Bun-Ichi Tamaoki

Relative in vitro effectiveness of 17α,20β-dihydroxy-4-pregnen-3-one and other pregnene derivatives on germinal vesicle breakdown in oocytes of ayu (Plecoglossus altivelis), amago salmon (Oncorhynchus rhodurus), rainbow trout (Salmo gairdneri), and goldfish (Carassius autatus)

The relative effectiveness of several pregnene derivatives, which had previously been identified in the ovaries of ayu (Plecoglossus altivelis), on germinal vesicle breakdown (GVBD) was investigated in vitro using folliculated oocytes of four species of teleosts, ayu, amago salmon (Oncorhynchus rhodurus), rainbow trout (Salmo gairdneri), and goldfish (Carassius auratus). Although some species differences existed in the relative effectiveness of each steroid on GVBD, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-diOHprog) was consistently the most potent inducer of final oocyte maturation. Both progesterone and 17 alpha-hydroxyprogesterone were effective at relatively high concentrations. Of the 5 beta-reduced metabolites, 17 alpha, 20 beta-dihydroxy-5 beta-pregnan-3-one was almost as effective as 17 alpha, 20 beta-diOHprog in oocytes of amago salmon and rainbow trout, while the other 5 beta-reduced compounds (3 alpha-hydroxy-5 beta-pregnan-20-one, 17 alpha-hydroxy-5 beta-pregnane-3,20-dione, 3 alpha, 17 alpha-dihydroxy-5 beta-pregnan-20-one, 5 beta-pregnane-3 alpha, 17 alpha, 20 beta-triol, and 5 beta-pregnane-3 beta, 17 alpha, 20 beta-triol) were almost or totally ineffective at the concentrations tested (1-0.001 micrograms/ml). These bioassay results, together with previous findings on the capacity of the ovary to produce 17 alpha, 20 beta-diOHprog, indicate that 17 alpha, 20 beta-diOHprog is the natural maturation-inducing steroid hormone common to three species of Salmoniformes, ayu, amago salmon, and rainbow trout. These results, however, only suggest that 17 alpha, 20 beta-diOHprog is involved in maturation of goldfish oocytes, since supportive physiological and biochemical data are lacking. Possible regulatory roles of 5 beta-reduced metabolites on steroid-induced oocyte maturation are 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

Abstract 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 14 C-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 pathway of formation of testosterone from 3β-hydroxypregn-5-en-20-one by rat testicular microsomes

Abstract 3β-Hydroxypregn-5-en-20-one (pregnenolone)-7α-3H was transformed to testosterone by rat testicular microsomes in the presence of NAD and NADPH. Addition of 105,000 × g supernatant fluid of the testicular homogenates signifincantly enhanced the formation of testosterone. Progesterone, 17α- hydroxyprogesterone and androst-4-ene-3, 17-dione (androstenedione) were obtained as the intermediates, whereas 3β, 17α-dihydroxypregn-5-en-20-one (17α-hydroxypregnenolone) and 3β-hydroxyandrost-5-en-17-one were found only in negligible amounts. A mixture of progesterone-4-14C and pregnenolone- 7α-3H was incubated with the microsomes for 5 to 20 minutes. The results showed that progesterone was more quickly converted to testosterone than was pregnenolone by the microsomes and that androstenedione was an intermediate substance between progesterone and testosterone as well as between pregnenolone and testosterone. A similar dual tracer study using 17α-hydroxyprogesterone-4-14C and 17α-hydroxypregrenolone-7α-3H showed that the former steroid was more quickly transformed to testosterone by the microsomes. It is concluded that testosterone is formed from pregnenolone by the microsomes mainly by way of progesterone and androstenedione.

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.

In vitro transformation of steroids by the head kidney, the body kidney, and the corpuscles of Stannius of the rainbow trout (Salmo gairdneri)

Abstract Incubation of 4- 14 C-labeled pregnenolone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, and androstenedione with homogenates of head kidneys containing interrenal tissue of the rainbow trout ( Salmo gairdneri ) resulted in the production of 17α-hydroxypregnenolone, 11-deoxycortisol, and cortisol from pregnenolone; 11-deoxycortisol, cortisol, and androstenedione from 17α-hydroxyprogesterone; corticosterone and 18-hydroxy-11-deoxycorticosterone from 11-deoxycorticosterone; and testosterone and 11β-hydroxyandrostenedione from androstenedione. After incubation of progesterone with homogenates of the body kidney, 5α- and 5β-pregnanedione, 3α- and 3β-hydroxy-5α-pregnan-20-one, and 3α- and 3β-hydroxy-5β-pregnan-20-one were obtained as the metabolites of progesterone. When homogenates of corpuscles of Stannius of the rainbow trout were incubated with progesterone or 11-deoxycorticosterone, no corticoid could be detected as metabolite of the substrates, but 5α-pregnanedione and its 5β-isomer were obtained as the main products from progesterone. These results demonstrated the presence of 11β-hydroxylase, 17α-hydroxylase, 18-hydroxylase, 21-hydroxylase, 17β-hydroxysteroid dehydrogenase, Δ 5 -3 β -hydroxysteroid dehydrogenase, C 17 C 20 lyase, and Δ 5 -Δ 4 isomerase activities in the head kidney of the rainbow trout. On the other hand, pregnene-5α- and 5β-hydrogenase activities were predominant in the corpuscles of Stannins as well as in the body kidney of the rainbow trout, while 3α- and 3β-hydroxysteroid dehydrogenase activities were also observed in the body kidney of the fish.

Submicrosomal distribution of adrenal enzymes and cytochrome P-450 related to corticoidogenesis

By density-gradient centrifugation in the absence of CsCl, the microsomal fractions obtained from porcine and rat adrenal glands were separated into two subfractions: one fraction consisted of smooth-surfaced membrane structures which bore no ribosomes on their outer surface, and the other consisted of abundant free ribosomes with membrane structures, according to electron microscopic observation. The activities of three microsomal enzymes related to corticoidogenesis, namely Δ5-3β-hydroxysteroid dehydrogenase (EC 1.1.1.51) coupled with the Δ5-Δ4 isomerase (EC 5.3.3.1), 17α-hydroxylase (EC 1.14.1.7) and 21-hydroxylase (EC 1.14.1.8), were predominantly located in the smooth-surfaced subfraction. In accordance with the distribution of the hydroxylases, adrenal microsomal cytochrome P-450 was concentrated exclusively in the smooth-surfaced subfraction. As 21-hydroxylation was significantly inhibited in a CO-containing atmosphere, the cytochrome P-450 in the smooth-surfaced subfraction was participating as the direct activating site of O2 during 21-hydroxylation. The Km values of the submicrosomal Δ5-3β-hydroxysteroid dehydrogenase for pregnenolone and of 17α-hydroxylase for progesterone were estimated as 0.2 and 0.13 mM, respectively. The Km of the adrenal 21-hydroxylase was evaluated as 99 μM for progesterone and as 0.22 mM for 17α-hydroxyprogesterone. Substrate specificities of the adrenal submicrosomal enzymes, dynamic analysis of the corticoidogenesis throughout the incubation time, and the relationship between the amounts of enzyme preparations and the products, were examined.

The effect of synthetic estrogen upon the biosynthesis in vitro of androgen and luteinizing hormone in the rat

Abstract Two-month-old male rats of the Wistar strain were treated with hexestrol (estrogen) for 4 weeks (2.5 mg each once a week) to examine the effect of synthetic estrogen on the biosynthesis of androgen in testes and of luteinizing hormone in anterior pituitary glands. Testicular homogenate and microsomal fractions obtained from these rats were incubated with radioactive precursors such as [4-14C]pregnenolone, 4-14C]progesterone, 17α-hydroxy-4-14C iprogesterone and [4-14C]androstenedione. The activities of enzymes related to androgen formation, apart from that of 17α-pregnene-C-17-C-20 lyase, were strongly reduced by hexestrol treatment. Testosterone formation from progesterone in testicular homogenate was severely inhibited by the hexestrol treatment and progesterone added as substrate was catabolized mainly to 3α-hydroxy-5α-pregnan-20-one, which had no androgenic activity. However, testosterone formation from progesterone incubated with intact testicular homogenate in hexestrol-containing medium remained at the level of a control lacking hexestrol in the incubation medium. On the basis of these results, the direct action of hexestrol upon testicular enzymes related to steroid biotransformation is discussed. Anterior pituitary lobes of hexestrol-treated and intact rats were incubated with [14C]leucine, and the luteinizing hormone fractions were separated by immuno-chemical methods. The incorporation of [14C]leucine into the luteinizing hormone fraction was measured. The biosynthetic activity of luteinizing hormone was significantly lowered per unit weight of the tissue, but the total activity per gland was nog significantly influenced by hexestrol treatment.

Studies on enzyme reactions related to steroid biosynthesis: I. Presence of the cytochrome P-450 in testicular tissue and its role in the biogenesis of androgens

Abstract Among the subcellular fractions of testicular homogenatcs of rats, the cytochrome P-450 was localized exclusively in the microsomal fraction (10,000–105,000 g precipitate) of the cells such as the interstitial cells, which were radio-resistant, and also responsive to gonadotrophin. In the atmosphere of CO:O 2 (95:5) mixture, the activities of the testicular microsomal 17α-hydroxylase and C 17 -C 20 lyase which cleaves the side-chain of 17α-hydroxyprogesterone were seriously decreased, in comparison with those exhibited in an Ar: O 2 (95:5) atmosphere, after the preparation had previously been gassed with CO or Ar. Therefore, the above mentioned two enzymes related to androgen biosynthesis were concluded to involve the cytochrome P-450 as the site of molecular oxygen activation which was required by the two enzymes.

In vitro bioconversions of steroids in the mature ovary of the hagfish, Eptatretus burgeri☆

Abstract 1. 1. Steroid hormone production in the mature ovary of the hagfish Eptatretus burgeri was demonstrated in vitro using labeled precursors of steroid hormones. 2. 2. Progesterone, 17α-hydroxyprogesterone, androstenedione and 11-deoxycortisol were identified as the metabolites, after incubation of the follicular tissue homogenate with 14 C-pregnenolone. Furthermore, 3α-hydroxy-5β-pregnan-20-one, as a metabolite of progesterone, and 5α-androstane-3,17-dione (5α-androstanedione) and 3β-hydroxy-5α-androstan-17-one (epiandrosterone), as metabolites of androstenedione, were identified respectively. 3. 3. From these identified metabolites, the metabolic pathways of steroids in the hagfish ovary are proposed. 4. 4. The relationship between the existence of 11-deoxycortisol and fish ovulation is discussed.

Side-chain cleavage as related to steroid hormone synthesis☆

The mechanism of side-chain cleavage during the course of steroid hormone biosynthesis by rat testicular and adrenal enzyme preparations has been investigated. 17α-Hydroxypregnene-C-17-C-20 lyase in testicular tissue required molecular oxygen in addition to NADPH. Progesterone and 17α-hydroxyprogesterone, under an 18O2-enriched atmosphere, and 17α-18O-hydroxylated progesterone under an 16O2 atmosphere were incubated with a rat testicular enzyme preparation, and the products were analyzed by high-resolution mass spectrometry. Progesterone-17α-hydroxylase required molecular oxygen, which was incorporated in the 17α-hydroxyl group of 17α-hydroxyprogesterone. However, in the case of the side-chain cleavage of 17α-hydroxyprogesterone, the molecular oxygen required was not incorporated into the direct steroid product, androstenedione, nor into the further product testosterone. It was thus established that the oxygen atoms of the 17-ketone and 17β-hydroxyl groups of androstenedione and testosterone, respectively, which were derived by testicular enzymes from progesterone, originated from the 17α-hydroxyl group of 17α-hydroxyprogesterone. The side-chain cleavage of cholesterol, in the synthesis of pregnenolone, was also studied, by incubation of 20α, 22R-dihydroxycholesterol with rat adrenal preparation under an 18O2 atmosphere. The immediate product pregnenolone and its further metabolite progesterone were isolated, and analysed by mass-spectrometry. No incorporation of molecular oxygen into the metabolites was observed, suggesting that the oxygen in the 20-ketone group of pregnenolone and progesterone is derived from the oxygen of the 20α-hydroxyl group introduced into cholesterol prior to the side-chain cleavage.