Personality traits change after an opportunity to mate.

There is growing evidence that personality traits can change throughout the life course in humans and nonhuman animals. However, the proximate and ultimate causes of personality trait change are largely unknown, especially in adults. In a controlled, longitudinal experiment, we tested whether a key life event for adults--mating--can cause personality traits to change in female threespine sticklebacks. We confirmed that there are consistent individual differences in activity, sociability and risk-taking, and then compared these personality traits among three groups of females: (i) control females; (ii) females that had physically mated, and (iii) females that had socially experienced courtship but did not mate. Both the physical experience of mating and the social experience of courtship caused females to become less willing to take risks and less social. To understand the proximate mechanisms underlying these changes, we measured levels of excreted steroids. Both the physical experience of mating and the social experience of courtship caused levels of dihydroxyprogesterone (17α,20ß-P) to increase, and females with higher 17α,20ß-P were less willing to take risks and less social. These results provide experimental evidence that personality traits and their underlying neuroendocrine correlates are influenced by formative social and life-history experiences well into adulthood.

Progesterone metabolism by the hypothalamus, pituitary, and uterus of the aged rat.

Progesterone metabolism was examined in tissues of rats in three stages of reproductive senescence (constant estrus, repeated pseudopregnancies, and anestrus) and in young rats. Metabolites were quantitated by reverse isotopic dilution analysis after incubation of the hypothalamus, pituitary, and uterus with [3H]progesterone. The metabolism of progesterone to 5 alpha-dihydroprogesterone and to 20 alpha-hydroxy-5 alpha-pregnan-3-one and the formation of total 5 alpha-reduced products was significantly reduced (by half) in pituitaries of constant estrous rats compared to rats in all other stages. The formation of 3 alpha-hydroxy-5 alpha-pregnan-20-one and total 3 alpha-reduced products was about 2-fold higher in pituitaries and hypothalami of pseudopregnant and anestrous rats than in constant estrous and young rats, but these differences were statistically significant only in the pituitary samples. In the uterus, progesterone metabolism to 20 alpha-dihydroprogesterone was significantly increased in anestrous rats compared to that in constant estrous and pseudopregnant rats. The results indicate that progesterone metabolism by target tissues, particularly the pituitary, is altered during reproductive senescence. They suggest the possibility that changes in the tissue metabolism of progesterone may be one means by which the effectiveness of progesterone is decreased during aging.

Microbial transformations of steroids-XI. Progesterone transformation by Streptomyces roseochromogenes-purification and characterisation of the 16alpha-hydroxylase system.

Streptomyces roseochromogenes, NCIB 10984, contains a cytochrome P450 which, in conjunction with two indigenous electron transfer proteins, roseoredoxin and roseoredoxin reductase, hydroxylates exogenous progesterone firstly to 16alpha-hydroxyprogesterone and thereafter in a second phase bioconversion to 2beta,16alpha-dihydroxyprogesterone. The progesterone 16alpha-hydroxylase P450 and the two electron transfer proteins have been purified to homogeneity. A reconstituted incubation containing these three purified proteins and NADH, the natural electron donor, produced identical hydroxy-progesterone metabolites as in intact cells. Peroxy and hydroperoxy compounds act in a shortened form of the cycle known as the 'peroxide shunt' by replacing the natural pathway requirement for the electron donor NADH, the electron transfer proteins and molecular O2, the terminal electron acceptor. In an NaIO4 supported incubation, the initial rate of progesterone hydroxylation was marginally higher (1.62 mmol progesterone/mmol P-450/h) than in the reconstituted natural incubation (1.18 mmol progesterone/mmol P-450/h) but the product yield was significantly lower, 0.45 mol hydroxyprogesterone produced/mol P-450 compared to 6.0 mol hydroxyprogesterone produced/mol P-450. These yield data show that in the reconstituted natural pathway, progesterone 16alpha-hydroxylase P450 supports multiple rounds of hydroxylation in contrast to a likely single oxygenation by a minority of P450s in the peroxide shunt pathway.

Two novel microbial conversion products of progesterone derivatives.

Two novel microbial steroid hydroxylations were found in a screening of 131 microorganisms under aerobic conditions. 17 alpha-Hydroxyprogesterone was hydroxylated in the 8 beta-position by Corynespora melonis CBS 16260, and fermentation of 17 alpha-acetoxyprogesterone with Pycnosporium species ATCC 12231 yielded 11 beta-hydroxy- and 11 beta, 12 beta-dihydroxy-17 alpha-acetoxyprogesterone. The known 11 beta-hydroxy compound could be obtained as a single product with Trichothecium roseum ATCC 12519.

In vitro-metabolism of 3H-progesterone in human testicular tissue: II Prepubertal and adolescent boys.

Normal sexual maturation in the human male depends upon a thorough regulation of the intratesticular androgen biosynthesis at various ages. We have systematically analysed the intratesticular steriod metabolic pathways in 11 boys, and 5-15 years, by means of incubation of minor testicular biopsy specimens with 3H-progesterone as substrate. Seven boys had abnormally situated testicles, and four had normally descended testicles. For comparison, 3 rats (10, 15 and 90 days old) were also investigated. Significant steroidogenic activity could be demonstrated in all prepubertal testicular tissue specimens. The steroid metabolic products recovered were identical with those found in a previously reported series of nine adult males (Kjessler & Berg, 1976), i.e., 20a-dihydroprogesterone, 17a-hydroxyprogesterone, 20a,17a-dihydroxyprogesterone, androstenedione and testosterone. No appreciable amounts of 5a-reduced substances were found. The steroid metabolic patterns observed in the present prepubertal and adolescent testicles differed from those found in adult males especially by their dissimilar proportions of newly synthesized 20a-dihydroprogesterone and 17a-hydroxyprogesterone. In prepubertal testicular tissue below the age of 11, 20a-dihydroprogesterone accounted for 45-82 per cent, and 17a-hydroxyprogesterone for only 3-6(-17) per cent of all metabolites formed. The steroid metabolic patterns displayed by abnormally situated testicles did not differ from those of normally descended testicles. The variation in steroid metabolic patterns observed between prepubertal and adult testicular tissue may be explained by the existing difference in physiologic gonadotrophic stimulation. It It may also be the result of an age-dependent maturation in various enzyme systems. The regulation of androgen biosynthesis with increasing age in the human male gonads does not seem to involve any significant reduction by 5a-reductase of continuously produced androgens, as in the immature rat. 2nstead, it appears to be brought about by a gradual shift in the metabolic pathways preferred by means of an increased activity of 17a-hydroxylase.

In vitro-metabolism of 3H-progesterone in human testicular tissue: III Males with extremely high and low levels of gonadotrophic hormones in the peripheral circulation; including one "XX-male".

Individual steroid metabolic patterns in testicular tissue seem to vary characteristically with increasing maturity, as previously demonstrated by means of in vitro incubation studies with 3H-progesterone as substrate (Kjessler & Berg 1976; Berg et al. 1976). In the present study, individual steroid metabolic patterns were determined in two hypergonadotrophic males, one of whom representing the first instance of an "XX-male" studied in detail with regard to intratesticular steroidogenesis, and one hypogonadotrophic, hypophysectomized male. The radiolabelled steroid metabolic products recovered in the present series of testicular incubates were identical with those earlier observed in various series of prepubertal, adolescent and adult testicular specimens, i.e., 20alpha-dihydroprogesterone, 17alpha-hydroxyprogesterone, 17alpha,20alpha-dihydroxy-progesterone, androstenedione and testosterone. Thus, again, no appreciable amounts of 5alpha-reduced compounds were found in the present hyper- and hypogonadotrophic individuals. However, the proportions of newly synthesized and recovered delta4-3-oxo-C21-steroids in this series, especially 20alpha-dihydroprogesterone and 17alpha-hydroxyprogesterone, were found to differ considerably with various gonadotrophic stimulations. Hence, the relative proportions of 17alpha-hydroxyprogesterone increased, whereas the relative proportions of 20alpha-dihydroprogesterone decreased, with increasing gonadotrophic stimulation. The present results sustain and further extend our previous assumption that the relative proportions of newly synthesized and recovered delta4-3-oxo-C21-steroids in testicular incubates in vitro, especially 20alpha-dihydroprogesterone and 17alpha-hydroxyprogesterone, are primarily related to the individual degree of physiologic gonadotrophic stimulation at the moment of testicular biopsy, and less related to an unspecific maturation of various steroid metabolic enzyme systems.

Increased substrate concentration causes a shift from production of 11-oxygenated androgens to 17,20-dihydroxyprogestogens during the in vitro metabolism of 17-hydroxyprogesterone by goldfish testes.

Goldfish testes were incubated with [3H]17-hydroxyprogesterone in the presence of 0 to 100 micrograms/ml of unlabeled substrate and metabolites examined by thin-layer and high performance liquid chromatography. Conjugated steroids, predominantly sulfates, accounted for 50% of recovered activity with radiolabeled substrate alone, but percentage yields decreased to very low levels with substrate concentrations of 1 micrograms/ml and above. The 11-oxygenated androgens, androstenetrione and 11-ketotestosterone, were the major products with 0 to 0.1 micrograms/ml substrate, but at concentrations of 1 to 100 micrograms/ml the major products were 17,20 alpha-dihydroxy-4-pregnen-3-one (30% of recovered activity) with smaller amounts of the 20 beta-epimer. 11-Deoxycortisol was a minor product at all substrate concentrations. Production of 11-oxygenated androgens in the medium reached a maximum value of 40 ng/100 mg tissue/3 hr with 2 micrograms substrate, but progestogen production continued to increase up to the maximum substrate used (30 micrograms at 200 micrograms substrate). The results demonstrate a clear switch from production of 11-oxygenated androgens to that of 20-reduced progestogens with increased substrate concentration. This switch shows similarities to that observed for in vivo plasma steroid concentrations during the prespawning period of many male teleosts and it is suggested that this, at least in part, may be due to increased substrate availability resulting from elevated gonadotropin secretion.

In situ subcellular distribution and metabolism of progesterone, estradiol and androstenedione in the vascularly separated and isolated hypothalamus of the female rhesus monkey.

A neurosurgical procedure has been developed for the vascular isolation of the hypothalamus-thalamus region of the rhesus monkey brain. The circulation to the left and right halves of the hypothalamus was also isolated and each half of the hypothalamus was perfused simultaneously, but separately, with a dextran-blood solution which contained radioactive gonadal steroids. The hypothalamus in situ efficiently converted [3H]androstenedione to [3H]estrone and this aromatization was inhibited by the presence of androsta-1,4,6-triene-3,17-dione (ATD) in the perfusate. [3H]Progesterone was metabolized predominantly to 5 alpha-pregnane-3,20-dione (5 alpha-DHP) and 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-OHP). Subcellular fractionation of the hypothalamus after the in situ perfusion with [3H]-progestin or [3H]estradiol to the hypothalamus of estrogen-treated ovariectomized monkeys or oil-treated ovariectomized monkeys, respectively, indicated that the retention of [3H]estradiol in the nucleus was a saturable, limited-capacity phenomenon. No saturable subcellular distribution of [3H]progesterone or [3H]R 5020 was observed. This latter observation might be attributable to the presence of a progesterone receptor in too small a concentration to be detected by the methods used.

Plasma membrane cholesterol is utilized as steroidogenic substrate in Y-1 mouse adrenal tumor cells and normal sheep adrenal cells.

Previous studies from this laboratory indicate that plasma membrane cholesterol acts as an important source of steroidogenic substrate for MA-10 Leydig tumor cells. The present studies were designed to generalize these findings to other steroidogenic cells and to another species. Studies were performed using the Y-1 murine adrenal tumor cell line and primary cultures of sheep adrenocortical cells. Treating Y-1 cells with the acyl coenzyme A:cholesterol acyltransferase inhibitor, 58-035, caused cellular cholesteryl ester depletion and rendered more apparent the effect of dibutyryl-cAMP to cause cellular free cholesterol depletion. Radioactive 20 alpha-dihydroprogesterone was synthesized by Y-1 cells that had been plasma membrane-labeled with [3H]-cholesterol. Primary sheep adrenal cultures that had been cholesteryl ester-depleted also demonstrated cellular free cholesterol depletion after stimulation with dibutyryl cAMP. Plasma membrane label was converted to steroid hormones in these cells as well. Taken together, these data indicate that the use of plasma cholesterol is not restricted to the MA-10 cells. The present data indicate that both neoplastic mouse adrenal tumor cells and normal sheep adrenal cells utilize plasma membrane cholesterol.

Expression of a full-length cDNA encoding bovine adrenal cytochrome P450C21.

Two full-length cDNA clones encoding bovine adrenocortical P450C21 have been constructed in a eukaryotic expression vector using partial-length cDNAs whose structures have been previously reported. Following expression of these cDNAs in COS 1 cells, the substrate specificity of P450C21 was determined. Of the 18 steroids tested, progesterone, 17 alpha-hydroxyprogesterone, and 11 beta,17 alpha-dihydroxyprogesterone were found to be the only steroids to serve as substrates for this adrenal enzyme, a much higher degree of substrate specificity than has been reported for a hepatic 21-hydroxylase. The Vmax for 17 alpha-hydroxyprogesterone was 2.5 times greater than that for progesterone, whereas delta 5-steroids were unable to serve as substrate for this enzyme. A difference between the two cDNAs is located at amino acid 401 where one resultant enzyme contains tyrosine while the other contains histidine. This amino acid difference appears to have no effect on the kinetic properties of adrenal P450C21.

Accumulation and mobilization of triglycerides and cholesteryl esters in Leydig tumor cells.

Incubating MA-10 Leydig tumor cells with sodium oleate led to the accumulation of triglyceride within the cells. Triglycerides were deposited in a time- and dose-dependent fashion. Cellular triglyceride promoted storage of cholesteryl ester. As much cholesteryl ester was stored in oleate-treated cells as in cells treated with saturating concentrations of low density lipoprotein. Addition of both oleate and low density lipoprotein resulted in additive accumulation of cholesteryl esters. Cholesteryl esters in cells loaded with triglyceride by oleate treatment were mobilized in response to dibutyryl-cAMP to an extent similar to that in cells containing low triglyceride concentrations. Dibutyryl-cAMP stimulated cholesteryl ester mobilization under all conditions, and stimulated triglyceride mobilization when adequate fatty acid acceptors were available. The results indicate that while triglyceride accumulation in MA-10 cells promoted cholesteryl ester deposition, it did not impair cAMP-dependent cholesteryl ester hydrolysis or steroid hormone production.

In vitro-metabolism of 3H-progesterone in human testicular tissue: I Adult males.

Androgen biosynthesis in the male gonads may be analysed in some detail by means of in vitro incubation of minor testicular biopsy specimens with various radiolabelled steroid precursors. We have investigated nine adult human male voluteers without apparent gonadal dysfunction with regard to their in vitro metabolism of 3H-progesterone. The following metabolic compounds were recovered: 20a-dihydroprogesterone, 17a-hydroxyprogesterone, 20a, 17a-dihydroxyprogesterone, androstenedione and testosterone. No significant amounts of 5a-reduced delta4-3-oxo-steroids or oestrogens were found. The major metabolites formed were 20a-dihydroprogesterone and 17a-hydroxyprogesterone, which accounted for 13-49 per cent and 17-47 per cent, respectively, of all newly synthesized steroid compounds. Radiolabelled delta4-3-oxo-C19-metabolites were recovered in minor amounts only, possibly due to metabolic interference with the endogenous pool of cold mass compounds. The individual steroid metabolic patterns were found to be poorly related to the individual levels of FSH, ICSH (LH) and testosterone in the peripheral circulation. Adequate knowledge of the steroid metabolic pathways generally utilized in ordinary testicular tissue in vitro is a prerequisite for the evaluation of steroid metabolism in males with various types of gonadal dysfunction.

Trout steroidogenic testicular cells in primary culture. II. Steroidogenic activity of interstitial cells, Sertoli cells, and spermatozoa.

Somatic cells (interstitial cells and Sertoli cells) were prepared either as single cells or in clusters, from spermatogenic and mature trout testes, according to Loir (1988), and cultured for 10-14 days. Sertoli cells are 3 beta-HSD negative when prepared from testes resuming spermatogenesis and from mature testes, but they are 3 beta-HSD positive in spermatogenic testes. Progesterone, 17 alpha-hydroxyprogesterone (17 alpha-OH-P), and free androgens are secreted by interstitial cells, 11-ketotestosterone (11KT) being the predominating steroid produced immediately after seeding. These cells also produce high levels of glucuronated androgens. At least in mature spermiating testes they do not secrete estradiol. After isolation, interstitial cells would lose most of their ability to secrete 17 alpha-hydroxy,20 beta-dihydroprogesterone (17 alpha 20 beta-OH-P) but they would recover it later. Testicular spermatozoa, which convert 17 alpha-OH-P independently of s-GtH, constitute a second source of this progestagen. In addition, our results suggest that Sertoli cells could be able to secrete 17 alpha-OH-P and also progesterone. A possible participation of the intralobular production of the former progestagen to the local regulation of germ cell maturation is evoked.

In vitro-metabolism of 3H-progesterone in human testicular tissue: IV Before and after long-term gonadotrophin treatment; including one 47, XYY-male.

Certain defects in the intratesticular androgen biogenetic system may lead to a significant impairment of gonadal function in total in the human male. In the present investigation, four males with impaired reproductive performance were analysed before and after long term treatment with gonadotrophic hormones with regard to their in vitro metabolism of 3H-progesterone in testicular incubates; one of whom representing the first instance of an XYY-male studied in some detail with regard to intratesticular steroidogenesis. A steroid metabolisc pattern of an "immature" type, i.e., possibly indicating a relative understimulation of the gonads by gonadotrophic hormones, in vivo, was found in one of the four patients, i.e., the XYY-male, inspite of normal levels of gonadotrophic hormones in the peripheral circulation. Gametic output was found to increase significantly during the course of gonadotrophin substitution therapy, and the individual steroid metabolic pattern also changed drastically towards a more mature type subsequent to therapy. In a second patients, who originally presented with completely immotile sperm, a certain shift in the steroid metabolic pattern was observed after the gonadotrophin therapy in favour of increasing relative amounts of 17a-hydroxyprogesterone, concurrent with a decreasing proportion of dead sperm, and a certain, though minimal, improvement in sperm kinetics. The last two patients, with originally mature types of in vitro steroid metabolism in testicular incubates, displayed no changes in steroid metabolic patterns or spermiogram qualities subsequent to the gonadotrophin therapy. However, one of them might have been azoospermic because of anatomical reasons. The present demonstration that an individual steroid metabolic pattern in testicular incubates under certain conditions may be deliberately modified as expected, by long term gonadotrophin therapy, further supports our previous suggestion that the relative proportions of newly synthesized and recovered deltal4-3-oxo-C21-steroids in testicular incubates, especially 20a-dihydroprogesterone and 17a-hydroxyprogesterone, may well reflect the actual gonadotrophic stimulation in a particular individual at the time of testicular biopsy. Investigations of individual steroid metabolic patterns in testicular incubates may therefore contribute useful information for the adequate selection of patients with disturbed gonadal function, who may benefit from gonadotrophin substitution therapy.

Luteolytic effect of the antiprogestin and antiglucocorticoid agent RU486 in rats.

Ovarian cells of pregnant rats were cultured with synthetic progestins (R5020, R2323), dexamethasone and RU486. Progesterone and 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) in the medium were measured by specific radioimmunoassay. Both R5020 and R2323 increased concentrations of these intrinsic progestins. RU486 decreased concentrations of progesterone, however, the addition of R5020 or R2323 counteracted this action. Immature hypophysectomized rats treated with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) were administered with RU486; the serum levels of progesterone and 20 alpha-dihydroprogesterone tended to decrease. R5020 and R2323 inhibited the effect of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), whereas RU486 did not. Inhibition of the cholesterol side chain cleavage enzyme (CSCC) by RU486 was more marked than that by R5020 or R2323. These results show that RU486 decreases progesterone synthesis in cultured ovarian cells. A part of the mechanism may involve an inhibition of CSCC.

Effects of peripheral-type benzodiazepine receptor antisense knockout on MA-10 Leydig cell proliferation and steroidogenesis.

The peripheral-type benzodiazepine receptor (PBR) is not only widely expressed throughout the body, but it is also genetically conserved from bacteria to humans. Many functions have been attributed to it, but its primary role remains a puzzle. In the current study, we stably transfected cultures of MA-10 Leydig cells with either control or 18-kDa PBR antisense knockout plasmids. The antisense knockout vector was driven by the human enkephalin promoter, which contains two cAMP response elements, such that cAMP treatment of transfected cells could superinduce 18-kDa PBR antisense RNA transcription and, hence, down-regulate endogenous 18-kDa PBR mRNA levels. Control and knockout MA-10 cell lines were then compared at the level of receptor binding, thymidine incorporation, and steroid biosynthesis. Eighteen-kilodalton PBR knockout reduced the maximal binding capacity of tritium-labeled PBR ligands, and the affinity of receptors to the ligands remained unaltered. Additionally, 24-h accumulation of progesterone was lower in the knockout cells. Exposure of the two cell types to 8-bromo-cAMP resulted in a robust increase in steroid production. However, a complex pattern of steroid accumulation was observed, in which further progestin metabolism was indicated. The later decline in accumulated progesterone as well as the synthesis of androstenedione were different in the two cell types. At the level of cell proliferation, reduction of 18-kDa PBR mRNA showed no effect. Thus, we conclude that the 18-kDa PBR may have a more important role in steroidogenesis than in proliferation in this Leydig cell line.