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 radiolabeled 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 microns). 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 on 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 metestrus and diestrus. In addition, [125I]rh-activin-A bound to the cumulus-oocyte 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-microns 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Woodruff
T K
TK
Department of Cell Culture Research and Development, Genentech, Inc., South San Francisco, California 94080.
Krummen
L
L
McCray
G
G
Mather
J P
JP
D028341:Activins; D000818:Animals; D003338:Corpus Luteum / Q000378:metabolism; D005260:Female; D017403:In Situ Hybridization; D007265:Inhibins / Q000235:genetics / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008024:Ligands; D009865:Oocytes / Q000378:metabolism; D006080:Ovarian Follicle / Q000378:metabolism; D010053:Ovary / Q000378:metabolism*; D010060:Ovulation; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D014599:Uterus / Q000378:metabolism
D007457:Iodine Radioisotopes; D008024:Ligands; D012333:RNA, Messenger; D011994:Recombinant Proteins; D028341:Activins; D007265:Inhibins