N. Takuma, T. Yamashita, K. Wada, and M. Ishikawa The soluble and membrane-anchored forms of heparin-binding epidermal growth factor-like growth factor appear to play opposing roles in the survival and apoptosis of human luteinized granulosa cells Mol. Hum. Reprod., August 1, 2002; 8(8): 734 - 741. [Abstract] [Full Text] [PDF]

				R. Sasson and A. Amsterdam Stimulation of Apoptosis in Human Granulosa Cells from in Vitro Fertilization Patients and Its Prevention by Dexamethasone: Involvement of Cell Contact and Bcl-2 Expression J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3441 - 3451. [Abstract] [Full Text] [PDF]

				M. Embree-Ku and K. Boekelheide Absence of p53 and FasL Has Sexually Dimorphic Effects on Both Development and Reproduction Experimental Biology and Medicine, July 1, 2002; 227(7): 545 - 553. [Abstract] [Full Text] [PDF]

				J.L. Benifla, C. Sifer, A.F. Bringuier, G. Blanc-Layrac, E. Camus, P. Madelenat, and G. Feldmann Induced apoptosis and expression of related proteins in granulosa cells from women undergoing IVF: a preliminary study Hum. Reprod., April 1, 2002; 17(4): 916 - 920. [Abstract] [Full Text] [PDF]

				R. Quintana, L. Kopcow, G. Marconi, C. Sueldo, G. Speranza, and R.I. Baranao Relationship of ovarian stimulation response with vascular endothelial growth factor and degree of granulosa cell apoptosis Hum. Reprod., September 1, 2001; 16(9): 1814 - 1818. [Abstract] [Full Text] [PDF]

				J. Prange-Kiel, C. Kreutzkamm, U. Wehrenberg, and G. M. Rune Role of Tumor Necrosis Factor in Preovulatory Follicles of Swine Biol Reprod, September 1, 2001; 65(3): 928 - 935. [Abstract] [Full Text] [PDF]