Effect of Inhibin from Primate Sertoli Cells and GnRH on Gonadotropin Subunit mRNA in Rat Pituitary Cell Cultures

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Abstract
Partially purified inhibin from primate Sertoli cell culture medium (pSCI) suppresses both LH and FSH secretion from cultured rat pituitary cells stimulated with GnRH. To examine the mechanism of action of pSCI, we have measured steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures exposed to 10 nM GnRH for 6 h in control or pSCI-containing medium (short term) and after 72-h pretreatment with pSCI or control medium (long term). Messenger RNA levels were determined by Northern analysis using specific cDNA probes for rat FSH.beta., LH.beta., and the common .alpha.-subunit. In the long term experiments, pSCI inhibited GnRH-stimulated release of FSH (47.4 .+-. 3.3% of control), LH (69.2 .+-. 2.3%), and free glycoprotein .alpha.-subunit (74.2 .+-. 4.5%), and intracellular FSH declined to 88.4 .+-. 3.5% of control. Concentrations of the subunit mRNAs were all decreased: FSH.beta. to 54.4 .+-. 5.0%, LH.beta. to 79.6 .+-. 9.4%, and .alpha. to 70.8 .+-. 8.7% of control. In the short-term experiments, pSCI also suppressed FSH, LH, and .alpha.-subunit secretion to 75.9 .+-. 3.6%, 79.5 .+-. 2.1%, and 90.9 .+-. 1.8% of control, respectively. Intracellular LH and .alpha.-subunit levels were significantly increased in cells treated for 6 h with GnRH and pSCI (155 .+-. 18%, 145 .+-. 14% of control), while FSH was comparable to control. After 6 h, pSCI selectively reduced the level of mRNA for FSH.beta. (56.5 .+-. 5.8% of control). In a separate series of experiments to assess the effect of GnRH on the subunit mRNAs, cultures received 10 nM GnRH alone, GnRH and pSCI, or vehicle for 6 h. FSH release was stimulated 3- to 4-fold by GnRH, LH release, 30- to 40-fold, and free .alpha.-subunit release, 2- to 3-fold. GnRH itself elevated levels of LH.beta. and .alpha. mRNAs to 113 .+-. 4% and 135 .+-. 7% of unstimulated controls; in contrast, FSH.beta. mRNA was decreased by GnRH (64.2 .+-. 8.5%), and this effect was enhanced by the simultaneous presence of pSCI (31.2 .+-. 4.3%). The effect of pSCI on GnRH receptor (GnRH-R) binding capacity was also determined by incubating cell homogenates with [125I]iodo-(D-Ala6,Des-Gly10)GnRH ethylamide: GnRH-R were decreased slightly (77% of control) at 24 h and extensively (17% of control) at 72 h. In conclusion, 1) both short- and long-term effects of pSCI on GnRH-induced FSH secretion can be accounted for partly by decreased levels of FSH.beta. mRNA; long-term effects may also involve reductions in GnRH-R; 2) long term effects of pSCI on LH and .alpha.-subunit secretion may involve decreases in LH.beta. and .alpha. mRNAs as well as GnRH-R; 3) GnRH itself differentially regulates the levels of mRNAs for the gonadotropin subunits.
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