Distribution and characterization of insulin and insulin-like growth factor I receptors in normal human ovary

Insulin-resistant hyperinsulinemic states are now widely known to be associated with ovarian hyperandrogenism, and this is thought to be due to an action of insulin on the ovary. However, the identity of the receptor that is responsible for insulin action in these patients, whose insulin receptors on classical target tissues are severely impaired, is unclear. We now report the presence of insulin receptors in stromal and follicular compartments as well as in granulosa cells obtained from normal ovaries. After 15-h incubations at 4 C with [125I]insulin and tissue fragments, specific insulin binding was 6-19% and 7-13%/mg protein (n = 8) to stroma and theca, respectively. Granulosa cells obtained in the course of in vitro fertilization were separated from red cells on a Percoll gradient; specific insulin binding ranged from 9-15%/10(6) cells. Insulin binding was characterized by sensitive insulin competition (half-maximal, 10 ng/ml), appropriately shifted proinsulin competition (20 times to the right), and complete inhibition by specific anti-insulin receptor antibodies (B-2). An antibody to the insulin-like growth factor I (IGF-I) receptor (alpha IR-3) that inhibits IGF-I binding to IGF-I receptors in other cell systems had no effect on insulin binding. Further proof that this binding is to classic insulin receptors was obtained from measurement of insulin-stimulated receptor autophosphorylation. When insulin receptors from stroma were extracted with Triton X-100 and incubated with [gamma-32P]ATP and Mn, insulin increased the incorporation of 32P into the beta-subunit of the receptor 5-fold. In parallel studies with [125I-]IGF-I and specific blocking antibodies to its receptor, no detectable IGF-I binding to stroma or follicles was found. We conclude that specific high affinity insulin receptors possessing tyrosine kinase activity are widely distributed in normal human ovary. IGF-I receptors in normal ovary are either absent or present at very low density. Binding of insulin to its own receptor (as opposed to IGF-I receptors) appears to be the most likely first step in the stimulation of ovarian steroidogenesis by insulin in normal ovaries and possibly in insulin-resistant states as well.