Validation of methods and quantification of luteal receptors for LH throughout the estrous cycle and early pregnancy in ewes

In order to understand better the role played by variation in the number of receptors for luteinizing hormone (LH) in the regulation of progesterone secretion from the ovine corpus luteum, methods were developed to quantify the total number of receptors for LH and the degrees to which these receptors were occupied by endogenous hormone. The number of unoccupied receptors for LH in the 30,000 x g fraction of ovine corpora lutea was determined using 1 2 5 I-hCG and Scatchard analysis. To establish the validity of the methods used for Scatchard analysis, the 1 2 5 I-hCG was characterized carefully for specific activity, maximal bindability to receptor and biological activity. The assay procedure used was also carefully evaluated to insure that background radioactivity was correctly monitored and that binding of hormone to receptor had reached equilibrium. The number of receptors occupied by endogenous LH was quantified by elution of the bound LH with formic acid and quantification of hormone by radioimmunoassay. The number of unoccupied and occupied receptors for LH in a 30,000 x g membrane fraction was determined for corpora lutea collected from 6 ewes each on Days 4, 6, 10, 12, 14 and 16 of the cycle and in 6 pools (3 corpora lutea/pool) collected from 18 ewes on Day 2 of the cycle. Both the number of unoccupied and the number of occupied LH receptors were highly correlated with serum and luteal progesterone concentrations and luteal weight (P