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
M. Eramaa et al., 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, The Journal of clinical endocrinology and metabolism, 79(6), 1994, pp. 1670-1677
We studied the effects of recombinant human FSH (rhFSH) and purified h
CG on the steady state messenger ribonucleic acid (mRNA) levels of inh
ibin alpha- and beta(A)-subunits in cultured granulosa-luteal cells of
preovulatory ovarian follicles obtained from women undergoing in vitr
o fertilization. Specific mRNA transcripts for the alpha- and beta(A)-
subunits were detected in Northern and dot blot filter hybridization a
nalyses, and the levels of these mRNAs were induced by rhFSH and hCG i
n a distinct concentration- and time-dependent manner. The basal and h
CG-stimulated alpha-subunit mRNA levels were first determined at 2- to
3-day intervals over a 3- to 10-day culture period after the initiati
on of the cultures. Both the basal and hCG-stimulated alpha-subunit mR
NA levels declined steadily during culture, but the maximal relative s
timulatory effect of hCG was observed on day 7 of culture. All subsequ
ent experiments, therefore, were performed on days 6-8 of culture. Bot
h 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 b
asal levels 24-48 h after stimulation. rhFSH and hCG induced beta(A)-s
ubunit mRNA levels with 3.0- to 5.8-fold and 2.3- to 8.6-fold increase
s 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 ac
tinomycin-D prevented the induction of alpha-subunit mRNA levels by hC
G, and no significant differences were detected in the stability of al
pha-subunit mRNA transcripts in hCG-treated cells vs. untreated cultur
es. 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 gonad
otropins were different, we examined how the inhibition of protein syn
thesis affects the induction of alpha- and beta(A)-subunit mRNAs by hC
G. 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-sub
unit by hCG. On the other hand, cycloheximide had no effect on basal o
r hCG-stimulated beta(A)-subunit mRNA levels at 2 h, but at the 24 h p
oint, it markedly induced both basal and hCG-stimulated beta(A)-subuni
t mRNA levels. Thus, the slower induction of the alpha-subunit mRNA ma
y require de novo synthesis of protein factors for the mediation of th
e effect of hCG. By contrast, protein synthesis inhibition did not aff
ect 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 tra
nsient induction of its levels. Taken together, our results suggest th
at 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 mec
hanisms.