Inhibition of follicle-stimulating hormone/diethylstilbestrol-stimulated ovarian growth by extracts of pregnancy urine.
We devised an in vivo biological assay for ovarian growth inhibiting activity to examine extracts of human pregnancy urine for the presence of ovarian growth inhibiting factor. Diethylstilbestrol (DES) capsules were implanted sc in immature hypophysectomized female rats; FSH was injected sc with or without test substance for 5 days. Rats with unstimulated ovaries were implanted with blank capsules and given the vehicle without FSH. Twenty four hours after the last injection, the ovaries were removed and weighed. The ovarian growth inhibition of the ovarian weight gain achieved in rats treated with DES and FSH. Crude commercial human CG (hCG) preparations, extracted from pregnancy urine, were chromatographed on Sephadex G-100, and the fractions were tested for ovarian growth inhibiting activity. The peak of ovarian growth inhibiting activity was found in fractions eluting from the column in the mol wt range of 12,000-20,000. Ovarian growth inhibiting activity was heat sensitive, not extracted by ether, and precipitated by acetone. Ovarian growth inhibiting activity was stable in acid at pH 2, but was inactivated by digestion with trypsin. The ovarian growth inhibiting activity was purified by chromatography on Concanavalin A-Sepharose and diethylaminoethyl-Sephacel. The active material contained hCG alpha, hCG beta, and beta-carboxyterminal peptide-immunoreactivity and its inhibiting activity could be removed from solution by immunoadsorption with antisera specific for hCG beta. The ovarian growth inhibiting activity was further purified on an anti-hCG alpha-immunoglobulin G affinity column. The activity was eluted from the affinity column at low pH, and eluted material contained all of the immunodeterminants of hCG. Virtually identical dose-response curves of ovarian inhibition were obtained using equivalent doses of beta-carboxyterminal peptide immunoreactivity of purified inhibitor and purified hCG (CR123). The inhibiting activity reached plateau of 80-90% at doses of 50-100 ng hCG/rat. Upon rechromatography on Sephadex G-100, the ovarian growth activity that was pooled from fractions corresponding to the 12,000-20,000 mol wt range was recovered in fractions corresponding to the elution position of hCG. We conclude that the low mol wt inhibiting activity observed in the crude pregnancy extracts is due to hCG and that hCG is a very potent inhibitor of FSH/DES stimulation of ovarian growth.
D000096:Acetone; D000818:Animals; D001681:Biological Assay; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D002846:Chromatography, Affinity; D004054:Diethylstilbestrol / Q000037:antagonists & inhibitors*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000037:antagonists & inhibitors*; D007163:Immunosorbent Techniques; D006080:Ovarian Follicle / Q000187:drug effects / Q000254:growth & development*; D011247:Pregnancy / Q000652:urine*; D051381:Rats; D011919:Rats, Inbred Strains
D006063:Chorionic Gonadotropin; D000096:Acetone; D004054:Diethylstilbestrol; D005640:Follicle Stimulating Hormone