Gonadotropin Binding and Aromatase Activity in Granulosa Cells of Young Proestrous and Old Constant Estrous Rats1

This study was designed to determine whether chronic anovulation in old constant estrous rats is associated with alternations in gonadotropin binding and/or aromatase enzyme activity of granulosa cells. Granulosa cells, isolated from mature Graafian follicles of old constant estrous rats (25–26 months of age) and young proestrous rats (4–5 months of age), were tested for their capacity of 1) bind human [125I] iodo-chorionic gonadotropin specifically and to 2) aromatize androstenedione into estrogens. Scatchard plot analysis of binding data indicated specific, high affinity, hCG receptor sites on granulosa cells of both young and old rats and there was no significant difference in the binding parameters between the two groups. The apparent dissociation constant (Kd) of the high affinity receptor was 4.3 (±0.6) × 10−11 M and 5.0 (±0.7) × 10−11 M for granulosa cells from young and old rats, respectively. The number of hCG binding sites/granulosa cell was determined to be 8,772 (±708) and 10,408 (± 1,854) from young and old rats, respectively. The aromatase activity was comparable in granulosa cells of young and old rats. After a 5 h incubation with increasing concentrations of androstenedione, a dose related increase in estrogen production was observed with 10−9 and 10−7 M androstenedione causing an approximate 2- and 30-fold increase in estrogen production. Lineweaver-Burk plot analysis of saturation curves gave similar Km (1 × 10−3 M) and Vmax (8 pmoles/5 h) values for the aromatase enzymes in both age groups. Addition of purified FSH and/or LH (200 ng/ml) did not enhance estrogen production suggesting the granulosa cell aromatase activity was maximal in mature follicles of young and old rats. Since no significant difference was found in gonadotropin binding and aromatase activity in granulosa cells from mature follicles of young proestrous and old constant estrous rats, it would appear that chronic anovulation in aging female rats is not related to deficiencies in the ability of the granulosa cell to produce estrogen and bind LH.