ACTIVIN-BINDING PROTEIN FOLLISTATIN MESSENGER-RIBONUCLEIC-ACID AND SECRETED PROTEIN-LEVELS ARE INDUCED BY CHORIONIC-GONADOTROPIN IN CULTURED HUMAN GRANULOSA-LUTEAL CELLS
T. Tuuri et al., ACTIVIN-BINDING PROTEIN FOLLISTATIN MESSENGER-RIBONUCLEIC-ACID AND SECRETED PROTEIN-LEVELS ARE INDUCED BY CHORIONIC-GONADOTROPIN IN CULTURED HUMAN GRANULOSA-LUTEAL CELLS, Endocrinology, 135(5), 1994, pp. 2196-2203
We studied the effect of hCG on follistatin (FS) messenger RNA (mRNA)
steady state levels and protein secretion in cultures of human granulo
sa-luteal (GL) cells obtained at oocyte harvest for in vitro fertiliza
tion. Three different overlapping FS complementary DNA (cDNA) fragment
s were generated by reverse transcription-polymerase chain reaction fr
om human GL cell RNA. Together, these fragments covered the open readi
ng frame, which appeared to be identical in sequence to previously iso
lated human testis-derived cDNAs. An alternative splicing event at the
3'-end of the FS transcript previously shown to give rise to transcri
pts encoding 344 amino acid (as) and 317- as proteins was also observe
d. In Northern analysis of human GL cell RNA, a major 2.5-kilobase tra
nscript and a minor 1.5-kilobase FS transcript were detected, and the
steady state levels of both mRNAs were induced by an 8-h stimulation w
ith hCG (30 ng/ml). Time and concentration dependence studies on the e
ffect of hCG were performed with cells cultured for 6-8 days before ho
rmone treatment. Time-course experiments indicated that hCG (30 ng/ml)
markedly induces FS mRNA levels as early as 2 h after stimulation. Th
e maximal response to hCG stimulation, about 9-fold (mean of five expe
riments) above basal levels, was observed at 6-8 h, and thereafter, on
ly moderate or no induction of FS mRNA levels could be detected at 24
or 48 h. Concentration dependence studies performed 8 h after stimulat
ion indicated that the maximal induction occurred with 30-100 ng/ml hC
G, with an ED(50) of about 3-10 ng/ml. When the cells were treated wit
h the protein synthesis inhibitor cycloheximide (20 mu g/ml) 20 min be
fore stimulation of the cells with hCG, both basal and hCG-stimulated
FS mRNA levels increased at 24 h, indicating stabilization of the tran
scripts. However, it did not affect the rapid induction of FS mRNA lev
els by hCG at 2 h. The decline in FS transcript levels in untreated an
d hCG-treated cells was studied by blocking the transcription with 5 m
u M actinomycin-D. The degradation rate of FS mRNA was increased in hC
G-treated compared to control cells. To study whether the transiently
induced FS mRNAs are translated to proteins in hCG-treated and untreat
ed human GL cells, metabolic labeling and immunoprecipitation experime
nts were performed to detect secreted [S-35]FS proteins with the speci
fic anti-FS antiserum Rb 32. Several immunoreactive proteins in the ra
nge of 32-39 kilodaltons, corresponding to the expected mol wt of vari
ably processed forms of FS, were detected by polyacrylamide gel electr
ophoresis. A clear increase in their levels was observed in medium of
hCG-treated cultures. The relatedness of the immunoprecipitated protei
ns to FS was verified by the ability of the recombinant human FS to di
splace their binding to anti-FS antiserum. We conclude that 1) the hum
an GL cell-derived FS cDNAs are indistinguishable from previously repo
rted cDNA clones derived from human testis; 2) the alternative splicin
g variant of FS transcript that encodes the 344-aa protein is more abu
ndantly expressed in human GL cells than that giving rise to the 317-a
a FS protein; 3) FS transcript levels are rapidly and transiently indu
ced by hCG in a time- and concentration-dependent manner; 4) the rapid
decline in FS mRNA levels after their initial induction by hCG is pro
bably explained by increased degradation of the transcripts in hCG-tre
ated cells compared to the controls; and 5) the induced mRNAs are tran
slated to secreted immunoreactive FS proteins. These results support a
physiological role for hCG (or LH) as a regulator of FS mRNA and prot
ein expression in the human ovary.