In order to elucidate the putative role of inhibin in regulation of spermatogenesis, expression of inhibin subunits was examined at defined stages of the cycle of the rat seminiferous epithelium. Twenty 2-mm segments of seminiferous tubules at stages XIII-I, II-VI, VII-VIII, and IX-XII were dissected using the transillumination technique and subunit specific messenger RNAs (mRNAs) were quantitated by filter hybridization. The alpha and beta-B subunit mRNAs varied significantly in different stages, the highest levels of both alpha and beta-B subunit expression were seen in stages XIII-I and the lowest in stages VII-VIII. The hybridization signals obtained with beta-actin probe were not significantly different between different stages indicating that the differences in the quantities of subunit mRNAs in different stages were not due to different amounts of RNA blotted. beta-A subunit mRNA levels were below the detection limit of the filter hybridization method. These data demonstrate that expression of inhibin alpha and beta-B subunits in the rat testis is stage dependent and suggest a paracrine role for inhibin-related peptides in regulation of spermatogenesis.

To examine the pretranslational regulation of inhibin subunits in the rat testis by FSH, we studied the effects of hypophysectomy with or without selective FSH replacement on testicular inhibin subunit mRNA levels in immature and adult animals. In the first experiment (Exp I), sexually immature (20-23 days old) intact and hypophysectomized male rats were killed 1, 3, and 7 days after surgery, and the testicular content of inhibin subunit mRNAs was determined by filter hybridization. A second group of immature, intact, or hypophysectomized rats was treated with saline or FSH for 7 days as follows: I) intact, saline; II) hypophysectomized, saline; III) hypophysectomized, FSH [0.05 microgram/100 g BW, sc, twice daily (BID)]; IV) hypophysectomized, FSH (0.50 microgram/100 g BW, sc, BID); V) hypophysectomized, FSH (5.0 micrograms/100 g BW, sc, BID); and VI) hypophysectomized, FSH (50.0 micrograms/100 g BW, sc, BID). In the second experiment (Exp II), adult (60 days old) intact or hypophysectomized animals were treated with saline, FSH, and/or testosterone for 7 days as follows: I) intact, saline; II) hypophysectomized; saline; III) hypophysectomized, 22-mm testosterone implant; IV) hypophysectomized, FSH (50.0 micrograms/100 g BW, sc, BID; and V) hypophysectomized, 22-mm testosterone implant plus FSH (50.0 micrograms/100 g BW, sc, BID. The effects of FSH and testosterone on testicular inhibin subunit mRNA levels were measured by filter hybridization. In Exp I, the level of inhibin alpha-subunit mRNA per testis was significantly lower in hypophysectomized rats than in intact controls at all time points after surgery. Replacement of FSH to hypophysectomized immature rats led to a dose-dependent increase in alpha-subunit mRNA per testis. However, hypophysectomy and FSH replacement had no significant effect on beta-B-subunit mRNA. In adult rats (Exp II), hypophysectomy significantly lowered and FSH replacement increased testicular inhibin alpha-subunit mRNA levels. Replacement of testosterone to adult animals, either alone or in combination with FSH, had no effect on expression of inhibin alpha-subunit mRNA. beta-B mRNA levels in adult testis were not significantly altered by any of the treatments. beta-A-Subunit mRNA levels were below the detection threshold of filter hybridization in both Exp I and II. Collectively, these data demonstrate that FSH regulates alpha- but not beta-B-subunit mRNA in the testis of both immature and adult rats in vivo. Differential regulation of inhibin subunits may provide a mechanism for creation and regulation of functional diversity of inhibin-related peptides in the testis.