Hypothalamic 5 alpha-reductase and 3 alpha-oxidoreductase activity in the male rat.

The aim of the present study was to assess the progesterone (Pr) transforming 5 alpha-reductase (5 alpha-R) and 3 alpha-oxidoreductase (3 alpha-OR) activities in the hypothalamus of the male rat as a function of age and following castration and/or adrenalectomy performed at the sixth day of life. The hypothalamic activity of these enzymes was estimated from the sum of the 5 alpha- or 3 alpha-reduced metabolites produced from 14C-labeled Pr incubated "in vitro" with hypothalamic tissue. Plasma levels of testosterone (T), progesterone (Pr), estrone (E1), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured simultaneously. Special attention was paid to the GC/MS analysis of the endogenous content of the hypothalamic Pr-metabolites 3 alpha-hydroxy-pregn-4-en-20-one (3 alpha-Pr), 5 alpha-pregnane-3,20-dione (5 alpha-Pr) and 3 alpha-hydroxy-5 alpha-pregnan-20-one (5 alpha,3 alpha-Pr). The high 5 alpha-R and 3 alpha-OR activities estimated in the hypothalamus of prepubertal rats are not related to the action of gonadal or adrenal steroids. Substantial and comparable endogenous 3 alpha- and/or 5 alpha-Pr-metabolites were found in hypothalami from both prepubertal and mature rats. The results of the present study do not provide evidence for a contributory role of the 3 alpha-hydroxylated Pr derivative to the regulation of gonadotropin secretion in the male rat.

Fat absorption and metabolism in bile duct ligated rats.

BACKGROUND: Bile excretion is obstructed in children with extrahepatic bile duct atresia (EHBA) resulting in fat malabsorption and disturbed lipid metabolism. AIM: Investigate if the bile duct ligated rat exhibits similar deviations as patients with EHBA under different feeding conditions. METHODS: 6 bile duct ligated Wistar rats and 12 matched paired controls were randomised over 3 feeding groups. Rats were killed 16 or 30 days postsurgery. Faeces, blood and livers were collected. Fat absorption was evaluated, markers for cholestasis and the fatty acid composition of serum phospholipids (PL) and cholesterol esters (CE) were determined. Fatty acid desaturation activities in liver microsomes were measured. RESULTS: Cholestatic bile duct ligated rats have a lower fat absorption coefficient and a lower fraction of 18:2n-6 and 18:3n-3 in serum triglycerides than their controls. This demonstrates that bile duct ligated rats suffer from fat malabsorption. In contrast to the observations in serum triglycerides, 18:2n-6 and 18:3n-3 were not reduced in serum PL and CE of cholestatic rats. Overflow of 18:2n-6 rich biliary PL in the general circulation could contribute to this observation. In agreement with what was found in man, serum PL of cholestatic rats have a higher 16:0/18:0 ratio, increased monoenes and reduced unsaturated fatty acids. However, no differences were observed in microsomal desaturation activities. CONCLUSION: Cholestatic bile duct ligated rats exhibit similar deviations in serum fatty acid composition as found in patients with EHBA, therefore they can be used as a model for this human disease.

The effects of combined conventional treatment, oral antioxidants and essential fatty acids on sperm biology in subfertile men.

We evaluated the effects of combined conventional treatment, oral antioxidants (N-acetyl-cysteine or vitamins A plus E) and essential fatty acids (FA) on sperm biology in an open prospective study including 27 infertile men. The evaluation included sperm characteristics, seminal reactive oxygen species (ROS), FA of sperm membrane phospholipids, sperm oxidized DNA (8-OH-dG), and induced acrosome reaction (AR). Treatment did not improve sperm motility and morphology, nor decrease the concentration of round cells and white blood cells in semen. Sperm concentration increased in oligozoospermic men (7.4+/-1.3 to 12.5+/-1.9 million/ml). Treatment significantly reduced ROS (mean+/-SEM) (775.3+/-372.2 to 150.3+/-105.2 x 10(3)counts/10 second) and 8-OH-dG (45.3+/-10.4 to 16. 8+/-3.3 fmol/microg DNA). Treatment increased the AR (55.1+/-2.2 to 71.6+/-2.2%), the proportion of polyunsaturated FA of the phospholipids, and sperm membrane fluidity. The overall pregnancy rate was 4.5% in 134 months. The per month pregnancy rate tended to be higher in partners of (ex)-smokers (7.15%, n=14,70 months) than in never-smokers (1.6%, n=13,64 months) (OR:4.57, 95% Cl:0.55-38.1).

Hormonal regulation of inhibin B secretion by immature rat sertoli cells in vitro: possible use as a bioassay for estrogen detection.

The influences of follicle-stimulating hormone (FSH), gonadal steroids, and culture time were studied in relation to inhibin B production by Sertoli cells of immature rats cultured in vitro. Sertoli cell-enriched cultures were established from 18-day-old rats and were maintained in medium supplemented with insulin, transferrin, and epidermal growth factor at 34 degrees C. A recently developed ELISA for the measurement of inhibin B was used to assess the effects of recombinant human FSH (rh FSH), testosterone (T), and estradiol (E2) on inhibin B production and accumulation in the culture media of Sertoli cell-enriched cultures and to optimize the cell culture system to serve as a bioassay for the detection and quantification of estrogens and estrogenlike substances. Prolonging the incubation time (24, 48, or 72 hours) of Sertoli cells with control medium without rh FSH, T, or E2 resulted in a time-dependent increase of inhibin B production. Incubation with rh FSH (1, 2.5, 5, or 10 U/L) caused a dose- and time-dependent increase of inhibin B production by Sertoli cells (but not by cultured Leydig cells), reaching a plateau at 5 U/L rh FSH. Addition of T in concentrations of 2.88, 5, or 50 ng/ml to medium without rh FSH and E2 significantly lowered the daily production rate of inhibin B (P < 0.05). In contrast, addition of E2 (0.01 and 0.1 ng/ml) caused a dose-responsive increase in inhibin B production after 24 and 48 hours. The relative increment of inhibin B production induced by E2 was maximal after 24 hours in the presence of 2.5 U/L rh FSH (acting synergistically) and in the absence of T. When these conditions are implemented, the Sertoli cell culture system may serve as a bioassay for estrogenic substances, and it may reflect the possibly harmful effect they may have on spermatogenesis.

Progesterone-transforming enzyme activity in the hypothalamus of the male rat.

The aim of the present study was to assess the activities of the progesterone (Pr) transforming enzyme systems 3alpha-oxidoreductase (3alpha-OR), 5alpha-reductase (5alpha-R) and 20alpha-oxidoreductase (20alpha-OR) in the hypothalamus of the male rat, at different stages of sexual maturation and following castration and adrenalectomy. Special attention was paid to transformation to 3alpha-reduced compounds previously shown to inhibit FSH synthesis and secretion. Homogenates of hypothalamic tissue were incubated with 14C-progesterone. Pr-metabolites were isolated, identified by gas chromatography/mass-spectrometry (GC/MS) and measured by liquid scintillation counting (LSC). In adult rats a ratio of 6:2.5:1 for 5alpha-R:3alpha-OR:20alpha-OR enzyme- activities was found. The hypothalamic 5alpha-R and particularly 3alpha-OR activities were considerably higher before puberty (10-20 day old rats) than in adulthood. Adrenalectomy in adult rats resulted in an increased activity of the three enzyme systems. No significant changes were seen following castration. Among the isolated metabolites, 3alpha-hydroxy-pregn-4-en-20-one (3alpha-Pr) and 3alpha-hydroxy-5alpha-pregnane-20-one (5alpha,3alpha-Pr) were identified. Conversion to both these neurosteroids was considerably higher during prepuberty than in adulthood. The finding that before puberty the hypothalamus has a markedly increased capacity to convert Pr to 3alpha-reduced compounds, such as 3alpha-Pr, known to effectively inhibit FSH release, warrants further research into the mechanisms regulating the hypothalamic formation of biologically active Pr derivatives and their role in the regulation of gonadotropin secretion.