Bizarre Chorionic-type Trophoblast in Second-trimester and Third-trimester Placentas: Clinicopathologic Characterization of a Placental Pseudoneoplastic Lesion.

Bizarre (atypical/symplastic) cells have been described in various gynecologic normal tissues and benign neoplasms. This type of bizarre cytologic change is usually an incidental finding and is regarded as a benign process. We describe 17 cases of bizarre chorionic-type trophoblast in second-trimester and third-trimester placentas that created concern for an underlying/undersampled or incipient intraplacental trophoblastic neoplasm, predominantly found in intervillous trophoblastic islands (11/17), placental septae (6/17), chorionic plate (1/17), and/or the chorion layer of fetal membranes (2/17). The bizarre trophoblastic cells exhibited sheet-like or nested architecture, had a multifocal/patchy distribution, and/or were present as individual cells within hyaline stroma; they were characterized by large nuclei with smudgy chromatin and occasional intranuclear pseudoinclusions. The degree of atypia was classified as mild (0/17), moderate (3/17), or severe (14/17). Mitotic figures and necrosis were not identified. A dual immunohistochemical stain for trophoblast (hydroxyl-delta-5-steroid dehydrogenase) and a proliferation marker (Ki-67), performed in 15 cases, demonstrated 0% to very low proliferative activity within the bizarre trophoblast (0% to 2% [10/15], 3% to 8% [5/15]). Immunohistochemical stains for fumarate hydratase showed intact/retained expression in the bizarre cells in 7 of 7 cases. Clinical follow-up ranged from 1 to 45 months, and all patients were alive and well without subsequent evidence of a gestational trophoblastic or other neoplasms. We conclude that bizarre chorionic-type trophoblast in second-trimester or third-trimester placentas have the potential to mimic an intraplacental trophoblastic neoplasm but are likely a benign degenerative change. This study expands the spectrum of bizarre cells that occur in the gynecologic tract.

Three populations of adult Leydig cells in mouse testes revealed by a novel mouse HSD3B1-specific rat monoclonal antibody.

Leydig cells play a pivotal function in the synthesis of a male sex steroid, testosterone. The ability of the steroid production is dependent on the expression of the steroidogenic genes, such as HSD3B (3ß-hydroxysteroid dehydrogenase/Δ5- Δ4 isomerase). It has been established that two different types of Leydig cells, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs), are developed in mammalian testes. FLCs and ALCs are characterized by different sets of marker gene expression. In the case of mouse Leydig cells, Hsd3b1 (Hsd3b type 1) is expressed both in FLCs and ALCs whereas Hsd3b6 (Hsd3b type 6) is expressed in ALCs but not in FLCs. However, because the antibodies established so far for HSD3B were unable to distinguish between the HSD3B1 and HSD3B6 isoforms, it remained unclear whether both of them are expressed in every ALC. Therefore, in the present study, we generated a rat monoclonal antibody specific for mouse HSD3B1. Intriguingly, this monoclonal antibody together with an antibody specific for HSD3B6 identified three populations of ALCs based on the expression levels of these HSD3Bs.

Regulation of steroidogenic genes by insulin-like growth factor-1 and follicle-stimulating hormone: differential responses of cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, and 3beta-hydroxysteroid dehydrogenase/isomerase in rat granulosa cells.

The present study sought to characterize the concerted action of FSH and insulin-like growth factor-1 (IGF-1) on functional differentiation of prepubertal rat ovarian granulosa cells in culture. To this end, we examined the regulation of three key genes encoding pivotal proteins required for progesterone biosynthesis, namely, side-chain cleavage cytochrome P450 (P450(scc)), steroidogenic acute regulatory (StAR) protein, and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD). Time-dependent expression profiles showed that P450(scc), StAR, and 3beta-HSD gene products accumulate in chronic, acute, and constitutive patterns, respectively. Each of these genes responded to FSH and/or IGF-1 in a characteristic manner: A synergistic action of IGF-1 was indispensable for FSH induction of P450(scc) mRNA and protein; IGF-1 did not affect FSH-mediated upregulation of StAR products; and IGF-1 alone was enough to promote expression of 3beta-HSD. The responsiveness of the genes to IGF-1 correlated well with their apparent susceptibility to the inhibitory impact of tyrphostin AG18, a potent inhibitor of protein tyrosine kinase receptors. Thus, IGF-1-dependent P450(scc) and 3beta-HSD expression was completely arrested in the presence of AG18, whereas StAR expression was unaffected in the presence of tyrphostin. These findings suggest that FSH/cAMP signaling and IGF-1/tyrosine phosphorylation events are interwoven in rat ovarian cells undergoing functional differentiation. We also sought the mechanism of IGF-1 synergy with FSH. In this regard, our studies were unable to demonstrate a stabilizing effect of IGF-1 on P450(scc) mRNA, nor could IGF-1 augment FSH-induced transcription examined using a proximal region of the P450(scc) promoter (-379/+6). Thus, the mechanism of IGF-1 and FSH synergy remains enigmatic and provides a major challenge for future studies.

Immunohistochemical study of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase in the rat cardiovascular system.

The enzyme complex 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is involved in the biosynthesis of all classes of active steroids. It is known that the enzymatic activity of 3beta-HSD is present not only in classical steroidogenic tissues, but also in many peripheral tissues including cardiac tissue. To determine whether 3beta-HSD is present in rat non-steroidogenic tissues, we examined cardiovascular tissues including the ventricle, atrium, aortic arch, abdominal aorta, and inferior vena cava by immunohistochemistry and Western blotting using polyclonal antibody raised against a synthetic peptide of human placental 3beta-HSD. By Western blotting, protein bands immunoreactive for anti-3beta-HSD were detected at molecular weights of 42 and 37 kDa in both the ventricle and atrium, whereas only a 37 kDa band was recognized in both the aortic arch and abdominal aorta. By immunohistochemistry, immunoreactivity for 3beta,-HSD was detected in both the ventricular and atrial cardiocytes, while immunostaining was also found, though faintly, in the smooth muscles of the aortic arch, abdominal aorta, and inferior vena cava. These results suggest that cardiocytes may synthesize the steroidogenic 3beta,-HSD enzyme.

Localization of testosterone and 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in cynomolgus monkey (Macaca fascicularis) testes.

The enzyme 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD) is essential for the biosynthesis of all classes of steroid hormones, including androgens. We localized testosterone and 3beta-HSD by light microscopic immunocytochemistry in the testes of adult cynomolgus monkeys. Immunoreactive testosterone was located as intense deposits in the labeled cytoplasm of Leydig cells, and located weakly in the interstitial tissues, basement membranes, and the regions near tubular walls within tubules. Immunoreactive 3beta-HSD was located in the cytoplasm of all Sertoli cells and was especially intense in the parts near tubular walls and located weakly to intensely in the cytoplasm of some Leydig cells. This is the first immunocytochemical evidence that Sertoli cells of cynomolgus monkeys, as well as Leydig cells, are involved in biosynthesis of androgens.

Subcellular localization of 3 beta hydroxysteroid dehydrogenase isomerase in testis of Bufo arenarum H.

3 Beta-hydroxysteroid dehydrogenase 5-ene isomerase (3 beta HSD/I) catalyzes an essential step in the biosynthesis of steroid hormones and is usually considered to be mainly microsomal, although there is a dual distribution of the enzyme in toad interrenals. The present study demonstrates that in the testicular tissue, as in interrenals of Bufo arenarum H., 3 beta HSD/I is both mitochondrial and microsomal. The conversion of dehydroepiandrosterone to androstenedione takes place only in microsomes while pregnenolone is converted to progesterone in both microsomes and mitochondria. Kinetic constants of 3 beta HSD/I were determined by the oxidation of pregnenolone and dehydroepiandrosterone. The preferred substrate of the microsomal 3 beta HSD/I enzyme was dehydroepiandrosterone (K(m) = 0.17 microM and 0.53 microM for dehydroepiandrosterone and pregnenolone, respectively) not only during the breeding season but also in the non-breeding period (K(m) = 0.49 microM and 2.9 microM for dehydroepiandrosterone and pregnenolone, respectively).

Mitochondrial localization of 3 beta-hydroxysteroid dehydrogenase 5-ene isomerase in interrenals of the toad Bufo arenarum H.

The enzymatic activity of 3 beta-hydroxysteroid dehydrogenase 5-ene isomerase (3 beta HSD/I) catalyzes an essential step in the biosynthesis of steroid hormones including progesterone, mineralocorticoids, glucocorticoids, estrogens, and androgens. Its subcellular localization in steroidogenic tissues is usually considered to be mainly microsomal. The present study demonstrates that in the interrenal of Bufo aernarum H., 3 Beta HSD/I activity localizes in mitochondria and micromes. It also shows that the two distinct pathways to aldosterone previously demonstrated for interrenals of B. arenarum H. exhibited differential subcellular localizations, microsomal for the 4-ene route and mitochondrial for the 5-ene route. Kinetic constants of 3 Beta HSD/I were determined for the oxidation of pregnenolone and the recently described 3 Beta-hydroxy analogue of aldosterone (3 Beta AA). The preferred substrate of the mitochondrial 3 Beta HSD/I enzyme was 3 Beta AA (Km = 0.7 microM and 14.0 microM for 3 Beta AA and pregnenolone, respectively). However, the microsomal enzyme has a greater affinity for pregnenolone (Km = 0.8 microM) than for 3 Beta AA (Km = 17.0). Enzymes from both localizations have similar nucleotide (NAD+) requirements, activities being higher in summer. This dual localization opens novel possibilities for the regulation of interrenal functions.

Altering culture conditions reveals strain-related differences in activity and immunoreactive isoforms of 3 beta-hydroxysteroid dehydrogenase-isomerase in mouse Leydig cells.

We previously reported that 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) activity is higher in Leydig cells from C57BL/6J mice than those from C3H/HeJ mice. This study examines whether the differences in 3 beta HSD activity in Leydig cells from the two strains of mice are due to the expression of different 3 beta HSD isoforms and if a specific isoform corresponds with the amount of 3 beta HSD activity under various culture conditions. Leydig cells were plated in Waymouth's +15% horse serum (HS) medium. Some cultures were terminated 24 h later after plating (day 1) and assayed for 3 beta HSD activity or immunoreactivity. The remaining cultures were maintained in HS or changed to serum-free medium. We demonstrate for the first time that two 3 beta HSD immunoreactive isoforms are expressed in freshly isolated Leydig cells and those cultured for 1 day. The same two 3 beta HSD isoforms are detectable in both strains. Thus, the previously reported strain-related differences in 3 beta HSD activity are not due to the expression of different isoforms. When cultured for 8 days, the higher molecular weight 3 beta HSD immunoreactive band is no longer detectable in Leydig cells from either strain. When maintained in HS, 3 beta HSD activity in C57BL/6J Leydig cells decreases significantly by day 8, while 3 beta HSD activity in C3H/HeJ Leydig cells does not change through day 8. When Leydig cells were cultured in serum-free medium, 3 beta HSD activity is maintained in cultured Leydig cells from C57BL/6J and significantly increases in C3H/HeJ 3 beta HSD by day 8. These data suggest that HS has a strain-specific inhibitory effect on 3 beta HSD activity, causing a significant decrease in C57BL/6J 3 beta HSD activity and preventing an increase in C3H/HeJ. Densitometric analysis reveals a correspondence between changes in 3 beta HSD activity and the lower molecular weight isoform but not the higher molecular weight isoform. Treatment with cAMP induces the immunoreactive mass of the lower molecular isoform but not the higher molecular isoform of 3 beta HSD. Currently, it is unclear what the function of the higher molecular weight 3 beta HSD isoform is in mouse Leydig cells.

Opposite effects of prolactin and corticosterone on the expression and activity of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase in rat skin.

In rat skin, type IV is the major 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) isoenzyme expressed. Although types I and II 3 beta-HSD mRNAs are also present in the skin, their level of expression is about two orders of magnitude lower than that of type IV. In this study, we have investigated the control of type IV 3 beta-HSD mRNA levels as well as 3 beta-HSD enzymatic activity in hypophysectomized adult rats of both sexes. Skin 3 beta-HSD activity was measured by the conversion of [14C]-dehydroepiandrosterone into [14C]-androstenedione, whereas ribonuclease protection assay using a specific type IV cRNA probe was used to assess mRNA levels. Intact male and female rats show a similar level of skin 3 beta-HSD activity, although hypophysectomy caused opposite effects, a decrease being observed in males while an increase was observed in hypophysectomized female animals. We next studied the effects of hyperprolactinemia, corticosterone and 1-thyroxine in hypophysectomized animals. L-thyroxine was found to stimulate 3 beta-HSD expression and activity in male rats whereas no significant effect was observed on the already elevated levels in hypophysectomized female rats. Corticosterone caused an inhibition of type IV 3 beta-HSD mRNA levels and activity in both male and female animals. Hyperprolactinemia achieved by pituitary implants inserted under the kidney capsule stimulated the expression of type IV mRNA as well as 3 beta-HSD enzymatic activity in hypophysectomized male and female animals. The present data demonstrate the multihormonal regulation of 3 beta-HSD/isomerase expression and activity in the rat skin.

Dual subcellular localization of the 3 beta-hydroxysteroid dehydrogenase isomerase: characterization of the mitochondrial enzyme in the bovine adrenal cortex.

The enzyme 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta-HSD/I) is an essential step in the biosynthesis of steroid such as progesterone, mineralo- and gluco-corticoids, estrogens and androgens in steroidogenic tissues. It is considered to be mainly localized in microsomes; however, 3 beta-HSD/I activity has also been described to be associated with mitochondrial preparations. In this study, we examined the subcellular distribution of 3 beta-HSD/I in bovine adrenocortical tissue and we characterized the catalytic properties of the enzyme present in the various cell compartments. About 30% of the total 3 beta-HSD/I activity was found to remain tightly associated with the purified mitochondrial pellet. The 3 beta-HSD/I and 3-ketoreductase activities were found in microsomes as well as in mitochondria. The 3 beta-HSD/I associated with the mitochondrial fraction did not require addition of exogenous NAD+. When the pyridine nucleotide was reduced following addition of substrates of the tricarboxylic acids cycle, the mitochondrial 3 beta-HSD/I activity decreased, suggesting that the enzyme utilizes NAD+ available from the matrix space. By contrast, the microsomal enzyme was inactive in the absence of exogenous NAD+. Submitochondrial fractionation disclosed that 3 beta-HSD/I was associated (i) with the inner membrane and (ii) with a particulate fraction sedimenting in a density gradient between inner and outer membranes. This fraction was characterized as contact sites between the two membranes. 3 beta-HSD/I specific activity was much higher in this fraction than in the inner mitochondrial membrane. Altogether, these observations suggest that these mitochondrial intermembrane contact sites may represent a special organization of functional significance, facilitating both the access of cholesterol to the inner membrane where cytochrome P-450scc is located and the rapid transformation of its product, pregnenolone, to progesterone, through 3 beta-HSD/I activity.

Immunohistochemical localization of 3 beta-hydroxysteroid/delta 5-delta 4-isomerase, tyrosine hydroxylase and phenylethanolamine N-methyl transferase in adrenal glands of sheep fetuses throughout gestation and in neonates.

Immunoreactive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) was localized in adrenal glands of sheep fetuses in cortical-type cells, but not in medullary-type cells, from day 43 of gestation to term and in 2-4-day-old neonates. From day 54 of gestation, the formation of distinct zones within the adrenal cortex was apparent and immunoreactive 3 beta-HSD was found in cortical cells in the zona fasciculata and in groups and cords of cortical cells within the developing medulla, with weak positive staining in the zona glomerulosa. At this stage, most medullary cells were positive for immunoreactive tyrosine hydroxylase, and some of these cells with a juxtacortical distribution also stained positively for immunoreactive phenylethanolamine N-methyl transferase (PNMT). Between days 65 and 130, the adrenal medulla increased in size with little change in the width of the cortex. Organization and zonation of immunoreactive 3 beta-HSD staining cells were evident in the zona fasciculata and in groups of cells in the medulla. Between day 130 and term, uniform immunoreactive 3 beta-HSD staining was found throughout the zona fasciculata, and there was also staining in single cells and small clusters of cells throughout the medulla. At this stage, immunoreactive tyrosine hydroxylase was distributed in most cells throughout the medulla, but in two distinct patterns: cells staining intensely for immunoreactive tyrosine hydroxylase in the central region of the medulla, and cells exhibiting weaker staining for immunoreactive tyrosine hydroxylase localized in a juxta-cortical position. These juxta-cortical cells were also positive for immunoreactive PNMT.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in human ovary.

The enzyme complex 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid delta 5----4-ene-isomerase (3 beta HSD) is involved in the biosynthesis of all classes of active steroids, namely glucocorticoids, mineralocorticoids, progesterone, and sex steroids. To obtain more information about the age-specific expression of 3 beta HSD in the human ovary, we have localized this enzyme by immunocytochemistry at the light microscopic level during fetal and postnatal periods of development in the human. Immunocytochemical localization was achieved using specific polyclonal antibodies developed against purified human placental 3 beta HSD. In the fetal ovary of 28-34 weeks, specific immunostaining for 3 beta HSD was exclusively detected in thecal cells surrounding primary follicles and in interstitial cells. From birth until puberty, no significant immunostaining for 3 beta HSD could be observed, while from puberty to menopause, staining was detected in theca interna cells as well as granulosa cells of growing follicles. The intensity of staining in the theca interna cells was always much higher than that in granulosa cells. Immunostaining was also found in the cytoplasm of luteinized granulosa and theca interna cells of the corpus luteum. Interestingly, there was an absence of immunoreactivity for 3 beta HSD in one to several layers of theca interna cells lying just beneath the basement membrane. These negative cells may correspond to fibroblast-like cells that are devoid of 3 beta HSD activity. In postmenopausal ovaries, immunolabeling was only found in dispersed interstitial cells, thus suggesting that the ovaries of postmenopausal women might be involved in sex steroid hormone secretion.

Expression of testicular 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase: regulation by luteinizing hormone and forskolin in Leydig cells of adult rats.

LH is required to maintain the activity of 3 beta-hydroxysteriod dehydrogenase/delta 5----4-isomerase (3 beta HSD) in testicular Leydig cells. The objective of the present study was to determine whether LH and effectors such as forskolin, which act via the intracellular cAMP signal transduction pathway, can regulate the expression of 3 beta HSD in rat Leydig cells in vitro. Primary cultures of Leydig cells were prepared from testes of adult rats and treated with oLH, forskolin, (Bu)2cAMP, or cholera toxin. The effects of treatment on 3 beta HSD activity were measured using [3 alpha-3H]dehydroepiandrosterone as substrate. Immunoreactive 3 beta HSD was quantified by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a polyclonal antiserum against 3 beta HSD. The synthesis of 3 beta HSD was quantified after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated cellular lysates of Leydig cells radiolabeled with L-[35S]methionine. The levels of 3 beta HSD mRNA were quantified by Northern analysis and hybridization with a cDNA encoding testicular 3 beta HSD (rat type I). A cell-free protein-synthesizing system was used to test the ability of 3 beta HSD mRNA to be translated into immunoreactive 3 beta HSD. 3 beta HSD activity increased 3.5- and 5.0-fold in Leydig cell cultures treated with forskolin (1 microM) and (Bu)2cAMP (1 mM), respectively, compared with control cultures. Maximal activity was attained after 48-72 h and maintained through 120 h of treatment. The increase in 3 beta HSD activity could be accounted for quantitatively by increases in the steady state levels and the rates of synthesis of 3 beta HSD. The cellular levels of immunoreactive 3 beta HSD increased 4.0- and 7.6-fold in Leydig cells treated with forskolin and (Bu)2cAMP, respectively. Moreover, both of these effectors increased by 6- to 8-fold the levels of newly synthesized 3 beta HSD after 24-72 h of treatment. Ovine LH, forskolin, cholera toxin, and (Bu)2cAMP increased the cellular levels of 3 beta HSD mRNA in a dose-dependent manner. The magnitude of the increases ranged from 2- to 42-fold, compared with that in control cultures, after 12 h of treatment. Maximal responses were effected by 1 ng/ml ovine LH, 1 microM forskolin, 1 ng/ml cholera toxin, and 1 mM (Bu)2cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgenic 17 beta-hydroxysteroid dehydrogenase activity of expressed rat type I 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase.

Transient expression in nonsteroidogenic mammalian cells of the rat wild type I and type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNAs shows that the encoded proteins, in addition to being able to catalyze the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into the corresponding delta 4-3-ketosteroids, interconvert 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). When homogenate from cells transfected with a plasmid vector containing type I 3 beta-HSD is incubated in the presence of DHT using NAD+ as cofactor, a somewhat unexpected metabolite is formed, namely 5 alpha-androstanedione (A-dione), thus indicating an intrinsic androgenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity of this 3 beta-HSD isoform. Although the relative Vmax of 17 beta-HSD activity is 14.9-fold lower than that of 3 beta-HSD activity, the Km value for the 17 beta-HSD activity of type I 3 beta-HSD is 7.97 microM, a value which is in the same range as the conversion of DHT into 3 beta-diol which shows a Km value of 4.02 microM. Interestingly, this 17 beta-HSD activity is highly predominant in unbroken cells in culture, thus supporting the physiological relevance of this "secondary" activity. Such 17 beta-HSD activity is inhibited by the classical substrates of 3 beta-HSD, namely pregnenolone (PREG), dehydroepiandrosterone (DHEA), delta 5-androstene-3 beta,17 beta-diol (delta 5-diol), 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) and DHT, with IC50 values of 2.7, 1.0, 3.2, 6.2, and 6.3 microM, respectively. Although dual enzymatic activities have been previously reported for purified preparations of other steroidogenic enzymes, the present data demonstrate the multifunctional enzymatic activities associated with a recombinant oxidoreductase enzyme. In addition to its well known 3 beta-HSD activity, this enzyme possesses the ability to catalyze DHT into A-dione thus potentially controlling the level of the active androgen DHT in classical steroidogenic as well as peripheral intracrine tissues.

Localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in rat gonads and adrenal glands by immunocytochemistry and in situ hybridization.

The enzyme complex delta 5-3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD) is involved in the biosynthesis of all classes of steroids, namely glucocorticoids, mineralocorticoids, progesterone, and sex steroids. To obtain information on the precise localization of 3 beta HSD in rat gonads and adrenal glands, two complementary cytochemical techniques were used; immunocytochemical localization was achieved with antibodies developed against purified human placental 3 beta HSD, while 3 beta HSD mRNA localization was achieved by in situ hybridization performed with a recently cloned rat 3 beta HSD cDNA. In the testis, specific immunostaining was restricted to the cytoplasm of the interstitial cells, while by in situ hybridization, specific silver grains were also seen over the interstitial cells. In the ovary, immunostaining was found in the cytoplasm of cells of the corpus luteum and theca interna, while the granulosa cells of the follicles showed no positive reaction. By in situ hybridization, a specific hybridization signal was observed over granulosa cells of the corpus luteum, which are mainly responsible for progesterone secretion, and to a lesser extent over theca interna cells, known for their role in secreting C19 androgens. In the adrenals, the three zones of the cortex were equally immunolabeled, whereas no staining could be detected in the medulla. Similarly, by in situ hybridization, silver grains were located over the zona glomerulosa, fasciculata, and reticularis, while no specific autoradiographic reaction could be observed on the chromaffin cells of the medulla. The present study provides new information about the precise cellular localization of 3 beta HSD in the adrenal glands and gonads in the rat, thus providing useful information about the site of action of 3 beta HSD, especially in the gonads. Moreover, the approaches used for localization studies, especially quantitative in situ hybridization, should provide a useful tool for assessing the role of hormones on 3 beta HSD expression in the different compartments of the gonads and adrenal glands.

In vitro inhibition by ketoconazole of human testicular steroid oxidoreductases.

An oral antimycotic agent, ketoconazole has been demonstrated to be an inhibitor of cytochrome P-450-dependent monooxygenases. To investigate its effect on steroid oxidoreductases, in vitro studies were carried out using subcellular fractions of human testes. Ketoconazole competitively inhibited activities of 3 beta-hydroxy-5-ene-steroid oxidoreductase/isomerase and NADH-linked 20 alpha-hydroxysteroid oxidoreductase for steroid substrate and the Ki values were 2.9 and 0.9 microM, respectively. In contrast, ketoconazole inhibited neither 17 beta-hydroxysteroid oxidoreductase nor NADPH-linked 20 alpha-hydroxysteroid oxidoreductase, indicating that the two 20 alpha-hydroxysteroid oxidoreductases are distinct. Further, ketoconazole inhibited non-competitively the above enzyme activities for the corresponding cofactors of NAD and NADH. From the binding mode of ketoconazole to cytochrome P-450 and the present findings, it appears likely that the agent binds to a site which is different from that of steroids or pyridine nucleotides.

Light microscopic immunocytochemical localization of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase in the gonads and adrenal glands of the guinea pig.

The enzyme complex 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is involved in the biosynthesis of all classes of active steroids, namely glucocorticoids, mineralocorticoids, progesterone, and sex hormone steroids. To obtain precise information about the cellular and subcellular localization of 3 beta HSD in the gonads and adrenals of the guinea pig, we have proceeded to immunocytochemical localization of the enzyme using antibodies developed against purified human placental 3 beta HSD. In the testis, specific immunostaining was restricted to the cytoplasm of the interstitial cells. In the ovary, on the other hand, immunostaining was found in the cytoplasm of the cells of the corpus luteum and theca interna. The granulosa cells of the follicles were not immunolabeled, with the exception of one layer of cells in contact with the zona pellucida. This restricted labeling was observed in growing and mature follicles, but not in primordial and small primary follicles. In the adrenals, the three zones of the cortex were labeled, whereas no staining could be detected in the medulla. Contrary to findings in the testis and ovary, staining was found in both the cytoplasm and nuclei of adrenocortical cells. The present data suggest a functional interaction between the ovocyte and the specialized layer of 3 beta HSD-containing granulosa cells which might play a role in ovocyte maturation.

3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland.

The fetal zone of the human fetal adrenal (HFA) gland is established to have decreased 3 beta-hydroxysteroid dehydrogenase/delta 4-5 isomerase (3 beta HSD) activity compared to the neocortex or definitive zone. 3 beta HSD activity, however, can be induced in primary cell culture through treatment with ACTH. Therefore, the HFA with two distinct steroidogenic zones with differences in 3 beta HSD activity as well as the capacity to increase 3 beta HSD activity in response to ACTH provides an excellent model to study the regulation of this enzyme. The presence of 3 beta HSD in the fetal and neocortex zones of the HFA was examined using a polyclonal antibody raised against purified human placental microsomal 3 beta HSD. After homogenates of the fetal and neocortical zones of the HFA were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and immunoblotted, the presence of the 3 beta HSD protein with a molecular size of 45 kDa could be demonstrated only in the neocortical zone. ACTH treatment (greater than 2 days) of fetal and neocortical zone explant cultures produced increases in cortisol secretion associated with the respective levels of immunodetectable 3 beta HSD protein. Cortisol and dehydroepiandrosterone sulfate were the respective principal steroid products of neocortical and fetal zone explants. After ACTH treatment, immunodetectable 3 beta HSD was induced to a greater magnitude in the neocortex. These findings provide evidence that the lack of 3 beta HSD activity in the fetal zone, previously considered to be the result of the presence of an endogenous inhibitor, is due to an absence of the protein in this portion of the gland. The lack or minimal expression of 3 beta HSD in the fetal zone of HFA may be due to the action (or lack thereof) of a tissue-specific factor regulating the synthesis of 3 beta HSD.

Light and electron microscopic immunocytochemistry on the localization of 3 beta-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells.

The localization of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3 beta-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3 beta-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3 beta-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.

Positioning of a spin-labeled substrate analogue into the structure of delta 5-3-ketosteroid isomerase by combined kinetic, magnetic resonance, and X-ray diffraction methods.

We have shown by kinetic and magnetic resonance measurements that a spin-labeled substrate analogue, spiro[doxyl-2,3'-5' alpha-androstan]-17'beta-ol, binds at the substrate site of crystalline delta 5-3-ketosteroid isomerase (steroid delta-isomerase; EC 5.3.3.1) of Pseudomonas testosteroni. The spin-labeled steroid is a linear competitive inhibitor with a Ki value (25 +/- 5 microM) that is consistent with dissociation constants obtained by direct binding measurements based on changes in the electron paramagnetic resonance spectrum of the nitroxide, longitudinal relaxation rates of water protons, and longitudinal and transverse relaxation rates of carbon-bound protons of the isomerase. These binding studies yield a stoichiometry for the nitroxide of 1 per subunit of the enzyme. Measurements of the longitudinal relaxation rates of water protons indicate that the 3-doxyl portion of the spin-label is highly immobilized yet is exposed to solvent. Paramagnetic effects of the nitroxide on T1 defined distances to several previously assigned [Benisek, W. F., & Ogez, J. R. (1982) Biochemistry 21, 5816-5825] and newly assigned protons of the enzyme. These distances were then used to locate (with an accuracy of +/- 2 A) the nitroxide moiety at a unique position in a partially refined 2.5-A resolution X-ray structure of native isomerase. Three of five additional proton resonance peaks, attributed to ring-shielded methyl groups, could be assigned to specific residues on the basis of distances from the spin-label in the X-ray structure. The remaining portion of the spin-labeled steroid was then docked into the X-ray structure in a hydrophobic cavity of the enzyme. This position of the steroid is consistent with the steroid binding site previously proposed [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103]. However, the rotational orientation of this steroid about its long axis could not be unambiguously established. If we assume that steroid substrates and the spin-labeled inhibitor bind to the same site, but with reversal of the 3- and 17-positions, then the phenolic hydroxyl of Tyr-55 is optimally positioned to function as the general acid that protonates the 3-keto group of the substrate, facilitated by the negative end of the dipole of a 10-residue alpha-helix, the only helix in the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)