James C. Coffey

Maturational changes in testicular steroidogenesis: Hormonal regulation of 5α-reductase

Abstract Studies were undertaken to compare the capacity of the human and rat testis to convert progesterone-3H (3H-P) both to testosterone (T) and 5α-androstane-3α,17β-diol (5α-diol) at various stages of development, and to determine the possible mechanisms regulating the maturational changes in 5a-reductase activity in the rat testis. Testes from 1- and 13-yr-old males converted 3H-P to T (7–13%) and 17-hydroxyprogesterone (3–20%) and several unidentified metabolites. A similar metabolic pattern was obtained when 3H-P was incubated with testicular homogenate of 20-yr-old subjects, with the exception that the formation of T (27%) was higher than in 13-yr-old testis. Very little 5α-reduced androstane products, however, accumulated in these three incubations. Under the same incubation conditions, 90-day-old rat testis converted 3H-P to T, while 28-day-old testis converted 3H-P mainly to 5α-diol (58%). To determine whether 5α-reductase activity is dependent on gonadotropic stimulation, 5α-reductase was measured in seminiferous tubules and interstitial cells following various periods of hypophysectomy. In both testicular compartments, 5α-reductase activity decreased rapidly, reaching a minimal level 7 days following hypophysectomy. Treatment with either FSH or LH (3 days) 2 days following hypophysectomy increased significantly 5α-reductase activity in whole testis. Large doses of testosterone propionate, however, did not appear to increase the activity above the level of the untreated hypophy-sectomized control, indicating that this effect of gonadotropins is not mediated through androgens. These studies clearly demonstrated that neither the immature nor the mature human testis possesses a high level of 5α-reductase activity as compared with the rat testis, and that 5oc-reductase activity in the rat testis is dependent on gonadotropic stimulation, in contrast to the androgen-dependent 5α-reductase in other target tissues.

Steroid metabolism by mouse submaxillary glands. I. In vitro metabolism of testosterone and 4-androstene-3,17-dione.

Abstract Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of 6 month old male mice. In 15 and 180 minute incubations fortified with NADPH, submaxillary tissue converted 4-androstene-3,17-dione predominantly to androsterone and, to a lesser extent, testosterone, 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α, 17β-diol. Testosterone was converted primarily to 5α-androstane-3α, 17β-diol when exogenous NADPH was available; trace amounts of 4-androstene-3,17-dione, 17β-hydroxy-5α-androstan-3-one and androsterone were also formed. When a NADPH-generating system was omitted from the incubation medium both 4-androstene-3,17-dione and testosterone were poorly metabolized by submaxillary tissue; the amounts of reduced metabolites accumulating were markedly reduced.

Steroid metabolism by testicular homogenates of the stanley-gumbreck pseudohermaphrodite male rat. I. increased formation of androsterone and androstanediol

Abstract Tritiated progesterone androstenedione and testosterone were incubated with testicular homogenates of 4- and 32-week-old Stanley-Gumbreck pseudohermaphrodite (Ps) and normal (N1) male littermate rats. In 15 and 180 minute incubations, both 4- and 32-week-old Ps testes converted all three substrates predominantly to androsterone and to a lesser extent androstanediol, while androstanediol in 4-week and testosterone in 32-week-old N1 testis were the major products. The addition of carrier testosterone (240 μg/g tissue) to 15 min incubations of testicular homogenates from 4- and 32-week-old N1 rats almost completely blocked the formation of androstanediol and markedly increased the accumulation of testosterone (47 and 41% from Progesterone-1,2-3 H; 66 and 92% from androstenedione-l,2-3H) indicating that androstanediol formed in the absence of carrier testosterone is, most likely, a product of testosterone reduction. When similar incubations were repeated using testicular homogenates from 4- and 32-week-old Ps rats, testosterone accumulation was not greatly increased (4–11%) by the addition of carrier testosterone, but androsterone formation was completely inhibited. However, when the incubations of Progesterone-1 ,2-3H with 4- and 32-week-old Ps and N1 testis in the presence of carrier testosterone were continued for 180 min, the major fraction of radioactivity from 32-week-old N1 testis was testosterone (79%) while that from 4-week-old N1 testis was androstanediol (60%) and from 4-and 32-week-old Ps testis was both androsterone (44–45%) and androstanediol (22–33%). The present data indicate that 4-week-old Ps testis, like the N1, has a high level of ring A reductase activity but forms androsterone rather than androstanediol as its major product. Unlike the normal mature male rat testis, in which ring A reductase activity diminishes allowing testosterone to become the major product, the 32-week-old Ps testis maintains a high level of reductase activity.

In Vitro Metabolism of 4-Androstene-3,17-Dione by Human Submaxillary Gland Homogenates

Tritiated 4-androstene-3,17-dione was incubated with human submaxillary gland homoaenates. In 180-minute incubations, human submaxillary gland converted 4-androstene-3, 17-dione predominately to testosterone and to a lesser extent, 5α-androstane-3α,17β-diol and androsterone.

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

Abstract Rat submaxillary gland homogenates incubated in vitro with progesterone-1, 2- 3 H 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.

Gonadotropic regulation of 5 alpha-reductase activity in the interstitial cells and whole testis homogenate of the immature rat.

As early as 1965, it was first reported (1) that radioactive progesterone is rapidly metabolized to testosterone (T) in homogenates of immature as well as mature rat testis, although T accumulation varies significantly with the age of the rat. It was observed that testosterone is rapidly formed in vitro but accumulates in inverse proportion to the rate of formation of 5α-androstane-3α, 17β-diol (3α-DIOL) (2,3). In the immature rat testis, the formation of 3α -DIOL was extremely rapid and little testosterone accumulated (3). At about the age of puberty, however, testosterone became the major product and the formation of 3α-DIOL was greatly reduced (3). Later studies in several laboratories confirmed these observations and extended them to all ages of the rat, from birth to adulthood (4–8). More recently, endogenous levels of 3α-DIOL were measured in testicular extracts of seminiferous tubules and interstitial cells from rats of various ages. 3α-DIOL concentrations in seminiferous tubules were found to be elevated between 25–35 days of age, at which time testosterone levels were minimal (9). Following the intravenous injection of 3H-progesterone and 3H-androstenedione into immature rats, radioactive 3α-DIOL was also found to accumulate as the major metabolite in testicular tissues (10).

In vitro metabolism of 4-androstene-3,17-dione and testosterone by rat submaxillary gland.

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of male and female rats. The metabolism was predominately reductive. In 15 and 180 min incubations submaxillary tissue converted 4-androstene-3,17-dione chiefly to androsterone. Less testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one, 5 alpha-androstane-3,17-dione, 5 alpha-androstane-3 alpha, 17 beta-diol, and 4-androstene-3 alpha, 17 beta-diol were also identified. Testosterone was converted to the same products plus 4-androstene-3,17-dione. 5 alpha-Androstane-3 alpha, 17 beta-diol was the major testosterone metabolite. Qualitatively the metabolism by male and female submaxillary gland was similar.

The in vitro metabolism of progesterone, 4-androstene-3,17-dione, testosterone and 17β-hydroxy-5α-androstan-3-one by fetal rhesus monkey testes

Homogenates prepared from fetal rhesus monkey testes were incubated with progesterone, 4-androstene-3,17-dione, testosterone and 17 beta-hydroxy-5 alpha-androstan-3-one. The major progesterone metabolite was 17-hydroxy-4-pregnene-3,20-dione. Testosterone also accumulated in the progesterone incubations. 4-Androstene-3,17-dione was converted chiefly to testosterone. Testosterone was not actively metabolized by the fetal monkey testis. 17 beta-Hydroxy-5 alpha-androstan-3-one was actively converted primarily to 5 alpha-androstane-3 beta,17 beta-diol.

Testosterone formation in testis of the immature androgen insensitive pseudohermaphrodite male rat (stanley-gumbreck rat)

Abstract Testosterone formation from pregnenolone (3β-hydroxy-5-pregnen-20-one) and progesterone in testis of the Stanley-Gumbreck pseudohermaphrodite (Ps) adult rat is greatly reduced in comparison to the normal (Nl) adult rat testis. In an attempt to determine whether this defect is congenital or acquired postnatally with increasing age, minced testis of 1-month-old Ps and Nl rats were incubated with progesterone, and the labeled metabolites identified. Almost equal amounts of progesterone were metabolized by both Ps and Nl testis. In mince incubations without NADPH nearly as much testosterone and 4-androstene-3,17-dione accumulated in the Ps as in the Nl testis. Very little androsterone and 5α-androstane-3α,17β-diol were formed in these incubations. When minces were incubated with progesterone in the presence of NADPH, testosterone and 4-androstene-3,17-dione accumulation was greatly reduced, and instead 5α-androstane-3α,17β-diol was formed as the major product by Nl testis and androsterone by Ps testis. Neither heparin, a 5α-reductase inhibitor, nor glucose-6-phosphate dehydrogenase alone significantly influenced progesterone metabolism or the accumulation of testosterone or 4-androstene-3,17-dione in either Ps or Nl testis. These results indicated that the 5α-reductase activity in both the Ps and N1 testis is dependent only on NADPH. Although studies were not carried out in younger rats (2–5 days of age), our results are in agreement with previous studies of Goldstein and Wilson who demonstrated equal accumulation of testosterone in incubations of testis from normal and Tfm/y mice. However, it is apparent that differences between Nl and Ps testis may be revealed only under conditions which allow maximum rates of 17-oxo- and 5α-reductions.

In vitro Androgen Metabolism in Submandibular Glands of Normal and Diabetic (C57BL/KsJ db/db) Mice

The mouse submandibular gland (SMG) is a sexually dimorphic tissue in which some morphological differentiation and some biochemical events are mediated by the action of androgens (Chretien, Int Rev Cytol 50:333, 1977). In mice with an autosomal recessive disease resembling maturity onset diabetes, Hanker et al. (Histochemistry, in press) described evoked feminization of the SMG of male mice and masculinization of the SMG of female mice. Feminization of the SMG of male diabetic mice was indicated by the greater proportion of striated ducts (characteristic of the postpubertal female) to granular tubules compared to the normal male. Masculinization of the SMG of female diabetic mice was indicated by a greater proportion of granular tubules (characteristic of the post-pubertal male) to striated ducts compared with the normal female. This finding prompted an examination of the androgen metabolizing capability of the SMG of normal and diabetic male and female mice.