Differences in steroidogenesis by the subcellular fractions of adrenocortical special zone and cortex proper of the female possum (Trichosurus vulpecula)

Steroidogenesis by subcellular fractions of adrenal cortex proper (C.P.) and special zone (S.Z.) of female possum (Trichosurus vulpecula) was studied. Mitochondrial, microsomal and cytosol cell fractions were incubated with appropriate substrates in the presence of an NADPH-generating system. The major products formed from [3H]progesterone and [3H]17 alpha-hydroxyprogesterone by the microsomal fraction of the C.P. were 11-deoxycortisol and 11-deoxycorticosterone and 3 alpha (beta)-hydroxy-5 alpha-androstan-17-one by the S.Z. The mitochondrial fraction converted [3H]11-deoxycortisol to cortisol in yields twenty times higher by the C.P. than by the S.Z. and to 17 alpha, 20 beta,21-trihydroxy-4-pregn-3-one thirty times higher by the S.Z. The conversion of [3H]androstenedione to 11 beta-hydroxyandrostenedione by the C.P. was approximately double that of the S.Z., while 18-hydroxyandrostenedione (tentatively identified) formed the highest yield in both zones. Incubation of the same substrates with cytosol formed two 5 beta-pregnane and two 5 beta-androstane derivatives in total yields less than 5% by C.P. and greater than 60% by S.Z. Aromatase activity, estimated by the release of [3H2O] from [1 beta 3H]testosterone, in the adrenals of 8 possums, was in each experiment negligibly low. Determination of total enzyme activities in the two zones revealed that 11 beta, 18 and 21-hydroxylases were higher in the C.P., while 17 alpha-hydroxylase was higher in the S.Z. Similar results were obtained when the rates of formation of hydroxylated products were estimated in the presence of saturating amounts of substrates. Active 5 alpha- and 5 beta-reductases, C17-20-lyase and 3 alpha (beta) and 20 beta-hydroxysteroid dehydrogenases were found almost exclusively in the S.Z. We conclude that the S.Z. at lower levels of activity than the C.P. could contribute to the basal secretion of corticosteroids. In addition, the S.Z. has a high capacity to form C19 steroids and 5 alpha- and 5 beta-reduced steroids. The possible role of the S.Z. in possum is discussed.     

Factors influencing prostatic 5 alpha-reductase activity in possum (Trichosurus vulpecula)

1. There were marked differences in prostatic wts among individual possums, but no evidence of a seasonally related change in wt could be established. It was concluded that the wt differences are mainly due to the changes in secretory activities. After castration the prostate wts fell while after administration of testosterone or oestradiol partially reversed this process. 2. Seven steroid conversion products were isolated from prostatic homogenates incubated with [3H] testosterone; 5 alpha-androstane-3 beta,17 beta-diol forming the highest yield. 3. While the 5 alpha-reductase activity of prostates from intact possums was very low (approx. 8% of the total yield), it increased to over 50% after castration. 4. Administration of testosterone or oestradiol partially reversed the post-castration rise in 5 alpha-reductase, while 17 beta-hydroxy-5 alpha-androstane-3-one (DHT) was ineffective. Administration of porcine FSH-NIH-P2 to both intact or castrated possums caused a marked rise in prostatic 5 alpha-reductase activity. 5. It was concluded that in possum, FSH may have a direct stimulatory effect on prostatic 5 alpha-reductase activity. The results are discussed in relation to placental mammals.     

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.     

The effects of various steroids on testosterone metabolism by the sexually mature rabbit epididymis

We examined the influences of steroids present in the epididymis on androgen metabolism by epididymal tissue and on the binding of androgen metabolites to the epididymal androgen receptor in castrated adult rabbit epididymides under in vitro conditions. The conversion of [3H]testosterone to [3H]17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-DHT) and to [3H]5 alpha-androstane-3 alpha (beta), 17 beta-diol was inhibited by unlabeled steroids in the following manner progesterone greater than testosterone greater than estradiol. Unlabeled 5 alpha-DHT did not inhibit [3H]testosterone metabolism indicating that product inhibition is not an important regulatory event. The antiandrogen cyproterone acetate did not inhibit the formation of 5 alpha-reduced metabolites of [3H]testosterone. All of the compounds used inhibited androgen binding to the classically defined cytoplasmic and nuclear androgen receptor.     

The effects of neonatal androgenization of male rats on testosterone metabolism by the hypothalamus-pituitary-gonadal axis

Male rats were androgenized on the third postnatal day by a single injection of 1 mg testosterone propionate. The in vitro metabolism of [4-14C]testosterone by pituitary and hypothalamus homogenates was investigated at the age of 90 days. The pituitary and hypothalamus homogenates from control and neonatally androgenized animals converted [4-14C]testosterone to the same metabolites, mainly 5 alpha-reduced derivatives; the quantitative yield of 5 alpha-reduced metabolites was much higher in the pituitary homogenates of androgenized rats. The hypothalamic homogenates showed no differences. In the androgenized rats a very significant increase of the plasma FSH levels was measured while the LH levels were also augmented. The plasma levels of testosterone were not different from the values in control rats, notwithstanding a 25% reduction in testes weight. The present experiments appear to indicate that the neonatal androgenization results in an accentuation of the sexual dimorphism which normally exists in the pituitary of adult rats for the 5 alpha-reductase activity.     

5alpha-reductase isoenzymes 1 and 2 in the rat testis during postnatal development

The pubertal initiation of spermatogenesis is reliant on androgens, and during this time, 5alpha-reduced androgens such as dihydrotestosterone (DHT) are the predominant androgens in the testis. Two 5alpha-reductase (5alphaR) isoenzymes (5alphaR1 and 5alphaR2) have been identified, which catalyze the conversion of testosterone to the more potent androgen DHT. The present study aimed to investigate the developmental pattern of 5alphaR isoenzymes and their relationship to the production of 5alpha-reduced androgens in the postnatal rat testis. Both 5alphaR1 and 5alphaR2 isoenzyme mRNAs were measured by real-time polymerase chain reaction, isoenzyme activity levels by specific assays, and testicular androgens by radioimmunoassay after high-performance liquid chromatographic separation. Both 5alphaR1 and 5alphaR2 mRNAs and activity levels were low in the 10-day-old (prepubertal) testis, peaked between Days 20 and 40 during puberty, and then declined to low levels at 60-160 days of age. The developmental pattern of both 5alphaR isoenzyme activity levels was mirrored by the testicular production of 5alpha-reduced metabolites. Although 5alphaR1 was greater than 5alphaR2 at all ages, it is likely, given the substrate preferences of the two, that both isoenzymes contribute to the pubertal peak of 5alpha-reduced androgen biosynthesis. The peak in 5alphaR isoenzymes and 5alpha-reduced metabolite production coincided with the first wave of spermatogenesis in the rat, suggesting a role for 5alpha-reduced metabolites in the initiation of spermatogenesis. This was explored by acute administration of a 5alphaR inhibitor (L685,273) to immature rats. The L685,273 markedly suppressed testicular 5alphaR activity during puberty by 75%-86%. However, a marked increase was observed in testicular testosterone levels (in the absence of changes in LH), and no decrease was observed in the absolute levels of 5alpha-reduced metabolites. Therefore, whether the formation of DHT in the presence of low testosterone levels in the pubertal testis is required for the initiation of spermatogenesis cannot be tested using 5alphaR inhibitors. We conclude that both 5alphaR1 and 5alphaR2 isoenzymes are involved in the peak of 5alpha-reduced androgen biosynthesis in the testis during the pubertal initiation of spermatogenesis.     

Galanin stimulates hCG induced testicular steroidogenesis in adult but not in immature male rats

The present study was undertaken to understand the role of galanin on testosterone secretion. Leydig cells from adult (60-80 days old) and immature (21-30 days old) rat testis were incubated with galanin (100 nM), galantide (100 nM) and Human Chorionic Gonadotropin (hCG, 25 I.U.) alone or in combinations and testosterone release was measured. It was observed that in adults, galanin failed to alter the basal testosterone release from the dispersed Leydig cells but potentiated the hCG induced testosterone release significantly. While galantide, prevented this galanin potentiating effect, but it did not alter the hCG alone induced testosterone release. On the other hand, the Leydig cells obtained from immature male rats were sensitive to hCG alone but not to galanin or galantide, both of which failed to alter the hCG induced testosterone release from these cells. Based on these results it can be postulated that galanin's role at the level of the male gonad is age dependent since its potentiating effects on hCG induced testosterone release were visible only in the adult and not in the immature male rats.     

Influence of age on the formation of 5alpha-androstanediol and 7alpha-hydroxy-testosterone by incubated rat testes.

Testes from rats of different ages were indubated with or without tritiated testosterone. The exogenously-added or endogenously-produced testosterone is mainly metabolized to 7alpha-hydroxylated testosterone in adult animals, and to 5alpha-reduced metabolites (especially 5alpha-androstanediol) in immature animals.     

Influence of rete testis fluid on the metabolism of testosterone by cultured principal cells isolated from the proximal or distal caput of the rat epididymis

Principle cells from 120 elutriations were used to improve procedures for culturing cells from the proximal or distal caput epididymidis. The criteria evaluated were metabolism of testosterone (T) to 5 alpha-reduced metabolites and cellular morphology after 6 days of culture. Isolated principal cells (greater than 90% viability) were cultured at 34 degrees C within a floating collagen matrix. Inclusion of transferrin or retinol in the culture medium increased the production of 5 alpha-reduced metabolites. Aggregation of principal cells before entrapment in the collagen matrix resulted in higher production of 5 alpha-reduced metabolites and more cells with a normal find structure than entrapment of dispersed cells in the matrix. Aggregated cells tended to form sheets or clusters, frequently arranged around a central lumen, with junctional complexes between adjacent cells. Cell polarity and morphologic features distinguishing principal cells from the proximal caput and distal caput epididymidis were retained. An average of 91% of the cells in aggregates were morphologically normal on Day 6 of culture in contrast to 5% for the single cells. Utilizing the improved culture procedure, we tested the hypothesis that ovine rete testis fluid (RTF) contains macromolecules which would aid in maintenance of a high rate of T metabolism. Principal cells were cultured in medium supplemented with 0 or 10% RTF, 10% ultrafiltrate of RTF (less than 10,000 daltons), or 10% newborn calf serum (NCS). Conversion of [3H]T to 5 alpha-reduced metabolites by cells from the proximal caput was twice that in cells from the distal caput on Day 6 of culture. Inclusion in the culture medium of 10% RTF or 10% NCS, but not 10% ultrafiltrate of RTF, increased (P less than 0.05) the production of 5 alpha-reduced metabolites by cells from both regions. We conclude that macromolecules in RTF or NCS are beneficial to maintenance of the ability to metabolize T by cultured principal cells, especially those from the proximal caput.     

Gonadotrophin induced development of the "special zone" in the adrenal cortex of immature female possums (Trichosurus vulpecula) with concomitant activation of steroid reductases

The effect of gonadotrophin and oestradiol administration on adrenocortical special zone (S.Z.) development and steroidogenesis was studied in immature female possums. Adrenals were examined histologically to determine S.Z. formation, and cell-free homogenates were incubated with 3H progesterone in the presence of an NADPH-generating system. Treatment with PMS + hCG, resulted in the development of S.Z.s. varying in volume from 10 to 60% of the total adrenal gland. This response was independent of ovarian status (i.e. immature or multifollicular). Treatment with porcine FSH (NIH-FSH-P2) also induced development of a S.Z. Oestradiol treatment was ineffective. The appearance of the S.Z. was associated with a change in steroidogenesis. The adrenals of controls produced cortisol and corticosterone in yields of approx. 70%, while these products were less than 22% in the animals with S.Z.s. The major conversion products in the treated animals were 5 beta-reduced pregnane derivatives, in yields ranging from 67 to 93%. The yields of products from the oestradiol treated animals closely resembled those of the controls. It was concluded that FSH is capable of inducing the development of an adrenocortical S.Z. in immature female possums and consequently stimulating adrenal steroid reduction. It appeared that oestradiol was not involved in this process.     

The biosynthesis of some androst-16-enes from C21 and C19 steroids in boar testicular and adrenal tissue

1. The formation of androst-16-enes from [4-(14)C]progesterone has been investigated with long-term incubations and short-term kinetic studies. After 4hr., 1.7 and 10.3% respectively of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes were formed in boar testis minces, but much smaller yields were obtained in boar adrenal. Both tissues formed small quantities of androsta-4,16-dien-3-one. 2. The amounts of androst-4-ene-3,17-dione and testosterone isolated were small, suggesting that androst-16-ene formation may occur preferentially in the boar testis. 3. In the absence of tissue no radioactive androst-16-enes were formed. 4. Incubation of both [4-(14)C]pregnenolone and [7alpha-(3)H]progesterone resulted in 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes containing (3)H/(14)C ratios of near unity and confirmed that both C(21) steroids were precursors. A similar incubation with 17alpha-hydroxy[4-(14)C]-progesterone and [7alpha-(3)H]progesterone gave the same Delta(16)-alcohols, but they contained only (3)H, indicating that side-chain cleavage of pregnenolone and progesterone occurred before 17alpha-hydroxylation. 5. Dehydroepiandrosterone, testosterone, testosterone acetate and 16-dehydroprogesterone were not found to be precursors of Delta(16)-steroids. 6. A pathway is proposed for the biosynthesis of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes from pregnenolone and progesterone; this may involve androsta-4,16-dien-3-one as an intermediate, but excludes 17alpha-hydroxyprogesterone, testosterone and dehydroepiandrosterone.     

Age-related changes in responsiveness of rat Leydig cells to hCG

The responsiveness of decapsulated testes and isolated Leydig cell preparations from rats (30-80 days of age) to a constant dose of 3 ng hCG/2 ml was assessed by comparison of the production of testosterone and "total 17beta-hydroxy androgen" (17beta-HA). When testosterone secretion was used as the index of response, there was a marked increase in the production with age by decapsulated testes and also by equal numbers of Leydig cells. When 17beta-HA was taken as the response parameter this increase was only marginal for the decapsulated testes and there was an age-dependent decrease when expressed per 10(6) cells. These differences probably reflect changes in the metabolism of testosterone to 5alpha-reduced products with increasing age because 80% of androgen secreted at 30 days is 3alpha-androstanediol and 86% is secreted as testosterone at 80 days. We conclude that for studies on hCG responsiveness and the steroidogenic capacity of immature rat Leydig cells (a) testosterone is an inappropriate response parameter and (b) this response undergoes a decrease rather than an increase during prepubertal development.     

Oxytocin receptors in the mammary gland and reproductive tract of a marsupial, the brushtail possum (Trichosurus vulpecula).

Previous studies of marsupial lactation have shown that the milk-ejection reflex changes in sensitivity, being greater in small mammary glands sucked by small pouch young and lesser in larger glands supplying milk to larger young. The involvement of oxytocin receptors in these changes was examined in the brushtail possum Trichosurus vulpecula. Oxytocin receptors were measured in the mammary glands, uterus, and medial vaginal sacs by radioreceptor assay, using [3H]oxytocin as radioligand. In the mammary gland, a single oxytocin binding site was found with an affinity and receptor concentration of 0.81 +/- 0.41 l/nmol and 10.2 +/- 4.8 pmol/g tissue respectively (SD, 10 possums). Competitive displacement curves with related peptides and analogs showed the following order of specificity: d(CH2)5[Tyr(Me)2,Thr4,Tyr9-NH2]-vasotocin much greater than vasotocin greater than oxytocin = Arg-vasopressin greater than mesotocin greater than [Thr4,Gly7]-oxytocin = Lys-vasopressin greater than [deamino-Pen1, O-methyl-Tyr2, Arg8]-vasopressin greater than isotocin much greater than [d(CH2)5, D-Phe2, Ile4, Ala9-NH2]-AVP. [3H]Oxytocin did not bind to vasopressin receptors in the thoracic aorta. The concentration of oxytocin receptors was very high in small mammary glands (18.6 pmol/g tissue in a 2-g gland) and decreased logarithmically as the size of the mammary gland increased. It is suggested that the changes in the sensitivity of milk ejection to oxytocin is related to the concentration of mammary oxytocin receptors. The presence of oxytocin receptors in both uterus and median vaginal sacs extends previous observations and supports the hypothesis that in marsupial parturition, the uterus and medial vaginal sacs respond as a single functional unit to oxytocin.     

5alpha-reduced C21 steroids are substrates for human cytochrome P450c17

The 5alpha-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5alpha-reduced C(19) steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate. We studied the metabolism of 5alpha-reduced C(21) steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C(21) steroids to C(19) steroids via its 17alpha-hydroxylase and 17,20-lyase activities. hCYP17 17alpha-hydroxylates 5alpha-pregnan-3,20-dione, but little androstanedione is formed by 17,20-lyase activity. hCYP17 also 17alpha-hydroxylates 5alpha-pregnan-3alpha-ol-20-one and the 5alpha-pregnan-3alpha,17alpha-diol-20-one intermediate is rapidly converted to androsterone by 17,20-lyase activity. Furthermore, 5alpha-pregnan-3alpha,17alpha-diol-20-one is a better substrate for the 17,20-lyase reaction than the preferred substrate 17alpha-hydroxypregnenolone and cytochrome b(5) stimulates androsterone formation only 3-fold. Both 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3alpha,17alpha-diol-20-one bind to hCYP17 with higher affinity than does progesterone. We conclude that 5alpha-reduced, 3alpha-hydroxy-C(21) steroids are excellent, high-affinity substrates for hCYP17. The brisk metabolism of 5alpha-pregnan-3alpha,17alpha-diol-20-one to androsterone by CYP17 explains how, when 5alpha-reductases are present, the testis can produce C(19) steroids androsterone and androstanediol from 17alpha-hydroxyprogesterone without the intermediacy of androstenedione and testosterone.     

Identification of a new Sertoli cell metabolite of testosterone: 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol, by HPLC and GC-MS

Testosterone metabolism by isolated rat Sertoli cells cultured in vitro was investigated using HPLC and GC-MS techniques. Monolayer cultures of Sertoli cells (greater than 90% pure and free of Leydig cells) were incubated for 3-day periods with a stable labeled [2,2,4,6,6-d5]testosterone prepared and used in a 1:1 proportion with unlabeled testosterone as the substrate (5 X 10(-7)M). After incubation, the metabolites were extracted from the media and reacted with oestradiol-antibodies. The antibody-bound components were separated on reverse phase HPLC and the fraction corresponding to oestradiol was analyzed by GC-MS in the form of TMS-ether. One of the metabolites whose mass spectrum contained d0 + d5 species was detected and interpreted to be a triol with a mol. wt of 308. Mass spectra data indicated that this testosterone metabolite is one of the sixteen possible isomers of 3,16,17-trihydroxy androstane. This substance was identified based on the Vm value (27.81) closely resembling that of 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol TMS-ether (Vm reported = 27.78) [1] and when compared directly with synthesized compounds [2-3]. Recently we have demonstrated that similar Sertoli cell preparations contain two 16 alpha-hydroxylases by their ability to convert oestradiol to oestriol [4] and 5 alpha-androstane-3 alpha,17 beta-diol to 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol [3], where the former conversion is not affected by FSH, the latter is significantly stimulated by the presence of FSH. Presence of this new product represents the first example of testosterone conversion to 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol and confirms our previous observation that 16 alpha-hydroxylation of 5 alpha-reduced androgens can occur in the rat testis.     

Metabolism of (3H) 5 alpha-androstane-3 alpha, 17 beta-diol by cultures of isolated rat sertoli cells and the effect of LH and FSH

Sertoli cells from immature rats metabolized (3H) 5 alpha-androstane-3 alpha, 17 beta-diol to (3H) 5 alpha-androstane-3 alpha, 16 alpha, 17 beta-triol and (3H) 3 alpha-hydroxy-5 alpha-androstan-17-one. This is the first report of 16 alpha-hydroxylation of 5 alpha-reduced androgens in the testis. FSH significantly stimulated 16 alpha-hydroxylation while LH significantly decreased this activity. 3 alpha-Hydroxy-5 alpha-androstan-17-one was the major metabolite formed and its production was significantly increased in the presence of both LH and FSH, although FSH stimulation was significantly more than LH. The possible role of 16 alpha-hydroxylase in androgen metabolism by immature rat Sertoli cells is discussed.     

Testosterone metabolism by incubated rat testes after chronic LHRH treatment

Adult male rats were injected 4 or 8 days with LHRH agonist. After sacrifice the testes were incubated in vitro with or without [4-14C]testosterone. After LHRH-administration the endogenously produced amounts of testosterone and of 7 alpha-hydroxytestosterone, the main testosterone metabolite normally found on incubation of adult rat testes, were drastically reduced when compared with controls. hCG, injected to rats 2 h before sacrifice, increased steroid production. In the LHRH-treated rats, however, the amounts of testosterone and of 7 alpha-hydroxytestosterone produced were much less while an important formation of 5 alpha-androstanediol was observed. The testes of LHRH treated rats metabolized [4-14C]testosterone to a large extent to 5 alpha-reduced and unextractable metabolites while the formation of 7 alpha-hydroxylated metabolites was much reduced. It is concluded that prolonged LHRH treatment provokes not only a depression of the testosterone production but has also an influence on the testicular metabolism pattern of testosterone resulting in a proportionally increased production of 5 alpha-reduced steroids and unextractable metabolites while the formation of 7 alpha-hydroxylated steroids is inhibited.     

Intestinal and hepatic microsomal metabolism of testosterone and progesterone by a 3 alpha-hydroxysteroid dehydrogenase to the 3 alpha-hydroxy derivatives in the channel catfish,Ictalurus punctatus

Intestinal or hepatic microsomes from channel catfish converted [4-14C]-testosterone to three major metabolites: 6 beta-hydroxytestosterone, androstenedione and a third metabolite. Formation of the unknown metabolite required NADPH as cofactor. When incubated with 200 microM testosterone, the rate of formation of the unknown metabolite was 265+/-158 pmol/(min mg) protein (mean+/-S.D.) in microsomes from the proximal intestine, 515+/-93 pmol/(min mg) protein in distal intestine and 226+/-42 pmol/(min mg) protein in hepatic microsomes. Comparison of the chromatographic and spectral properties of the unknown metabolite with those of authentic testosterone derivatives showed that this metabolite corresponded to 4-androstene-3 alpha,17 beta-diol. No 3 alpha-reduced metabolite was formed in incubations of testosterone with catfish intestinal cytosol. Testosterone was reduced to 5 alpha-dihydrotestosterone primarily in the cytosolic fraction and not in microsomes. Incubation of progesterone with intestinal microsomes resulted in the formation of a metabolite with properties similar to that of the 3 alpha-reduced testosterone, and this metabolite was identified by co-chromatography with authentic standard as 3 alpha-reduced progesterone. Thus, 3 alpha-hydroxysteroid dehydrogenase is an important pathway in intestinal microsomes of the channel catfish.     

Biosynthesis of testicular steroids in the immature, adult and senescent guinea-pig

The potential biosynthetic capacity of testicular hormones was studied in immature, pubertal and aging guinea-pig. In their sexual development towards puberty, changes in the relationship of the steroids involved in the steroidogenic pathways were observed. The testosterone/androstenedione ratio changes markedly, showing an important increase with pubertal proximity. The testosterone in equilibrium androstenedione sequence, reversibly catalyzed by 17 beta-hydroxysteroid oxidoreductase (17 beta-oxido-reductase), clearly shifted towards androstenedione in immature animals irrespective of the precursor utilized. Post-pubertal animals showed a greater enzymatic activity in the 5-ene and 4-ene testicular synthesis pathways, testosterone production being greatest. In the aging animal, hormonal biosynthetic capacity falls. Reversion of the 17 beta-oxido-reductase activity could be one of the mechanisms responsible for the decrease in testosterone, as in immature guinea-pigs. In order to investigate the in vitro steroidogenic capacity of glands at different ages, minces of testicular tissue were incubated with labelled precursors. The studies were conducted in triplicate at 35 degrees C. For equal quantities of incubated tissue the non-metabolized amount of [3H]pregnenolone and [14C]progesterone, utilized as precursors, was different in post-pubertal and senescent animals: 55.7 +/- 3 vs 59.3 +/- 2.3% (P less than 0.01) for pregnenolone, and 50.1 +/- 3.3 vs 56.3 +/- 2.9% (P less than 0.01) for progesterone, respectively. Testosterone production was 12 +/- 2% in adult and 6.7 +/- 2.7% in senescent animals (P less than 0.01). The testosterone/androstenedione ratio was not significantly different in post-pubertal and senescent animals: 2.8 +/- 0.5 vs 2.4 +/- 0.4, but consistently higher than found in immature animals: 0.3 +/- 0.1. The lesser potential capacity of the aging tissue to synthesize testosterone could be explained by a decline in the glands capacity to metabolize the hormonal precursors.     

Appearance of the rat testicular receptor for calcitriol (1,25-dihydroxyvitamin D3) during development

In the present study we have examined the developmental changes in the concentration of receptors for calcitriol in high-salt cytosol from the rat testis. Receptors for calcitriol were undetectable (less than 0.4 fmol/mg protein) until day 24, after which there was a rapid increase to reach adult levels (6-8 fmol/mg protein) between day 50-60. The lack of receptors in high-salt cytosol from the immature rat testis is not due to degrading enzymes, since cytosols prepared from the combination of equal volumes of testis homogenates from immature and adult rats had binding levels exactly half of that found in "adult controls". Furthermore, the increase in specific binding of [3H]calcitriol during development is due to an increase in the number of receptor sites, and is not due to a change in the apparent affinity of the receptors (Kd approximately equal to 1 X 10(-11) M at 0 degrees C). These results may explain why we previously were unable to demonstrate calcitriol receptors in cultured Sertoli cells and peritubular cells isolated from 19-day old rats. Furthermore, they indicate that calcitriol may be of minor importance for testicular function in the immature rat. The role of calcitriol in the pubertal and adult testis remains to be established.