REGULATION OF INHIBIN ALPHA-SUBUNIT AND BETA(A)-SUBUNIT MESSENGER-RIBONUCLEIC-ACID LEVELS BY CHORIONIC-GONADOTROPIN AND RECOMBINANT FOLLICLE-STIMULATING-HORMONE IN CULTURED HUMAN GRANULOSA-LUTEAL CELLS

We studied the effects of recombinant human FSH (rhFSH) and purified hCG on the steady state messenger ribonucleic acid (mRNA) levels of inhibin alpha- and beta(A)-subunits in cultured granulosa-luteal cells of preovulatory ovarian follicles obtained from women undergoing in vitro fertilization. Specific mRNA transcripts for the alpha- and beta(A)-subunits were detected in Northern and dot blot filter hybridization analyses, and the levels of these mRNAs were induced by rhFSH and hCG in a distinct concentration- and time-dependent manner. The basal and hCG-stimulated alpha-subunit mRNA levels were first determined at 2- to 3-day intervals over a 3- to 10-day culture period after the initiation of the cultures. Both the basal and hCG-stimulated alpha-subunit mRNA levels declined steadily during culture, but the maximal relative stimulatory effect of hCG was observed on day 7 of culture. All subsequent experiments, therefore, were performed on days 6-8 of culture. Both gonadotropins induced alpha-subunit mRNA levels with slower kinetics than those of the beta(A)-subunit. Varying between experiments, rhFSH and hCG increased the expression of the alpha-subunit with a maximal effect of 2.5- to 5.7-fold and 1.7- to 7.2-fold, respectively, above basal levels 24-48 h after stimulation. rhFSH and hCG induced beta(A)-subunit mRNA levels with 3.0- to 5.8-fold and 2.3- to 8.6-fold increases above basal levels, respectively, at 2 h; thereafter, only moderate or no stimulation of the beta(A)-subunit mRNA levels could be detected at 7-48 h. Treatment of the cells with the RNA synthesis inhibitor actinomycin-D prevented the induction of alpha-subunit mRNA levels by hCG, and no significant differences were detected in the stability of alpha-subunit mRNA transcripts in hCG-treated cells vs. untreated cultures. This indicates that hCG induces transcription of the alpha-subunit gene rather than maintains the levels of preexisting transcripts. As the kinetics of induction of alpha- and beta(A)-subunit mRNAs by gonadotropins were different, we examined how the inhibition of protein synthesis affects the induction of alpha- and beta(A)-subunit mRNAs by hCG. Cycloheximide had no effect on basal alpha-subunit mRNA levels at 2 or 24 h. However, it inhibited at 24 h the induction of the alpha-subunit by hCG. On the other hand, cycloheximide had no effect on basal or hCG-stimulated beta(A)-subunit mRNA levels at 2 h, but at the 24 h point, it markedly induced both basal and hCG-stimulated beta(A)-subunit mRNA levels. Thus, the slower induction of the alpha-subunit mRNA may require de novo synthesis of protein factors for the mediation of the effect of hCG. By contrast, protein synthesis inhibition did not affect the rapid induction of beta(A)-subunit by hCG, but it may prevent the degradation of the beta(A)-subunit mRNA seen after the initial transient induction of its levels. Taken together, our results suggest that both gonadotropins are potent inducers of inhibin alpha- and beta(A)-subunit mRNAs in human granulosa-luteal cells and that the induction of the mRNAs of these two subunits is regulated through different mechanisms.