Leon Milewich

Conversion of plasma progesterone to deoxycorticosterone in men, nonpregnant and pregnant women, and adrenalectomized subjects.

During the third trimester of human pregnancy the concentrations of deoxycorticosterone (DOC) in maternal plasma are 4-50 times those in nonpregnant women and men. It has been suggested that the increased amount of DOC in maternal plasma originates in the fetal compartment. We considered an alternate explanation for the high levels of DOC in plasma or near-term pregnant women, viz., that DOC may be derived in part from 21-hydroxylation of maternal plama progesterone. To test this hyposthesis we measured the fractional conversion of plasma progesterone to DOC from the relationship between the 3H:14C ratio of the infused tracers, [3H]progesterone and [14C]-DOC, and the 3H:14C ratio or urinary 3 alpha,21-dihydroxy-5 beta-pregnan-20-one (tetrahydro-DOC). The fractional conversion of plasma progesterone to DOC ([rho](BU)P-DOC), measured in this manner, was 0.007 +/- 0.001 (mean +/- SEM, n = 26) in the subjects of this study. The values for [rho](BU)P-DOC varied widely among subjects (0.002-0.022) but the range of values for [rho](BU)P-DOC was similar among women pregnant with an anencephalic or dead fetus, nonpregnant and adrenalectomized women, and men. The transfer constant of conversion of progesterone to DOC in plasma, [rho](BB)P-DOC, remained constant in a nonpregnant woman during the infusion of nonradiolabeled progesterone at rates of 0-14 mg/h. Based on the results of these studied, we conclude that DOC is formed by extra-adrenal 21-hydroxylation of plasma progesterone and that the rate of formation of DOC by this pathway is proportional to the concentration of progesterone in plasma.

Peroxisome proliferation and induction of peroxisomal enzymes in mouse and rat liver by dehydroepiandrosterone feeding

Dehydroepiandrosterone (DHEA) treatment is effective in the prevention of various genetic and induced disorders of mice and rats. In studies designed to define some of the basic mechanisms that underline the beneficial chemopreventive effects exerted by the action of this steroid, we found that the liver undergoes profound changes that result in: (i) hepatomegaly; (ii) color change from pink to mahogany; (iii) proliferation of peroxisomes; (iv) increased cross-sectional area and volume density of peroxisomes; (v) increased or decreased number of mitochondria per cell; (vi) decreased mitochondrial cross-sectional area; (vii) marked induction of the peroxisomal bifunctional protein enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; (viii) increased activities of enoyl-CoA hydratase and other peroxisomal enzymes assayed in this study, viz. catalase, carnitine acetyl-CoA transferase, carnitine octanoyl-CoA transferase, and urate oxidase; and (ix) increased activity of mitochondrial carnitine palmitoyl-CoA transferase. In addition, feeding DHEA to mice resulted in increased plasma cholesterol levels in two strains of mice evaluated in this study, and either slightly decreased or markedly increased plasma triglyceride levels, depending on the strain. Whether liver peroxisome proliferation, induced by DHEA feeding to mice and rats, plays a role in the chemopreventive effects elicited by this steroid remains to be established.

Compartmentalization of type 117β-hydroxysteroid oxidoreductase in the human ovary

Abstract The steroid-metabolizing enzyme, type I 17β-hydroxysteroid oxidoreductase (17β-HSOR) also called 17β-hydroxysteroid dehydrogenase (17β-HSD) plays a key role in ovarian synthesis of 17β-estradiol. This is the only enzyme in the steroid-metabolizing pathway which has not been localized in the human ovary by immunohistochemistry. In this study, using antibody directed against human placenta! cytosolic 17β-HSOR (type I), a single protein band with a relative molecular mass of approximately 34 kDa was demonstrated by Western analysis in both human luteinized granulosa cells and placental tissue. In placental tissue, immunoreactive type I 17β-HSOR was demonstrated within the syncytiotrophoblast using immunohistochemistry. In human ovary, immunoreactive type I 17β-HSOR was localized exclusively in granulosa cells of developing follicles, ranging from primary follicles with a single layer of cuboidal-shaped granulosa cells, preantral follicles with multiple layers of granulosa cells, and large antral follicles. No immunoreactivity was detected in spindle-shaped granulosa cells of primordial follicles, theca interna, theca externa or surrounding stroma. In the corpus luteum, type I 17β-HSOR immunoreactivity was localized solely in granulosa-lutein cells. For comparison, immunoreactive 3β-hydroxysteroid dehydrogenase (3β-HSD) was examined in the same tissues. Both theca interna and granulosa cells of preantral and antral follicles exhibited 3β-HSD staining. Primary follicles did not exhibit detectable 3β-HSD in either granulosa or theca cells. This study serves to demonstrate that in the human ovary, type I 17β-HSOR is compartmentalized in granulosa cells of the developing follicles and granulosa-lutein cells. In addition, the expression of type I 17β-HSOR appears to be present prior to that of 3β-HSD at the stage of primary follicle. With the strategic location of type I 17β-HSOR and the ability to aromatize theca-derived androstenedione to estrone, granulosa cells have the capacity to synthesize and maintain the high intrafollicular levels of 17β-estradiol, which are essential for normal follicular development.

Regulation of expression of the 3β-hydroxysteroid dehydrogenases of human placenta and fetal adrenal

The appropriate expression of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3 beta-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3 beta-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3 beta-HSD expression and, moreover, that 3 beta-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3 beta-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3 beta-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3 beta-HSD expression. Moreover, 3 beta-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3 beta-HSD expressed in these cytotrophoblastic populations was the 3 beta-HSD type I gene product (M(r), 45K) and not 3 beta-HSD type II (M(r), 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3 beta-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3 beta-HSD type II activity and mRNA expression. The higher level of 3 beta-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.

Steroid Metabolism by Epidermal Keratinocytesa

The metabolism of various radiolabeled steroids by cultured human epidermal keratinocytes was studied in an attempt to identify the steroid-metabolizing enzymes present in these cells. Sulfatase activity was demonstrated in keratinocytes with either E1S or DS as substrates. The products of sulfatase action were E1 and DHEA, respectively. The specific activity of the enzyme was approximately 5- to 14-fold greater with E1S as the substrate compared with DS, and the rates of hydrolysis were linear with incubation time up to 3 h. The metabolism of DHEA by the keratinocyte 17 beta-HSOR-catalyzed reaction resulted in the predominant formation of 5-androstene-3 beta,17 beta-diol. The rate of formation of 5-androstene-3 beta,17 beta-diol was linear with time of incubation up to 18 h, and the specific activity of 17 beta-HSOR, with DHEA as the substrate, was greater in keratinocytes maintained in culture for 4 weeks compared with keratinocytes kept in culture for 1 week. Androstenedione was a minor product of DHEA metabolism. The metabolism of DHT by epidermal keratinocytes resulted in the formation of 5 alpha-androstanedione, 5 alpha-androstane-3 alpha,17 beta-diol, 5 alpha-androstane-3 beta,17 beta-diol, androsterone, and isoandrosterone: the rates of formation of 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol were linear with incubation time up to 24 h, and the specific activities of 3 alpha-HSOR and 3 beta-HSOR did not appear to change with keratinocyte time in culture up to 3 weeks. The metabolism of DOC by epidermal keratinocytes resulted in 5 alpha-dihydrodeoxycorticosterone production: the rate of formation of this metabolite was linear with incubation time up to 4 h. The metabolism of E1 by epidermal keratinocytes yielded E2, and that of E2 resulted in the formation of E1. The rate of E1 formation from E2, was approximately 10-fold greater than the rate of formation of E2 from E1; these rates were linear with incubation time up to 4 h. Epidermal keratinocytes maintained in culture did not metabolize androstenedione to either E1 or E2, and pregnenolone was not metabolized by these cells. This study serves to ascertain that epidermal keratinocytes express steroid 5 alpha-reductase, 17 beta-HSOR, 3 beta-HSOR, 3 alpha-HSOR, 3 beta-hydroxysteroid oxidoreductase-delta 5----4-isomerase, and sulfatase activities.

Metabolism of dehydroisoandrosterone and androstenedione by the human lung in vitro.

Abstract 3β-Hydroxysteroid oxidoreductase-Δ 5→4 -isomerase and 7α-hydroxylase activities are present in human lung and this is reflected in the rates of formation at initial velocities of androstenedione (∼110 pmol/100mg protein/h) and of 7α-hydroxydehydroisoandrosterone (∼100 pmol/100mg protein/h) as the principal metabolites obtained from incubations of human lung slices with [7- 3 H]-dehydroisoandrosterone. The 5α-reduced metabolites 5α-androstanedione and androsterone were formed with total conversion rates of ∼75 pmol/100 mg protein/h, and in addition, the 17β-hydroxysteroids 5-androstene-3β, 17β-diol and testosterone were also formed (∼25 pmol/100 mg protein/h and ∼5 pmol/100 mg protein/h, respectively). The incubation of human lung slices with [1,2,6,7- 3 H]-androstenedione resulted in the formation of 5α-androstanedione, androsterone and isoandrosterone with total conversion rates of ∼ 44 pmol/100 mg protein/h, and of testosterone (∼ 22 pmol/100 mg protein/h). 5β-Reduced-C 19 -steroids were not identified among the metabolites. The human lung is a potential site for the conversion of circulating dehydroisoandrosterone to androstenedione, to testosterone and to 5α-reduced steroids. The resulting steroids may exert their activities in situ or elsewhere, conceivably after further metabolism to more potent androgens.

5α-Reductase activity in human placenta

Abstract The concentration of 5α-pregnane-3,20-dione in peripheral blood of pregnant women is higher than that found in blood obtained from nonpregnant women throughout the luteal phase of the ovulatory cycle. The in vitro synthesis of 5α-reduced pregnanes from [ 3 H]progesterone by placental tissue was investigated using tissue minces and homogenates in the presence of added nicotinamide-adenine dinucleotide phosphate. The metabolites, [ 3 H]5α-pregnane-3,20-dione and [ 3 H]3β-hydroxy-5α-pregnan-20-one, were isolated and characterized employing a combination of chromatographic techniques, derivative formation, and crystallization with authentic [ 14 C]5α-pregnane-3,20-dione and [ 14 C]3β-hydroxy-5α-pregnan-20-one to constant 3 H: 14 C ratios. In short-term incubations with placenta homogenates, the apparent pH optimum for the synthesis of 5α-pregnane-3,20-dione was 8.8. There was no evidence for the formation of 5β-reduced pregnanes from [ 3 H]progesterone by placental tissue. The in vitro metabolism of [ 3 H]progesterone by minces of five term placentas, during 1 hour incubations at pH 7.4, was studied. The rate of formation of 5α-pregnane-3,20-dione varied from 109 to 197 pmoles/gm placental tissue/hr.

Dehydroisoandrosterone sulfate in peripheral blood of premenopausal, pregnant and postmenopausal women and men

Abstract A radioimmunoassay specific for the direct measurement of dehydroisoandrosterone sulfate in 0.5 or 1 μl of human serum was developed using an antibody directed against dehydroisoandrosterone hemisuccinate coupled to thyroglobulin. Dehydroisoandrosterone sulfate levels were measured in pregnant women throughout gestation starting with the 6th week of pregnancy, and in young ovulatory women, postmenopausal women and in men. During pregnancy the concentrations of dehydroisoandrosterone sulfate in maternal serum decreased steadily with advancing gestation [1825 ± 68 ng/ml (mean and S.E.) at 6–8 weeks gestation vs 706 ± 141 ng/ml at 36–41 weeks gestation], and the most striking decrease was observed between 14 and 16 weeks of gestation. The mean concentrations of dehydroiso-androsterone sulfate were higher in men than in young nonpregnant women (2679 ±196 ng/ml vs 2020 ± 108 ng/ml respectively). In postmenopausal women, mean serum dehydroisoandrosterone sulfate concentrations (764 ± 28 ng/ml) were considerably lower than those in young premenopausal women P

17β-Hydroxysteroid oxidoreductase: A ubiquitous enzyme. Interconversion of estrone and estradiol-17β in BALBc mouse tissues

A survey was conducted to define the sites of 17 beta-hydroxysteroid oxidoreductase activity in organs and tissues of male and female BALB/c mice, as well as the favored direction of the oxidoreductase reaction in intact tissues. The enzyme activity was assayed by use of radiolabeled estrone and estradiol-17 beta as substrates. Estrone formation from estradiol-17 beta was demonstrated in all tissues. The formation of estradiol-17 beta from estrone was demonstrated in most tissues, however, it was barely detected or was undetectable in the glandular stomach, small intestine, cecum, and large intestine. Thus, 17 beta-hydroxysteroid oxidoreductase activity is expressed in all BALB/c mouse organs and tissues. Approximately two-thirds of the tissues and organs examined, including those of the reproductive tracts, favored the conversion of estrone to estradiol-17 beta rather than the reverse reaction. The results of this study, however, represent qualitative estimates of 17 beta-hydroxysteroid oxidoreductase activity in BALB/c mouse tissues that are uncorrected for conversion to hydroxylated metabolites. These in vitro findings suggest that the 17 beta-hydroxysteroid oxidoreductase catalyzed reduction of estrone may contribute to the maintenance of physiologic levels of estradiol-17 beta in estrogen responsive tissues.

Steroid secretion by ACTH-stimulated human fetal adrenal tissue during the first week in organ culture

Fetal adrenal tissue has been reported to lose its in vivo secretory pattern by virtue of a loss of fetal zone cells after the first week in culture. Consequently, we studied the steroidogenic capacity and the responsiveness to ACTH of human fetal adrenal tissue during the first week in organ culture. The culture medium was removed daily and assayed for cortisol and dehydroisoandrosterone sulfate (DS). First, as the concentration of ACTH in the medium was increased from 0 to 1 micrograms/ml steroid secretion increased. When tissue fragments were maintained in the absence of ACTH for 3 to 4 days, there was a striking increase in steroid secretion upon addition of ACTH to the medium, with larger rates of secretion of cortisol than DS being observed. Second, the steroidogenic capacity of the separate zones of the fetal adrenal gland was assessed. Tissue from the fetal zone secreted large amounts of DS and small amounts of cortisol, whereas neocortex tissue secreted similar quantities of DS and cortisol. Third, fetal zone tissue was maintained the absence of ACTH for 4 days and thereafter ACTH was added to the media for an additional 6 days. In this experiment, there was a marked increase in DS secretion rate after the addition of ACTH and a smaller increase in cortisol secretion.