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The follicle stimulating activity (FSA) and interstitial cell stimulating activity (ICSA) of highly purified human chorionic gonadotropin (hCG), its alpha and beta subunits, and hCG generated by subunit recombination were determined by ovarian weight and ventral prostate weight bioassays. Whereas highly purified hCG exhibited both FA and ICSA, its separated subunits were essentially devoid of both activities. ICSA and FSA, indistinguishable from that of the highly purified hCG, were restored by recombination of the hCG subunits. These observations are consistent with the hypothesis that the FSA and ICSA found in highly purified hCG preparations are properties of the hCG molecule.

In connection with systematic studies of steroid and peptide hormone interactions during follicular growth, we have measured ovarian weight responses to graded doses of highly purified human chorionic gonadotropin (hCG), human interstitial cell stimulating hormone (hICSH)in hypophysectomized immature female rats (HIFR) treated with diethylstilbestrol in silastic capules (desc) implanted subcutaneously. Our results are consistent with earlier reports of enhancement of ovarian weight responses to hCG and FSH. Contrary to results of similar experiments reported by others, we have found that estrogen treatment of HIFR enhanced ovarian weight response to ICSH. In addition, we report for the first time that small doses of hCG and hICSH inhibit ovarian weight responses to estrogen in HIFR. Our observations on effects of small doses of hCG and hICSH and the long-known fact that ovarian interstitial cells are stimulated in HIFR given similar doses of these hormones lead us to hypothesize that ovarian interstitial cell stimulation is involved in the control of follicular maturation.

Inhibition by antiestradiol serum of ovarian weight gain and follicular growth in hypophysectomized immature rats given FSH and hCG suggested that gonadotrophin induced endogenous estrogen secretion plays a role in the ovarian augmentation reaction. We have studied the effects of a chemical estrogen antagonist, cis-clomiphene, on ovarian weight response to gonadotrophins in hypophysectomized immature female rats. We found that this antiestrogen inhibits the ovarian response to FSH and hCG. To our knowledge, this is the first demonstration of a direct effect of a chemical antiestrogen on the ovary, a result consistent with a role for intraovarian estrogen in follicular growth.

In hypophysectomized immature female rats (HIFR), the ovarian weight response to subcutaneously implanted diethylstilbestrol capsules (DESC) is inhibited by small doses of human chorionic gonadotropin (hCG). This effect, reproduced by equivalent doses of interstitial cell stimulating hormone (ICSH) but not by follicle-stimulating hormone (FSH), is inhibited by treatment with antiandrogens. These data implicate gonadotropic stimulation of interstitial cell androgen production in the control of follicular maturation in rats.

The concentration of testosterone (T) and 5alpha-dihydrotestosterone (DHT) in plasma, seminal fluid, and epididymis of male Wistar rats have been determined by radioimmunoassay. The DHT/T ratio rose from 0.1 in blood to 1 in testicular fluid and 4 in epididymal fluid. This ratio is 2 in tissues of "caput" epididymis and 1 in "cauda" epididymis. These data indicate a transformation of T to DHT in the epididymal tissue, particularly at the "caput" level.

We have investigated folluclar atresia by giving hypophysectomized immature female rats (HIFR) diethylstibestrol or gonadotrophins with and without the chemical antiestrogen CI-628, making total counts of normal and atretic follicles greater than 125 muM in diameter, and using a simple model to analyze data. Our results show an antiatretic effect of estrogen, independent of its well-documented mitogenic effect on preantral follicles. We have also shown that CI-628 acts as an anti-estrogen to block follicular proliferation, while acting as an estrogen to inhibit atresia. In addition, we have observed an increase in atresia caused by gonadotrophins, in opposition to their estrogen-mediated positive effect on follicular growth.

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.

Estrogen pretreatment of hypophysectomized immature rats stimulated granulosa cell proliferation and enhanced FSH binding to the ovary in vivo. The present studies were undertaken to ascertain whether estrogen pretreatment altered the number of specific FSH receptor sites per ovarian cell, resulting in increased FSH binding. Cell suspensions were prepared from the ovaries of groups of rats pretreated with graded doses of 17beta-estradiol. The isolated cells contained specific FSH receptors with high affinity for FSH. The results of these studies clearly showed that the number of specific FSH receptors per ovarian cell was unaffected by pretreatment with graded doses of 17beta-estradiol or with diethylstilbestrol. The enhanced in vivo FSH binding was solely the result of estrogen-induced proliferation of granulosa cells with a fixed number of specific FSH receptors per cell.