Luteinizing hormone activity in plasma during the menstrual cycle.

Daily determinations of luteinizing hormone activity in plasma throughout a menstrual cycle in ten young women showed a sharp peak of activity lasting less than 48 hours around midcycle and higher mean values during the follicular phase than during the luteal phase in nine instances.

Pregnancies after chemotherapy of trophoblastic neoplasms.

We have analyzed 88 pregnancies in 50 women who had previously been treated for gestational trophoblastic neoplasms with chemotherapeutic agents. No increase in fetal wastage, congenital abnormalities or complicated pregnancies was noted, suggesting that these drugs do not damage human oocytes in the doses and time periods used. The possibility that recessive mutations have been induced but were undetected cannot be evaluated definitively at present.

Monthly gonadotropin cycles in premenarcheal girls.

Patterns of nocturnal excretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were investigated in 11 girls. Autoregressive digital filtering of low- and high-frequency variations was used to make patterns more apparent. Coincident FSH and LH surges, separated by an interval of 20 to 40 days, were seen in specimens from three of six postmenarcheal girls and three to five premenarcheal girls. This suggests that cyclic hypothalamic-pituitary-ovarian interactions occur before menarche.

Radioimmunoassay for luteinizing hormone in human plasma or serum: physiological studies.

It is not practical to quantitate gonadotropin in the blood of normal men and women by utilizing bioassays. We have developed a method for sensitive, precise, and specific radioimmunoassay of luteinizing hormone (LH) in human serum or plasma. Antisera were developed against human chorionic gonadotropin, and one of these was selected for extensive cross-reaction with human LH. Highly purified LH was radioiodinated by the method of Greenwood, Hunter, and Glover. Separation of antibody-bound from free LH-(131)I was accomplished by a double antibody technique. Dose-response curves for the purifed LH, an impure urinary LH preparation, pituitary powder, and LH in plasma were all identical. Immunoassay and bioassay of impure urinary and pituitary gonadotropin preparations in terms of a common standard resulted in an index of discrimination of close to unity. LH levels in plasma from 32 adult men and 30 women outside the midcycle ranged from 0.6 to 3.2 mmug per ml (1 mmug of our laboratory LH standard is equivalent to 8 mU of the Second International Reference Preparation of Human Menopausal Gonadotropin). Levels were remarkably constant in men from day to day and in women except at midcycle, when a sharp peak occurred lasting less than 24 hours. In all women studied who had a midcycle LH peak, mean plasma LH levels during the follicular phase of the menstrual cycle were higher than mean values obtained during the luteal phase. Prepubertal children had detectable plasma LH, and mean values were only slightly less than in adults. Plasma from castrate men or women or postmenopausal women contained 4.5 to 10.5 mmug per ml. Clomiphene treatment of four men resulted in a doubling of plasma LH in 5 days.

Follicle-stimulating hormone (FSH) and luteninizing hormone (LH) in the urine of prepubertal children.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have been measured by specific bioassays in pooled urine samples from prepubertal children, aged 2-6 yr, and from male adults. For children the mean urinary excretion of FSH was 2.2 U 2nd International Reference Preparation (2nd IRP) per liter and the mean urinary excretion of LH was 0.44 U 2nd IRP per liter. For adults the mean FSH excretion was 5.6 U 2nd IRP per liter and the mean LH excretion was 4.7 U 2nd IRP per liter. Our data show a 2.5-fold increase of FSH, a 10.7-fold increase of LH, and a consequent decrease in the FSH: LH ratio from 5 to 1 between childhood and adulthood. FSH and LH in urine from three patients with gonadal abnormalities have also been studied. The results from normal children, adults, and abnormal patients form a spectrum and reveal that sexual maturity is accompanied by a marked increase in the excretion of LH with relatively smaller increases in FSH.

Metabolic clearance and production rates of human luteinizing hormone in pre- and postmenopausal women.

Metabolic clearance rates and production rates of human luteinizing hormone (HLH) were determined in pre- and postmenopausal women by the constant infusion technique. Highly purified HLH-(131)I was infused into the fasting subjects at a constant rate. Serial plasma samples were obtained and the radioactive hormone was precipitated by a double antibody technique. Plasma HLH-(131)I levels reached equilibrium by 4 hr after the infusion started. Metabolic clearance rates were: 24.4 +/- 1.8 (mean +/- SE) ml/min in five normal premenopausal women; 23.3 +/- 1.1 ml/min in five normal women taking norethinodrel and mestranol; and 25.6 +/- 4.1 ml/min in four postmenopausal women. Endogenous plasma HLH levels measured in the same subjects by radioimmunoassay immediately before infusion were 32.0 +/- 9.6 mU/ml in the normal women, 16.8 +/- 3.2 mU/ml in the women on oral contraceptives, and 99.2 +/- 23.2 mU/ml in the postmenopausal women. The corresponding HLH production rates were: 734 +/- 170 mU/min in the normal women: 387 +/- 86 mU/min in the women on norethinodrel and mestranol; and 2400 +/- 410 mU/min in the postmenopausal women. The metabolic clearance rate did not change after ovariectomy in one women, but the production rate rose from 583 to 1420 mU/min. Based on previously reported bioassay values for pituitary content and urinary excretion of HLH, the estimated turnover of HLH in the pituitary is about once per day and less than 5% of the total HLH produced appears in the urine in a biologically active form.

Radioimmunoassay for human follicle-stimulating hormone: physiological studies.

Most of the information concerning secretion changes in follicle-stimulating hormone (FSH) in humans has been gained with relatively insensitive bioassays of concentrates of pools of urine. We have developed a sensitive and specific radioimmunoassay for FSH that is 500-1000 times more sensitive than the rat ovarianweight augmentation assay and which is capable of quantifying FSH in small volumes of serum. Anti-FSH was prepared by immunizing rabbits with an impure FSH preparation. The majority of antisera showed complete inability to distinguish LH, TSH, and FSH, illustrating the immunological similarities of these hormones. One antiserum was specific when used in a radioimmunoassay. Potency estimates by bioassay were in good agreement, with a single exception, with those obtained with the radioimmunoassay for 10 FSH-containing preparations. Highly purified LH gave a higher potency by immunoassay than by bioassay. Sera from eugonadal men contained 5-25 mIU/ml; sera from castrate men contained over 30 mIU/ml. Sera from eugonadal women contained 7-25 mIU/ml during the follicular phase and 5-15 mIU/ml during the luteal phase of the menstrual cycle. Sera from castrate or postmenopausal women contained 40-250 mIU/ml. FSH was measured throughout the menstrual cycle in 19 women. The general pattern that emerged is summarized as follows: there is a small early follicular phase rise in FSH, and then FSH is relatively constant until mid-cycle; in the majority of women a mid-cycle rise of FSH occurs coincidentally to the mid-cycle LH ovulatory peak; during the luteal phase FSH levels are relatively constant and lower than during the follicular phase. Nonsequential oral contraceptives containing estrogen and progestogen abolish these changes and FSH concentrations remain low throughout treatment. Treatment of castrate men and castrate or postmenopausal women with high doses of oral estrogens results in a fall of FSH to levels found in eugonadal men or women, but not to undetectable levels. Children less than 5 yr of age had undetectable FSH (< 5 mIU/ml).

Production rates and metabolic clearance rates of human follicle-stimulating hormone in premenopausal and postmenopausal women.

The production rates (PR) and the metabolic clearance rates (MCR) of human follicle-stimulating hormone (HFSH) were determined in six pre- and five postmenopausal women. Human FSH (PER-780) labeled with (131)I to specific activities of 50-150 muc/mug was used as a tracer. Both double antibody and trichloroacetic acid (TCA) precipitation techniques were used to determine HFSH-(131)I levels in infusate and plasma. In four of the subjects MCRs measured by both constant infusion and single injection techniques were the same. By constant infusion, plasma HFSH-(131)I levels reached equilibrium between 4-5 hr.MCRs in six premenopausal women were 14.2+/-1.1 (mean +/-SE) ml/min. MCRs in five postmenopausal women were 12.6 +/-1.1 ml/min. Simultaneous HFSH and human luteinizing hormone (HLH) MCRs were determined in a single patient using HFSH-(125)I and HLH-(131)I as tracers by both constant infusion and single injection methods. These studies showed that the MCR of HFSH was 10.8-11.1 ml/min, and the MCR of HLH was 18.5-19.4 ml/min. From these data and previous MCR and PR studies of HLH from this laboratory, it appears that the MCR of HFSH is about one-half that of HLH. Endogenous HFSH and HLH levels were measured by radioimmunoassay. The PRs of HFSH, calculated by the product of endogenous level and MCR, were 146 +/-27mU/min in the premenopausal women and 2141 +/-264 mM/min in the postmenopausal women. 24-hr PRs, based on these results, compared with reports of 24-hr urinary excretions of biologically active HFSH indicate that 3-5% of production is found in urine in biologically active form. After our single injections of HFSH-(131)I, 8-29% was recovered in urine over 24 hr.

Studies of the pituitary-Leydig cell axis in young men with hypogonadotropic hypogonadism and hyposmia: comparison with normal men, prepuberal boys, and hypopituitary patients.

Pituitary and gonadal function was studied in seven chromatin-negative men, ages 15-27 yr, with retarded sexual and somatic development, skeletal anomalies, and hyposmia. These hyposmic patients were compared with normal men, prepuberal boys and hypogonadal patients with hypopituitarism. The urinary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels of hyposmic subjects were the same as those of normal boys and hypopituitary patients but significantly lower than those of normal men. Clomiphene citrate did not cause an increase in plasma FSH and LH levels in either hypogonadal group as it does in normal men. In contrast to hypopituitary patients, thyroid and adrenocortical function and release of growth hormone in the hyposmic subjects were normal. The plasma testosterone levels were equally low in prepuberal, hypopituitary, and hyposmic patients but were increased to a greater extent by human chorionic gonadotropin (HCG) treatment in prepuberal and hypopituitary subjects than in the hyposmic patients. Prolonged treatment with HCG has failed to return plasma testosterone levels to normal in two hyposmic patients. These observations suggest that there are defects of both pituitary and Leydig cell function in men with the syndrome of hypogonadism, skeletal anomalies, and hyposmia. They have impaired secretion of FSH and LH and a Leydig cell insensitivity to gonadotropin.

Further characterization of a rat ovarian testosterone receptor with evidence for nuclear translocation.

We have recently described receptor like testosterone (T)-binding protein in the cytosol of ovaries from estrogen-primed hypophysectomized immature female rats (HIFR). This binding protein is thermolabile, saturable, specific for T, and sediments at 7-8 S on sucrose gradients. We now report the further characterization of this protein. It is present in the cytosol of isolated granulosa cells, has a molecular weight of 240,000, a frictional ratio of 1.8, and a mean Stokes radius of 73 A. The binding protein is acidic and cysteine residues are necessary for binding. We have also demonstrated the selective nuclear accumulation of T by granulosa cells after the in vivo administration of (3H)T to estrogen primed HIFR. After the in vitro incubation of the ovaries with (3H)T, a thermolabile protein specific for T was extracted from nuclei under high-salt conditions. The cytosol and nuclear T-binding proteins described in this report have many characteristics of an androgen receptor, and may play a role in the regulation of granulosa cell proliferation during preantral follicular growth.

A receptor-like testosterone-binding protein in ovaries from estrogen-stimulated hypophysectomized immature female rats.

A soluble thermolabile protein with many characteristics of an androgen receptor has been demonstrated for the first time in the cytosol (100,000 X g supernatant) of estrogen-stimulated ovaries from hypophysectomized immature female rats (HIFR) treated with diethylstilbestrol in Silastic capsules (DESC). This binding protein is organ-specific and androgen-specific, has a high affinity with a Kd of 2.4 X 10(-9)M for testosterone, and is saturable with 2.1 X 10(-13) moles of binding sites per mg cytosol protein. The number of binding sites is linear with cytosol protein concentration and the binding protein sediments at 7-8 S on sucrose gradients. Estradiol is an effective inhibitor of testosterone binding. A role for this testosterone-binding protein as an effector of ovarian morphologic change is hypothesized.

Androgenic stimulation of progesterone production by granulosa cells from preantral ovarian follicles: further in vitro studies using replicate cell cultures.

The influence of testosterone (T) on progesterone (P) production by isolated rat ovarian granulosa cells was studied in vitro using a new replicate culture technique. Preantral granulosa cells from ovaries of estrogen-primed hypophysectomized immature female rats were cultured in the presence of graded concentrations of T, diethylstilbestrol (DES), cyproterone acetate (CPA), flutamide and the hydroxylated derivative of flutamide, Sch 16423. The accumulation of P in medium collected from granulosa cell cultures was measured by specific radioimmunoassay. Maximal P production by cultured granulosa cells was attained during the second day of culture and declined markedly thereafter. The presence of 10(-9), 10(-8) or 10(-7)M T elicited increases in P production 2.4, 8 and 11 times that of controls, respectively, during the initial 48 h of culture. Each concentration of T elicited enhanced P production within the first 24 h of culture. Granulosa cells cultured in control medium for 2 days did not respond to 10(-7)M T during the subsequent 3 days. DES at a high concentration in the medium (10(-5)M) markedly suppressed the response to 10(-9) and 10(-8)M T. At a lower concentration (10(-9)M) DES significantly enhanced the stimulatory effect of 10(-9)M T but did not alter the response to higher concentrations of T. Neither high nor low concentrations of DES influenced P production in response to 10(-7) M T. The stimulatory effects of T on P production were suppressed in the presence of a 100-fold molar excess of the anti-androgens, CPA or Sch 16423. The present data indicate that androgenic stimulation of P production by preantral granulosa cells is a specific receptor mediated event which is modulated by the presence of estrogen in vitro. It is suggested that androgen-responsive P production is a functional capacity which granulosa cells acquire at a very early stage of hormonal differentiation and may be of physiological consequence in the intraovarian control of follicular maturation in vivo.

Independence of steroidogenic capacity and luteinizing hormone receptor induction in developing granulosa cells.

The relationship between FSH-induced acquisition of LH/hCG receptors and the steroidogenic capacity of granulosa cells from estrogen-primed hypophysectomized rat ovaries has been examined. Granulosa cells harvested from the immature preantral follicles of animals not treated with FSH (controls) displayed negligible specific human [125I]iodo-hCG binding and produced only minimal amounts of progesterone during 48 h of culture in vitro. Addition of highly purified hFSH or prostaglandin-E2 (PGE2) to the culture medium elicited substantial increases in progesterone production which were not accompanied by measurable increases in [125I]iodo-hCG binding. Treatment with oFSH in vivo for 24 h led to the initiation of antrum formation in many follicles and was accompanied by an 8-10-fold increase in hCG binding by freshly isolated granulosa cells. Basal, hFSH-, and PGE2-stimulated progesterone production during culture was also greater than controls. In contrast, cells from animals receiving oFSH in vivo for only 12 h showed no increase in hCG binding either before or after culture, yet basal and stimulated progesterone production in vitro was significantly greater than controls, indicating that the initiation of steroidogenesis was antecedent to LH/hCG receptor induction. Only those cells obtained after the 24-h in vivo treatment with oFSH produced elevated amounts of progesterone when incubated in the presence of hCG, thereby showing that the observed increases in [125I]iodo-hCG binding reflected the induction of functionally active LH/hCG receptors. Pharmacological stimulation of steroidogenesis by cell suspensions with N,O'-dibutyryl cAMP resulted in consistently high levels of progesterone production irrespective of previous treatment with FSH in vivo. This uniform expression of in vitro steroidogenic capacity occurred in the complete absence of measurable increases in LH/hCG receptors, suggesting that these two fundamental developmental processes are independent phenomena which may be under separate regulation in vivo.

Inhibition of the ovarian augmentation reaction by a chemical antiestrogen.

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.

Interaction of estrogen and gonadotrophins on follicular atresia.

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.

Effects of human chorionic gonadotropin, human interstitial cell stimulating hormone and human follicle-stimulating hormone on ovarian weights in estrogen-primed hypophysectomized immature female rats.

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.

Production of long term steroid-producing granulosa cell cultures by cell hybridization.

Primary cultures of rat ovarian granulosa cells have been used extensively to study hormonal regulation of cellular function. To date, no long term cultures of ovarian cells which retain their differentiated functions have been developed. Hypoxanthine guanine phosphoribosyl transferase-deficient simian virus 40-transformed rat ovarian granulosa cells were fused with freshly prepared rat granulosa cells using inactivated Sendai virus. Putative hybrid cell strains obtained after selection in medium containing hypoxanthine, aminopterin, and thymidine were analyzed for progesterone synthesis. Neither the original simian virus 40-transformed granulosa cell nor its hypoxanthine guanine phosphoribosyl transferase-deficient derivative produced progesterone, but three of the hybrid strains produced progesterone at basal levels and in response to dibutyryl cAMP. One of these strains produced progesterone in a dose-responsive fashion when exposed to prostaglandin E2, cholera toxin, dibutyryl cAMP, and 2-chloroadenosine. Cell strains obtained by hybridization were remarkably similar to primary cultures of granulosa cells with respect to both the magnitude and temporal aspects of progesterone production in response to dibutyryl cAMP.

Follicle stimulating activity of human chorionic gonadotropin: effect of dissociation and recombination of subunits.

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.

Evidence of a role of adrogens in follicular maturation.

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.

Progesterone production by cultured preantral rat granulosa cells? Stimulation by androgens.

Progesterone (P) production by isolated rat granulosa cells from preantral follicles was enhanced by addition of androgens to the tissue culture medium. Testosterone (T) at 10(-7), 10(-6), and 10(-4)M as well as 10(-6)M dihydrotestosterone (DHT) increased P production 400 to 700% over paired control cultures. Human chorionic gonadotropin (100 mIU/ml) and 17beta-estradiol (7.8 X 10(-10M) had no effect on P production. P was identified by both a specific radioimmunoassay and sephadex LH-20 column chromatography. The stimulatory influence of T and DHT on these preantral follicular cells is consistent with a direct role for androgens in granulosa cell differentiation.