In situ ligand binding of recombinant human [¹²⁵I] activin-A and recombinant human [¹²⁵I]inhibin-A to the adult rat ovary

Inhibin and activin are hormones produced by ovarian follicles. Specific ovarian cells that bind iodinated recombinant human (rh)-activin-A and rh-inhibin-A were identified by in situ ligand binding. Iodinated rh-molecules were incubated with tissue sections from ovaries, uterus, and oviduct, collected on the mornings of metestrus, diestrus, proestrus, and estrus and the evening of proestrus. Additionally, inhibin/activin subunit mRNA and follistatin mRNA accumulation was examined by in situ hybridization of radiolabled antisense riboprobes. The cellular site of activin/inhibin binding could thus be colocalized to the cellular site of ligand mRNA and binding protein mRNA production. The association of both [¹²⁵I]rh-activin-A and [¹²⁵I] rh-inhibin-A with specific cell types varied across the rat estrous cycle. Nonspecific binding was evaluated by competition with a 1000-fold excess of homologous ligand, and low affinity association of the heterologous ligand was evaluated by competition with a 1000-fold excess of heterologous ligand. [¹²⁵I]rh-activin-A binding was more widespread than was [¹²⁵I]rh-inhibin-A binding under our experimental conditions. [¹²⁵I]rh-Activin-A bound to the granulosa cells of all stages of follicles: unrecruited, growing, and Graafian follicles. Thecal cell binding was found in developing follicles (350-500 μm). The granulosa cells of stimulated follicles (evening of proesterus) bound less [¹²⁵I]rh-activin-A than those of unstimulated follicles. [¹²⁵I]rh-Activin-A binding was also associated with antral fluid of follicles in each size class. Although early atretic follicles retained some [¹²⁵I]rh-activin-A binding, late atretic follicles did not bind [¹²⁵I]rh-activin-A. Corpus luteum present on metestrus and diestrus bound [¹²⁵I]rh-activin-A; however, corpus lutea present or proestrus and estrus bound little or no [¹²⁵I]rh-activin-A. [¹²⁵I]rh-Activin-A-binding sites were also present in the uterus and oviduct in a cycle-dependent manner. The highest levels of binding were found in the muscle wall of the uterus and the epithelial lining of the thick- | walled portion of the uterus on etestrus and diestrus. In addition, [¹²⁵I]rh-activin-A bound to the cumulus-ooctye complex present in the oviduct on metestrus, but did not bind to the oocytes present in developing follicles. Binding of [¹²⁵I]rh-inhibin-A was restricted to the antral granulosa cells of 450- to 500-μm follicles. No other ovarian, uterine, or oviduct cells bound [¹²⁵I]rh-inhibin-A. [¹²⁵I]rh-Activin-A and [¹²⁵I]rh-inhibin-A ligand binding was associated primarily with follicles coexpressing inhibin/activin subunit and follistatin mRNA. The current data support the hypothesis that inhibin/activin have paracrine activities in the ovaries.