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Inhibition of Luteinizing Hormone/Human Chorionic Gonadotropin-Stimulable Adenylyl Cyclase Activity in Rat Luteal Membranes by Nonsteroidal Component(s) in Human Follicular Fluid^*

Departments of Obstetrics and Gynecology and Biochemistry, The University of Texas Health Science Center at San Antonio San Antonio, Texas 78284

Address all correspondence and requests for reprints to: Dr. F. J. Rojas, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284.

We examined the ability of nonsteroidal components of human follicular fluid (hFF) to alter gonadotropin responsiveness using the LH/hCG-sensitive adenylyl cyclase system of rat luteal membranes. Follicular aspirates were obtained from regularly ovulatory women (n = 10) whose follicles were stimulated by human menopausal gonadotropin and hCG as part of an in vitro fertilization program. hFF from large follicles was pooled and extracted with 10% (wt/vol) activated charcoal. Maximal hCG stimulation of adenylyl cyclase activity obtained with 10 µg/ml hCG and 100 µM of the hydrolysisresistant GTP analog guanyl 5 '-yl-imidodiphosphate was significantly inhibitedby hFF in a dose-dependent manner. Addition of about 500 µg hFF protein caused inhibition of 70% compared to the control value. Fractionations of hFF by ultrafiltration using membranes of precalibrated pore size demonstrated that the inhibitory activity was associated with a less than 10,000 mol wt fraction; 3 µg protein/assay of this fraction resulted in 50% inhibition (IC₅₀) of maximal hCG stimulation. The inhibitory activity also passed through an Amicon YM-2 membrane (mol wt retention, 1,000), but not through an Amicon YC-05 membrane (mol wt retention, 500). An IC₅₀ of about 0.01 µg protein/assay was found for both the 500-1,000 and the 1,000- 5,000 mol wt fractions. NaF or forskolin-stimulated adenylyl cyclase activity was not altered by unfractionated hFF or by the 500–10,000 mol wt subfractions, suggesting that inhibition was limited to LH/hCG stimulation. Further analysis of the effects of low mol wt fraction on hCG stimulation of adenylyl cyclase indicated that enzyme inhibition was not accompanied by a shift in the hCG concentration required for half-maximal stimulation (the apparent activation constant) compared to dose-response curves obtained in the absence of added fraction. Equilibrium binding studies showed that [¹²⁵I]hCG interaction with luteal membranes was significantly inhibited by hFF; 7 µg protein/ assay of the less than 10,000 mol wt fraction reduced specific binding by 60%. Moreover, kinetic analysis carried out in the absence or presence of a fixed amount of low mol wt fractions revealed a competitive type of binding inhibition. Our data demonstrate that a nonsteroidal component(s) of hFF has a direct inhibitory effect on LH/hCG-responsive luteal adenylyl cyclase and that the inhibitor(s) exerts its actions through a mechanism involving competition with LH/hCG for the same binding sites.

^* Presented in part at the 67th Annual Meeting of The Endocrine Society, Baltimore, MD, June 1985.

Received October 29, 1985.