Peripubertal decline in ovarian LHRH receptor content: Characterization and distribution

In the rat, ovarian LHRH receptor content, as measured by the binding of ¹²⁵I-D-Ala⁶, Pro⁹-LHRH (A-LHRH) to ovarian membranes, decreases markedly during the days preceding the first preovulatory gonadotropin surge. The present study shows that the decrease in LHRH receptor number occurs exclusively in granulosa cells, and that the greatest rate of decline takes place between the juvenile period and the early proestrous phase of puberty. In contrast to granulosa cells, membranes prepared from ovarian tissues after removal of these cells demonstrated no significant change in LHRH binding capacity throughout puberty. Scatchard analysis of competition curves, constructed using membranes from whole ovaries, revealed that receptor affinity remains unchanged (K(a) = 9 x 10⁹ M^-1) as the receptor number decreases. Kinetic binding studies demonstrated that LHRH binding to its receptor reaches equilibrium within 40 min and can be displaced by addition of unlabeled hormone. A kinetic estimate of receptor affinity provided values similar to those obtained by Scatchard analysis. Treatment of ovarian membranes with MgCl² to uncover occupied binding sites results in more than 70% dissociation of bound labeled ligand. Dissociation of endogenous LHRH using this procedure resulted in some increase in available binding sites only in juvenile rats, but not in first proestrous or first estrous animals in which receptor content was low. The results demonstrate that the decline in ovarian LHRH receptors observed during peripubertal days is confined to granulosa cells, is not due to changes in receptor affinity, and represents a true loss of binding sites.