We showed previously that inhibin, partially purified from cynomolgus monkey Sertoli cell culture medium (primate Sertoli cell inhibin referred to as pSCI), selectively suppressed basal FSH secretion from dispersed rat pituitary cells and decreased total cellular FSH, but not LH content, suggesting a decrease in FSH biosynthesis. In order to investigate the mechanism of action of inhibin at the molecular level, we have now examined the effects of pSCI on steady state levels of the subunit mRNAs encoding LH and FSH and correlated these with release and intracellular content of LH, FSH, and glycoprotein α-subunit. Dispersed pituitary cells from 7- to 8-week-old adult male rats were cultured in the presence of pSCI or control medium for 2-72 h. FSH secretion was reduced significantly by 6 h (P < 0.05) and reached a nadir (38% of control) by 48 h. LH secretion was unchanged, while release of the α-subunit was decreased to 89% of control at 72 h (P < 0.05). Also by 72 h, cell content of both FSH (73% of control) and α-subunit (81% of control) were significantly suppressed (P < 0.001, P < 0.01), while LH was slightly affected. Total RNA was extracted from the pituitary cell cultures, electrophoresed in 1.2% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with32P-labeled cDNA probes for the ratα-, LHβ-, and FSHβ-subunits. In order to normalize for differences in amounts of RNA in each lane, blots were rehybridized to a chick β-actin cDNA probe. pSCI had a rapid and profound effect on levels of FSH-β mRNA: 2 h after inhibin treatment FSHβ mRNA was reduced to 38% of control, and at 6 h this RNA was almost undetectable. FSHβ mRNA levels remained significantly suppressed (P < 0.001) but increased gradually to 51% of control at 72 h. Concentrations of mRNA encoding the a-subunit also decreased (P < 0.01), but to a lesser extent (67% of control at 72 h) and with a slower time course. In contrast, LHβ mRNA was slightly increased (109%) at 24 h and slightly decreased (84%) at 72 h. In conclusion, our study indicates that the action of inhibin on FSH synthesis and secretion can be accounted for, at least in part, by decreased levels of mRNA for the FSHβ-subunit and possibly also the α-subunit.
ASJC Scopus subject areas
- Molecular Biology