FSH stimulation promotes progesterone synthesis and output from human granulosa cells without luteinization

Abstract

STUDY QUESTION: Can granulosa cells produce progesterone (P) in response to FSH stimulation? SUMMARY ANSWER: FSH actively promotes P synthesis and output from granulosa cells without luteinization by up-regulating the expression and increasing enzymatic activity of 3 beta-hydroxysteriod dehydrogenoase (3 beta-HSD), which converts pregnenolone to P. WHAT IS KNOWN ALREADY: Serum P level may rise prematurely prior to ovulation trigger in stimulated IVF cycles and adversely affect implantation and clinical pregnancy rates by impairing endometrial receptivity. STUDY DESIGN, SIZE, DURATION: A translational research study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human ovarian cortical samples (n = 15) and non-luteinizing FSH-responsive human mitotic granulosa cell line (HGrC1) were stimulated with rec-FSH at 12.5, 25 and 50 mIU/ml concentrations for 24 and 48 h. FSH receptor expression was knocked-down and up-regulated in the granulosa cells using short hairpin RNA (shRNA) technology and activin-A administration, respectively. The expressions of the steroidogenic enzymes were analyzed at mRNA level by real-time quantitative RT-PCR, and protein level by western blot and immunoprecipitation assay. The enzymatic activity of 3 beta-HSD was measured using a spectrophotometric method. In vitro estradiol (E2) and P productions of the cells before and after FSH stimulation were measured by electrochemiluminescence immunoassay method. MAIN RESULTS and THE ROLE of CHANCE: Stimulation of the HGrC1 cells with FSH resulted in a dose-dependent increase in the mRNA and protein level of 3 beta-HSD. Overall, when all time points and FSH doses were analyzed collectively, FSH significantly up-regulated the mRNA expression of its own receptor (3.73 +/- 0.06-fold, P < 0.001), steroidogenic acute regulatory protein (stAR, 1.7 +/- 0.03-fold, P < 0.01), side-chain cleavage enzyme (SCC, 1.75 +/- 0.03-fold, P < 0.01), aromatase (4.49 +/- 0.08-fold, P < 0.001), 3 beta-HSD (1.68 +/- 0.02-fold, P < 0.01) and 17 beta-hydroxy steroid dehydrogenase (17 beta-HSD, 2.16 +/- 0.02-fold, P < 0.01) in the granulosa cells. Expression of 17 alpha-hydroxylase (17 alpha-OH, 1.03 +/- 0.01-fold P > 0.05) did not significantly change. Similar changes were observed in the protein expression analysis of these enzymes on western blotting after FSH stimulation. FSH significantly increased 3a-HSD, 17 beta-HSD and aromatase in a dose-dependent manner but did not affect 17 alpha-OH. Protein expression of P was increased along with 3a-HSD after FSH stimulation, which was further evidenced by immunoprecipitation assay. Enzymatic activity of 3 beta-HSD was significantly enhanced by FSH administration in the HGrC1 cells in a dose-dependent manner. In line with these findings P output (1.05 +/- 0.3 vs. 0.2 +/- 0.1 ng/ml, respectively, P < 0.001) from the samples stimulated with FSH were significantly increased along with E2 (1918 +/- 203 vs. 932 +/- 102 pg/ml, respectively, P < 0.001) compared to unstimulated controls. FSH-induced increase in 3 beta-HSD expression was amplified and reversed in the HGrC1 cells when FSH receptor expression was up-regulated by activin-A and down-regulated with shRNA, respectively. LIMITATIONS and REASONS FOR CAUTION: As only the effect of FSH was studied we cannot extrapolate our findings to the potential effects of HMG and recombinant LH. WIDER IMPLICATIONS of THE FINDINGS: This data provides a molecular explanation for the largely unexplained phenomenon of P rise during the follicular phase of gonadotropin stimulated IVF cycles. Our findings may progress the research to uncover potential mechanisms for preventing premature P rise that appears to be associated with inferior outcomes in women undergoing IVF.