In-vitro synthesis of androgen from pregnenolone in the testes of the goat (Capra hircus) and identification of 5-pregnene-3beta,17alpha,20alpha-triol as an intermediate in the metabolic pathway of pregnenolone

When [4(-14)C]pregnenolone was aerobically incubated in vitro in the presence of NAD+ and NADPH with cell-free homogenates of testicular tissue of adult domestic goats (Capra hircus), progesterone, 17alpha-hydroxypregnenolone, 17alpha-hydroxyprogesterone, 17alpha,20alpha-dihydroxy-4-pregnen-3-one, dehydroepiandrosterone, androstenedione and testosterone were identified as its known metabolites. Time-course studies on this metabolism showed that the production of 17alpha,20alpha-dihydroxy-4-pregnen-3-one and testosterone constantly increased up to the end of incubation, suggesting that these are both end-products of pregnenolone metabolism in this system. The other metabolites behaved as intermediates and were ultimately converted, in part, to testosterone by the testicular homogenates, indicating that testosterone was synthesized through both 4-ene and 5-ene-pathways. Furthermore, besides these metabolites, 5-pregnene-3beta,17alpha,20alpha-triol was also identified as an intermediary metabolite, formed from pregnenolone through 17alpha-hydroxypregnenolone in the presence of NADPH, and further convertible into 17alpha,20alpha-dihydroxy-4-pregnen-3-one by the microsomal fraction and into 17alpha-hydroxypregnenolone by the cytosol fraction in the presence of NAD+ and NADP+. It was not, however, significantly transformed into C19-steroids. Furthermore, 17alpha,20alpha-dihydroxy-4-pregnen-3-one, which was formed either from 5-pregnene-3beta,17alpha,20alpha-triol or from 17alpha-hydroxyprogesterone, remained almost unchanged without conversion to C19-steroids when incubated with the caprine testicular homogenates.     

Biosynthesis of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one in the ovaries of grey mullet (Mugil cephalus) during induced ovulation by carp pituitary homogenates and an LHRH analogue.

Three female mullets received a priming injection of carp pituitary homogenate followed by a resolving injection of an LHRH analogue 24 hr later. Ovarian biopsies were obtained just prior to the first injection (phase I), 24 hr after the first injection (i.e., immediately before the second injection, phase II), and 8 hr after the second injection (phase III). Two fish (Nos. 1 and 3) spawned approximately 12 hr after the second injection. Serum levels of testosterone increased to some extent during phase II in all of the fish. Testosterone levels decreased abruptly during phase III in both fish Nos. 1 and 3. In contrast the concentration of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) was undetectable in phases I and II, and increased dramatically during phase III in the same fish. In fish No. 2, which did not spawn, neither the decrease of testosterone nor the increase of 17 alpha,20 beta-diOHprog was observed. Ovarian tissues of all the fish were pooled in each phase and incubated with 14C-labeled progesterone or 17 alpha-hydroxyprogesterone to investigate steroid metabolism. During phases I and II progesterone was converted to 17 alpha-hydroxyprogesterone, androstenedione, and testosterone. During phase III, production of these steroids decreased drastically, and in turn, synthesis of 20 beta-hydroxy-4-pregnen-3-one and 17 alpha,20 beta-diOHprog was induced. Using 17 alpha-hydroxyprogesterone as a substrate, androstenedione and testosterone were produced during phases I and II, whereas they decreased considerably during phase III. This was followed by the production of 17 alpha,20 beta-diOHprog as the major metabolite.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lunar synchronization of in vitro steroidogenesis in ovaries of the golden rabbitfish, Siganus guttatus (Bloch)

To assess the relationship between lunar cycle and steroidogenesis in the ovaries of the golden rabbitfish, Siganus guttatus, the intact follicles of oocytes were incubated in vitro with human chorionic gonadotropin (hCG) and seven steroid hormones, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), 17alpha-hydroxyprogesterone (17alpha-OHP), progesterone (P), cortisol, estradiol-17beta (E2) and testosterone, during the two lunar phases, the new moon (1 week before spawning) and the first lunar quarter (just before spawning). Around the new moon, germinal vesicle breakdown (GVBD) could not be induced by addition of hCG or any steroid hormones. Around the first lunar quarter, GVBD was induced by addition of hCG, DHP, 20beta-S, 17alpha-OHP, P, and cortisol. DHP was the most potent steroid hormone. When the intact follicles of oocytes were incubated with hCG in both lunar phases, the production of E2 and DHP measured by enzyme immunoassay decreased and increased significantly from the new moon to the first lunar quarter, respectively. These results suggest that the ovarian follicles produce E2 around the new moon and DHP around the first lunar quarter and that the production/conversion of the steroid hormones is under the influence of gonadotropin(s). The synchronous increase in ovarian activity supports the hypothesis that lunar periodicity is a major factor for the ovarian development of S. guttatus.     

Kinetic studies on ovarian C-17,20-lyase activity: effect of luteinizing hormone surge

It has been shown that 3 h after the preovulatory gonadotropin surge, an abrupt decrease occurs in follicular C-17,20-lyase (lyase) activity which causes a decrease in C19-steroid production. To determine the reason for the reduced lyase activity, we used rats that were induced to ovulate by means of PMSG administration. In these rats, a 54% decrease in lyase activity occurred at the peak of the LH surge. When the gonadotropin surge and ovulation were blocked by pentobarbitone the decrease was prevented. Administration of LH to the pentobarbitone-blocked rats reduced lyase activity to well below the level reached after the endogenous gonadotropin surge. In cycling proestrous rats as well, human CG (hCG) decreased the lyase activity, as measured in isolated follicles 3 h after hCG administration. Out of three potential inhibitory steroids for lyase activity; progesterone, 3 alpha,17 alpha-dihydroxy-5 alpha-pregnen-20-one, and 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, only the last compound inhibited competitively ovarian lyase activity. The inhibition constant (Ki) value was 29 microM. In order to explore which of the two activities of the lyase complex is regulated by the gonadotropin, a double label double substrate experiment was conducted using [14C]progesterone with [3H]17 alpha-hydroxyprogesterone (17 alpha-OHP). With this assay procedure, we could determine in the same experiment the site of stimulation, the preferred substrate, and the amount of conversion. The conversion of progesterone to 17 alpha-OHP, as well as the conversion to androstenedione were significantly inhibited throughout the reaction by the gonadotropin. Thus, the changes in ovarian lyase after hCG mimic those of 17 alpha-hydroxylase. The labeling pattern of androstenedione showed that the ovarian lyase complex catalyzed the conversion preferentially from progesterone. Whereas the 3H/14C ratio in androstenedione varied between 0.29 to 0.76, the ratios in the 17 alpha-OHP were from 5 to 22. Thus, the exogenous 17 alpha-OHP did not equilibrate with the product formed from progesterone. The effect of the hCG was to decrease the preference of progesterone over 17 alpha-OHP as substrate. It is concluded that: the LH of the surge inhibits both the 17 alpha-hydroxylase and lyase activities. The ovarian lyase complex shows a preference for progesterone as a substrate rather than 17 alpha-OHP. 17 alpha-OHP is not an obligatory intermediate in androstenedione production in ovarian tissue. hCG affects the ovarian lyase complex by shifting the relative preference of substrates towards 17 alpha-OHP.     

Germinal vesicle breakdown in oocytes of catfish, Mystus vittatus (Bloch): relative in vitro effectiveness of estradiol-17 beta, androgens, corticosteroids, progesterone, and other pregnene derivatives

The relative in vitro effectiveness of estradiol-17 beta, androgen, corticosteroids, progesterone, and other pregnene derivatives on germinal vesicle breakdown (GVBD) using folliculated oocytes of an Indian catfish Mystus vittatus was investigated. 17 alpha,20 beta-Dihydroxyprogesterone was found to be the most effective maturation-inducing steroid (MIS). Estradiol-17 beta and testosterone were not effective but androsterone was found to be fairly effective in final oocyte maturation. Deoxycorticosterone (DOC), hydrocortisone, progesterone, and 17 alpha-hydroxyprogesterone were also effective at their higher concentrations. Among 5 beta-reduced pregnenes, all but 5 beta-pregnene-3 alpha-ol-20-one could induce oocyte maturation. The significance of these findings in relation to the progestagens as natural MIS and the present controversial position of another Indian catfish Heteropneustes fossilis is discussed in the light of available literature.     

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.     

The course of steroid release by intact ovarian follicles of Atlantic salmon (Salmo salar) incubated in vitro with and without gonadotrophin

Intact ovarian follicles were dissected from a single ovary and groups of follicles incubated in balanced saline at 6 degrees with and without a pacific salmon gonadotrophin (GTH) preparation. Aliquots of the incubation media were withdrawn at intervals, the steroids were extracted with solvent, and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha 20 beta P), 17 alpha-hydroxyprogesterone, progesterone, androstenedione, testosterone, and oestradiol were separated chromatographically and measured by radioimmunoassay. The main findings were (1) germinal vesicle breakdown commenced on Day 8 under the influence of GTH, (2) after an induction period of about 2 days there was a dramatic and continuing release of 17 alpha 20 beta P in the presence of GTH, (3) GTH promoted a moderate release of 17 alpha-hydroxyprogesterone, (4) GTH stimulated testosterone release up to Day 6 but thereafter levels fell, reflecting either absorption or catabolism of the released steroid, (5) GTH induced a precipitous release of androstenedione during the first few hours of incubation, and (6) progesterone and oestradiol release were little affected by GTH. Steroid concentrations were also measured in ova stripped from naturally ovulating wild fish and the relative amounts were shown to bear some similarity to those released into the incubation medium in the presence of GTH. The routes of biosynthesis and functions of these steroids are considered in light of the results.     

Steroid production by ovarian follicles of the viviparous guppy (Poecilia reticulata) and its regulation by precursor substrates, dibutyryl cAMP and forskolin.

Production in vitro of estradiol-17 beta, testosterone, 17 alpha-20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P), 17 alpha-hydroxyprogesterone, and progesterone by follicles of the guppy at various stages of oocyte growth and gestation was investigated. Basal production of estradiol-17 beta was highest in 0.8- and 1.2-mm follicles, whereas that of testosterone and 17 alpha,20 beta-P was highest in 1.6-mm (postvitellogenic) follicles. Levels of these steroids declined after fertilization and were undetectable in late gestation and postpartum follicles. 17 alpha-Hydroxyprogesterone and progesterone levels were low at all stages. Thus, none of these steroids appears to be involved in maintaining gestation. Regulation of estradiol-17 beta and 17 alpha,20 beta-P secretion by vitellogenic (1.0 mm) and postvitellogenic follicles by precursor substrates, dbcAMP (0.1 to 10 mM) and forskolin (1 to 100 microM), was also investigated. Vitellogenic follicles synthesized increased quantities of estradiol-17 beta in the presence of exogenous testosterone, whereas estradiol-17 beta production by postvitellogenic follicles was not altered by testosterone. These results suggest decreased aromatase activity in the postvitellogenic follicles. Dibutyryl cAMP and/or forskolin stimulated testosterone and estradiol-17 beta production by vitellogenic follicles but did not stimulate conversion of testosterone to estradiol-17 beta, suggesting that the adenylate cyclase system stimulates estradiol-17 beta production by stimulating testosterone production but does not mediate conversion of testosterone to estradiol-17 beta. Postvitellogenic follicles synthesized increased quantities of 17 alpha,20 beta-P in response to 17 alpha-hydroxyprogesterone in a dose-dependent manner. Although 1 microM of forskolin stimulated 17 alpha,20 beta-P production by postvitellogenic follicles in the absence of exogenous 17 alpha-hydroxyprogesterone, 100 microM of forskolin inhibited 17 alpha,20 beta-P production. Dibutyryl cAMP, however, did not affect 17 alpha,20 beta-P production. In the presence of 50 ng of 17 alpha-hydroxyprogesterone, dbcAMP (10 mM) and forskolin (1 to 100 microM) suppressed 17 alpha,20 beta-P production. It is suggested that cAMP mediates 17 alpha,20 beta-P production up to a certain threshold level, beyond which it inhibits 17 alpha,20 beta-P production.     

Steroidogenesis by gonads of a viviparous teleost, the sailfin molly (Poecilia latipinna), in vitro and in vivo

Gonads of Poecilia latipinna transformed [3H]testosterone into a number of reduced and conjugated metabolites in high yield in vitro. In the male 5 beta-androstane-3 alpha, 17 beta-diol, 5 beta-androstane-3 beta, 17 beta-diol, and their "sulphates" were identified. The only 11-oxygenated androgen detected was a compound tentatively identified as 5 beta-androstane-3 beta, 11 beta, 17 beta-triol. In ovarian incubates androstenedione, 5 beta-androstane-3 alpha, 17 beta-diol and its glucuronide, testosterone glucuronide, and 5 beta-androstane-3 alpha, 17 beta-diol glucuronide were identified. Highest yields of the ovarian glucuronides coincided with the termination of vitellogenesis which may indicate a possible pheromonal role of these conjugates. In vivo plasma levels of estradiol in the female were correlated with vitellogenesis and fell markedly after castration or hypophysectomy. In males the plasma concentrations of testosterone, 11-ketotestosterone and 11 beta-hydroxytestosterone and their conjugates were variable and not apparently correlated with testicular weight, but they were reduced to undetectable levels by castration and hypophysectomy. The results suggest that 5 alpha- and 5 beta-reduced steroids may play a role in the reproductive endocrinology of P. latipinna and that measurements of only the "classical" steroid hormones in this and possibly other species may give only a partial and misleading picture of endocrine changes.     

Synthesis of 20 alpha-hydroxylated steroids by ovaries of the dab (Limanda limanda)

Dab (Limanda limanda) ovarian fragments were incubated in vitro with either [4,7-3H]pregnenolone or 17 alpha-hydroxy[1,2,6,7-3H]progesterone to investigate the pattern of steroidogenesis. A major enzyme found in the dab ovary was 20 alpha-hydroxysteroid dehydrogenase. Among the steroids that were tentatively identified in ovarian incubates were 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P). 17 alpha,20 alpha-dihydroxy-5 beta-pregnan-3-one, 3 beta, 17 alpha,20 alpha-trihydroxy-5 beta-pregnane (3 beta,17,20 alpha-P-5 beta), and 3 alpha,17 alpha,20 alpha-trihydroxy-5 beta-pregnane. The presence of these steroids in plasma of mature female and male dabs was studied by radioimmunoassay. The antiserum was raised against 17,20 alpha-P. The 17,20 alpha-[3H]P label was produced by incubating place milt with 17 alpha-hydroxy [3H]progesterone. The radioimmunoassay was shown to have a high cross-reaction with the 5 beta-reduced analogues of 17,20 alpha-P and was therefore used, in conjunction with thin-layer chromatography, to measure the steroids. High concentrations of both 17,20 alpha-P and 3 beta,17,20 alpha-P-5 beta found in female and male dab plasma. The possible role of these steroids is discussed.     

In vitro metabolism of 4-pregnenes in ovaries of a freshwater teleost,the ayu (Plecoglossus altivelis): Production of 17α,20β-dihydroxy-4-pregnen-3-one and its 5β-reduced metabolites,and activation of 3β- and 20β-hydroxysteroid dehydrogenases by treatment with a fish gonadotropin

In vitro steroidogenesis in the mature ovaries of a freshwater teleost, the ayu (Plecoglossus altivelis), was examined. Cell-free homogenates of the ovaries of untreated fish or fish treated with a salmon gonadotropin (SG-G100) were aerobically incubated with ¹⁴C-labeled progesterone, 17α-hydroxyprogesterone, or 17α,20β-dihydroxy-4-pregnen-3-one, in the presence of NADPH. (1) Untreated fish: Progesterone was converted to 3α,17α-dihydroxy-5β-pregnan-20-one (the major product), 17α-hydroxyprogesterone, testosterone, 3α-hydroxy-5β-pregnan-20-one, 17α-hydroxy-5β-pregnane-3,20-dione, 17α,20β-dihydroxy-5β-pregnan-3-one, and 5β-pregnane-3α,17α,20β-triol. 17α-Hydroxyprogesterone was also transformed to 3α,17α-dihydroxy-5β-pregnan-20-one (the major product), in addition to the above-stated metabolites. 17α,20β-Dihydroxy-4-pregnen-3-one was metabolized into 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol. (2) Fish treated with the gonadotropin: Besides the metabolites mentioned above, 17α,20β-dihydroxy-4-pregnen-3-one was confirmed as a metabolite of progesterone, and enhanced production of 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol was observed. 17α-Hydroxyprogesterone was transformed to 17α,20β-dihydroxy-4-pregnen-3-one and 5β-pregnane-3β,17α,20β-triol, in addition to the metabolites obtained by the untreated fish, and formation of 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol was increased. 17α,20β-Dihydroxy-4-pregnen-3-one was converted into 5β-pregnane-3β,17α,20β-triol besides 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol. From these results it is concluded that, by the treatment of the fish with gonadotropin, ovarian 3β- and 20β-hydroxysteroid dehydrogenases were selectively activated.     

The in vitro metabolism of radioactive progesterone and testosterone by the gonads of the protandrous Rhabdosargus sarba at various sexual phases

The in vitro steroidogenic capacity of the gonadal tissue in the protandrous Rhabdosargus sarba was studied. Testicular and ovarian tissues from various sexual phases were used either separately or combined. With progesterone as precursor, high yield of 5 beta-reduced metabolites, and no 11-ketotestosterone or 11 beta-hydroxytestosterone were found. The production of 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnane-3,20-dione was very high in incubations with testicular tissue from intersexes or males and was low in those with ovarian tissue only. The production of 17 alpha-hydroxyprogesterone was high in the female but was low in other sexual phases. With testosterone as precursor, 11-ketotestoterone and 11 beta-hydroxytestosterone were identified. The latter was the most abundant and prominent polar steroid in all incubations. 5 beta-Reductase activity was high in the male and relatively low in the female. 5 alpha-Reduced products, however, were absent. There was an increase in the production of androstenedione as the animal underwent sex reversal. The significance of this change in steroidogenesis in this protandrous fish is at present under investigation. Experimental results also indicated that in the intersexual gonad there may be interaction between the testicular component and the ovarian component in steroidgenesis.     

Aging and ovarian steroidogenesis in the rat

Ovaries obtained from rats of 4 and 18 mo of age were incubated in vitro with 3H-pregnenolone as the added precursor. Incorporation of radioactivity into progesterone (P), 20alpha-dihydroprogesterone (20alpha-P), 17alpha-hydroxyprogesterone (17alpha-OHP), androstenedione, and testosterone were studied by serial paper and thin-layer chromatography. Significantly more P, 17alpha-OHP and androgen was produced by the older ovaries. Addition of hCG (100 I.U.) to the incubate increased 20alpha-P production in the ovaries of both age groups, depressed 17alpha-OHP and androgen formation in the ovaries of older rats, and had no effect on progesterone synthesis in either age group. Serum progesterone in 4-mo-old rats was 13.5 +/- 1.91 ng/ml at estrus and 3.69 +/- 0.9 ng/ml at diestrus, whereas serum progesterone in 18-mo-old rats was 0.85 +/- 0.15 ng/ml at constant estrus and 1.23 +/- 0.54 ng/ml at constant diestrus. Treatment with hCG for 20 days increased serum progesterone to 29.6 ng/ml in young rats and to 4.21 +/- 2.0 ng/ml in older rats. Shorter treatments were less effective. These data reveal a definite change in steroidogenic competence in older rat ovaries.     

Partial 17, 20-desmolase and 17 alpha-hydroxylase deficiencies in a 16-year-old boy

Thirteen plasma steroids as well as ACTH, LH and FSH were measured by specific RIAs under basal and dynamic conditions in a 16-year-old boy (normal external genitalia, 46, XY karyotype) who presented slowness and unachievement of pubertal development. On the delta 4-pathway: basal levels of testosterone and dihydrotestosterone were low- with a normal ratio-, delta 4-androstenedione and 11 beta-hydroxyandrostenedione were in the low normal range. Meanwhile, 17 alpha-hydroxyprogesterone and progesterone levels were markedly elevated. On the delta 5-pathway: dehydroepiandrosterone was extremely low while 17 alpha-hydroxypregnenolone and pregnenolone were almost normal; dehydroepiandrosterone sulfate was subnormal while pregnenolone sulfate was normal. Cortisol, aldosterone were normal while ACTH was moderately increased. Basal and responsive levels of LH and FSH were markedly increased. ACTH stimulation induced a subnormal rise of cortisol and 11 beta-hydroxyandrostenedione, a low or absent rise of dehydroepiandrosterone, 17 alpha-hydroxypregnenolone, androstenedione and 17 alpha-hydroxyprogesterone contrasting with a marked rise of pregnenolone and progesterone. After hCG stimulation, responses were low for testosterone, extremely high for 17 alpha-hydroxyprogesterone with a normalisation of the 17 alpha-hydroxyprogesterone/progesterone ratio. Fluoxymesterone dramatically reduced the pathologically high basal levels of progesterone and 17 alpha hydroxyprogesterone. Dexamethasone induced only a minute decrease in the delta 4-progestagens, a marked decrease in pregnenolone, with a more than 80% reduction of 17 alpha- hydroxypregnenolone, dehydroepiandrosterone, dehydroepiandrosterone sulfate and androstenedione. These data suggest a defect involving the cytochrome P450 common to both 17 alpha-hydroxylase and 17, 20-desmolase activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood steroid levels in the goldfish: measurement of six ovarian steroids in small volumes of serum by reverse-phase high-performance liquid chromatography and radioimmunoassay

A reliable and rapid technique for the measurement of estradiol-17 beta, testosterone, progesterone, 17 alpha-hydroxyprogesterone, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, and cortisol by reverse-phase high-performance liquid chromatography and radioimmunoassay in single female goldfish serum samples of 50 microliters was developed. The steroids were extracted with Sep-Pak C18 cartridges after heat treatment. While estradiol-17 beta was assayed directly after extraction, the other steroids were separated on a mu Bondapak C18 stainless-steel column with acetonitrile:water (53:47, v/v) in 17 min under isocratic conditions, and then quantitated by specific radioimmunoassays. The technique was validated and was shown to be highly accurate, precise, sensitive, and specific for measuring those particular ovarian steroids in goldfish serum. The steroid levels measured in this way were not significantly different from those measured after separation on a Nova-Pak C18 column with methanol:water (56:44, v/v), where all the individual steroids could be completely resolved during a 40-min run. With this technique, the steroid levels in the serum of the goldfish during the secondary yolk stage, the tertiary yolk stage, and at 0 hr after ovulation were determined. While estradiol-17 beta was found to be significantly higher in the tertiary yolk stage, testosterone was significantly higher in the secondary yolk stage than in the other two stages. There was no significant difference in the levels of cortisol, 17 alpha-hydroxyprogesterone,17 alpha,20 beta-dihydroxy-4-pregnen-3-one, and progesterone among the three stages. We conclude that the technique is particularly useful for assaying multiple steroids in very small volumes of biological fluids.     

Estrogen regulation of thecal cell steroidogenesis and differentiation: thecal cell-granulosa cell interactions

Estrogen regulation of thecal cell steroidogenesis and differentiation was investigated with cells from ovarian antral follicles. Bovine theca interna cells were isolated and cultured in serum-free conditions to evaluate the effects of estradiol on thecal cell production of androstenedione, testosterone, and progesterone. Estradiol increased thecal cell androgen production throughout a 6-day culture period; however, the basal and stimulated levels of androgen production diminished after day 3 of culture. Androstenedione accumulation was approximately 10-fold greater than that of testosterone. In contrast to the stimulatory effects that estradiol had on androgen production, estradiol suppressed progesterone production throughout the 6-day culture period. Comparison of the effects of estradiol and hCG on thecal cells from small (less than 5 mm), medium (5-10 mm), and large (greater than 10 mm) antral follicles demonstrated that estradiol stimulated androgen production to a greater extent than hCG with cells from all of these stages of follicle development. Estradiol stimulation of androstenedione was greater in theca from small follicles than in theca from medium or large follicles. In contrast, suppressive effects of estradiol on progesterone were most apparent on thecal cells from medium and large follicles and less apparent on theca from small follicles. Estradiol stimulated androstenedione production in a dose-dependent fashion, with a minimum effective concentration of 10(-9) M and a maximum effective concentration of 10(-7)-10(-6) M. Concentrations greater than 10(-6) M estradiol resulted in a decline in the stimulatory response and may be important in the preovulatory follicle to suppress thecal cell androgen production and initiate the process of luteinization. Progesterone production was slightly stimulated by 10(-9) M estradiol, whereas higher concentrations (10(-7)-5 x 10(-6) M) resulted in a dose-dependent suppression of progesterone production. Interestingly, combined treatment of thecal cells with estradiol and hCG resulted in a greater than additive stimulation of androstenedione production, and estradiol decreased the ability of hCG to stimulate progesterone production. Observations demonstrate that estradiol can dramatically alter thecal cell production of steroids and support a hypothesis that steroid-mediated interactions between granulosa and thecal cells play an important role in regulating cellular function within follicles. The data provide evidence that a local feedback loop may exist in ovarian follicles, where androgens produced by thecal cells are used as a substrate for granulosa cell aromatization into estrogens, which, in turn, may feed back to stimulate thecal cell production of androgens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Steroid biosynthesis by the testes of the dogfish Scyliorhinus caniculus

Dogfish testes were incubated with radioactive progesterone, pregnenolone, and testosterone, and both free and conjugated metabolites were examined. In the free fraction, which contained 42–70% of the incubated radioactivity, progesterone, androstenedione, and testosterone were identified as incubation products of both progesterone and pregnenolone. In addition, a small amount of 17α-hydroxyprogesterone was identified as a metabolite of progesterone in one fish. Testosterone and androstenedione were the only free steroids isolated from incubations of testosterone. Although steroid glucuronide formation was insignificant, very large amounts of solvolysable steroids were isolated from all incubations. With pregnenolone and progesterone, 10–30% of the incubated radioactivity was recovered in this solvolysable fraction, in which the major products were identified as testosterone and 17α,20β-dihydroxy-4-pregnen-3-one. With two fish incubated with [¹⁴C]testosterone, 5α-androstane-3β, 17β-diol was isolated in low yield from the solvolysable fraction in addition to testosterone, but in one incubation with [³H]testosterone, the sole component of this fraction was testosterone which accounted for 21% of the initial radioactivity.     

The in vitro synthesis of final maturational steroids by ovaries of brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens)

The steroidogenic profile produced by ovaries of brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens) was examined using radioactive incorporation incubations. The radioactive metabolites produced were identified and were tested for their ability to induce germinal vesicle breakdown (GVBD) in oocytes in in vitro bioassays. Brook trout ovarian tissue converted [14C]progesterone to five metabolites that were effective in inducing GVBD in the bioassays: 5 beta-pregnanedione, 17 alpha-hydroxyprogesterone, deoxycorticosterone, and two more polar metabolites that were not identified. One of these two metabolites comigrated with 11-deoxycortisol and 17 alpha-hydroxy,20 beta-dihydroprogesterone (17 alpha, 20 beta prog) in thin layer chromatography. Androstenedione, testosterone, and estradiol-17 beta were also identified as metabolites of progesterone. Brook trout ovarian tissue did not extensively metabolize [14C]pregnenolone. Dehydroepiandrosterone and 17 alpha-hydroxypregnenolone were tentatively identified as metabolites of pregnenolone. Yellow perch ovarian tissue converted [14C]progesterone or [14C]pregnenolone to six metabolites that were effective in inducing GVBD in the bioassays: 5 alpha-pregnanedione, 17 alpha-hydroxyprogesterone, 20 alpha-dihydroprogesterone, 17 alpha-hydroxy, 20 alpha-dihydroprogesterone (tentatively identified), and two other unidentified metabolites. One of these two metabolites comigrated with 11-deoxycortisol and 17 alpha, 20 beta prog in thin layer chromatography. The only metabolite that induced ovulation of yellow perch oocytes in vitro was the metabolite comigrating with 11-deoxycortisol and 17 alpha, 20 beta prog. Androstenedione and testosterone were also identified as metabolites produced by yellow perch ovarian tissue. The results show that the ovaries of both species produce steroids that are active in the induction of oocyte final maturation.     

Plasma levels of ovarian steroids, including 17 alpha,21-dihydroxy-4-pregnene-3,20-dione and 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one, in female plaice (Pleuronectes platessa) induced to mature with human chorionic gonadotrophin

In one previous paper we reported on the identification of 11-deoxycortisol and 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one in the ovaries and plasma of mature female plaice and also described the development of radioimmunoassays for these two steroids. The present paper describes temporal changes in plasma levels of the free and conjugated forms of these and of some other steroids (17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one, 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, 17 alpha-hydroxy-4-pregnen-3-one, testosterone, and 17 beta-oestradiol) in female plaice injected with and without human chorionic gonadotrophin (HCG). Oocyte final maturation, but not ovulation, was induced by HCG injections. Levels of most of the steroids were also elevated by the HCG injections and were significantly higher than in control fish throughout the experiment (112 hr). The two most abundant steroids were 11-deoxycortisol and 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one (up to 600 ng ml-1). Only relatively small amounts of 17 alpha-hydroxy-4-pregnen-3,20-dione (less than 15 ng ml-1), 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, and 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (less than 5 ng ml-1) were found. 17 alpha,20 beta,21-Trihydroxy-4-pregnen-3-one was not present. Testosterone and 17 beta-oestradiol levels rose briefly, in response to the first of the two HCG injections, and then fell significantly. The ratio of conjugated to free steroids (except for 17 alpha-hydroxy-4-pregnene-3,20-dione and 17 beta-oestradiol) was almost always greater than 1. In the HCG-injected fish, there was a significant negative correlation between the response of 17 beta-oestradiol levels and the response of 11-deoxycortisol and 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one levels. This further confirms that, as teleosts approach the time of full maturity, there is switch-over in the ovaries from predominantly C19 and C18 steroid production to predominantly C21 steroid production.     

The skin of the male African catfish, Clarias gariepinus: a source of steroid glucuronides

Steroid metabolism in the skin of mature male African catfish, Clarias gariepinus, reared in the laboratory, was studied in vitro by tissue incubations with [3H]pregnenolone, [3H]dehydroepiandrosterone, [3H]17 alpha-hydroxyprogesterone, [3H]androstenedione, [14C]11 beta-hydroxyandrostenedione, and [3H]testosterone as precursors. While pregnenolone was not converted to any other steroid, dehydroepiandrosterone was transformed mainly to 5-androstene-3 beta, 17 beta-diol. The products of 17 alpha-hydroxyprogesterone incubations were 5 beta-pregnane-3 alpha,17 alpha-diol-20-one, 5 beta-pregnane-3 alpha,17 alpha, 20 beta-triol, and 5 beta-pregnan-17 alpha-o1-3,20-dione. The major steroids of androstenedione incubations were etiocholanolone, testosterone, and androsterone. Testosterone was converted mainly to etiocholanolone and androstenedione, and only small quantities of 11 beta-hydroxytestosterone, 11-ketotestosterone, and 11-ketoandrostenedione were the metabolites found in 11 beta-hydroxyandrostenedione incubation. These results demonstrated the presence of the enzymes 5 alpha- and 5 beta-reductases and 3 alpha-, 11 beta-, 17 beta-, and 20 beta-hydroxysteroid dehydrogenases in the skin. From enzymehistochemical results it appeared that the steroid conversions take place in the epithelial cells. Moreover, the presence of UDP-glucose dehydrogenase, an enzyme involved in the synthesis of glucuronic acid, in these cells indicates the possibility of steroid glucuronide formation. Indeed significant amounts of water-soluble steroid conjugates, particularly 5 beta-dihydrotestosterone- and testosterone-glucuronide, were found in the incubations with androstenedione and testosterone, indicating the presence of the UDP-glucuronosyl transferase in the catfish skin. In the light of these results, a role of the skin of African catfish in the production of semiochemicals having pheromonal properties is discussed.