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Gonadotropin Receptors of the Primate Corpus Luteum. I. Characterization of ¹²⁵I-Labeled Human Luteinizing Hormone and Human Chorionic Gonadotropin Binding to Luteal Membranes from the Rhesus Monkey^*

JUDY L. CAMERON and RICHARD L. STOUFFER

Department of Physiology, University of Arizona Health Sciences Center, Tucson, Arizona 85724

Abstract

Gonadotropin-binding sites of the primate corpus luteum were characterized via specific [¹²⁵I]iodohuman LH ([¹²⁵I] iodo-hLH)/hCG binding to 15,000 x g particulate fractions of luteal tissue removed from rhesus monkeys on days 17–19 of the nonfertile menstrual cycle. [¹²⁵I]iodo-hLH/hCG uptake was directly proportional to tissue concentration and was a function of the pH of the incubation medium. Binding was maximal between pH 6.0-7.5 and diminished sharply above and below this range. Labeled hLH and hCG bound specifically to corpora lutea, but not to the oviduct or prepubertal ovary of the macaque. Unlabeled hLH and hCG inhibited [¹²⁵I]iodo-hLH/hCG uptake in a similar fashion, whereas ovine, bovine, and porcine LH were much less effective. Human FSH, PRL, and the - and/?-subunits of hCG did not compete readily for LH/CG-binding sites. Gonadotropin binding to luteal membranes was time and temperature dependent. The association process followed second order kinetics, with an association rate constant of 0.3 x 10⁵ M"sec"1 at 24 C. The dissociation of gonadotropin from luteal membranes was a slow process, with only 40% of specifically bound [¹²⁵I]iodo-hCG removed within 8 h after a 100-fold buffer dilution. Dissociation did not follow first order kinetics, but was biphasic; the dissociation rate constant was 6.4 x 10^-5 sec"1 for the fast phase and 6.4 x 10^-6 sec"1 for the slow phase. Although excess unlabeled hCG did not alter the dissociation rate, acidification of the buffer to pH 3.5 or the presence of 4 M MgCU dissociated 95% of the bound gonadotropin within 15 min. Specific [¹²⁵I]iodo-hLH/hCG uptake increased in a dose-dependent manner until saturation occurred. Scatchard analysis of equilibrium binding data yielded linear plots. The equilibrium dissociation constants for hLH and hCG were 0.71 x 10^-10 and 0.60 x 10 < up>^-10 M, respectively, when corrected for hormone degradation during incubation. The maximum binding capacities for hLH (8.0 fmol/mg tissue) and hCG (7.1 fmol/mg) were comparable.

The data indicate that during the midluteal phase of the nonfertile menstrual cycle, the macaque corpus luteum contains a homogeneous population of gonadotropin-binding sites that bind the primate gonadotropins hLH and hCG with equally high affinity. These sites display binding properties consistent with those of gonadotropin receptors. We conclude that particulate preparations of macaque corpus luteum provide a novel system for detailed investigation of primate gonadotropin receptors, particularly with regard to potential changes in the receptor population during the lifespan of the corpus luteum of the menstrual cycle and gonadotropin receptor activation of adenylyl cyclase.

Footnotes

^* This work was supported by NIH Grants HD-12333, BRSG RO- 5675, and HL-07249 and an Upjohn graduate fellowship in physiology.

resent address: Department of Physiology and Biophysics SJ-40, School of Medicine, University of Washington, Seattle, Washington 98195.

To whom requests for reprints should be addressed

Received July 2, 1981.