Identification of proteins in pooled human follicular fluid which suppress follicular response to gonadotropins.
To evaluate the role of nonsteroidal, follicular fluid proteins in folliculogenesis, the 10-55% saturated ammonium sulfate fraction of pooled human follicular fluid was dialyzed against 0.025 M Tris/HCl (pH 7.5) using 10,000 molecular weight exclusion membranes, then passed through agarose immobilized textile dye. Activity was determined by test fraction inhibition of human menopausal gonadotropin (2 U human LH/FSH . day), induced ovarian weight, and serum estradiol increase in hypophysectomized, diethylstilbesterol-treated, 25-day-old female rats. Specific inhibition (89 +/- 6.8% SEM) of ovarian weight increase was found in the material (2 ml) eluted from an Orange A column with KCl (1.5 M, pH 6.8). Inhibitory activity of the Orange A-bound material, which eluted through a standardized Sephadex G-50 column, corresponded to a molecular weight of 13,000-25,000. Isoelectric focusing on a Sephadex G-15 support bed or ampholyte displacement chromatography of Orange A bound material demonstrated inhibitory activity at pH 3.5-4.5 and 6.5-7.0. No demonstrable activity was found in similar fractions eluted through a Concanavalin A-Sepharose 4B column before or after addition of alpha-methyl-D-mannoside (2 M, pH 7). When active fractions were heated (56 C, 1 h) or exposed to trypsin (10 mg/100 ml), activity was lost. When aliquots of the saturated ammonium sulfate-extracted, dialyzed, Orange A-bound eluent were separated by high performance liquid chromatography using gel exclusion columns, activity in the bioassay was recovered in the 13,000-35,000 molecular weight range. Although confirmatory data await further studies, it is tempting to speculate that this protein(s) may be an important inter- and/or intraovarian regulator of follicular response to gonadotropins.
D000328:Adult; D000818:Animals; D001681:Biological Assay; D001826:Body Fluids / Q000032:analysis*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004958:Estradiol / Q000097:blood; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing; D008596:Menotropins / Q000494:pharmacology*; D009929:Organ Size / Q000187:drug effects; D006080:Ovarian Follicle / Q000032:analysis* / Q000502:physiology; D010053:Ovary / Q000033:anatomy & histology / Q000187:drug effects*; D011506:Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains
D011506:Proteins; D004958:Estradiol; D008596:Menotropins