Investigation of the role of sex hormones in alcoholics' visuospatial deficits.

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.

Motility and fertilizing ability of rat epididymal spermatozoa washed by a continuous gradient of Percoll.

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.

Gonadotropin stimulated protein phosphorylation in porcine granulosa cells.

Treatment of granulosa cells with luteinizing hormone (LH) or follicle-stimulating hormone (FSH) stimulated the phosphorylation of a 58,000 molecular weight protein found in the 900 x g pellet. The phosphorylation of this protein was rapid, being significant at 1 min. LH treatment for 30 min induced greater phosphorylation of this protein than did FSH. LH and FSH also appeared to stimulate the phosphorylation of different 900 x g pellet proteins. Since both are known to utilize cAMP as a second messenger, the finding of these unique gonadotropin-induced phosphorylations may point to an additional regulatory mechanism.

Effects of aging and testosterone administration on liver alcohol dehydrogenase activity in male Fischer 344 rats.

The activity of alcohol dehydrogenase was measured in liver cytosolic 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 mumol/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 mumol/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.

Effect of vitamin E deficiency on serum concentrations of follicle-stimulating hormone and testosterone during testicular maturation and degeneration.

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.

Inhibition of placental aromatase activity in a cell free assay by ovarian protein.

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)

Identification of a porcine follicular fluid fraction which suppresses follicular response to gonadotropins.

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)

Identification of proteins in pooled human follicular fluid which suppress 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.

Quantitative separation of estrogens, androgens and progestogens using reverse phase high pressure liquid chromatography.

Dual UV wavelength scanning at 206 and 254 nm was used to develop a sensitive, separation and quantitation procedure for estrone, estradiol-17 beta, testosterone, 4-androstene-3,17,-dione, progesterone and 17-hydroxyprogesterone using reverse phase high pressure liquid chromatography. The difference in UV absorption of the estrogens from the androgens and progestogens allows for correction of the co-elution of testosterone with estradiol-17 beta and 4-androsten-3,17,-dione with estrone. Incorporation of an isocratic step-gradient provides improved separation while maintaining shortened elution times for the less polar steroids.

Effect of surgical technique on peritoneal adhesion reformation after lysis.

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.

Relationship of estradiol to luteal function in the cycling baboon.

Since exogenous 17 beta-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 less than 0.01). Clomiphene blocked the luteolytic effect of exogenous E2, resulting in P levels which were not significantly different (P greater than 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 clomiphene alone maintained circulating LH at early to midluteal phase levels but did not alter luteal phase P concentrations or prolong the length of the luteal phase (15.6 +/- 0.4 days). The results suggest that E2-induced luteolysis is a result of the withdrawal of luteotropic support from the (corpus) luteum, while spontaneous luteolysis is a result of some other mechanism which may not involve endogenous E2.

Fertility in the male gorilla (Gorilla gorilla): Relationship to semen parameters and serum hormones.

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.

Studies on serum apolipoproteins and lipids in amniotic fluid and neonatal urine.

Amniotic fluid and neonatal urine were examined for the presence of lipids and serum apolipoproteins. Human apolipoproteins A-I, A-II, and ApoD found principally in serum high density lipoproteins were identified in both neonatal urine and amniotic fluid. A lecithin/sphingomyelin ratio of greater than 5 associated with fetal lung maturity was accompanied by the disappearance of A-II from amniotic fluid. Dissimilarities of total fatty acid composition of amniotic fluid when compared to cord serum or neonatal urine indicate other tissue sources for fatty acids found in amniotic fluid. In addition, the presence of serum apolipoproteins and lipids in both amniotic fluid and neonatal urine suggests that a least a portion of these constituents could be derived from fetal urine.

Copper and ceruloplasmin activity in human amniotic fluid.

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.

Influence of glucocorticoid administration and inhibition on fetal baboon pulmonary maturity and the amniotic fluid L/S ratio.

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.

Escherichia coli shock in the baboon and the response to adrenocorticosteroid treatment.

Results of recent studies from this laboratory have demonstrated that methylprednisolone sodium succinate increases the survival rate of dogs given LD100 Escherichia coli endotoxin. This study was undertaken to determine the effects of methylprednisolone on the baboon infused with live Escherichia coli organisms. Awake baboons were paired by infusing intravenously comparable doses of Escherichia coli during a five hour period. Baboons given methylprednisolone received bolus injections of 30 milligrams per kilogram at 15 minutes after beginning the infusion of Escherichia coli and two hour infusions of 15 milligrams per kilogram at two hour intervals until death or for a 24 hour period. The mortality was unaltered by methylprednisolone. Six of seven baboons that were dying became progressively hypoglycemic, while hypoinsulinemia occurred in all baboons within six hours and was sustained until death. Systemic hypotension was observed. although pressures were variable. Potassium and lactate concentrations increased, while pH remained relatively constant in most baboons. Serum glutamic-pyruvic transaminase and arginase concentrations rose in most baboons dying with 18 hours. Results of morphologic studies revealed the presence of fibrin thrombi in the liver, kidney and adrenal tissue in most baboons. No significant differences in physiologic, metabolic, hematologic or morphologic parameters were observed between treated and untreated baboons.