Identification of Proteins in Pooled Human Follicular Fluid which Suppress Follicular Response to Gonadotropins*

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Abstract
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 KC1 (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 a-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 Abound 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 interand/or intraovarian regulator of follicular response to gonadotropins.