Follicle-stimulating hormone-stimulated secretion of an immunoreactive 29 kDa inhibin alpha-subunit complex, but not of 32 kDa bioactive inhibin, from cultured immature rat Sertoli cells.

The medium of cultured Sertoli cells from immature rat testes contains 29 and 32 kDa proteins, which are recognized by an antiserum against the 22 N-terminal amino acids of the inhibin alpha-subunit. These proteins were detected by immunoprecipitation of labelled proteins after incubation of Sertoli cells with [35S]methionine, and by Western blotting. The amount of the 32 kDa protein was not affected by the addition of follicle-stimulating hormone (FSH) to the culture medium of the Sertoli cells, whereas FSH induced a large increase of the amount of the 29 kDa protein. Finally, the 29 and 32 kDa proteins in the medium from control and FSH-stimulated Sertoli cells were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis, and inhibin bio- and immunoactivity were determined in eluates of the slices of the gel. Equal amounts of bioactivity were found in control and FSH-stimulated samples at 32 kDa, while the amount of immunoactivity at 29 kDa was increased; no bioactivity was detected in the eluates of these slices. It is concluded that FSH stimulates the secretion of a 29 kDa inhibin-like protein, which does not contain inhibin bioactivity. This indicates that results of experiments, in which antibodies against N-terminal peptides of the inhibin alpha-subunit are used to detect inhibin, do not necessarily reflect the amount of bioactive inhibin produced.

Inhibin and related proteins: localization, regulation, and effects.

Inhibin has originally been defined as a gonadal hormone that exerts a specific negative feedback action on the secretion of FSH from the gonadotropic cells of the pituitary gland. The existence of inhibin was postulated by Mottram and Cramer (15) as early as 1923. However, only after reliable and sensitive bioassay systems had been developed for detection and estimation of inhibin and an ample source of inhibin was found in the form of ovarian follicular fluid, was progress made on the isolation and characterization of the hormone. It is apparent now that inhibin, which itself consists of a dimer of two different subunits, alpha and beta, is a member of a much larger family of (glyco)protein hormones and growth factors that includes Müllerian inhibiting substance, transforming growth factor-beta, activin/erythroid differentiation factor, bone morphogenetic proteins, and an insect and a Xenopus protein. All play important roles in cell differentiation. Gonadal inhibin is produced in the Sertoli cells in the testis and in the granulosa cells in the ovary. The production of inhibin is stimulated by FSH, but controversy exists about other factors that might play a role in the regulation of the production of inhibin. It appears likely that inhibin plays an important role in the feedback regulation of peripheral concentrations of FSH during the period in which Sertoli cells and granulosa cells--the target cells for FSH--divide, i.e., during puberty in male animals and during the development of ovarian follicles in female animals. In this way, inhibin may be an important regulator of the number of developing Sertoli cells and of the length of the seminiferous tubuli in the testis and of the number of developing follicles in the ovary. Apart from its function in the pituitary-gonadal axis, inhibin and activin may be produced and act in a number of other organs such as the placenta, hypothalamus, adrenal, and bone marrow. Investigation of the role of the members of the inhibin family in these systems has only begun, but will certainly be a field of major interest in the near future.

Further characterization of the effects of hypophysectomy, FSH and estrogen on LH stimulation of testosterone production in Leydig cells isolated from immature rats.

Hypophysectomy of immature rats results after 5 days in a loss of LH responsiveness of Leydig cells. LH responsiveness can be partly maintained by treatment with FSH for 5 days. When estradiol benzoate was administered together with FSH to hypophysectomized rats the maintenance of LH responsiveness was not observed. The loss in LH responsiveness after hypophysectomy in terms of testosterone production could not be explained by either a change in the amount of Leydig cells present in the Leydig cell preparation or to a higher conversion of testosterone. The LH-stimulated cAMP production in cells from hypophysectomized rats was very low compared to cells from intact rats. There was no difference between cAMP production of Leydig cells from untreated, FSH-treated or FSH plus estradiol benzoate treated hypophysectomized rats. During the first 2 days after hypophysectomy LH responsiveness in both untreated and FSH-treated rats showed a comparable decrease. From day 2 after hypophysectomy LH responsiveness remained at a constant level in cells from rats treated with FSH, but declined further in cells from untreated rats. A single injection of estradiol benzoate to hypophysectomized rats treated with FSH counteracted the effect of FSH on LH responsiveness, but only when estradiol was administered at that time after hypophysectomy, when the effect of FSH on LH responsiveness was clear.

Hormonal regulation of LH stimulation of testosterone production in isolated Leydig cells of immature rats: the effect of hypophysectomy, FSH, and estradiol-17beta.

Testosterone production in isolated Leydig cells from testes of immature and adult rats was stimulated by addition of LH in a dose dependent way. Hypophysectomy of adult rats had no influence on LH-stimulated testosterone production in isolated Leydig cells after 5 days. In contrast hypophysectomy of immature rats resulted after 5 days in an almost complete loss of LH sensitivity of isolated Leydig cells. Daily adminitration of FSH during 5 days starting immediately after hypophysectomy maintained LH responsiveness of isolated Leydig cells of immature rats. Also FSH administration starting on day 5 after hypophysectomy resulted in a restoration of LH responsiveness. Estradiol benzoate, injected simultaneously with FSH, abolished the FSH-induced LH responsiveness.