Inhibition of testosterone biosynthesis by ethanol: relation to the pregnenolone-to-testosterone pathway

The concentrations of metabolites in the pregnenolone → testosterone pathway were determined in freezestopped testes in control rats and during ethanol intoxication (2 h after injection of 1.5 $\text{g ethanol}\text{kg}$ body wt). Ethanol lowered the mean testicular concentrations of testosterone (by 63–74%), androstenedione (49–81%), 17-hydroxyprogesterone (60–76%), progesterone (29–67%) and pregnenolone (12–25%). 4-Methylpyrazole had no effect on the ethanol-induced changes. The present results reveal no inhibition at the 17-hydroxyprogesterone → androstenedione → testosterone steps, but do not exclude inhibition before the step yielding pregnenolone and at the pregnenolone → progesterone → 17-hydroxyprogesterone steps.     

Plasma trace element (Se,Zn, Cu) concentrations in maternal and umbilical cord blood in Poland

Selenium (Se), copper (Cu), and zinc (Zn) concentrations were determined in plasma of 64 mothers at delivery, 58 nonpregnant women, 64 neonates, and 12 infants, aged 2–12 mo. Se and Zn concentrations in mothers at delivery were significantly lower, and Cu higher than in nonpregnant women. Mean Se and Cu concentrations in newborns were statistically lower than those in mothers at delivery, and Zn and Cu concentrations in preterm infants (n=13) were significantly higher than in fullterm infants (n=51). Maternal parity had no significant influence on the distribution of plasma trace element levels. No significant differences were observed in Se and Zn levels in maternal and cord blood plasma according to birth weight, contrary to maternal Cu concentration. Significant correlations were found between maternal and cord blood Se content, and between maternal plasma Cu concentration and birth weight of neonates.     

Androgens in the bovine fetus and dam (38567).

Umbilical arterial and venous blood, and fetal testes were taken from 38 bovine fetuses at 90, 180 or 260 days of gestation. Concurrently blood also was taken from the jugular, and from the uterine artery and vein of the dams. Testosterone and androstenedione were determined by radioimmunoassays. Fetal testicular homogenates had 0.96 and 0.35 mug/g of testosterone and 0.39 and 0.50 mug/g of androstenedione at 180 and 260 days of gestation, respectively. Males had five to tenfold more serum testosterone and about twofold more androstenedione than female fetuses at each trimester of gestation. Male fetal blood testosterone decreased (P less than 0.01) from 2.7 to 0.3 ng/ml between 90 and 260 days of gestation. But, maternal testosterone and androstenedione increased (P less than 0.05) during gestation in cows with males, but not in cows with female fetuses. Testosterone was higher (P less 0.05) in cows carrying males than in cows with female fetuses. Androstenedione was higher in blood leaving the placenta on both the maternal and on the vetal sides suggesting placental synthesis of androstenedione.     

Peripheral plasma levels of progesterone, oestradiol-17 beta, oestrone, testosterone, androstenedione and chorionic gonadotrophin during pregnancy in the marmoset monkey, Callithrix jacchus.

Concentrations of LH/CG, androstenedione and testosterone rose in early pregnancy to maximum values at 6--10 weeks. Thereafter LH/CG levels declined and androstenedione and testosterone levels remained at plateau values or declined until term. Progesterone, oestradiol-17 beta and oestrone increased after ovulation and remained high throughout pregnancy. At 12 weeks, when LH/CG levels were falling, progesterone and oestradiol rose well above the luteal-phase levels which were maintained for the first 12 weeks. Progesterone declined in the 2 weeks before birth, while oestradiol and oestrone remained high. Pregnancies of an unknown stage were dated by reference to a graph of uterine diameter, measured by abdominal palpation, in animals at known times after conception. Measurement of progesterone concentrations during the conception cycle gave more accurate dating and showed that the gestation length was 144 days.     

Plasma sex hormone concentrations during the reproductive cycle in the male lizard, Podarcis s. sicula

Progesterone, 17-hydroxyprogesterone, androstenedione, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, testosterone and oestradiol concentrations in the plasma were measured by simultaneous radioimmunoassay in males of the lizard Podarcis s. sicula. Hormonal determinations were performed at monthly intervals from January to December (except for August). Testosterone and androstenedione reached peak values of 174.8 ng/ml and 21.4 ng/ml in the mating season (spring) and then testosterone fell abruptly to 5.9 ng/ml in June remaining at this level during hibernation when dehydroepiandrosterone (DHA) reached a maximal level of 28.5 +/- 9.3 ng/ml. Castration resulted in a marked decrease of testosterone, androstenedione, dihydrotestosterone and DHA values, with DHA being significantly lowered only during the winter season. In castrated animals, however, testosterone and androstenedione persisted conspicuously in the plasma during the breeding period, suggesting that adrenal sex steroid output may change during the annual reproductive cycle. In intact animals, progesterone and oestradiol exhibited peak values during the refractory period after the mating season. We suggest a probable role of oestradiol in the induction of the refractory period in this lizard.     

Identification and quantification of C21 and C19 steroids in the haemolymph of Leptinotarsa decemlineata,a phytophagous insect

o-Pentafluorobenzyloxime (OPFB)-heptafluorobutyrylester (HFB)-derivatives were prepared from extracts of haemolymph from last instar larvae of Leptinotarsa decemlineata and subjected to negative ion chemical ionization capillary gas chromatography-mass spectrometry (NCI/GC-MS). Ten C21 and C19 steroids could be positively identified: testosterone, dehydroepiandrosterone, 5α-dihydrotestosterone, 11-ketotestosterone, 11β-hydroxytestosterone, androstenedione, progesterone, 17α-hydroxyprogesterone, pregnenolone and 17α,20β-dihydroprogesterone. No estrogens could be found in these larvae. Radioimmunoassay of chromatographed extracts of haemolymph taken from the larval and pupal stages showed fluctuations in testosterone (and 5α-dihydrotestosterone) titer.     

Testicular steroidogenesis in the baboon Papio anubis.

We recently reported that the baboon testis converts pregnenolone to testosterone through the delta-4 pathway. The present studies were to determine the metabolism of intermediates of the delta-4 and delta-5 pathway by the baboon testis. Fragments (50 mg) were incubated for 3 hr with 10 muCi of the following tritium-labelled substrates: pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, or testosterone. Pregnenolone was converted to testosterone primarily through the delta-4 pathway, with accumulation of progesterone, 17-hydroxyprogesterone and 20alpha-dihydroprogesterone as predominant intermediates. Similar results were obtained in progesterone incubations. 17-hydroxyprogesterone was not efficiently metabolized by the fragments, while 17-hydroxypregnenolone and dehydroepiandrosterone were efficiently converted into testosterone and androstenedione. Androstenedione was metabolized primarily to testosterone, while testosterone was not a suitable substrate. Some 5alpha-androstanediol was identified in each incubate. These results suggest that although testosterone is formed from pregnenolone through the delta-4 pathway, the delta-5 intermediates are more suitable substrates for testosterone synthesis in the baboon testis.     

Biosynthesis and metabolism of testosterone by sertoli cell-enriched seminiferous tubules

Sertoli cell-enriched tubules isolated from rats which had been treated with 1,4-dimethyl sulfonyloxybutane were incubated with either [¹⁴C] progesterone or [¹⁴C] testosterone for 2 hours. Tubules of normal rats and fragments of Sertoli cell-enriched testes were incubated under the same conditions. Sertoli cell-enriched tubules converted progesterone to 20α-dihydroprogesterone, 17α-hydroxyprogesterone, androstenedione and testosterone. The major metabolite was 20α-dihydroprogesterone. The percentage conversion of progesterone into testosterone corresponded to a production of 10–20 ng testosterone. Sertoli cell-enriched tubules converted testosterone to dihydrotestosterone, androstenedione, 3α-androstanediol and 3β-androstanediol. Under our experimental conditions, dihydrotestosterone was the major 5α-reduced metabolite. Normal tubules converted progesterone and testosterone to the same metabolites as Sertoli cell-enriched tubules. Fragments of Sertoli cell-enriched testes were much more active than isolated tubules in metabolizing progesterone. They produced the same amounts of 5α-reduced metabolites of testosterone.     

Testicular steroidogenesis in the rhesus monkey (Macaca mulatta.

Studies were undertaken to investigate testicular steroidogenesis in the Rhesus monkey Macaca mulatta. Testicular fragments (50 mg) were incubated for 3 hr with pregnenolone-7-3H or with progesterone-7-3H. The major metabolite of pregnenolone was progesterone (70.1%), with a lesser conversion to 17-hydroxyprogesterone (1.6%), androstenedione (3.3%), and testosterone (7.2%). The delta-5 intermediates 17-hydroxypregnenolone (4.6%) and dehydroepiandrosterone (8.6%) were also identified in the pregnenolone incubates. A majority of the progesterone substrate was not metabolized by the testicular fragments (80.1%), while some conversion to 17-hydroxyprogesterone (3.4%), androstenedione (4.8%), and testosterone (11.7%) occurred in the incubates. These results suggest that testicular fragments from the Rhesus monkey may convert pregnenolone to testosterone through both the delta-4 and the delta-5 pathways.     

Effects of chronic ethanol ingestion on steroid profiles in the rat testis

Testosterone, seven of its potential precursors, three of its metabolites and estradiol were analyzed in testes from rats given ethanol for 23 days in a nutritionally adequate liquid diet. The results were compared to those obtained with pair-fed control rats. The concentrations of pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were markedly lowered in four of the five rats given ethanol. The concentrations of the other 3 beta-hydroxy-delta 5 steroids and estradiol were unchanged, resulting in significantly increased ratios between 17-hydroxypregnenolone and 17-hydroxyprogesterone (P less than 0.025) and between androstenediol and testosterone (P less than 0.025) in the ethanol-treated rats. The results indicate that chronic ethanol administration reduces formation of testosterone by affecting a step prior to pregnenolone. There may also be an effect on the conversion of some 3 beta-hydroxy-delta 5 to the corresponding 3-oxo-delta 4 steroids. The levels of testosterone and three other steroids in testes of rats given the liquid diet were significantly lower than those in testes of animals fed a standard rat chow. This indicates a dietary influence on testicular steroid concentrations.     

Identification by capillary gas chromatography-mass spectrometry of eleven non-ecdysteroid steroids in the haemolymph of larvae of Sarcophaga bullata

$\text{O-}\text{Pentafluorobenzyloxime}$ (OPFB)-heptafluorobutyrylester (HFB) derivatives and $\text{OPFB}\text{-O-}\text{methyloxime}$ (MO)-trimethylsilylether (TMS) derivatives of non-ecdysteroid steroids were prepared from haemolymph extracts of last instar larvae of the fleshfly Sarcophaga bullata. Using a negative ion chemical ionization capillary gas chromatography-mass spectrometry (NCI/GC-MS) technique the following steroids could be identified: progesterone, testosterone, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, androst-5-ene-3β,17β-diol, androstenedione, 5α-dihydrotestosterone, 11-ketotestosterone, 11β-hydroxytestosterone, 17α-hydroxyprogesterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxyprogesterone. Although the technique is very sensitive, estrogens could not be detected. These results suggest an active metabolism of progesterone and testosterone.     

Plasma concentrations of testosterone,androstenedione, progesterone,oestradiol-17β, LH and FSH during the preovulatory period in the ewe

Seven mature ewes were synchronized to oestrus by two injections of 125 μg Cloprostenol given 9 days apart. Blood samples, collected for 72 h at 4-h intervals beginning 16 h after the second Cloprostenol injection, were assayed for testosterone, androstenedione, progesterone, oestradiol-17β, LH and FSH. For the testosterone measurements, two radioimmunoassays, using two different antisera, were validated and used. A typical pattern of release was observed for progesterone, oestradiol-17β, LH and FSH, with a preovulatory gonadotrophin peak recorded 16.1 ± 2.1 h after the observed oestrous behaviour. In two of the experimental animals, an extra oestradiol peak was recorded before the usual preovulatory rise. The changes in the concentrations of testosterone and androstenedione during the same period were not synchronous. The levels of the two androgens fluctuated considerably with occasional peaks of 150–250 pg/ml and even 900–1400 pg/ml. Although a tendency towards an increase in the levels of both androgens was observed during the period of oestrous behaviour, the individual variations were significant.     

Hormonal correlates of 'masculinization' in female spotted hyaenas (Crocuta crocuta). 2. Maternal and fetal steroids.

Concentrations of androgens (androstenedione, testosterone, 5 alpha-dihydrotestosterone), oestrogen and progesterone were measured in relation to pregnancy in the spotted hyaena (Crocuta crocuta). The gestation period was estimated to be about 110 days. There was a marked progressive rise in all the steroids starting in the first third of gestation. Chromatographic separation of plasma showed that much of the oestrogen is not oestradiol (only 12% of total measured) and that a significant fraction of the 'testosterone' may be dihydrotestosterone. In the final third of pregnancy, concentrations of androgen (especially testosterone plus dihydrotestosterone) in the female circulation reached the maximal values of adult males; the percentage of dihydrotestosterone relative to total testosterone plus dihydrotestosterone was higher in females (44 +/- 3.9%, n = 20) than in males (29.5 +/- 3.5%, n = 17). Plasma androstenedione was also significantly higher in females, but the increment was less than for oestrogen, testosterone and progesterone, and the temporal pattern was less clear. Samples from the maternal uterine and ovarian circulation showed that androstenedione is largely of ovarian origin and metabolized by the placenta, while testosterone, progesterone and oestrogen are primarily of placental or uterine origin. Fetal samples were taken from two mixed-sex sets of twins and one male singleton. Gradients across the placenta measured in the fetal circulation confirmed that the placenta metabolizes androstenedione and is a source of testosterone for the female fetus; there were no consistent differences in androgens between male and female fetuses. It is suggested that the conspicuous masculinization of the female spotted hyaena, especially evident in the external genitalia at birth, is a result, at least in part, of high placental production of testosterone or dihydrotestosterone derived from the metabolism of high maternal androstenedione.     

Progesterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and androsterone concentrations in specific regions of the human brain

The concentrations of progesterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and androsterone were determined in tissue samples from the human hypothalamus, anterior pituitary, pineal, amygdala and parietal cortex, taken at autopsy from male (n = 4) and female cadavers (n = 4) of various ages. The measurements were performed using radioimmunoassays for the individual steroids after the chromatographic purification of solvent extracts of tissue samples on Lipidex-5000TM. Preliminary qualitative analyses of the chromatographic profiles of various steroids by radioimmunoassay demonstrated the presence of these steroids in various regions of the brain, but an immunoreactive peak corresponding to 17-hydroxyprogesterone was not found. The concentrations (ng/g tissue wet wt.) of all steroids measured were either very low or below the limit of detection in brain tissues taken from male and female infants. In the adult brain, there was no difference in the distribution of steroids between the various regions studied. There was no sex difference in the brain tissue steroid concentrations, with the exception of testosterone which was clearly much higher in brain tissues from men as compared to women. Although testosterone was undetectable in most samples taken from adult women. 5 alpha-dihydrotestosterone could be measured in almost all samples, which suggests that this is the most important androgen in the human brain. When brain tissue steroid levels are compared with serum concentrations, it can be postulated that a state of equilibrium exists between the fraction of serum steroids which are not bound to high-affinity binding proteins and the amount of steroids in brain tissues.     

Urinary excretion of free progestins, androgens, and estradiol after injection of (1-24)ACTH in the Mongolian gerbil

Injection of a "long-acting" synthetic adrenocorticotrophin [(1-24)ACTH, 20 IU/animal] into Mongolian gerbils resulted in a 3.1 fold increase of urinary free testosterone excretion over 2 days. It was accompanied by an elevation of urinary free progesterone (2.1 fold), 17-hydroxyprogesterone (2.5 fold), DHEA (2.8 fold) and androstenedione (3.0 fold) excretion. Similarly, administration of human chorionic gonadotrophin (HCG, 100 IU/animal) increased urinary excretion of free testosterone (2.3 fold), progesterone (4.1 fold), 17-hydroxyprogesterone (2.9 fold), DHEA (4.6 fold), androstenedione (5.4 fold) and of estradiol (2.9 fold). Data presented in this work show that the measurement of urinary free steroid excretion represents a reliable index for the secretory activity of the adrenal-gonadal-axis, and that it may in some aspects be more practicable than the measurement of steroid plasma levels, especially in small laboratory animals, enabling us to monitor the excretion of various steroids over longer time periods without stressing the animals by handling/or blood sampling.     

Endocrine correlates of pregnancy in the ring-tailed lemur (Lemur catta): implications for the masculinization of daughters

Female ring-tailed lemurs (Lemur catta) are Malagasy primates that are size monomorphic with males, socially dominate males, and exhibit a long, pendulous clitoris, channeled by the urethra. These masculine traits evoke certain attributes of female spotted hyenas (Crocuta crocuta) and draw attention to the potential role of androgens in lemur sexual differentiation. Here, hormonal correlates of prenatal development were assessed to explore the possibility that maternal androgens may shape the masculine morphological and behavioral features of developing female lemurs. Maternal serum 17α-hydroxyprogesterone, dehydroepiandrosterone sulphate (DHEA-S), ?⁴ androstenedione (androst-4-ene-3,17,dione), testosterone, and 17β-estradiol were charted throughout the 19 pregnancies of 11 ring-tailed lemurs. As in spotted hyenas, lemur pregnancies were associated with an immediate increase in androgen concentrations (implicating early maternal derivation), followed by continued increases across stages of gestation. Pregnancies that produced singleton males, twin males, or mixed-sex twins were marked by greater androgen and estrogen concentrations than were pregnancies that produced singleton or twin females, especially in the third trimester, implicating the fetal testes in late-term steroid profiles. Concentrations of DHEA-S were mostly below detectable limits, suggesting a minor role for the adrenals in androgen biosynthesis. Androgen concentrations of pregnant lemurs bearing female fetuses, although less than those of pregnant hyenas, exceeded preconception and postpartum values and peaked in the third trimester. Although a maternal (and, on occasion, fraternal) source of androgen may exist for fetal lemurs, further research is required to confirm that these steroids would reach the developing female and contribute to her masculinization.     

Serum HPL level in maternal vein, umbilical cord artery and vein in term and preterm birth

The serum HPL level in maternal vein and the cord vein and artery in term (n = 34) and preterm (n = 74) birth was tested to classify the hormonal changes and the correlation between the maternal and fetal HPL values. It was found that between the 28th and the 40th weeks of gestation the maternal level tended to increase while in the cord vein and artery it decreased significantly in negative correlation with the weeks of pregnancy. The maternal serum HPL level was significantly higher than that of the cord vein and artery. Between the 28th and 40th week the value in the cord artery divided by the value in the cord vein multiplied by 100 tended to decrease. While the HPL concentration in maternal serum did not correlate with its level in the cord vein and artery the levels in the cord vein and artery were significantly correlated. The characteristic and independent change in the fetal serum HPL concentration raised the possibility of a change in the transport function of the placenta and of an autonomous fetal HPL metabolism.     

Relationship between functional activity of the adrenal glands and gonads in monkeys during puberty]

Testicular and adrenal cortex endocrine functions were simultaneously tested in pubertal baboons (papio hamadryas) during one year. Concentrations of androgens (testosterone, 5 alpha-dihydrotestosterone, dehydroepiandrosterone), corticosteroids (cortisol, progesterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone) and aldosterone were determined in blood of the experimental monkeys by radioimmunoassay. It was shown that with increasing androgen (testosterone and dihydrotestosterone) levels in blood of the pubertal monkeys there was a significant decrease of corticosteroid concentration which was most pronounced in the monkeys with maximum increase of testosterone level. Described hormonal changes did not influence aldosterone concentration.     

Identification of phospholipids capable of modulating the activities of some enzymes involved in androgen and 16-androstene biosynthesis in the immature pig testis.

Testicular steroidogenic enzymes in the microsomal fraction from immature pigs were investigated for the effects of phospholipids of known structure on androgen and 16-androstene biosynthesis. Untreated (control) microsomes metabolized pregnenolone to 17-hydroxypregnenolone, DHA and small quantities of progesterone, 17-hydroxyprogesterone, androstenedione and testosterone; and to 5,16-androstadien-3 beta-ol (andien-beta) and 4,16-androstadienone (dienone) in the 16-androstene pathway. Phosphatidyl(P)-serine, P-glycerol, P-ethanolamine, P-inositol, P-choline and phosphatidic acid did not significantly alter the 17-hydroxylase/C-17,20 lyase or "andien-beta-synthetase" activities. Thus, the C21 side-chain cleavage reactions appeared not to be dependent upon phospholipids for optimal activity. The conversion of pregnenolone to 4-ene steroids (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) was inhibited by dilinoleoyl-phosphatidyl-choline, but other phospholipids tested were without effect. On the other hand, the conversion of andien-beta to dienone was inhibited by P-serine, P-inositol and P-cholines with short saturated or long polyunsaturated acyl chains. Therefore, the presence of these phospholipids in pregnenolone incubations had different consequences for 3 beta-hydroxysteroid dehydrogenase-isomerase activities. It is concluded that substrate specific 3 beta-HSD-isomerases exist for androgen and 16-androstene biosynthesis and that phospholipids may play an intrinsic role in their catalytic activity.     

The conversion of pregnenolone-7alpha-3H and progesterone-4-14C to estrogens by corpora lutea of menstrual cycles and pregnancy.

The metabolism of pregnenolone-7alpha-3H and progesterone-4-14C by human corpora lutea tissue of menstrual cycles and pregnancy was studied. In the incubations, equimolar mixtures of pregnenolone-7alpha-3H and progesterone-4-14C were used as substrates. Three corpora lutea of cycles were used as minced tissue. From those corpora lutea progesterone, 17-hydroxyprogesterone and androstenedione were formed, although no estrogens were formed. One corpus luteum of cycle and one corpus luteum of pregnancy were used as homogenated tissue, and those formed estrone and estradiol as well as the same three delta4-metabolites. The corpus luteum of cycle also formed testosterone. All metabolites including estrogens showed the lower 3H to 14C ratio than the starting ratio. 17-hydroxypregnenolone in only one corpus luteum, and no delta5-metabolites in the other four corpus luteum were identified. It is therefore proposed that the major pathway for estrogen formation in human corpus luteum is pregnenolone yields progesterone yields 17-hydroxyprogesterone yields androstenedione (or testosterone) yields estrone and estradiol.