Formation of 5alpha-reduced C19-steroids from progesterone in vitro by a pathway through 5alpha-reduced C21-steroids in ovaries of late prepubertal rats.

Ovarian homogenates from 10-150-day-old rats were incubated with [3H]progesterone and NADPH. Also, ovarian homogenates from 28-day-old rats were incubated for 5-180 min with either [14C]progesterone, [3H]5alpha-pregnane-3,20-dione or [14C]progesterone plus [3H]5alpha-pregnane-3,20-dione. Following incubation, radioactive metabolites were isolated, identified, and measured by column and paper chromatography, with derivative formation and recrystallizations to constant specific activity. Prepubertal ovaries (10, 20, and 28 days of age) converted 15-60% of progesterone to C21-17-hydroxysteroids and C19-steroids. At 40 and 150 days of age (postpubertal), the formation of these steroids decreased to less than 2%. At 10 and 150 days of age, the major C19-steroids formed from progesterone were androstenedione and testosterone. At 20 and 28 days of age, however, no accumulation of these C19-delta4-3ketosteroids was found (less than 0.1% of each), at which time the conversion of progesterone to 5alpha-reduced C19-steriods, such as androsterone and 5alpha-androstane-3alpha,17beta-diol, reached 30%. In ovaries of 28-day-old rats, the results from incubation studies for the detection of metabolic pathways indicated two biosynthetic pathways leading to 5alpha-reduced C19-steroids, one from progesterone via 5alpha-reduced C21 steroids, such as 3alpha-hydroxy-5alpha-pregnan-20-one and 3alpha,17alpha-dihydroxy-5alpha-pregnan-20-one, and a second via 17-hydroxyprogesterone, androstenedione, and testosterone. It seems that the active 5alpha-reduction of C19-delta4-3-ketosteroids and the formation of 5alpha-reduced C19-steroids by the pathway through 5alpha-reduced C21-steroids, are present in the ovaries of older prepubertal rats and may be the biological significance.     

Relationship between the structures and steroidogenic functions of the testes of the urohaze-goby (Glossogobius olivaceus)

The testis of the brackishwater goby (Glossogobius olivaceus, the urohaze-goby in this text) consists of two main components, the glandular and the seminiferous tissue. After manual separation of the two tissues, in vitro steroidogenesis in each tissue was examined using testes from mature males in the breeding season. Cell-free homogenates (800g supernatant fluid) of each tissue were aerobically incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, testosterone, or 5 alpha-pregnane-3,20-dione in the presence of NAD+ or NADPH. (1) Glandular tissue: Pregnenolone and dehydroepiandrosterone were converted to progesterone and androstenedione, respectively, in the presence of NAD+. In the presence of NADPH, the following metabolism of steroids was established. Progesterone was transformed to 5 alpha-pregnane-3,20-dione (main product), 17 alpha-hydroxyprogesterone, 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, and androstenedione. 17 alpha-Hydroxyprogesterone was metabolized into 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione (main product), 3 beta, 17 alpha-dihydroxy-5 alpha-pregnan-20-one, androstenedione, and 5 alpha-androstane-3,17-dione. From androstenedione, 5 alpha-androstane-3,17-dione (main product) and epiandrosterone were obtained. Testosterone was transformed to 5 alpha-dihydrotestosterone (main product), 5 alpha-androstane-3 beta, 17 beta-diol, epiandrosterone, and 5 alpha-androstane-3,17-dione. 5 alpha-Pregnane-3,20-dione was metabolized into 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, 5 alpha-androstane-3,17-dione, epiandrosterone, and 5 alpha-dihydrotestosterone. (2) Seminiferous tissue: Almost all of the above metabolites were obtained, but the yield was much smaller, especially for 5 alpha-reduced metabolites, compared with that for glandular tissue. From these results, it is concluded that steroidogenesis in the testis of G. olivaceus is characterized by the predominant activity of 5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase and that these are localized mainly in glandular tissue, together with delta 5-3 beta-hydroxysteroid dehydrogenase + delta 5-delta 4 isomerase, 17 alpha-hydroxylase, and C-17-C-20 lyase.     

Progesterone metabolism in vitro by rabbit testes at different stages of development.

Testicular homogenates from white rabbits of 0.4, 1, 3, 4, 5, 8, 18, and 24 months of age were incubated with [3H]progesterone and NADPH. At 12 days of age, major C21-17-OH-and C19- steroids formed from progesterone were 17alpha-hydroxyprogesterone and testosterone. However, at 4-24 months of age, 17alpha-hydroxyprogesterone, 3beta,17alpha-dihydroxy-5alpha-pregnan-20-one, and 5alpha-reduced C19-steroids such as 17beta-hydroxy-5alpha-androstan-3-one, 3beta-hydroxy-5alpha-androstan-17-one, and 5alpha-androstane-3beta,17beta-diol were the major products. The formation of significant quantities of 5alpha-reduced C19-steroids, which had been demonstrated previously only in prepubertal testes of rats and mice, was present in prepubertal as well as adult testes of rabbits. These findings clearly indicate that the metabolic patterns of progesterone in the adult rabbit differ from those in the adult rat and mouse. The major C19-steroids formed by testes are testosterone in the adult rat and mouse, but 5alpha-reduced C19-steroids in the adult rabbit.     

Seasonal changes in testicular steroidogenesis in the toad Bufo arenarum H

The biosynthesis of androgens in Bufo arenarum takes place through the 5-ene pathway that includes 5-androstane-3beta,17beta-diol as intermediate in testosterone biosynthesis. Besides testosterone and 5alpha-dihydrotestosterone, testes are able to synthesize 5alpha-pregnan-3,20-dione and several 3alpha- and 20alpha-reduced derivatives. Steroid biosynthesis changes during the breeding period (spring and early summer), turning from androgen to C21 steroid production. During the reproductive season, the production of progesterone, 5alpha-pregnan-3alpha,20alpha-diol, 3alpha-hydroxy-5alpha-pregnan-20-one, and 5alpha-pregnan-3,20-dione increases significantly. The function of most of these steroids in amphibians remains unknown. However, 5alpha-androstan-3alpha,17beta-diol and 3alpha-hydroxy-5alpha-pregnan-20-one were shown to be neuroactive in mammals, modulating sexual behavior. Thus, 5alpha/3alpha-reduced steroids could be involved in the regulation of the reproductive behavior in B. arenarum, a species with a dissociated reproductive pattern. Percentage contribution of each enzymes to the total metabolism reveals that neither 3beta-hydroxysteroid dehydrogenase/isomerase nor 5alpha-reductase change throughout the reproductive cycle. However, a strong reduction in 17-hydroxylase-C(17-20) lyase activity occurs in the reproductive season, suggesting that this enzyme could represent a key enzyme in the regulation of the seasonal change of steroidogenesis. Also, 3alpha-hydroxysteroid dehydrogenase and 20-hydroxysteroid dehydrogenase activities increase during the reproductive period, implying that steroid metabolism is clearly focused on C21-reduced steroids.     

Structure and steroidogenic enzymes of the seminal vesicles of the urohaze-goby (Glossogobius olivaceus)

The in vitro steroid metabolism in the seminal vesicles of the brackish water goby (urohaze-goby, Glossogobius olivaceus) was studied using males in the breeding season. The moderate activity of delta 5-3 beta-hydroxysteroid dehydrogenase was histochemically detected only in the epithelial cells of the organ, though these cells have the characteristics of secretory cells ultrastructurally. Cell-free homogenates (800 g supernatant fluid) of the whole tissue were aerobically incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, or testosterone in the presence of NAD+ or NADPH. Pregnenolone and dehydroepiandrosterone were converted to progesterone and androstenedione, respectively. Progesterone was transformed to 5 alpha-pregnane-3,20-dione (main product) and 17 alpha-hydroxyprogesterone. 17 alpha-Hydroxyprogesterone was metabolized into androstenedione (main product) and 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione. From androstenedione, 5 alpha-androstane-3,17-dione (main product) and epiandrosterone were obtained. Testosterone was transformed to 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 beta, 17 beta-diol, 5 alpha-androstane-3,17-dione, and androstenedione. These results indicate that the steroid metabolic patterns in the seminal vesicles of G. olivaceus are closely resembled to those in the testes.     

Characterization of Binding Components for Progesterone and 5α-Pregnane-3,20-dione in the Hamster Uterus

In the hamster uterus, a specific progesterone (pregn-4-ene-3,20-dione) receptor has been identified in the cytosol fraction. In the present study, we examined hamster uterine cytosol for the possible existence of specific macromolecules that bind the progesterone metabolite, 5alpha-pregnane-3,20-dione. When cytosol was analyzed by density-gradient centrifugation with sucrose-glycerol gradients and by Scatchard plot analysis of [(3)H]5alpha-pregnane-3,20-dione binding data, there was no evidence of specific binding components for this metabolite. In vivo treatment of proestrous hamsters with unlabeled progesterone, 5alpha-pregnane-3,20-dione, or cortisol for 1 hr revealed that only progesterone caused the depletion of progesterone-receptor sites from the uterine cytosol fraction. Incubation of uterine strips which had been preloaded with two different concentrations of [(3)H]-progesterone demonstrated that progesterone was metabolized to 5alpha-pregnane-3,20-dione and to a greater extent to 3alpha-hydroxy-5alpha-pregnan-20-one. The accumulation of 5alpha-pregnane-3,20-dione during progesterone metabolism appeared to be related to the availability of nonspecifically bound hormone. These studies (i) strongly suggest there is no specific receptor system for 5alpha-pregnane-3,20-dione in the uterine cytosol fraction, (ii) confirm the existence of a specific progesterone receptor in uterine cytosol, and (iii) provide evidence that progesterone itself mediates the uterine progestational response via interaction with a specific receptor system.     

Metabolism of (4-14C)progesterone and (7alpha-3H)pregnenolone by human ovaries perfused in vitro.

Nine human ovaries were perfused in vitro with [4-14C]progesterone and in addition one ovary with [7-3H]pregnenolone. From the perfusate unchanged progesterone and five different metabolites were isolated: 20alpha-dihydroprogesterone, 17alpha-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 5alpha-pregnane-3,20-dione and 4-androstene-3,17-dione. In the ovarian tissue saturated pregnane derivatives were the main metabolites. When [3H]pregnenolone and [14C]progesterone were perfused simultaneously a stimulation of delta4-5-isomerase and 3beta-dehydrogenase activity by HCG was demonstrated.     

Reduction of 3-oxosteroids in human liver microsomes.

The 3alpha- and 3beta-reduction of the following steroids was studied in human liver microsomes: 5alpha-androstane-3,17-dione, 17beta-hydroxy-5alpha-androstan-3-one, 5alpha-pregnane-3,20-dione, 5beta-pregnane-3,20-dione, 3-oxo-5beta-cholanoic acid and 7alpha-hydroxy-5alpha-cholestan-3-one. With NADH as cofactor there was a preferential 3alpha-reduction of the C19- and C21-3-oxo-steroids and a preferential 3beta-reduction of the C24- and C27-3-oxo-steroids. Substitution of NADH with NADPH influenced reduction of the substrates in different ways, indicating the presence of several 3alpha- and 3beta-hydroxysteroid dehydrogenases with different substrate specificity and specificity towards NADH and NADPH. Only small or insignificant sex differences could be observed in the reductions studied.     

Steroid metabolism in normal mammary gland and in the dimethylbenzanthracene-induced mammary tumor of rats.

After incubation of [4-14C]progesterone with cell-free homogenates of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced mammary tumor of rats, 20 alpha-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione, 20 alpha-hydroxy-5 alpha-pregnan-3-one, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 5 alpha-pregnane-3 alpha, 20 alpha-diol were identified as the metabolites. In normal mammary tissue, however, 4-pregnene-3 alpha-diol was isolated in addition to 5 alpha-reduced, and 3 alpha- and 20 alpha-hydroxy metabolites. When radioactive testosterone was employed as a substrate, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were obtained as the metabolites of the mammary tumor. In the normal mammary gland, only 4-andorstene 3 alpha, 17 beta-diol was formed as its metabolite. Although the enzyme activities relevant to the metabolism varied among the tumor examined, the activity of 20 alpha-hydroxysteroid dehydrogenase in the mammary tumor was significantly lower than that in the normal mammary gland, whereas the activity of 5 alpha-reductase was higher in some of the mammary tumors than in the normal gland. The 5 alpha-reductase activity in the normal mammary gland was mostly localized in the crude microsomal fraction, whereas the same enzyme activity in the tumor was detected in all the organelle fractions. The activities of 20 alpha-hydroxysteroid dehydrogenase and NADPH-linked 3 alpha-hydroxysteroid dehydrogenase were found mainly in the cytosol fractions of the tumor and the normal tissue. The NADH-linked 3 alpha-hydroxysteroid dehydrogenase activity was detected only in the cytosol fraction of the normal mammary gland, but in the tumor studied, the activity of this enzyme was detected in all the subcellular fractions examined.     

Selective suppression of follicle-stimulating hormone secretion in anterior pituitary cells by the gonadal steroid 3 alpha-hydroxy-4-pregnen-20-one

The effect of 3 alpha-hydroxy-4-pregnen-20-one (3HP), a Sertoli cell steroid, on the secretion of gonadotropins from rat anterior pituitary cells in culture was examined and subsequently compared with the action of other gonadal steroids and steroids structurally related to 3HP. Pituitary cells from randomly cycling, sexually mature female rats were isolated and maintained in culture 72 h before use. On the day of treatment, medium was changed, steroids (10(-16)-10(-4) M) and/or LHRH (10(-8) M) were added, and cells were allowed to incubate for a further 24 h. Medium was then examined for gonadotropin content by RIA. 3HP treatment of anterior pituitary cells resulted in a significant reduction of both basal and LHRH-induced FSH secretion, while LH secretion was unaffected. The lowest effective dose of 3HP (10(-16) M) significantly decreased basal FSH secretion to 65.6% of control levels. The lowest effective dose of 3HP that significantly inhibited (by 31%) LHRH-induced FSH secretion was 10(-14) M 3HP. Maximum suppression by 3HP of basal FSH secretion occurred between 10(-10)-10(-8) M, and maximum suppression of LHRH-induced secretion occurred at 10(-12) M. None of the other gonadal steroids tested (progesterone, testosterone, 5 alpha-dihydrotestosterone, 17 beta-estradiol, 20 alpha-hydroxy-4-pregnen-3-one, and 5 alpha-pregnane-3,20-dione) had a similar selective effect on FSH secretion; progesterone, testosterone, and 17 beta-estradiol actually resulted in increased FSH release, and 5 alpha-pregnane-3,20-dione resulted in significant increase in basal LH. A number of metabolites and structural variations of 3HP were examined in this in vitro system at concentrations of 10(-12)-10(-6) M, and none exhibited a similar selective FSH-suppressing activity as 3HP. The data suggest that the selective FSH-suppressing effect of 3HP seen previously in vivo and here in vitro is due to 3HP itself and not the result of a metabolite of this molecule.     

Steroid 5alpha-reductase 1 promotes 5alpha-androstane-3alpha,17beta-diol synthesis in immature mouse testes by two pathways

5alpha-Androstane-3alpha,17beta-diol (androstanediol) is the predominant androgen in immature mouse testes, and studies were designed to investigate its pathway of synthesis, the steroid 5alpha-reductase isoenzyme involved in its formation, and whether testicular androstanediol is formed in embryonic mouse testes at the time of male phenotypic development. In 24-26-day-old immature testes, androstanediol is formed by two pathways; the predominant one involves testosterone --> dihydrotestosterone --> androstanediol, and a second utilizes the pathway progesterone --> 5alpha-dihydroprogesterone --> 5alpha-pregnane-3alpha-ol-20-one --> 5alpha-pregnane-3alpha,17alpha-diol-20-one --> androsterone --> androstanediol. Formation of androstanediol was normal in testes from mice deficient in steroid 5alpha-reductase 2 but absent in testes from mice deficient in steroid 5alpha-reductase 1, indicating that isoenzyme 2 is not expressed in day 24-26 testes. The fact that androstenedione and testosterone were the only androgens identified after incubation of day 16 and 17 embryonic testes with [3H]progesterone implies that androstanediol formation in the testis plays no role in male phenotypic differentiation in the mouse.     

Effects of multiple injections of LH on the secretion of pregnane compounds from polycystic ovaries of androgen-sterilized rats

Differences in the secretion of pregnane compounds by follicular polycystic ovaries of androgen-sterilized rats and by normal preovulatory ovaries of early pro-oestrous rats were compared. Some rats were injected i.v. with LH 30 min before bleeding, in order to stimulate the secretion of steroids. This injection of LH greatly increased the secretion of progesterone, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one by both types of ovaries. The response of the two progesterone metabolites in the polycystic ovaries was low, suggesting low 5 alpha-reductase activity. Because it is known that the preovulatory LH surge is absent in androgen-sterilized rats, a classical approach was taken to circumvent the probable deficit in cyclic release of LH by giving an i.v. injection of LH (25 micrograms) every 4 days for 16 days. Ovarian venous blood was collected 4 days after the last injection. The mean secretion of 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one from the ovaries of such androgen-sterilized rats became much (P less than 0.01) higher than that of multiple saline-treated controls. These results suggest that low 5 alpha-reductase activity of polycystic ovaries in androgen-sterilized rats may be due to the absence of cyclic release of LH from the pituitary gland.     

Biosynthetic pathways of testosterone and estradiol-17 beta in slices of the embryonic ovary and testis of the chicken (Gallus domesticus)

To elucidate synthetic pathways of testosterone and estradiol-17 beta in embryonic gonads of the chicken, metabolism of various 14C-labeled steroids in slices of the left ovaries and paired testes of 15- and 9-day-old chicken embryos was examined. (1) Fifteen-day-old chicken embryos: From pregnenolone, more 17 alpha-hydroxypregnenolone was produced than progesterone in the ovary, while more progesterone was produced than 17 alpha-hydroxypregnenolone in the testis. From 17 alpha-hydroxypregnenolone, however, only dehydroepiandrosterone was detected as a product in both gonads. Dehydroepiandrosterone was converted mainly into androstenedione and its 5 beta-reduced derivatives by both gonads. Progesterone was converted into 5 beta-pregnane-3,20-dione more than into 17 alpha-hydroxyprogesterone by both gonads. Both gonads metabolized 17 alpha-hydroxyprogesterone, androstenedione, and testosterone predominantly into their corresponding 5 beta-reduced steroids, while production of androstenedione from 17 alpha-hydroxyprogesterone and of testosterone from androstenedione was limited. Estradiol-17 beta was produced from androstenedione and testosterone only by the ovary. (2) Nine-day-old chicken embryos: From pregnenolone, production of progesterone and 17 alpha-hydroxypregnenolone was similar in the ovary. On the other hand, in the testis, more progesterone was produced than 17 alpha-hydroxypregnenolone from pregnenolone. For delta 4-3-oxo steroids, strong activity of 5 beta-reductase was demonstrated in both gonads. From these results, both delta 4- and delta 5-pathways are involved in the formation of testosterone and then finally of estradiol-17 beta by the embryonic gonads of the chicken, and relative preference for the pathway seems to depend on sexes and embryonic ages. In addition, it is suggested that steroidogenesis in these embryonic gonads is characterized by marked activity of 5 beta-reductase, irrespective of sexes or ages.     

Influence of Ethynodiol Diacetate on the Formation of A-Homo-3Oxa-5?-Pregnane-4,20-Dione in Female Rats

OBJECTIVE: To give more insight in the progesterone metabolism in rat after the treatment with the progestin ethynodiol diacetate. METHODS: Urinary excretion of the metabolites of subcutaneously administred (4-14C)-progesterone was studied in female rats. After an acid hydrolysis and extraction of urine the metabolites were analysed by thin layer chromatography and by gas chromatography-mass spectrometry. RESULTS: The most of radioactivity was excreted during the first 24 h, and total of 8.36 % has been recovered within four days. The excreted metabolites in urine were found as glucuronides and free steroids (80.72 %), and 19.28 % were determined as sulphates. Among detected metabolites, 5alpha-pregnane-3,20-dione, 3alpha-hydroxy-5alpha-pregnan-20-one and A-homo-3-oxa-5alpha-pregnane-4,20-dione were determined in the urinary extracts. The last one has not yet been identified before in rat urine. CONCLUSIONS: Consecutive injections of progestin ethynodiol diacetate (6 mg/kg b.w. daily) to adult female rats during 10 days (short-term treatment), or during 70 days (long-term treatment), starting on the 21st day of life, caused significant differences in the amounts of excreted 3alpha-hydroxy-5alpha-pregnan-20-one and A-homo-3-oxa-5alpha-pregnane-4,20-dione. Significant increase in the weights of pituitary, liver and kidneys were noted in rats treated with ethynodiol diacetate. The short-term treatment caused an increase, while after the long-term treatment a decrease of the ovarian weight was observed.     

Modulation of the firing activity of female dorsal raphe nucleus serotonergic neurons by neuroactive steroids

Important gender differences in mood disorders result in a greater susceptibility for women. Accumulating evidence suggests a reciprocal modulation between the 5-hydroxytryptamine (5-HT) system and neuroactive steroids. Previous data from our laboratory have shown that during pregnancy, the firing activity of 5-HT neurons increases in parallel with progesterone levels. This study was undertaken to evaluate the putative modulation of the 5-HT neuronal firing activity by different neurosteroids. Female rats received i.c.v. for 7 days a dose of 50 micro g/kg per day of one of the following steroids: progesterone, pregnenolone, 5beta-pregnane-3,20-dione (5beta-DHP), 5beta-pregnan-3alpha-ol,20-one, 5beta-pregnan-3beta-ol,20-one, 5alpha-pregnane-3,20-dione, 5alpha-pregnan-3alpha-ol,20-one (allopregnanolone, 3alpha,5alpha-THP), 5alpha-pregnane-3beta-ol,20-one and dehydroepiandrosterone (DHEA). 5beta-DHP and DHEA were also administered for 14 and 21 days (50 micro g/kg per day, i.c.v.) as well as concomitantly with the selective sigma 1 (sigma1) receptor antagonist NE-100. In vivo, extracellular unitary recording of 5-HT neurons performed in the dorsal raphe nucleus of these rats revealed that DHEA, 5beta-DHP and 3alpha,5alpha-THP significantly increased the firing activity of the 5-HT neurons. Interestingly, 5beta-DHP and DHEA showed different time-frames for their effects with 5beta-DHP having its greatest effect after 7 days to return to control values after 21 days, whereas DHEA demonstrated a sustained effect over the 21 day period. NE-100 prevented the effect of DHEA but not of 5beta-DHP, thus indicating that its sigma1 receptors mediate the effect of DHEA but not that of 5beta-DHP. In conclusion, our results offer a cellular basis for potential antidepressant effects of neurosteroids, which may prove important particularly for women with affective disorders.     

Nuclear progesterone-binding protein in the guinea pig adrenal cortex: distinction from the classical progesterone receptor

Nuclei purified from the guinea pig adrenal cortex contain a specific progesterone-binding activity which, based on enzyme degradation studies, appears to be proteinaceous. Saturation analysis revealed a Kd of about 15 nM and a binding capacity of about 33 pmol/mg DNA. The activity of the nuclear binding protein was specific essentially for progestational steroids; the two most potent progesterone competitors were 5 alpha-pregnane-3,20-dione and medroxyprogesterone (17 alpha-hydroxy-6 alpha-methylprogesterone), while 17 beta-estradiol, testosterone, cortisol, and other related steroids were poor competitors. The adrenocortical nuclear progesterone-binding protein was present to an equal extent in both male and female guinea pigs. The adrenocortical nuclear progesterone-binding protein differed from the classical progesterone receptor in that 1) the affinity of the adrenocortical binding protein for progesterone is an order of magnitude lower; 2) the potent synthetic progestin R5020 binds less tightly to the adrenocortical progesterone-binding protein; 3) the adrenocortical progesterone-binding protein is not modulated by estrogenic activity; 4) the adrenocortical progesterone-binding protein is more stable at 37 C; 5) the adrenocortical nuclear progesterone-binding protein is not salt extractable; and 6) Western blot analysis has revealed that an antiprogesterone receptor monoclonal antibody, which recognizes the guinea pig uterine classical nuclear progesterone receptor, does not recognize the adrenocortical nuclear progesterone-binding protein. Thus, the guinea pig adrenocortical nucleus contains a type of progesterone-binding protein that appears to be clearly different from the classical progesterone receptor.     

5alpha-androstane-3alpha,17beta-diol is formed in tammar wallaby pouch young testes by a pathway involving 5alpha-pregnane-3alpha,17alpha-diol-20-one as a key intermediate

The synthetic pathway by which 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in the testes of tammar wallaby pouch young was investigated by incubating testes from d 20-40 males with various radioactive precursors and analyzing the metabolites by thin-layer chromatography and HPLC. [(3)H]Progesterone was converted to 17-hydroxyprogesterone, which was converted to 5alpha-adiol by two pathways: One involves the formation of testosterone and dihydrotestosterone as intermediates, and the other involves formation of 5alpha-pregnane-3alpha,17alpha-diol-20-one (5alpha-pdiol) and androsterone as intermediates. Formation of 5alpha-adiol from both [(3)H]testosterone and [(3)H]progesterone was blocked by the 5alpha-reductase inhibitor 4MA. The addition of nonradioactive 5alpha-pdiol blocked the conversion of [(3)H]progesterone to 5alpha-adiol, and [(3)H]5alpha-pdiol was efficiently converted to androsterone and 5alpha-adiol. We conclude that expression of steroid 5alpha-reductase in the developing wallaby testes allows formation of 5alpha-reduced androgens by a pathway that does not involve testosterone as an intermediate.     

Identification and quantitative determination of steroids in bovine corpus luteum during oestrous cycle and pregnancy.

Neutral steroids in bovine corpus luteum were isolated by liquid-gel chromatography on hydrophobic Sephadex, and were analyzed by computerized gas chromatography-mass spectrometry. The presence of progesterone and 20beta-hydroxy-4-pregnen-3-one was confirmed. In addition, 3beta-hydroxy-5-pregnen-20-one, 5-pregnene-3beta,20beta-diol, 3beta-hydroxy-5alpha-pregnan-20-one and 5alpha-pregnane-3beta,20beta-diol were fully identified, and 3-hydroxy-4-pregnen-20-one, 4 pregnene-3,20-diol, 22-hydroxycholesterol and 20,22-dihydroxycholesterol were partially characterized. Steroid sulphates were not detected. Quantification of the six fully identified steroids was based on peak areas in specific fragment ion current chromatograms constructed by the computer. During the 4th-19th day of the oestrous cycle the steroid concentrations varied as follows: progesterone 6.0-36.7 mug/g wet luteal weight, 20beta-hydroxy-4-pregnen-3-one 0.8-5.5 mug/g, 3beta-hydroxy-5-pregnen-20-one 1.0-7.1mug/g, 5-pregnene-3beta,20beta-diol smaller than 0.2-0.9 mug/g, 3beta-hydroxy-5alpha-pregnan-20-one 1.7-8.6 mug/g, and 5alpha-pregnane-3beta,20beta-diol smaller than 0.2-1.2 mug/g. The concentrations of progesterone and 3beta-hydroxy-5-pregnen-20-one seemed to vary in parallel and were low during days 11-17. During this period the concentrations of 5-pregnene-3beta,20beta-diol and 5alpha-pregnane-3beta,20beta-diol were highest as was the relative contribution of all three 20beta-hydroxysteroids to the total amount of steroids. The relative amount of 3beta-hydroxy-5alpha-pregnan-20-one seemed to be highest during days 4-6. The total steroid concentration in corpora lutea taken in early pregnancy (75-105 days) was 18-47 mug/g. In the period 75-90 days, progesterone constituted only 35-42% of the total steroids, 3beta-hydroxy-5alpha-pregnan-20-one as much as 23-40% and the total 20beta-hydroxysteroids 18-30%. The total steroid concentration in corpora lutea taken in midterm and late pregnancy was 21-77 mug/g. In this period progesterone was by far the predominant steroid and constituted about 80-90% of the total steroids in corpora lutea taken between days 150 and 240. Possible correlations between luteal growth, steroid oxidoreductases and steroid concentrations are discussed.     

The characterization of polar corticosteroids in the urine of the macaque monkey (macaca fascicularis) and the baboon (papio hamadryas).

Computerised gas chromatography-mass spectrometry was employed in the identification of polar corticosteroid metabolites excreted in the urine from the macaque monkey (Macaca fascicularis) and the baboon (Papio hamadryas). The following steroids were identified in significant amounts in the urine from both species: 3alpha,17alpha,20alpha, 21-tetrahydroxy-5beta-pregnan-11-one; 3alpha,17alpha,20beta,21-tetrahydroxy-5beta-pregnan-11-one; 5beta-pregnane-3alpha,11beta,17alpha,20alpha,21-pentol; 5beta-pregnane-3alpha,11beta,17alpha,20beta-pentol; 5alpha-pregnane-3beta,11beta,17alpha,20beta,21-pentol. 11beta,17alpha,21-Trihydroxy-4-pregnene-3,20-dione (cortisol), 11beta,17alpha,20beta,21-tetrahydroxy-4-pregnen-3-one and 11beta,17alpha,20beta,21-tetrahydroxy-5xi-pregnan-3-one were identified in macaque monkey urine. Two steroids, 17alpha,20beta,21-trihydroxy-4-pregnane-3,11-dione and 17alpha,20alpha,21-trihydroxy-4-pregnene-3,11-dione were excreted as major C21 metabolites in the baboon but were not identified in the urine from the macaque monkey. 3beta-Hydroxy-5alpha-pregnane metabolites were identified in the urine from both species. All these steroids were excreted conjugated to glucuronic acid, evidenced by their recovery after hydrolysis with beta-glucuronidase enzyme. An efficient 20beta-reduction of corticosteroids in both species is apparent, and the excretion pattern of polar steroid metabolites in the two species was shown to be similar.     

Progesterone metabolism in vitro in the decapod crustacean, Penaeus monodon.

Thin-layer chromatography (TLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) with on-line detection of radioactive steroids were applied to identify metabolites of [4-14C]progesterone incubated in vitro with prawn ovary. There was extensive metabolism of progesterone by stage II (vitellogenic) ovary of Penaeus monodon. The most abundant metabolites were 5 alpha-pregnane derivatives together with two minor metabolites, 20 alpha-hydroxypregn-4-en-3-one and 1,4-pregnadiene-3,20-dione. In contrast, a much lower level of progesterone metabolism was observed in stage 0 (immature) ovary of this species. The hepatopancreas, gill, and abdominal muscle of P. monodon all metabolised [4-14C]progesterone to varying degrees, generating materials similar to those produced by the ovary. A comparative study of progesterone metabolism in stage II ovary of Nephrops norvegicus indicated that one metabolite, 20 alpha-hydroxypregn-4-en-3-one, was produced.