Steroid metabolism in gonads of turtle embryos as a function of the incubation temperature of eggs

In embryos of many reptiles, the sexual differentiation of gonads is temperature-dependent. In the turtle Emys orbicularis, all individuals become phenotypic males at 25 degrees C, whereas 100% phenotypic females are obtained at 30 degrees C. Steroid metabolism in embryonic gonads was studied at both temperatures, during and after the thermosensitive period for sexual differentiation. Pools of gonads were incubated for various times, with 3 beta-hydroxy-5-pregnen-20-one (pregnenolone), progesterone, dehydroepiandrosterone or 4-androstene-3,17- dione as substrates. The analysis of metabolites combined two successive chromatographies (HPLC and TLC) and autoradiography. Conversion of pregnenolone to progesterone and of dehydroepiandrosterone to 4-androstene-3,17-dione was more important in testes at 25 degrees C than in ovaries at 30 degrees C. In ovaries, a large amount of 5-pregnene- 3 beta,20 beta-diol was formed from pregnenolone, and 5-androstene-3 beta,17 beta-diol was produced from dehydroepiandrosterone. In both testes and ovaries, 5 alpha-pregnane and 5 alpha-androstane derivatives were the main metabolites obtained from progesterone and 4-androstene-3,17-dione, respectively. Progesterone was also converted to 20 beta-hydroxy-4-pregnen-3-one. Dehydroepiandrosterone and 4-androstene-3,17-dione were also metabolized into 11 beta-hydroxy-4-androstene-3,17-dione (only in testes), testosterone, 11 beta,17 beta-dihydroxy-4-androstene-3-one, 17 beta-hydroxy-4-androstene-3,11-dione (low amounts in testes, traces in ovaries), 17 alpha-hydroxy-4-androstene-3-one, estrone and estradiol-17 beta (traces).     

Steroid metabolism by rat nasal mucosa: studies on progesterone and testosterone

The metabolism of [4-14C]progesterone and [4-14C]testosterone by slices of the nasal mucosa from rats was studied. As shown by gas chromatography-mass spectrometry there was a preferential formation of reduced progesterone-metabolites (5 alpha-pregnane-3,20-dione, 3 alpha- and 3 beta-hydroxy-5 alpha-pregnane-20-one, 20 alpha- and 20 beta-hydroxypregn-4-en-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha-pregnane-20-one, 3 alpha,16 alpha-dihydroxy-5 alpha-pregnane-20-one) and reduced testosterone-metabolites (4-androstene-3,17-dione, 5 alpha-dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstane-17-one, and 5 alpha-androstane-3 alpha, 17 beta-diol, 2 alpha-hydroxy-5 alpha-dihydrotestosterone, 5 alpha-androstane-2 alpha,3 alpha, 17 beta-triol) indicating the presence of 5 alpha-reductase, 3 alpha-, 3 beta-, 17 beta-, 20 alpha- and 20 beta-hydroxysteroid oxidoreductase activities in this tissue. Progesterone-metabolites hydroxylated at positions 2 alpha, 6 alpha, 6 beta, 15 alpha and 16 alpha and testosterone-metabolites hydroxylated at positions 1 beta, 2 alpha, 6 beta, 15 beta and 16 alpha were also identified, indicating the presence of several steroid hydroxylases in the nasal mucosa. Autoradiography of the nasal region of rats injected with [4-14C]progesterone or [4-14C]testosterone showed a selective localization of radioactivity in the mucosa covering the olfactory region of the nasal cavity.     

Characterization of delta 4-3-ketosteroid-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase in cell extracts of Clostridium innocuum

Cell extracts prepared anaerobically from Clostridium innocuum and Clostridium paraputrificum reduced delta 4-3-ketosteroids to 3 beta 5 beta and 3 alpha 5 beta derivatives, respectively. delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) from both organisms required NADH for activity. 5 beta-Reductase from C. innocuum had a pH optimum of 5.0. The substrate concentration at half-maximal reaction velocity was 4.2 microM, and a specific activity of 17 nmol product formed/h per mg protein was determined using 4-pregnen-3,20-dione (progesterone) as a substrate. delta 4-3-Ketosteroid-5 beta-reductase from C. innocuum reduced progesterone and testosterone, but not 4-cholesten-3-one, to corresponding 3-keto-5 beta derivatives. A relative molecular (Mr) weight of 80 000 was estimated for 5 beta-reductase using HPLC-gel filtration chromatography. 3 beta-Hydroxysteroid dehydrogenase in cell extracts of C. innocuum was oxygen sensitive and required NADH for activity. An Mr of 80 000 was estimated for 3 beta-hydroxysteroid dehydrogenase. However, 5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase activities were separated using an HPLC-DEAE chromatography technique.     

Structural requirements for progesterone binding to the hepatic endoplasmic reticulum in the female rat

Microsomes isolated from the liver of the female rat specifically bind progesterone. The progesterone-microsomal complex shows highly specific characteristics. The binding is probably associated with the carbonyl groups at positions C-20 and C-3. Other steroids compete for microsomal binding sites less effectively. Competition for progesterone binding sites by other steroids in percentages: testosterone 33; testosterone propionate, 9; 17-methyltestosterone, 23.2; cortisol, 6.4; estradiol-17 beta, 1.8; 17 alpha-ethynyl estradiol, 4.7; mestranol, 1.0; norethynodrel, 4.5; ethisterone, 7.1; lynestrenol, 4.3; medroxyprogesterone, 23.3; medroxyprogesterone acetate, 15.2; 5 alpha-pregnane-3,20-dione, 47.6; 5 beta-pregnane-3,20-dione, 20.7; pregnenolone, 14.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8%; 20 beta-hydroxy-4-pregnen-3-one, 2.8; 3 beta-hydroxy-5 alpha-pregnan-20-one, 5.2; 4-pregnene-3 beta, 20 beta-diol, 2.1; 11 alpha-hydroxyprogesterone 21.0; 16 alpha-hydroxyprogesterone, 7.9; 17-hydroxyprogesterone, 26.7; 16 alpha, 17-epoxyprogesterone, 2.7; 16 alpha-methylprogesterone, 3.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8; promegestone, 27.0. 3 beta-Hydroxy-5 beta-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 5-pregnene-3 beta,20 beta-diol, 5-pregnene-3 beta, 20 alpha-diol; 5 alpha-pregnane-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol diacetate, 5 beta-pregnane-3 alpha, 20 beta-diol, 3 alpha, 17-dihydroxy-5 beta-pregnan-20-one, 17-hydroxypregnenolone, 6-methyl-17-hydroxypregnenolone, pregnenolone-16 alpha-carbonitrile, dihydrotestosterone and cholesterol show no competition at all. The varying degree of competition by different steroids is unrelated to their lipid solubility.     

Inhibition of uterine contractility by progesterone and progesterone metabolites: mediation by progesterone and gamma amino butyric acidA receptor systems.

Progesterone and several progesterone metabolites are capable of inhibiting uterine contractility. Some progesterone metabolites have shown little or no affinity for the progesterone receptor but have been found to be potent modulators of the GABAA receptor system. This study examined whether the inhibition of uterine contraction by progesterone and its metabolites was progesterone receptor-mediated or gamma amino butyric acidA (GABAA) receptor-mediated. Uterine contractions were measured in annular rings of uterine tissue, 5 mm in length, from diestrous II rats, under a fixed tension of 1 gram. The steroids tested were 3 beta-hydroxy-5 beta-pregnan-20-one (6 micrograms/ml), 5 beta-pregnane-3,20-dione (10 micrograms/ml), 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha,5 alpha-THP, 27.5 micrograms/ml), and progesterone (40 micrograms/ml). All compounds significantly inhibited spontaneous uterine contractions when compared to controls. No effect was seen by either 16 micrograms/ml of the progesterone antagonist, RU486, or 32 micrograms/ml of the GABAA antagonist, pictrotoxin, when administered alone. However, when uterine tissues were exposed to a combination of the steroid and the antagonist, the effect of 3 beta-hydroxy-5 beta-pregnan-20-one and 3 alpha,5 alpha-THP was blocked by picrotoxin but not by RU486, indicating that the action of these steroids was mediated through the GABAA system. The effect of 5 beta-pregnane-3,20-dione and progesterone was effectively blocked by RU486 but not by picrotoxin, suggesting that their actions were mediated through the progesterone receptor system. These results indicate that multiple mechanisms exist in the uterus for inhibiting uterine contractility by progesterone and its metabolites.     

Microbial transformation of 5alpha,6alpha-epoxy-3beta-hydroxy-16-pregnen-20-one by Trichoderma viride

Fermentation of 5alpha,6alpha-epoxy-3beta-hydroxy-16-pregnen-20-one (4) with Trichoderma viride under aerobic condition yielded 3beta,5alpha,6beta-trihydroxy-16-pregnen-20-one (5) and 3beta,5alpha,6beta,15beta-tetrahydroxy-16-pregnen-20-one (6). Each microbial metabolite was characterized by spectroscopic methods. Compounds 6 and 3beta,5alpha,15beta-trihydroxy-16-pregnen-6,20-dione (7) are reported for the first time.     

The in vitro biosynthesis and metabolism of progesterone and estrogens by chimpanzee placental tissue

The biosynthesis and metabolism of progesterone and estrogens have been studied in chimpanzee placental tissue in vitro. The conversion of androstenedione-4-14C to estrone and estradiol-17β and of pregnenolone-7α-3H to progesterone has been demonstrated. In addition, the following metabolites were isolated following incubation of either pregnenolone-7α-3H or progesterone-4-¹⁴C: 20α-dihydroprogesterone, 20β-dihydroprogesterone, 6β-hydroxyprogesterone, 5α-pregnane-3,20 dione. The compound 5α-pregnan-3β o1-20-one was identified only after incubation with pregnenolone-7α-3H, while 5β-pregnane-3, 20 dione was identified only after incubation with progesterone-4-¹⁴C. No estrogens could be demonstrated following the incubation of placental preparations with either of the C₂₁ substrates.     

In vitro progesterone metabolism in rat submaxillary gland: The formation of 20α-hydroxy-4-pregnen-3-one and other substances

Rat submaxillary gland homogenates incubated $\text{in vitro}$ with progesterone-1, 2-³H converted the substrate to several products. Three products, 20α-hydroxy-4-pregnen-3-one, 5α-pregnane-3,20-dione and 17α-hydroxy-4-pregnene-3,20-dione, were characterized by thin-layer chromatography and recrystallization to constant specific activity.     

Progesterone metabolism in the gastric mucosa microsomes of guinea pig

The microsomes from guinea pig gastric mucosa were found to convert [4-14C]progesterone to two major metabolites in the presence of NADPH. The gastric metabolizing activity was the highest among the gastrointestinal tissues of guinea pig. 5 alpha-Pregnane-3,20-dione and 3 beta-hydroxy-5 alpha-pregnan-20-one were identified as the major metabolites by thin-layer chromatography and crystallization to constant specific activity, suggesting the presence of steroid 5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase activities in the gastric mucosa microsomes. Furthermore, time course of progesterone metabolism and analysis of 5 alpha-pregnane-3,20-dione metabolites suggest that the gastric progesterone metabolism is initiated by 5 alpha-reductase and followed by 3 beta-hydroxysteroid dehydrogenase. The progesterone-metabolizing activity was strongly inhibited by SKF 525-A and disulfiram. The activity was also inhibited by methyrapone to a somewhat lesser extent than the above inhibitors. From gastric mucosa microsomes, the progesterone-metabolizing activity was successfully solubilized with 2% digitonin using 0.1 M potassium chloride and 1 mM dithiothreitol, 0.4 mM NADPH and 20% glycerol as stabilizers for the solubilized activity. Among these stabilizers, glycerol was found to be most effective for stabilizing the activity of the solubilized microsomes.     

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.     

Identification of 2 alpha-hydroxy-4-pregnene-3,20-dione in human pregnancy urine.

2 alpha-Hydroxyprogesterone (2 alpha-hydroxy-4-pregnene-3,20-dione) was identified in human late pregnancy urine by liquid-gel chromatography, GLC and GC-MS. In addition, the following 2-hydroxylated C21 steroids were found and identified as 2 zeta-hydroxy-5 zeta-pregnane-3,20-dione, 2 zeta,20 zeta-dihydroxy-4-pregnen-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha- (and 5 beta)-pregnan-20-one, two isomers of pregnane-2,3,20-triol and 2 zeta,3 zeta,16 zeta-trihydroxy-5 zeta-pregnan-20-one.     

Saturated metabolites of progesterone in human myometrium during pregnancy.

A method for the separation and quantitative determination of epimeric 3alpha/beta-hydroxy-5alpha/beta-pregnan-20-ones by gas liquid chromatography and electron capture detection is presented. Reliability of the method and applicability to biological material was tested. In one total human uterus and 20 samples of myometrium the concentration of epimeric pregnanol-ones was determined. 3beta-hydroxy-5alpha-pregnan-20-one and 3alpha-hydroxy-5alpha-pregnan-20-one were present in similar quantitative range as progesterone and 20alpha-hydroxy-4-pregnen-3-one. A correlation between progesterone and concentration of metabolites could not be established. 3beta-hydroxy-5beta-pregnan-20-one was not detected in the tissue.     

Formation of 5alpha-reduced C19 steroids from progesterone in vivo by 5alpha-reduced pathway in older prepubertal rat testis.

Either [3H] progesterone (0.5 or 5 nmol/5 muCi), 5alpha-[3H] pregnane-3,20-dione (5 nmol/5 muCi) or [14C] progesterone (6.6 nmol/0.2 muCi) plus 5alpha-[3H]-pregnane-3,20-dione (1 or 6.6 nmol/0.6 muCi), suspended in 0.05 ml of physiological saline solution, was injected into each testis of 32- and 90-day-old rats. Following injection, radioactive metabolites in testis and spermatic vein blood were extracted, isolated, measured and identified by column and paper chromatographies, with derivative formation and recrystallization to constant specific activity. In the blood and testis of older prepubertal rats, major 17-OH-C21 and C19 metabolites of progesterone were 5alpha-reduced steroids such as 3alpha, 17alpha-dihydroxy-5alpha-pregnan-20-one, 5alpha-androstane-3alpha,17beta-diol and androsterone. Following injection of [14C] progesterone plus 5alpha-[3H] pregnane-3,20-dione into 32-day-old rat testis, no significant augmentation of the isotope from progesterone was observed in 5alpha-reduced C19 steroids as compared with 5alpha-reduced 17-OH-C21 steroids, indicating that 5alpha-reduced C19 steroids were mainly formed from 5alpha-reduced 17-OH-C21 steroids in older prepubertal testis. In the blood and testis of adult rats, small amounts of 5alpha-reduced metabolites were shown to be produced from progesterone, while active 17alpha-hydroxylation of 5alpha-pregnane-3,20-dione followed by C17-C20-lyase reaction was demonstrated. These findings seem to indicate that formation of 5alpha-reduced C19 steroids from progesterone by the 5alpha-reduced pathway is a major pathway of androgen biosynthesis in older prepubertal rat testis in vivo.     

Conversion of 17 alpha-hydroxyprogesterone to 5 alpha, 3 alpha, and 20 alpha-reduced metabolites by female rat anterior pituitary and hypothalamus

The metabolism of 17 alpha-[3H]hydroxyprogesterone was examined in female rat anterior pituitary and hypothalamic tissues. After reverse isotopic dilution analysis and purification to constant specific activity, the following 5 alpha-, 3 alpha- and 20 alpha-reduced products were detected in both tissues: 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione; 3 alpha,17 alpha-dihydroxy-5 alpha-pregnan-20-one; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one and 5 alpha-pregnane-3 alpha,17 alpha,20 alpha-triol. While the metabolites formed were qualitatively the same, there were quantitative differences between the two tissues. The 3 alpha,5 alpha-reduced metabolite, 3 alpha,17 alpha-dihydroxy-5 alpha-pregnan-20-one, was the principal product in the anterior pituitary while the 5 alpha-reduced metabolite, 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, was produced in largest amount by the hypothalamus. With both tissues, the aforementioned four products plus starting substrate accounted for nearly all of the starting radioactivity. There was no evidence for the formation of C19 steroids (androgens) despite the presence of the 17 alpha-hydroxy group.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

The effects of progesterone and its metabolites on the induction of sexual receptivity in rats

Estrogen-primed female rats were administered progesterone, 5 a-pregnan-3,20-dione, 5a-pregnan-3a-ol-20-one, 5a-pregnan-3β-ol-20-one, 4-pregnen-20a-ol-3-one, and 5a-pregnan-20a-ol-3-one in oil or oil alone and tested for the display of lordosis behavior 3 and 5 hr after progestin treatment. Progesterone was the most effective progestin in facilitating lordosis behavior. 5a-Pregnan- 3,20-dione and 5a-pregnan-3a-ol-20-one were partially effective, while the other progestins were no more effective than oil. The data suggest that the neuronal system which controls lordosis behavior is not entirely specific for progesterone.     

Formation of 5-[17beta-2H]androstene-3beta,17alpha-diol from 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one by the microsomal fraction of boar testis

After incubation of 3beta-hydroxy-5-[17,21,21,21-2H]-pregnen-20-one with the microsomal fraction of boar testis, the metabolites were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following metabolites were identified: 3beta,17alpha-dihydroxy-5-[21,21,21-3H]pregnen-20-one, 3beta-hydroxy-5-androsten-17-one, 5-androstene-3beta,17beta-diol, and 5-[17beta-2H]androstene-3beta,17alpha-diol. The presence of a 2H atom at the 17beta position of 5-androstene-3beta,17alpha-diol was confirmed by oxidizing the steroid with 3beta-hydroxy-steroid dehydrogenase of Pseudomonas testosteroni to obtain 17alpha-hydroxy-4-[2H]androsten-3-one and then by oxidizing the latter steroid with chromic acid to obtain nonlabeled 4-androstene-3,17-dione. Among these metabolites, the first three can be interpreted to be synthesized by a well documented pathway, including 17alpha-hydroxylation followed by side chain cleavage as follows: 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one leads to 3beta,17alpha-dihydroxy-2-[21,21,212H]-pregnen-20-one leads to 3beta-hydroxy-5-androsten-17-one leads to 5-androstene-3beta,17beta-diol. On the other hand, 5-androstene-3beta,17alpha-diol, which contained a 2H atom at the 17beta position, is not likely to be synthesized via above mentioned pathway in which nonlabeled 3beta-hydroxy-5-androsten-17-one is formed as the first C19-steroid. It seems that an alternate side chain cleavage mechanism leading from pregnenolone to 17alpha-hydroxy-C19-steroid exists in boar testis.     

Alterations of 20 alpha-hydroxysteroid dehydrogenase activity in cultured rat granulosa cells by follicle-stimulating hormone and testosterone

The effect of follicle-stimulating hormone (FSH) and testosterone (T) on rat granulosa cell progestin metabolism was investigated by incubation of the cells for 24 h with FSH and/or T and subsequent reincubation with an appropriate rabiolabeled steroid for 3 h. Exposure to varying concentrations of FSH (8-1000 ng/ml) and T (4-500 nM) decreased overall 4-[14C] progesterone utilization and accumulation of 20 alpha-reduced metabolites of progesterone in a dose-related manner. The accumulation of 5 alpha-reduced metabolites was not markedly changed by FSH and T treatments. Treatments with FSH and/or T decreased utilization of all progestins studied: progesterone by 30-50%, 20 alpha-hydroxy-4-pregnen-3-one by 23-31%, 3 alpha-hydroxy-5 alpha-pregnan-20-one by 41-64%, and 5 alpha-pregnane-3 alpha,20 alpha-diol by 26-34%. The greatest effects were observed following FSH + T treatments. Decreased utilization of substrates was associated with the decrease of 20 alpha-hydroxy-steroid dehydrogenase activity; the conversion of progesterone to 20 alpha-hydroxy-4-pregnen-3-one was decreased by 44-62%, the conversion of 20 alpha-hydroxy-4-pregnen-3-one to progesterone was decreased by 41-61%, the conversion of 3 alpha-hydroxy-5 alpha-pregnan-20-one to 5 alpha-pregnane-3 alpha,20 alpha-diol was decreased by 42-69%, and the conversion of 5 alpha-pregnane-3 alpha,20 alpha-diol to 3 alpha-hydroxy-5 alpha-pregnan-20-one was decreased by 53-60%. The incubation of granulosa cells with cyanoketone (10(-6)M), an inhibitor of delta 5,3 beta-hydroxysteroid dehydrogenase, virtually eliminated de novo progesterone production but did not alter the inhibitory effect of FSH and T on radiolabeled progesterone utilization and accumulation of 20 alpha-reduced metabolites, indicating that the observed effects are not influenced by endogenous production of progesterone. It was concluded from these studies that both FSH and testosterone inhibit the 20 alpha-hydroxysteroid dehydrogenase activity and consequently decrease progesterone catabolism by granulosa cells.     

Metabolism of progesterone by avian granulosa cells in culture

Previous studies have demonstrated that progesterone is the primary product of steroidogenesis in avian granulosa cells during short-term incubation. However, during more prolonged culture, lasting several days, the progesterone content in the medium was found to decrease progressively, indicating in vitro metabolic conversion. In the present study we have isolated and identified a number of progesterone metabolites. Granulosa cells, isolated from mature ovarian follicles of laying hens, were cultured in medium 199 supplemented with fetal calf serum and containing [14C]progesterone. After 4 days in culture, cells + media were extracted and the radioactive metabolites separated and identified by TLC, HPLC and GC-MS. Several of the metabolites were further characterized by derivatization and crystallization to constant specific activity. A total of 24 radioactive substances was detected. Of these, 15 have been positively identified, 5 tentatively and the remaining 4 are unidentified. The principal metabolite, representing more than 45% of the total radioactivity, was identified as 3 alpha-hydroxy-5 beta-pregnan-20-one. In addition, significant amounts of 3 alpha-hydroxy-5 alpha-pregnan-20-one (5.76%), 5 beta-pregnane-3,20-dione (3.05%), and 5 alpha-pregnane-3,20-dione (2.95%) were detected and identified. The results indicate that avian granulosa cells possess 3 alpha-hydroxy-steroid dehydrogenase (3 alpha-HSD), 17 beta-HSD, 20 alpha-HSD, 20 beta-HSD, 17 alpha-hydroxylase, C17-20-lyase and 5 alpha- and 5 beta-reductase activities. These enzyme activities may convert progesterone to biologically inactive or less active metabolites. However, a functional role for some of these metabolites cannot be ruled out.     

Progesterone biotransformation by plant cell suspension cultures.

Progesterone was converted to 5alpha-pregnane-3alpha-ol-20-one, delta4-pregnene-20alpha-ol-3-one, delta4-pregnene-14alpha-ol-3,20-dione, delta4-pregnene-7beta,14alpha-diol-3,20-dione, and delta4-pregnene-6beta,11alpha-diol-3,20-dione by cell cultures of Lycopersicon esculentum. Cell cultures of Capsicum frutescens (green) metabolized progesterone to delta4-pregnene-20alpha-ol-3-one in very high yield, and Vinca rosea yielded delta4-pregnene-20beta-ol-3-one and delta4-pregnene-14alpha-ol-3,20-dione. A stereospecific reduction of the keto groups and a double bond and stereospecific introduction of hydroxyl groups at the 6, 11, and 14 positions have been observed. The mono- and dihydroxylated progesterones have not previously been reported as metabolic products of progesterone by plant cell systems and represent de novo hydroxylation of a nonglycosylated steroid.