Binding characteristics of 125I-labelled human FSH to granulosa cells from Booroola ewes which were homozygous, heterozygous or non-carriers of a major gene(s) influencing their ovulation rate

Abstract

At 37.degree.C 125I-labelled human (h) FSH (NIAMDD-hFSH-I-3) bound rapidly to granulosa cells from Booroola and Romney ewes with 50% maximum binding achieved after 3 min and equilibrium being reached within 45 min, irrespective of whether the cells were obtained from the FF, F+ or ++ Booroola genotypes or from Romney ewes. Bidning of 125I-labelled FSH followed second order kinetics and there was no effect of follicle diameter (1-2.5 mm vs .gtoreq. 3 mm). Irrespective of breed, genotype or follicle size, the mean (.+-. s.e.m.) calculated association rate constant, (ka) was 7.3 (.+-.0.8) .times. 105 liters mol-1 sec-1 (n = 12). Dissociation of receptor bound 125I-labelled hFSH was <5% after 30 min and low but variable (i.e. between 0 and 30%) after 2-6 h irrespective of breed, genotype or follicle size. No gene-specific differences were noted in binding specificity between F+ and ++ genotypes: studies were not performed with cells from FF ewes because of insufficient cells. The binding of 125I-labelled hFSH could be displaced with sheep FSH (NIH-FSH-S16; 10% cross-reaction) and FSH-P (2.5% cross-reaction) but other sheep pituitary hormones and hCG showed little or no cross-reaction (.gtoreq. 0.1%). The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hFSH binding to granulosa cells did not differ between the Booroola genotypes or between Booroola or Romney follicles of different diameter (i.e. 1-2.5 mm; or .gtoreq. 3 mm). The overall mean .+-. s.e.m. (n = 24) Bmax and Kd values were 16.7 .+-. 0.8 fm/mg protein (i.e. .apprx. 800 available receptor binding sites/cell) and 1.1 .+-. 0.1 nM respectively. Collectively, these findings suggest that the earlier maturation of follicles in FF or F+ ewes compared to ++ ewes is unlikely to be due to gene-specific differences in the FSH binding characteristics of the granulosa cells.