11β-HSD1 Research Articles

Expression of steroid receptors and steroidogenic enzymes in the baboon (Papio anubis) corpus luteum during the menstrual cycle and early pregnancy.

As estrogen and progesterone are proposed regulators of luteal function, this study was undertaken to correlate the presence of receptors for these steroids with luteal function during early pregnancy. Corpora lutea (CL) were obtained from nonpregnant baboons during the midluteal [ML; days 7-8 postovulation (PO)] and late luteal (LL: days 11-12 PO) phases of the menstrual cycle or from pregnant baboons on days 18, 25, 29, or 31-33 PO. Estrogen and progestin receptors (ER and PR, respectively) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were detected by immunocytochemistry using specific monoclonal (H222 for ER; JZB39 for PR) or polyclonal (S683 for 3 beta HSD) antibodies. In addition, ribonucleic acid (RNA) was extracted from CL, processed for Northern blot analysis, and probed with complementary DNAs to human PR, human 3 beta HSD, and rat aromatase. Levels of messenger RNA (mRNA) for 3 beta HSD were quantified by laser densitometric scanning, and the data were normalized to the expression of a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase) to correct for loading differences. CL did not demonstrate specific nuclear, stain for ER at any stage of the menstrual cycle or pregnancy. In contrast, PR-positive cells were present during the ML phase, but decreased during the LL phase (P < 0.05). PR-positive cells were maintained during early pregnancy at levels comparable to the ML phase (P < 0.05). Staining for 3 beta HSD was present at all stages of the cycle and pregnancy. Although the percent of 3 beta HSD-positive cells appeared to decrease as pregnancy proceeded, this was not statistically different (P > 0.05). The complementary DNA to PR hybridized to multiple transcripts (approximately 4.4, 3.1, 1.6, and 0.95 kilobases) in CL of the cycle. A single transcript (approximately 1.8 kilobases) for 3 beta HSD was present in CL at all stages of the cycle and pregnancy. The level of 3 beta HSD mRNA was highest during the ML phase and declined significantly (P < 0.05) during the LL phase and early pregnancy. Three transcripts (approximately 3.6, 3.0, and 1.7 kilobases) for aromatase were detected in CL of the cycle and pregnancy. Aromatase mRNA increased during early pregnancy. These results support the concept of PR-mediated events, but not ER-regulated processes in the primate CL. Further-more, the data suggest that the steroidogenic enzymes 3 beta HSD and aromatase are differentially regulated during early pregnancy.

Localization of 11/β-Hydroxysteroid Dehydrogenase: Specific Protector of the Mineralocorticoid Receptor in Mammalian Olfactory Mucosa

The enzyme 11/β-hydroxysteroid dehydrogenase (11β HSD) plays a major role in the protection of the mineralocorticoid (type 1) receptor. The cellular mechanism of aldosterone selectivity relies on the coexpression of mineralocorticoid receptors and 11β HSD in the same cells. In the current study, 11β HSD activity was localized in the mammalian olfactory mucosa by a histochemical technique which links steroid metabolism with the deposition of formazan. The histochemical reaction results from oxidation of the synthetic substrate β-hydroxyandrostenedione and is dependent on nicotine-adenine dinucleotide (NAD). This demonstrates the presence of a dehydrogenase activity separate from the nicotineamide-adenine dinucleotide phosphate (NADP)-dependent 11βHSD. In the olfactory mucosa, the presence of NAD-dependent 11βHSD activity is localized to the sustentacular cells and acinar cells of Bowman's glands. No definite NAD-dependent activity was demonstrated in the olfactory receptor neurons. These data suggest that mineralocorticoid receptors present in acinar cells and sustentacular cells are aldosterone selective.

Seasonal changes in spermatogenesis and testicular steroidogenesis in the male black bear Ursus americanus.

American black bears, Ursus americanus, are seasonal breeders with a mating season in late spring to early summer. The objectives of this study were to determine whether there are seasonal changes in spermatogenesis and immunolocalization of testicular steroidogenic enzymes, and to correlate these changes with peripheral steroid concentrations. Three captive mature bears were maintained in open cages during the summer season and provided with chambers for denning during the winter. Testicular biopsies and blood samples were obtained from anaesthetized bears on 12 March, 15 June, 12 October and 15 January. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), porcine testicular 17 alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Spermatogenesis changed seasonally: spermatogonia and degenerating spermatocytes were observed in October; spermatogonia and primary spermatocytes were present in January; spermatogonia, spermatocytes and round spermatids were present in March; and spermatogonia through spermatozoa were present in June. P450scc and P450c17 were immunolocalized in spermatids and Leydig cells in June, whereas in October these enzymes were present only in Leydig cells. 3 beta HSD was localized in Leydig cells in June and October with more intense staining in June. Localization of P450arom changed seasonally: no immunostaining in October; positive immunostaining in Sertoli cells in January; more extensive immunostaining in Sertoli cells, peritubular-myoid cells and round spermatids in March; and strong immunostaining in Sertoli cells and round and elongating spermatids in June. Serum testosterone and oestradiol concentrations changed seasonally: testosterone and oestrogen were low in October and January, slightly higher in March, and high in June. The present study demonstrates that in the black bear seasonal changes in spermatogenesis are accompanied by changes in the immunolocalization of testicular steroidogenic enzymes that are correlated with changes in serum testosterone and oestradiol concentrations. The presence of P450arom in Sertoli cells at the beginning of testicular recrudescence suggests that aromatase and oestrogen may play a role in re-initiating spermatogenesis.

Seasonal changes in the immunolocalization of steroidogenic enzymes in the testes of the Japanese black bear (Ursus thibetanus japonicus).

Seasonal changes in sites of immunostaining of steroidogenic enzymes were examined in testes of the Japanese black bear, Ursus thibetanus japonicus. In addition, serum concentrations of testosterone and estradiol-17beta were investigated by radioimmunoassay, and the seasonal changes were compared with the results of immunostaining. On the basis of morphological observations of spermatogenic activity, the reproductive cycle was divided into five periods: an active period in May and June; a degenerative period in November; a resting period in January; an early-resumptive period in March; and a late-resumptive period in April. Serum concentrations of testosterone differed with season accompanied by differences in spermatogenic activity, with baseline levels in November and January, increasing levels in March and April, and high levels in May, and April and June of the next year. Immunoreactivities specific for cholesterol side-chain cleaving cytochrome P450, 17alpha-hydroxylase cytochrome P450 and 30-hydroxysteroid dehydrogenase (3beta HSD) were observed in Leydig cells throughout the year. Only the percentages of Leydig cells immunopositive for 3beta HSD exhibited seasonal differences that correlated with serum concentrations of testosterone. Aromatase cytochrome P450 (P450arom) was immunolocalized in Leydig and Sertoli cells throughout the year, in spermatids in May, and April and June of the next year and in myoid cells in January and March. The percentages of Leydig cells immunopositive for this enzyme increased in May, and January, March and June of the next year. On the other hand, no pattern of seasonal change in serum estradiol-17beta concentration was observed. These results suggest that 3beta HSD is a key enzyme in the regulation of the testosterone production in Leydig cells. Furthermore, estrogen derived from Leydig and myoid cells seems to play a role in the regulation of Leydig cells by negative feedback as a paracrine and/or autocrine mediator.

Differential expression of 11 beta-hydroxysteroid dehydrogenase types 1 and 2 in human placenta and fetal membranes.

Two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) are present in mammals. 11 beta HSD1 interconverts biologically active cortisol and inactive cortisone, whereas 11 beta HSD2 only converts cortisol to cortisone. Placental 11 beta HSD has been proposed to protect the fetus from high level of maternal glucocorticoids. Although bidirectional activity of 11 beta HSD has been demonstrated in homogenized human placental tissues, the tissue and cellular distribution of 11 beta HSD1 has not been resolved. In this study, the cellular localization of 11 beta HSD1 protein and levels of its messenger ribonucleic acid (mRNA) in human placenta and fetal membranes were determined by immunohistochemistry and Northern blot analysis, respectively. We found that 11 beta HSD1 immunoreactivity was present in the placental extravillous intermediate trophoblasts, chorion trophoblasts, amnion epithelial cells, and stromal cells of the decidua vera. Positive staining was also observed in the endothelium of the blood vessels in both placental villous tissue and umbilical cord. However, in contrast to previous reports of immunoreactive 11 beta HSD2 localization, 11 beta HSD1 immunoreactivity was undetectable in placental syncytiotrophoblast. Using a human 11 beta HSD1 complementary DNA as probe, a 1.5-kilobase mRNA transcript was detected in the chorion, amnion, and placental tissue, with the greatest amount in the chorion. In contrast, the 1.9-kilobase mRNA of 11 beta HSD2 was observed only in the placenta, not in the chorion and amnion. The process of labor had no significant effect on levels of 11 beta HSD1 or 11 beta HSD2 mRNA in the chorion or placenta. We conclude that there is a striking difference in the tissue localization of 11 beta HSD1 and 11 beta HSD2 expression in the late gestation human placenta and fetal membranes, which may discretely determine the accessibility of bioactive glucocorticoid to specific cell types.

Identification of 3 beta-hydroxysteroid dehydrogenase as a novel target of steroid cell autoantibodies: association of autoantibodies with endocrine autoimmune disease.

Autoantibodies directed against steroid hormone-producing cells (SCA) detectable by immunofluorescence are typically found in a small proportion of patients with premature ovarian failure (POF) as well as in other endocrine autoimmune diseases. The SCA pattern stains cells in the outer zones of the adrenal cortex, ovary, and testis. To identify the molecular target of SCA, an adrenal complementary DNA expression library was screened using SCA-positive serum, and the steroid enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was identified. Only 1 of 48 (2%) patients with idiopathic POF, not pre-selected for the presence of other autoimmune diseases, had SCA by immunofluorescence, whereas 10 of 48 (21%) had anti-3 beta HSD autoantibodies detectable by immunoblot using recombinant human enzyme compared with 6 of 115 (5%) control subjects (P = 0.002). Absorption of SCA-positive serum with recombinant human 3 beta HSD abolished the immunofluorescence pattern. We also examined the prevalence of anti-3 beta HSD autoantibodies in other endocrine autoimmune diseases. Two of 112 (2%) diabetic patients, but none of the thyroid or Addisonian patients, had SCA by immunofluorescence. Twenty-six (23%) diabetic subjects (P < 0.001 vs. controls), 3 of 18 thyroid patients (P > 0.05 vs. controls), and none of 4 Addisonian patients had anti-3 beta HSD autoantibodies. 3 beta HSD is the first steroid cell autoantigen defined at the molecular level to be associated with idiopathic POF occurring in the absence of other polyglandular diseases. Autoantibodies to 3 beta HSD in patients with other organ-specific autoimmune diseases indicate that the enzyme behaves as a typical target of polyendocrine autoimmunity. Anti-3 beta HSD autoantibodies in patients with POF may provide a marker of those subjects whose ovarian failure is autoimmune in origin and, as recent studies suggest, may be salvageable with glucocorticoid treatment.

Functional maturation of the primate fetal adrenal in vivo. II. Ontogeny of corticosteroid synthesis is dependent upon specific zonal expression of 3 beta-hydroxysteroid dehydrogenase/isomerase.

Cortisol, produced by the primate fetal adrenal, regulates the maturation of organ systems necessary for extrauterine life. During most of primate pregnancy, however, the fetal adrenal lacks the enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), which is essential for cortisol synthesis. Therefore, we used immunohistochemistry and in situ hybridization techniques to investigate the developmental expression of 3 beta HSD in the fetal rhesus monkey adrenal from 109 days' gestation until term (165 +/- 5 days) and assessed the role of ACTH in the induction of its expression and localization. We also examined whether ACTH regulates the expression of two other steroidogenic enzymes, cytochrome P450 cholesterol side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase, 17/20-lyase (P450c17), in the fetal rhesus monkey adrenal. To stimulate ACTH secretion from the fetal pituitary in vivo, we administered metyrapone to late gestation fetal rhesus monkeys for 3-7 days. Adrenals were collected from untreated fetuses at 109-125 days (n = 5), 130-148 days (n = 7), 155-172 days (n = 4), and after metyrapone treatment at 135-137 days (n = 4). The cortical width and total amount of 3 beta HSD staining were measured using an image analysis system. 3 beta HSD was localized primarily in the definitive zone cells of the adrenal from fetuses between 109-148 days, whereas at term (155-172 days), 3 beta HSD was localized in both definitive and transitional zone cells. The cortical width and total amount of 3 beta HSD staining in the adrenal increased significantly (P < 0.05) between 148 days (137 +/- 14 microns and 3,689 +/- 522 grains) and 155 days (315 +/- 61 microns and 7,321 +/- 2,008 grains). Interestingly, in metyrapone-treated fetuses at 135-137 days, 3 beta HSD messenger RNA (mRNA) and protein were localized extensively in both the definitive and transitional zones, a pattern seen only in term fetal adrenals in untreated animals. In addition, metyrapone treatment significantly (P < 0.05) increased cortical width (386 +/- 95 microns) and total 3 beta HSD immunostaining (29,063 +/- 13,692 grains) compared with age-matched controls. In contrast to 3 beta HSD, P450scc mRNA was detected in the definitive, transitional, and fetal zones, and its expression was not altered after metyrapone treatment. P450c17 mRNA was detected in the transitional and fetal zones, and the relative abundance was greater in the transitional zone. The relative abundance of P450c17 mRNA was increased in the fetal zone after metyrapone treatment. In summary, at term or after metyrapone treatment, expression of 3 beta HSD is induced in the transitional zone of the fetal rhesus monkey adrenal gland, an indication of functional maturation of the primate adrenal cortex. These data suggest that the ontogenetic increase in fetal pituitary ACTH secretion plays an important role in the induction of 3 beta HSD expression in the transitional zone.

Immunohistochemical analysis of AT1 receptor versus P450c17 and 3 beta HSD expression in ovine adrenals.

Previous studies in ovine adrenocortical cells in vitro have shown angiotensin II (AII) receptors are expressed on the zona fasciculata (ZF) cells and are functionally coupled to phosphoinositidase C and increased [Ca2+]i, but AII stimulation does not cause an acute change in cortisol biosynthesis. AII can, however, chronically regulate differential expression of P450c17 and 3 beta HSD in ovine adrenocortical cells in vitro. We have stained ovine adrenal sections with specific antisera to the angiotensin II Type-1 receptor (AT1-R), as well as P450c17 and 3 beta HSD in order to further test the hypothesis that changes in AT1-R expression underlie changes in zonal expression of P450c17 and 3 beta HSD in vivo. AT1-R expression was found to be highest in the outermost layer of cells (zona glomerulosa, ZG) which stained negatively for P450c17 and only faintly positive for 3 beta HSD, as expected. The adjacent layer of cells (ZF) stained much less strongly for AT1-R but stronger for P450c17 and 3 beta HSD. These findings are consistent with our previously reported in vitro expression data, and suggest that the transition from ZG to ZF phenotype, i.e. increased P450c17 and 3 beta HSD expression, may require reduced expression of AT1-R, but maintenance of reduced levels of AT1-R expression in the ovine ZF still allows for differential control of the P450c17: 3 beta HSD ratio. Thus, even though there is no acute cortisol response to AII alone in these cells, AII stimulation can oppose C19 steroid production in the face of cortisol biosynthesis by the ZF in response to agonists such as ACTH.

Studies of 3 beta-hydroxysteroid dehydrogenase genes in infants and children manifesting premature pubarche and increased adrenocorticotropin-stimulated delta 5-steroid levels.

Classic 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) deficiency congenital adrenal hyperplasia (CAH) results from a mutation in the type II 3 beta HSD gene encoding adrenal and gonadal 3 beta HSD. We investigated the type II and type I 3 beta HSD gene sequences in 15 infants and children with premature pubarche (PP; mean/range of age at PP, 4/0.08-9 yr) and elevated ACTH-stimulated delta 5 precursor steroid levels. Compared to Tanner I control subjects of similar age, ACTH-stimulated hormonal levels were at 2.3-10.7 SD for 17-hydroxypregnenolone (delta 5-17P) in all PP subjects, at 2.2-17 SD for dehydroepi-androsterone (DHEA) and 2.4-5.6 SD for the delta 5-17P/cortisol (F) ratio in all PP subjects except 1 infant, and at 2.3-10 SD for the DHEA/ androstenedione (delta 5-A) ratio in 8 PP subjects. Compared to Tanner II normal children, the hormonal levels were at 3-8 SD for delta 5-17P in all 13 PP children, at 2.3-4.7 SD for the delta 5-17P/F ratio in 6 PP children, and at 2.3-6.5 SD for DHEA and 3.5-9 SD for the DHEA/delta 4-A ratio in 7 PP children. Type II 3 beta HSD gene sequences, including regions of a putative promoter, all exons (I, II, III, and IV), and exon-intron boundaries, were normal in all subjects. Sequences of the type I 3 beta HSD gene encoding extraadrenal and extragonadal 3 beta HSD were normal in the 6 patients tested. The ACTH-stimulated delta 5-17P levels and delta 5-17P/F ratios in the PP children without type II 3 beta HSD gene mutation were exceedingly lower than the respective reported hormonal data for children with 3 beta HSD deficiency CAH with proven type II 3 beta HSD gene mutation. The ACTH-stimulated DHEA levels and DHEA/delta 4-A ratios were not exceedingly different between the children with and without type II 3 beta HSD gene mutation. These findings suggest that the degree of ACTH-stimulated delta 5 precursor steroid abnormality, such as delta 5-17P levels up to 10 SD above the normal mean level found in our PP patients, is not caused by a mild variant of 3 beta HSD deficiency CAH resulting from type II or type I 3 beta HSD gene mutation. The hormonal criterion for ACTH-stimulated delta 5-17P levels in patients with mild variant 3 beta HSD deficiency, therefore, is predicted to be higher than 10 SD above the normal mean value.

Sympathetic effects on the steroidogenesis and proliferation of adrenocortical cellsin vitro.

Recent evidence supports the hypothesis that sympathetic neurons play a significant role in the regulation of adrenocortical cell proliferation and steroidogenesis. Co-cultures of rat adrenocortical and sympathetic ganglion cells have been established to study sympatho-adrenal interactions. In these studies we have compared differentiation, growth and secretion of adrenocortical cells grown in co-culture with those grown alone. Adrenocortical cells were identified using 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) histochemistry or immunocytochemistry; sympathetic neurons were identified using immunocytochemical localization of tyrosine hydroxylase. The sympathetic neurons usually form clusters of 3-10 cells, and extend neurites to adrenocortical cells. Adrenocortical cells continue to proliferate, express 3 beta HSD and sequester lipid droplets. In the co-cultures, the adrenocortical cells are smaller, form larger clusters and show denser 3 beta HSD staining than the adrenocortical cells alone. The presence of the sympathetic neurons enhances adrenocortical cell proliferation as shown by a 2 fold increase in the number of 3 beta HSD(+) cells as well as increased BrdU incorporation after 48 hrs. Steroidogenesis appeared to be enhanced in the presence of sympathetic neurons as demonstrated by 3 beta HSD(+) staining and a 2-fold greater corticosterone and aldosterone secretion. However, when secretion is expressed per number of adrenocortical cells, the rates are comparable, indicating that secretion rate per cell remains unaltered by the presence of neurons. The effects of sympathetic neuron activation on adrenocortical cells remain to be determined.

Adrenarche is associated with decreased 3 beta-hydroxysteroid dehydrogenase expression in the adrenal reticularis.

The increased production of adrenal dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) which occurs during the prepubertal period is known as adrenarche. One hypothesis for adrenarche is that alterations in intra-adrenal expression of steroidogenic enzymes within the inner reticularis zone leads to the increased production of 19-carbon steroids. We tested the hypothesis that at the time of adrenarche there is decreased expression of 3 beta HSD in the reticularis. Immunohistochemical localization of 3 beta HSD was performed and staining intensities compared between adrenal glands from children ages 4 months to 7 years (N = 11) and ages 8 to 11 years (N = 6). No difference was observed between the levels of staining in the glomerulosa and fasciculata from either age group. However, the reticularis from the older children exhibited diminished 3 beta HSD immunoreactivity. These findings suggest that as children mature there is a decreased level of 3 beta HSD in the adrenal reticularis which may contribute to the increased production of DHEA and DHEAS seen during adrenarche.

The zona reticularis is the site of biosynthesis of dehydroepiandrosterone and dehydroepiandrosterone sulfate in the adult human adrenal cortex resulting from its low expression of 3 beta-hydroxysteroid dehydrogenase.

Based on indirect evidence, it has often been assumed that the zona reticularis of the adult human adrenal cortex is the source of the adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), but direct tests of this concept have been few. Using the techniques of cell culture, Northern blotting, and RIA, we compared the properties of separated adult zonal cells to those of fetal zone cells, a cell type well known to secrete large amounts of DHEA(S) due to its low expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). In nine glands from donors of a wide age range, the zona fasciculata and zona reticularis were separated and dissociated, and the cells were placed in culture. After 5 days, serum was removed by a 24-h period in serum-free defined medium followed by a 24-h exposure to cAMP analogs, with the optional addition of insulin, also in serum-free medium. The separated fasciculata and reticularis cells showed large differences in the DHEA(S)/cortisol (F) production ratios from added pregnenolone precursor, consistent with the synthesis of only F and essentially no DHEA(S) by fasciculata cells and with the synthesis of mostly DHEA(S) with little or no F by both reticularis cells and fetal zone cells. The different patterns of steroidogenesis were accompanied by a much lower level of expression of type II 3 beta HSD in reticularis cells, similar to that in fetal zone cells. In contrast, other genes were similarly regulated in the two adult zones and in the fetal zone by both cAMP and insulin. The levels of messenger ribonucleic acids for 17 alpha-hydroxylase, cholesterol side-chain cleavage enzyme, 21-hydroxylase, and 11 beta-hydroxylase responded to cAMP and insulin in both reticularis cells and fetal zone cells in the same pattern as that previously established in fasciculata cells. The central role of the limited expression of 3 beta HSD in the DHEA(S)-synthesizing property of reticularis cells was established by inhibition of 3 beta HSD in fasciculata cells with trilostane, which caused them to increase their DHEA/F production ratio to a level exceeding even that in fetal zone cells. There did not appear to any age-related changes in gene expression that could account for the large age-related decline in DHEA(S) biosynthesis in humans in either reticularis or fasciculata cells. Thus, the most likely cause of the age-related decline in adrenal androgen biosynthesis is an age-related decline in the number of functional reticularis cells, without a major change in the differentiated properties of the zonal cells as a function of age.

Human ovarian expression of 17 beta-hydroxysteroid dehydrogenase types 1, 2, and 3

Three isozymes of 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) have been cloned and characterized as distinct gene products (17 beta HSD1, 17 beta HSD2, and 17 beta HSD3). The presence and location of these isozymes in the human ovary have not been defined. In this study, we utilized Northern analysis and RT-PCR to examine transcripts for the three isozymes of 17 beta HSD. RNA was isolated from ovarian cortex, stroma (pre- and postmenopausal), hilum, follicles, and corpora lutea obtained from adult women, as well as whole fetal ovaries. By Northern analysis, high levels of 17 beta HSD1 messenger RNA were found in follicles, corpora lutea, and cortex, whereas low levels were detected in the postmenopausal stroma and in fetal ovaries by RT-PCR. 17 beta HSD1 messenger RNA was not detected in hilar tissue by either Northern analysis or RT-PCR. Utilizing RT-PCR, transcripts for 17 beta HSD2 were not detectable in cortex, stroma, (pre-or postmenopausal), hilum, or follicles, but were present in RNA derived from the corpora lutea and fetal ovary. The androgenic isozyme 17 beta HSD3 was not detectable in any of the ovarian compartments examined by either Northern analysis or RT-PCR. These data provide additional insight into the mechanism of testosterone and estradiol synthesis within the ovary. Specifically, the high level of 17 beta HSD1 is clearly localized to follicles and corpora lutea indicating involvement in the synthesis of estradiol. Secondly, androgenic 17 beta HSD3 is not expressed in the human ovary. Thus testosterone production within the human ovary, occurring under physiological conditions, arises from either the 17 beta HSD1 or an uncharacterized isozyme.

Human ovarian expression of 17 beta-hydroxysteroid dehydrogenase types 1, 2, and 3.

Three isozymes of 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) have been cloned and characterized as distinct gene products (17 beta HSD1, 17 beta HSD2, and 17 beta HSD3). The presence and location of these isozymes in the human ovary have not been defined. In this study, we utilized Northern analysis and RT-PCR to examine transcripts for the three isozymes of 17 beta HSD. RNA was isolated from ovarian cortex, stroma (pre- and postmenopausal), hilum, follicles, and corpora lutea obtained from adult women, as well as whole fetal ovaries. By Northern analysis, high levels of 17 beta HSD1 messenger RNA were found in follicles, corpora lutea, and cortex, whereas low levels were detected in the postmenopausal stroma and in fetal ovaries by RT-PCR. 17 beta HSD1 messenger RNA was not detected in hilar tissue by either Northern analysis or RT-PCR. Utilizing RT-PCR, transcripts for 17 beta HSD2 were not detectable in cortex, stroma, (pre-or postmenopausal), hilum, or follicles, but were present in RNA derived from the corpora lutea and fetal ovary. The androgenic isozyme 17 beta HSD3 was not detectable in any of the ovarian compartments examined by either Northern analysis or RT-PCR. These data provide additional insight into the mechanism of testosterone and estradiol synthesis within the ovary. Specifically, the high level of 17 beta HSD1 is clearly localized to follicles and corpora lutea indicating involvement in the synthesis of estradiol. Secondly, androgenic 17 beta HSD3 is not expressed in the human ovary. Thus testosterone production within the human ovary, occurring under physiological conditions, arises from either the 17 beta HSD1 or an uncharacterized isozyme.

Regulation by gonadotropins of the messenger ribonucleic acid for P450 side-chain cleavage, P450(17) alpha-hydroxylase/C17,20-lyase, and 3 beta-hydroxysteroid dehydrogenase in cultured pig Leydig cells.

The Leydig cell from the immature pig provides a good model for studying testicular steroidogenesis. Regulation of the enzymes involved, which has been well studied in rodents, has not been characterized in the pig. The objectives of this study were to examine the regulation of three steroidogenic enzymes in pig Leydig cells by LH/hCG and testosterone. The mRNA for P450 side-chain cleavage and P450(17) alpha-hydroxylase/C17-20-lyase, although constitutively expressed, decreased over time in culture, while that for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) remained relatively constant. The mRNA for all three enzymes was increased in a dose- and time-dependent manner by treatment with hCG. Run-on experiments demonstrated that the main effect of the hormone was at the level of transcription. Treatment with hydroxyflutamide, either alone or in combination with hCG, had no effect on the mRNA for these enzymes. Treatment with hCG plus aminoglutethimide, an inhibitor of steroidogenesis, had no effect on the mRNA for the two P450 enzymes, but resulted in an increase in mRNA for 3 beta HSD when compared to treatment with hCG alone. However, exogenous testosterone could not block the effect of aminoglutethimide. Therefore, the steroidal regulation of 3 beta HSD in pig Leydig cells may act through a mechanism separate from the androgen receptor. While aspects of the regulation of these enzymes are similar to those seen in rodents, some significant differences exist. Our results support the concept that regulation of steroidogenic enzymes in Leydig cells is species-specific.

Differential patterns of expression of DAX-1 and steroidogenic factor-1 (SF-1) in the fetal rat testis.

Gonadal differentiation is dependent upon a cascade of molecular and morphological events. Steroidogenic factor 1 (SF-1) and DAX-1 have been implicated in this process. A SF-1 binding site has been reported to be present on the DAX-1 gene. We therefore used immunocytochemistry to determine whether these two transcription factors are co-expressed in the fetal testis. DAX-1 was immunolocalised to the cytoplasm of interstitial cells from fetal testis of rat and human from day 15.5 and 16 weeks of gestation respectively. SF-1 was detected in nuclei of fetal Sertoli and interstitial cells. In the fetal rat expression of SF-1 in interstitial cells was not uniform; cells with abundant SF-1 all contained 3-beta HSD and were classified as Leydig cells. DAX-1 expression was not exclusive to cells which contained SF-1 and SF-1/DAX-1 co-expressing cells were not exclusively fetal Leydig cells. We conclude that in the fetal testis expression of the DAX-1 protein is not dependent upon the presence of SF-1 although SF-1 is present at an earlier stage of gestation in the gonad. DAX-1 may therefore play a separate or complementary role to that of SF-1 in the modulation of testicular gene expression and differentiation.

11β‐HYDROXYSTEROID DEHYDROGENASE TYPE I ENZYME IN THE HEARTS OF NORMOTENSIVE AND SPONTANEOUSLY HYPERTENSIVE RATS

1. The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) converts glucocorticoids to their inactive 11-keto metabolites. The ubiquitous expression of the NADP-dependent isoform (11 beta HSD1) suggest an important role in modulating glucocorticoid action, but little is known about 11 beta HSD1 gene expression and enzymatic activity in the rat heart. 2. In the present study rat cardiac 11 beta HSD1 activity and ontogeny of gene expression have been characterized. The addition of NADP, but not NAD, to heart homogenates resulted in significant increases in the metabolism of both corticosterone and cortisol, with the former substrate displaying far greater metabolism. Both 11 beta HSD1 gene expression and enzyme activity increased in parallel from low levels at 1 week of age to maximal levels at 8 weeks, with no further change by 16 weeks of age. 3. We also compared the activity of 11 beta HSD1 in the hearts of male and female spontaneously hypertensive rats (SHR) with normotensive Wistar-Kyoto (WKY) controls. Enzyme activity in the pooled atria of female SHR was significantly higher than in male SHR atria (7.6 +/- 0.6% conversion of corticosterone vs 4.5 +/- 0.5%; P < 0.05). The left ventricles of female WKY rats contained significantly less 11 beta HSD activity than either male WKY rats or female SHR (8.6 +/- 0.8% conversion vs 17 +/- 1.4 and 13.6 +/- 0.5%, respectively; P < 0.05). In the right ventricle, female WKY rats also had significantly less enzyme activity than either female SHR or male WKY rats (4.9 +/- 0.7 vs 10.0 +/- 1.7 and 10.2 +/- 1.4%; P < 0.05). 4. These results clearly show that the rat heart contains significant amounts of the 11 beta HSD1 enzyme and that this activity is sexually dimorphic. Furthermore, significant differences were observed between a normotensive and hypertensive strain of rat. The relevance of these observations to the aetiology and maintenance of hypertension remains to be explored.

A (R80Q) mutation in 17 beta-hydroxysteroid dehydrogenase type 3 gene among Arabs of Israel is associated with pseudohermaphroditism in males and normal asymptomatic females.

Four isozymes of steroid 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) encoded by different loci catalyze the reversible conversion of androstenedione to testosterone and that of estrone to estradiol. The 17 beta HSD type 3 (17 beta HSD3) isozyme is encoded by the 17 beta HSD3 gene on chromosome 9q22 and expressed only in testes. Inherited defects in the 17 beta HSD3 isozyme cause a form of male pseudohermaphroditism that is rare within the general population, but frequent among a highly inbred Arab population in the Gaza strip. A point mutation in exon 3, codon 80 of the 17 beta HSD3 gene, R80Q, caused by a single base substitution from CGG to CAG was identified in both alleles of 24 individuals from 9 extended Arab families from Gaza, Jerusalem, and Lod-Ramle. Twenty-one homozygotes were male pseudohermaphrodites (46,XY) with testicular 17 beta HSD3 deficiency, born with either female-looking external genitalia or various degrees of genital ambiguity. If not reassigned in infancy, they were reared as females until puberty, when marked virilization occurred, often leading to the spontaneous adoption of a male gender role. In contrast, the 3 homozygote females (46,XX) were asymptomatic, had normal internal and external genitalia and normal sexual development, and revealed no biochemical evidence of 17 beta HSD3 deficiency. The molecular pattern in these families is compatible with an autosomal recessive mode of inheritance that is sex dependent.

Substrate and inhibitor specificity of the cloned human 11 beta-hydroxysteroid dehydrogenase type 2 isoform.

The 11 beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) enzyme is thought to confer specificity on the mineralocorticoid receptor by inactivating glucocorticoids in mineralocorticoid target organs. The cloned 11 beta HSD2 displayed Michaelis constant values for corticosterone and cortisol of 5.1 and 61 nM, respectively. Linearity in the dose-response curve ranged between 1 and 200 nM for corticosterone and 25 and 2,000 nM for cortisol, with no evidence for complex kinetics. Inhibition of cortisol oxidation by other steroids was purely competitive in nature. Inhibition of 11 beta HSD2 activity by the end product or aldosterone occurred only at supraphysiological levels, whereas corticosterone and deoxycorticosterone displayed significant inhibition at physiological concentrations and progesterone at concentrations that occur during pregnancy. In intact transfected CHOP cells, dexamethasone was converted to 11-dehydrodexamethasone by 11 beta HSD2 but not type 1 11 beta-hydroxysteroid dehydrogenase, an aspect that may be useful in evaluating 11 beta HSD activity in intact cells.

A role for epidermal growth factor in the morphological and functional maturation of the adrenal gland in the fetal rhesus monkey in vivo.

We determined the effects of epidermal growth factor (EGF) and beta-methasone on the growth and development of the adrenal gland of the fetal rhesus monkey in vivo between 121-128 days of gestation. The adrenal to body weight ratio was significantly greater (P < 0.05) in EGF-treated fetuses (0.988 +/- 0.046 x 10(-3) g/g) and significantly reduced (P < 0.05) in beta-methasone-treated fetuses (0.401 +/- 0.056 x 10(-3) g/g) compared with that in control fetuses (0.689 +/- 0.050 x 10(-3) g/g). The increase in adrenal weight with EGF administration was due to hypertrophy of definitive zone cells of the adrenal cortex, whereas the reduction in adrenal weight after beta-methasone treatment was due to a decrease in the size of definitive and fetal zone cells of the adrenal cortex. By Western analysis, EGF treatment induced a significant (P < 0.05) 2.8-fold increase in the amount of protein for 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD) in the fetal adrenal. EGF also stimulated the induction of immunocytochemical staining for 3 beta HSD in transitional zone cells of the adrenal cortex. In contrast, beta-methasone resulted in 2.6-, 4.5-, and 6.6-fold significant decreases (P < 0.05) in the amount of protein for cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17 alpha-hydroxylase/17,20-lyase, and 3 beta HSD in the fetal adrenal. After beta-methasone treatment. 3 beta HSD staining was detected in some of the definitive zone cells, with no 3 beta HSD staining in the transitional zone. In conclusion, growth and functional differentiation of fetal primate adrenal gland can be accelerated prematurely by EGF and inhibited by glucocorticoid negative feedback.