Validation of Methods and Quantification of Luteal Receptors for LH throughout the Estrous Cycle and Early Pregnancy in Ewes1 (1978)

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 × g fraction of ovine corpora lutea was determined using 125I-hCG and Scatchard analysis. To establish the validity of the methods used for Scatchard analysis, the 125I-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 × 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<0.01). The number of LH receptors (both occupied and unoccupied) was less during the early (Days 2–6) and late (Day 16) luteal phases than during the mid-luteal (Days 10–14) phase of the estrous cycle (P<0.05). The total number of LH receptors per corpus luteum increased approximately 40-fold from Day 2 to Day 10 of the cycle while the number of receptors occupied by LH increased 6-fold. This represents an increase of 3.3 × 1010 molecules of LH bound to receptors per corpus luteum. On Day 10 of the cycle, 0.6% of the total LH receptors were occupied by hormone. The affinity constant of the receptors for LH was the same on each day of the cycle. These data indicate that the number of receptors occupied by LH increases during the mid-luteal phase of the cycle due to increased total receptors and that this increase in occupied receptors correlates well with enhanced progesterone secretion. During regression of the corpus luteum, both the total number of receptors and the number occupied by LB decreases. However, it was not possible to determine if a decrease in LH receptors was involved in the mechanism of luteal regression. The number of unoccupied and occupied receptors for LH was also determined in corpora lutea from 6 ewes each on Days 12, 16 and 20 of pregnancy. There were no differences in the total number of receptors, the number of occupied receptors or the affinity constant on any of the days of pregnancy. In addition, there was no difference in these parameters when luteal tissue from ewes on Day 12 of the cycle was compared to tissue from ewes on Day 12 of pregnancy. Therefore, neither increased number of LH receptors, increased binding of LH to receptor nor an increase in the affinity of the receptor for LH appears to be the signal for the maintenance of the corpus luteum of pregnancy.