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Photoperiodic and hormonal modulation of mRNAs for testicular inhibin/activin subunits and follistatin were studied in a seasonally breeding rodent, the bank vole (Clethrionomys glareolus). Photoperiod-induced testicular regression had no effect on the relatively low steady-state levels of follistatin mRNA. Inhibin alpha (I alpha) and beta B (I beta B) mRNA levels were significantly higher in regressed than in active gonads, but inhibin beta A was undetectable. The effect of gonadotropin administration on testicular weight and mRNA concentrations differed between the sexually active and quiescent voles. Neither FSH (1.2 U/kg; s.c. for 5 days) nor hCG (600 IU/kg; s.c. for 5 days) affected testicular weight in sexually active voles, whereas both gonadotropins significantly increased testicular weight in photo-regressed individuals. FSH had no effect on I alpha or I beta B mRNA concentrations in the active testes, whereas excessive hCG challenge induced a decrease in the steady-state levels of these mRNAs. FSH induced an increase in I alpha mRNA concentrations in the regressed gonad, whereas both gonadotropins concomitantly down-regulated I beta B mRNA levels. In conclusion, the high expression of I alpha and I beta B mRNA in the regressed testis imply autocrine and paracrine roles for inhibin/activin in the quiescent gonad of seasonal breeders. Inhibin alpha-subunit expression is at least partly under the control of FSH in the bank vole testis.

It has recently been shown that mice deficient in activin-beta A subunits and follistatin exhibit major defects in dentition. To increase understanding of the roles played by these molecules during tooth development, we determined the temporospatial expression of activin-beta A subunit and follistatin messenger RNA and their corresponding proteins in developing murine molars (between day E 14 and 2 days after birth). The effects of recombinant human activin A and its binding protein follistatin on odontoblast differentiation were also studied in cultures of dental papillae (DP) isolated from the mandibular first molars of E-17-day mice. In situ hybridization indicated that transcripts for activin-beta A subunit were abundant in pre-odontoblasts at the tips of forming cusps prior to odontoblast terminal differentiation, and transcripts for follistatin in overlying inner enamel epithelial cells (pre-ameloblasts). Pre-odontoblasts were also weakly immunoreactive in relation to activin-beta A subunit, pre-ameloblasts in relation to follistatin. When follistatin was added at different concentrations to a DP culture model (2-14 nmol/DP) together with heparin at constant concentration, differentiation of odontoblast-like cells was induced, as evidenced by polarization and deposition of extracellular matrix in vitro, to extents depending on the follistatin concentration. In contrast, the addition of activin A (2 nmol/DP) had no effect on the differentiation parameters studied. These findings suggest that the activin-follistatin system regulates odontoblast differentiation during tooth development. In particular, we suggest that binding of endogenous activin A by follistatin may allow odontoblast terminal differentiation to occur.

Granulosa cell-derived inhibin A (a dimer of alpha- and beta A-subunits), activin A (a homodimer of beta A-subunits) and the activin-binding protein follistatin are important regulators of human ovarian steroidogenesis. We here studied how 8-bromo-cAMP (8br-cAMP), a protein kinase A activator, and 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, affect the steady-state levels of alpha- and beta A-subunit and follistatin mRNAs in cultured human granulosa-luteal cells. 8br-cAMP induced alpha- and beta A-subunit and follistatin steady-state mRNA levels in a time- and concentration-dependent manner. The levels of alpha-subunit mRNAs were stimulated by 8br-cAMP in a sustained manner with a maximal induction seen at the time points 24 and 48 h. By contrast, beta A-subunit and follistatin mRNA levels were rapidly and transiently induced by 8br-cAMP with maximal effects observed at 3 h and 8 h, respectively. TPA did not affect basal alpha-subunit mRNA levels but it rapidly induced beta A-subunit mRNAs at 3 h and the stimulation was still evident at 48 h. TPA induced follistatin mRNA levels with kinetics similar to 8br-cAMP but to a lesser extent. Moreover, 8br-cAMP and TPA stimulated beta A-subunit and follistatin mRNA levels synergistically at 3 h. By contrast, TPA had a potent inhibitory effect on 8br-cAMP- and hCG-induced alpha-subunit levels. Neither 8br-cAMP nor TPA regulated inhibin/activin beta B-subunit mRNA levels. Taken together the activation of protein kinase-A and -C by 8br-cAMP and TPA, respectively, lead to clearly differential responses in the steady-state levels of inhibin activin alpha- and beta A-subunit and follistatin mRNAs. These results suggest that the inhibin A vs. activin A ratio as well as follistatin levels are regulated by multiple second-messenger pathways in the human ovary.

Regulation of the activin-binding protein follistatin (FS) by recombinant human (rh) FSH, prostaglandin E2 (PGE2), and several polypeptide growth factors was examined in cultures of human granulosa-luteal (GL) cells obtained from in-vitro fertilization patients at oocyte retrieval. Northern and dot blot hybridization analyses demonstrated that both rhFSH and PGE2 caused stimulatory effects on FS mRNA levels in a culture stage-, time-, and concentration-dependent manner. An 8-h stimulation with rhFSH (100 ng/ml) significantly increased FS mRNA levels on Days 5 and 7 of culture and PGE2 (10(-6)M) on Days 2, 4, and 7. The stimulatory effect of rhFSH and PGE2 on FS mRNA levels were rapid and transient. Maximal inductions occurred 8 h after stimulation, whereas weak or no stimulatory effects were seen at 24 or 48 h. PGF2 alpha did not affect FS mRNA levels at any time point studied. Treatment of the cells with the protein synthesis inhibitor cycloheximide prior to rhFSH stimulation did not inhibit the rapid induction of FS mRNAs, but it prevented the decline at 24 h. Both rhFSH and PGE2 clearly also increased the levels of secreted FS proteins are detected by immunoprecipitation studies with a specific antibody. The effects of the polypeptide growth factors epidermal growth factor (EGF); transforming growth factor beta 1 (TGF beta 1), and activin A on FS mRNA levels were also examined. TGF beta 1 and activin A had no effect on basal FS expression at any concentration or time point studied. An 8-h stimulation with EGF increased FS mRNA levels, but the effect was weaker than those caused by rhFSH and PGE2. We conclude that rhFSH and PGE2 induce FS mRNA and protein in human cultured GL cells. EGF is able to induce FS mRNA to a lesser extent than are rhFSH and PGE2, whereas PGF2 alpha, TGF beta 1, and activin A do not affect basal FS mRNA levels in human cultured GL cells. This study together with our previous report on the stimulatory effect of hCG on FS levels suggest that in the luteal phase of the human menstrual cycle, FS expression in granulosa cells is likely to be positively controlled by luteotropic factors such as gonadotropins and PGE2. Consequently, elevated FS levels may support the survival of the human CL since FS is known to prevent the antisteroidogenic effects of activin in human GL cells.

We studied the effect of hCG on follistatin (FS) messenger RNA (mRNA) steady state levels and protein secretion in cultures of human granulosa-luteal (GL) cells obtained at oocyte harvest for in vitro fertilization. Three different overlapping FS complementary DNA (cDNA) fragments were generated by reverse transcription-polymerase chain reaction from human GL cell RNA. Together, these fragments covered the open reading frame, which appeared to be identical in sequence to previously isolated human testis-derived cDNAs. An alternative splicing event at the 3'-end of the FS transcript previously shown to give rise to transcripts encoding 344 amino acid (aa) and 317-aa proteins was also observed. In Northern analysis of human GL cell RNA, a major 2.5-kilobase transcript 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 with hCG (30 ng/ml). Time and concentration dependence studies on the effect of hCG were performed with cells cultured for 6-8 days before hormone treatment. Time-course experiments indicated that hCG (30 ng/ml) markedly induces FS mRNA levels as early as 2 h after stimulation. The maximal response to hCG stimulation, about 9-fold (mean of five experiments) above basal levels, was observed at 6-8 h, and thereafter, only moderate or no induction of FS mRNA levels could be detected at 24 or 48 h. Concentration dependence studies performed 8 h after stimulation indicated that the maximal induction occurred with 30-100 ng/ml hCG, with an ED50 of about 3-10 ng/ml. When the cells were treated with the protein synthesis inhibitor cycloheximide (20 micrograms/ml) 20 min before stimulation of the cells with hCG, both basal and hCG-stimulated FS mRNA levels increased at 24 h, indicating stabilization of the transcripts. However, it did not affect the rapid induction of FS mRNA levels by hCG at 2 h. The decline in FS transcript levels in untreated and hCG-treated cells was studied by blocking the transcription with 5 microM actinomycin-D. The degradation rate of FS mRNA was increased in hCG-treated compared to control cells. To study whether the transiently induced FS mRNAs are translated to proteins in hCG-treated and untreated human GL cells, metabolic labeling and immunoprecipitation experiments were performed to detect secreted [35S]FS proteins with the specific anti-FS antiserum Rb 32.(ABSTRACT TRUNCATED AT 400 WORDS)

Activins have potent effects on early morphogenetic events during amphibian embryogenesis but no evidence for their role during human development other than their expression in steroidogenic tissues has been reported. We previously showed the expression of the activin type II and IIB receptor mRNAs in several tissues of the mid-gestational human fetus with highest expression levels in developing neural, muscular and exocrine glandular organs. We now report that the mRNA transcripts for activin beta A- and beta B-subunits and for the activin-binding protein follistatin are found co-expressed in several of these extragonadal tissues. Their mRNAs were detected by Northern analyses using specific single-stranded 32P-labeled cDNA probes. In the nervous system, both activin beta A- and beta B-subunit transcripts were expressed in the cerebrum and spinal cord. Follistatin was abundantly expressed in the spinal cord whereas weaker signals where observed in the cerebrum and cerebellum. In the muscular system, beta A-subunit was abundantly expressed in the heart but to a lesser extent in the skeletal muscle while the opposite was observed for follistatin. Follistatin, and activin beta A- and beta B-subunit mRNAs were also detected in developing kidney, salivary gland, liver, and adrenal. The predominance of beta A-subunit mRNAs in the bone marrow and beta B-subunit mRNAs in the salivary gland suggests specific roles for activin A and B, respectively, in these tissues. No hybridization signal was detected for the inhibin alpha-subunit in non-steroidogenic tissues indicating that, in contrast to activins and follistatin, the effects of inhibins may be restricted to the gonads and adrenals which are known to express high levels of the alpha-subunit transcript. Taken together, our results suggest that the activin-follistatin system regulates the development of several organ systems in the mid-gestational human fetus.