Previously we identified a fraction of follicular fluid (follicle regulatory protein: FRP) which inhibits granulosa cell aromatase activity. During the course of these studies the question of FRP acting via autocrine as well as paracrine mechanisms arose in addition to the need for a more efficient method of screening for aromatase inhibitory activity during the purification of FRP. Accordingly, we assessed the effects of FRP on aromatase activity in a microsomal assay. Placental microsome preparations were preincubated for 20 minutes with or without FRP prior to a 20 minute incubation with testosterone. Significantly less conversion of testosterone to estrogen occurred with FRP compared to control preincubation. When follicular protein was added without pre-incubation, there was no apparent change in microsomal aromatase activity, whereas after a 20 minute pre-incubation with the follicular protein fraction, significantly less testosterone was converted into estrogen. When various concentrations of FRP were assayed in the placental aromatase assay, a dose-response curve demonstrated a 50% inhibitory dose (ID50) of approximately 400 micrograms/ml. To further purify the aromatase inhibitory activity, 5 mg of the crude follicular fluid preparation was eluted through an anion exchange column via HPLC using a sodium acetate gradient. The fractions in the central elution peak contained aromatase inhibitor activity with ID50 values of 25-160 micrograms/ml. Thus Fractions were further purified by elution through a gel exclusion column via HPLC which demonstrated inhibition of cell free placental aromatase activity in the 15,000-18,000 molecular weight range with an ID50 of 5 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

This study investigated whether an alcohol-related disturbance in the functioning of the hypothalamic-pituitary-gonadal axis was a factor in alcoholics' visuospatial impairment. One month detoxified male alcoholics (n = 58) performed significantly poorer than peer controls (n=30) on a battery of visuospatial and verbal tests. Serum concentrations of testosterone, estradiol, luteinizing hormone and follicle stimulating hormone were obtained at three times during cognitive testing. Previously reported correlations in healthy young male adults between visuospatial performance with testosterone and follicle stimulating hormone concentrations were replicated in control subjects. These correlations were not significant in the alcoholics. The results suggest that chronic alcoholism may disrupt the normal relationships between the hypothalamic-pituitary-gonadal axis and cognitive functioning.

Removal of epididymal fluids from epididymal sperm suspension is an important step for the study of sperm motility, capacitation, and the acrosome reaction. The technique of washing should minimize damage to viable spermatozoa but at the same time efficiently remove debris, non-sperm cells, and biological fluids. We examined sperm motility and fertilizability in vitro of rat epididymal spermatozoa after washing with Percoll continuous gradient. Nine milliliters (ml) of 50% N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid (HEPES) buffered Percoll solution was centrifuged at 20,000 g for 45 minutes to form a continuous gradient. One hundred to 300 microliters of sperm suspension was loaded onto the surface of the gradient and centrifuged at 150 g and 1,500 g for 10 minutes. Two main layers of spermatozoa were formed, one of high (lower layer) and one of low (upper layer) motility. At centrifugation 1,500 g, the sperm density and motility in the lower layer were greater than at 150 g. Spermatozoa from both layers at 150 g and at 1,500 g were diluted with modified Krebs-Ringer's bicarbonate solution (mKRB) and preincubated for 5 hours. Superovulated eggs collected from 21-25-day-old Wistar strain immature rats were introduced into the preincubated sperm suspension for insemination and fixed 5-5.5 hours later for observation of fertilization. Spermatozoa from both layers, 150 g and 1,500 g, showed the same fertilizability in vitro as control spermatozoa. From these results we conclude that Percoll gradients can be used for washing rat epididymal sperm for the study of sperm physiology including fertilization.

Although vitamin E has been recognized as a requirement for normal testicular function for more than 50 yr, the functional role of this fat soluble vitamin in the maintenance of spermatogenesis has not been clarified. Vitamin E deficiency has a deleterious effect on germ cell proliferation and differentiation in the rat and a variety of other animal species. The potential effect of vitamin E on the sensitivity of the hypothalmo-hypophyseal-gonadal feedback system has not been previously evaluated. Therefore, serum testosterone concentrations, androgen function in maintaining seminal vesicle weight, and citrate synthesis, as well as the circulating amounts of FSH, were determined in rats fed semipurified diets with vitamin E, control, and without vitamin E, vitamin E-deficient. To assess dietary effects before, during, and after testicular degeneration, analyses were carried out on animals fed the diets starting at 3 weeks of age and continuing for 1 yr. The defect produced by vitamin E deficiency does not reflect impaired gonadotropin function. There are no effects of vitamin E deprivation on either serum levels of testosterone or on seminal vesicle weight and citrate content suggesting that the LH-testosterone feedback loop is not impaired. The feedback loop of FSH and inhibin also appears to be normal. Serum FSH concentrations are not elevated, although the testis is germ cell depleted. Comparison of the vitamin E-deficient model to other models of testicular degeneration suggests that the effect of vitamin E occurs directly or indirectly on the regulation of intratesticular factors which regulate specific steps of germ cell development.

The activity of alcohol dehydrogenase was measured in liver cyto‐solic fractions of male Fischer 344 rats at ages representing young adulthood, middle age, and old age. The activities were 1.7 ± 0.1, 2.3 ± 0.1, and 2.6 ± 0.2 μmol/min/g liver in rats aged 4–5, 14–15, and 24–25 months, respectively. Hepatic alcohol dehydrogenase activity in female rats (3.4 ± 0.2 μmol/min/g liver) was the same in young as in old rats. Castration increased alcohol dehydrogenase activity in young males to levels found in females, and testosterone administration reversed the effect. However, neither physiological nor pharmacological doses of the hormone restored the elevated enzyme activities of old male rats to levels found in young male rats.

To evaluate the role of nonsteroidal, follicular fluid proteins in folliculogenesis, the 10-55% saturated ammonium sulfate fraction of pooled porcine follicular fluid (PFF) was dialyzed against 0.025 M Tris/HCl, pH 7.5, using 10,000 molecular weight exclusion membranes, then passed through agarose-immobilized textile dye. Activity was determined by test fraction inhibition of human menopausal gonadotropin (hMG) [2 U human luteinizing hormone (LH)/follicle-stimulating hormone (FSH) per day] induced ovarian weight and serum estradiol increase in hypophysectomized, diethylstilbestrol (DES)-treated, 25-day-old female rats. Specific inhibition (84 +/- 7.4%) of ovarian weight increase was found in the material (5 ml) eluted from the orange A column with KCl (1.5 M, pH 6.8). Inhibitory activity of the orange A-bound material which eluted through a standardized Sephadex G-100 column corresponded to a molecular weight of 12,000-30,000. Isoelectric focusing (IEF) on a Sephadex G-15 support bed of orange A-bound material demonstrated inhibitory activity at pH 3.7-4.0. Serial dilutions of active material from IEF preparations demonstrated a dose-response relationship in the bioassay. No demonstrable activity was found in similar fractions eluted through a Concanavalin A-Sepharose 4B column with or without addition of alpha-methyl mannoside (2 M, pH 7). When active fractions were heated (56 degrees C, 1 h) or exposed to trypsin (10 mg%), activity was lost. When aliquots of the saturated ammonium sulfated precipitated, dialyzed, orange A-bound, Sephadex G-100 (Ve/Vo 1.3-1.7) eluent were separated by high-performance liquid chromatography (HPLC) using gel exclusion columns, activity in the bioassay was recovered in the 18,000-35,000 molecular weight range.(ABSTRACT TRUNCATED AT 250 WORDS)

Recently, we reported the identification of a heat- and trypsin-labile substance recovered directly from the venous drainage of the human preovulatory ovary and pooled follicular fluid which suppressed follicular response to gonadotropins. Since many of the steroidal components of follicular fluid are derived from granulosa cells, we questioned whether granulosa cells secrete a substance that inhibits follicular response to gonadotropins. Follicular fluid was aspirated from regularly menstruating women (25–35 yr old) undergoing clomiphene citrate (150 mg/ day for 5 days, beginning 3–8 days after the onset of spontaneously occurring menses) and hCG (4000 IU, 36 h before aspiration) treatment (n = 7). Follicles were characterized by quantitation of viable granulosa cells and steroid concentration. Individual follicular fluids were precipitated by 10–55% ammonium sulfate, dialyzed with 10,000 mol wt exclusion membranes, lyophylized, and eluted by Orange A dye matrix chromatography and high performance liquid chromatography. The granulosa cells from these follicles were cultured with or without varying doses of LH/FSH from 1–4 days. Extracted follicular fluids and tissue culture media were evaluated in an LH/FSH-challenged, immature, hypophysectomized, diethylstilbesterol treated rat (HIFR-hMG) for inhibition of ovarian weight and trunk serum estradiol concentrations. All of the antral fluid/s steroid concentrations suggested premature luteinization. After extraction, all seven follicular fluids contained inhibitory activity, as evidenced by reduction of both rat ovarian weight (45–85%) and trunk serum estradiol concentrations (85–100%) in the HIFR-hMG bioassay. Bioassay of these follicles' granulosa cell culture media indicated inhibitory activity present during the first 48 h, while no inhibitory activity was noted after 72 h of culture. Spent culture media derived from granulosa cells cultured without additional gonadotropins contained inhibitory activity in the HIFR-hMG bioassay throughout the first 2 days in vitro. Thereafter (days 3 and 4), inhibitory activity of media from unstimulated cultures declined. After coincubation of the granulosa cells with varying doses of LH/ FSH, inhibitory activity was markedly suppressed even after the first 24 h. An inverse correlation was apparent between inhibitory activity in the bioassay and the culture medium progesterone level. Although FSH binding of granulosa cells derived from rats used in the HIFR-hMG bioassay was similar with or without injection of test fractions, their aromatase activity was markedly inhibited by treatment with human granulosa cell culture medium. These data suggest that although no marked inhibition in rat granulosa cell FSH binding was induced by the human granulosa cell culture medium, a clear disruption of aromatase activity occurred, which may account for the decreased rat ovarian weight and serum estradiol concentrations found in the bioassay. Here, we have identified from aspirates of individual human follicles obtained during clomiphene- and hCG-stimulated ovarian cycles as well as from culture media derived from these follicle's granulosa cells a substance that actively suppresses follicular responsivity to gonadotropins. When taken together, these data suggest that the human granulosa cell secretes a protein that inhibits follicular response to gonadotropins.

To evaluate the role of nonsteroidal, follicular fluid proteins in folliculogenesis, the 10-55% saturated ammonium sulfate fraction of pooled human follicular fluid was dialyzed against 0.025 M Tris/HCl (pH 7.5) using 10,000 molecular weight exclusion membranes, then passed through agarose immobilized textile dye. Activity was determined by test fraction inhibition of human menopausal gonadotropin (2 U human LH/FSH . day), induced ovarian weight, and serum estradiol increase in hypophysectomized, diethylstilbesterol-treated, 25-day-old female rats. Specific inhibition (89 +/- 6.8% SEM) of ovarian weight increase was found in the material (2 ml) eluted from an Orange A column with KCl (1.5 M, pH 6.8). Inhibitory activity of the Orange A-bound material, which eluted through a standardized Sephadex G-50 column, corresponded to a molecular weight of 13,000-25,000. Isoelectric focusing on a Sephadex G-15 support bed or ampholyte displacement chromatography of Orange A bound material demonstrated inhibitory activity at pH 3.5-4.5 and 6.5-7.0. No demonstrable activity was found in similar fractions eluted through a Concanavalin A-Sepharose 4B column before or after addition of alpha-methyl-D-mannoside (2 M, pH 7). When active fractions were heated (56 C, 1 h) or exposed to trypsin (10 mg/100 ml), activity was lost. When aliquots of the saturated ammonium sulfate-extracted, dialyzed, Orange A-bound eluent were separated by high performance liquid chromatography using gel exclusion columns, activity in the bioassay was recovered in the 13,000-35,000 molecular weight range. Although confirmatory data await further studies, it is tempting to speculate that this protein(s) may be an important inter- and/or intraovarian regulator of follicular response to gonadotropins.

Since exogenous 17β-estradiol (E2) is luteolytic in some primates, it is reasonable to conclude that endogenous E2 is the naturally occurring luteolytic substance. However, a link between endogenous E2 and spontaneous luteolysis has not been demonstrated. In an attempt to understand the function of E2 in spontaneous luteolysis, we employed the antiestrogen clomiphene. We administered E2, clomiphene, or E2 plus clomiphene to baboons during the luteal phase and measured systemic levels of estrone, E2, progesterone (P), and LH. E2 alone depressed the concentrations of P and bioassayable LH prematurely. The length of the luteal phase was significantly shortened (12.8 ± 0.9 vs. 15.8 ± 0.4 days for sham controls; P 0.01) from control and a luteal phase of normal length (15.0 ± 1.0 days). Treatment with E2 alone caused serum LH concentrations to decrease. The administration of clom...

The effectiveness of macrosurgical and microsurgical techniques for adhesion management was evaluated in nonpregnant female baboons that had significant pelvic adhesions as a sequela of previous surgery. Two weeks after the initial laparotomy the animals were reoperated upon and adhesion reformation assessed. Mean adhesion scores were not significantly different between the macrosurgery and microsurgery groups before or after lysis. However, the reduction in the mean adhesion score following lysis approached significance (P less than 0.1) in the microsurgical group, whereas it did not in the macrosurgical group. This study does not confirm that the use of microsurgery in adhesion management is clearly superior but does suggest it may be slightly more effective in reducing adhesion reformation.

Circulating levels of steroid and protein hormones were measured in 17 adult male lowland gorillas (Gorilla gorilla). The population included both fertile and infertile males as identified by previous siring of offspring and presence or absence of spermatozoa in the ejaculate obtained by rectal probe electrostimulation. Correlations were sought between levels of testosterone, dihydrotestosterone, androstenedione, estrone, estradiol, progesterone, 170H-progesterone, dihydroepiandrosterone, luteinizing hormone, follicle-stimulating hormone (FSH), and potential fertility status. The results identify normal circulating levels of these hormones, and indicate that aspermatogenesis and infertility are not necessarily associated with any alteration in levels of gonadal steroids. There is an association of aspermatogenesis with elevation of FSH. Levels of adrenal androgens are more similar to other non-human primates than to the human, which is of interest because in other aspects of reproductive physiology so far investigated the gorilla has proved to resemble the human more closely than it does the other nonhuman primates.

The variation in the level of copper and ceruloplasmin oxidase activity in human amniotic fluid from 20 weeks' gestation to term was reported. The protein content of amniotic fluid decreased toward term. A definite decreasing trend of concentration of copper, expressed both as nanograms of copper per milliliter of amniotic fluid and nanograms of copper per milligram of protein, was also observed from midgestation toward term. Ceruloplasmin, on the other hand, demonstrated a significant increase from the period 20 38 weeks' gestation, with a subsequent decline after 38 weeks.

The pregnant baboon feto-placental unit responded to intra-amniotic dexamethasone by a rise in the lecithin-sphingomyelin ratio (L/S ratio) and accelerated pulmonary maturity with evidence of increased amounts of pulmonary surfactant. Metyrapone 11 beta-hydroxylase inhibition of corticoid synthesis prevented the expected rise in the L/S ratio without influencing the normal development of pulmonary maturity. The L/S ratio did not accurately reflect pulmonary maturity when metyrapone was administered to the pregnant baboon. Prudently, model systems such as the baboon should be used for more detailed studies of endocrine effects on pulmonary development because of the reported untoward effects of exogenous steroid administration on the human neonatal nervous system.