Localization of Inhibin and Activin Binding Sites in the Testis during Development by in Situ Ligand Binding

Abstract

Inhibin and activin are related proteins thought to be potential paracrine regulators of testicular development and maintenance of spermatogenesis. Messenger RNA and proteins immunologically related to both factors have been identified in the adult testis. However, the role(s) of these factors in paracrine regulation of testicular function is poorly understood. To identify potential targets for inhibin and activin in immature and adult testis, we used in situ binding of [125I]-labeled ligands to localize and describe the distribution of binding sites for inhibin and activin in testes of 15-, 18-, 21-, 30-, 45-, and 60-day-old rats. Nonspecific binding was defined as that occurring in the presence of a 1000-fold excess of unlabeled recombinant human (rh) inhibin or activin. [125I]-Inhibin was found to bind to interstitial cells throughout development. Inhibin binding was shown to co-localize with cells that showed positive staining for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). Competition studies demonstrated that this binding was indeed specific for inhibin. In contrast, [125I]-activin showed two distinct patterns of binding. First, [125I]-activin was shown to bind in a non-stage-dependent manner to cells located in the basal compartment of the seminiferous tubules in testis obtained from animals of all ages studied. Binding of [125I]-activin in the periphery of the tubule could be inhibited entirely by coincubation with excess unlabeled activin and partially with excess unlabeled inhibin. The ability of inhibin to compete with activin for binding appeared to be more pronounced in younger animals. In 45- and 60-day-old animals, a second stage-dependent component of [125I]-activin binding was also apparent. This binding was localized to spermatids found in stage VII-VIII tubules and was inhibited by the presence of excess activin, but not inhibin. These results indicate that inhibin can bind specifically to testicular interstitial cells throughout development and may be an important regulator of Leydig cell testosterone production or interstitial cell function. In contrast, activin appears to bind in a specific and stage-dependent manner to receptors or high-affinity binding proteins on spermatids as well as to sites on the periphery of all seminiferous tubules. These results support the hypothesis that both activin and inhibin may act at several levels to regulate proliferation or differentiation of germ and Sertoli cell function as well as to modulate interstitial cell activity.

Age-Related Changes in the Serum Levels of Inhibin, FSH, LH and Testosterone in Holstein Bulls.

“…On the other hand, serum inhibin levels were low and were relatively constant in mature bulls tested in the present study. Thus, although recent evidence supports an autocrine or paracrine role for inhibin in regulating testicular function [12,13], inhibin does not seem to be directly involved in the improvement of the quantity and quality of semen. In summary, the present study showed agerelated changes in serum concentrations of inhibin, gonadotropins and testosterone.…”

Expression of Inhibin a-Subunit in Horse Testis.

“…In testis, it is reported that inhibin produced in Sertoli cells affects spermatogonial proliferation [9,30] and is related to the modulation of LH-stimulated androgen biosynthesis [3,27,33]. In fact, inhibin was taken up by spermatocytes in human testis [31], and inhibin-binding sites are localized in rat Leydig cells [13]. These reports suggest that inhibin produced in Leydig cells might play a paracrine/autocrine role in the regulation of androgen production.…”

The role of inhibins and activins in prostate cancer pathogenesis.

“…Delineation of an intracellular signalling pathway for inhibin awaits unequivocal proof of a receptor for inhibin, about which controversy regarding its very existence persists. The evidence presented supporting the existence of an inhibin receptor is based on the identification of specific binding sites for inhibin on ovarian granulosa cells and Leydig cells (Woodruff et al 1990, Krummen et al 1994. Additional supporting data for the existence of a specific receptor include the demonstration of an inhibin-specific protein complex, k562, in haematopoietic cell lines (Lebrun & Vale 1997) and tumour-derived inhibin receptor proteins in the Matzuk inhibin knockout mice (Draper et al 1998).…”