The pineal gland, but not melatonin, is associated with the termination of seasonal testicular activity in an annual reproductive cycle in roseringed parakeet Psittacula krameri

The role of the pineal gland and its hormone melatonin in the regulation of annual testicular events was investigated for the first time in a psittacine bird, the roseringed parakeet (Psittacula krameri). Accordingly, the testicular responsiveness of the birds was evaluated following surgical pinealectomy with or without the exogenous administration of melatonin and the experimental manipulations of the endogenous levels of melatonin through exposing the birds to continuous illumination. An identical schedule was followed during the four reproductive phases, each characterizing a distinct testicular status in the annual cycle, namely, the phases of gametogenic quiescence (preparatory phase), seasonal recovery of gametogenesis (progressive phase), seasonal initiation of sperm formation (pre-breeding phase), and peak gametogenic activity (breeding phase). In each reproductive phase, the birds were subjected to various experimental conditions, and the effects were studied comparing the testicular conditions in the respective control birds. The study included germ cell profiles of the seminiferous tubules, the activities of steroidogenic enzymes 17β-hydroxysteroid dehydrogenase (17β-HSD), and Δ⁵3β-hydroxysteroid dehydrogenase (Δ⁵3β- HSD) in the testis, and the serum levels of testosterone and melatonin. An analysis of the data reveals that the pineal gland and its hormone melatonin may play an inhibitory role in the development of the testis until the attainment of the seasonal peak in the annual reproductive cycle. However, in all probability, the termination of the seasonal activity of the testis or the initiation of testicular regression in the annual reproductive cycle appears to be the function of the pineal gland, but not of melatonin.     

Stress hormone and male reproductive function

The Leydig cell is the primary source of testosterone in males. Levels of testosterone in circulation are determined by the steroidogenic capacities of individual Leydig cells and the total numbers of Leydig cells per testis. Stress-induced increases in serum glucocorticoid concentrations inhibit testosterone-biosynthetic enzyme activity, leading to decreased rates of testosterone secretion. It is unclear, however, whether the excessive glucocorticoid stimulation also affects total Leydig cell numbers through induction of apoptosis and thereby contributes to the stress-induced suppression of androgen levels. Exposure of Leydig cells to high concentrations of corticosterone (CORT, the endogenously secreted glucocorticoid in rodents) increases their frequency of apoptosis. Studies of immobilization stress indicate that stress-induced increases in CORT are directly responsible for Leydig cell apoptosis. Access to glucocorticoid receptors in Leydig cells is modulated by oxidative inactivation of glucocorticoid by 11β-hydroxysteroid dehydrogenase (11βHSD). Under basal levels of glucocorticoid, sufficient levels of glucocorticoid metabolism occur and there is likely to be minimal binding of the glucocorticoid receptor. We have established that Leydig cells express type 1 11βHSD, an oxidoreductase, and type 2, a unidirectional oxidase. Generation of redox potential through synthesis of the enzyme cofactor NADPH, a byproduct of glucocorticoid metabolism by 11βHSD-1, may potentiate testosterone biosynthesis, as NADPH is the cofactor used by steroidogenic enzymes such as type 3 17β-hydroxysteroid dehydrogenase. In this scenario, inhibition of steroidogenesis will only occur under stressful conditions when high input amounts of CORT exceed the capacity of oxidative inaction by 11βHSD. Changes in autonomic catecholaminergic activity may contribute to suppressed Leydig cell function during stress, and may explain the rapid onset of inhibition. However, recent analysis of glucocorticoid action in Leydig cells indicates the presence of a fast, non-genomic pathway that will merit further investigation.     

Steroid metabolism in Antarctic soft corals

Whole body tissue preparations of the Antarctic soft corals Alcyonium paessleri and Clavularia frankliniana were incubated in vitro with the radiolabelled precursors ³H-progesterone and ³H-androstenedione to determine steroidogenic capacity. Steroidal metabolites were identified using TLC, derivitization, and recrystallization techniques. The Antarctic soft corals converted labelled precursors (³H-progesterone and ³H-androstenedione) into a maximum of five metabolites, potentially indicating the activity of the following enzymes: 5α-reductase, 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, and acyl transferase. Both species exhibited similar steroidogenic capacity. Radioimmunoassays verified the presence of relevant concentrations of progesterone, androstenedione, testosterone, and estradiol in whole body extracts from each species of soft coral. Alcyonium paessleri and Clavularia frankliniana actively converted precursors at temperatures up to 10°C above the ambient encountered by these species. Although similar steroidal compounds are produced in other phyla of benthic invertebrates, conversion rates for these soft corals are substantially lower. The role of these steroids is as yet unidentified; however they may be related to reproduction, and be important in chemical signaling or as defensive metabolites, or they may serve as transient intermediates to the production of other bioactive derivatives. Received: 2 September 1996 / Accepted: 10 January 1997     

Exposure to high fluoride concentration in drinking water will affect spermatogenesis and steroidogenesis in male albino rats

Sodium fluoride (NaF) administered orally to adult male rats at a dose level of 4.5 ppm and 9.0 ppm for 75 days caused significant decrease in the body weight, brain index and testicular index. A significant decrease in sperm count, sperm motility, sperm viability and sperm function (HOS positive) with increased sperm abnormalities was also observed in NaF-exposed male rats. The activity levels of testicular steroidogenic marker enzymes 3-hydroxysteroid dehydrogenase (3-HSD) and 17-hydroxysteroid dehydrogenase (17-HSD) were significantly decreased in NaF-treated rats indicating decreased steroidogenesis and in turn spermatogenesis in rats exposed to NaF.     

Δ5-3β-Hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. – a multifunctional enzyme in steroid metabolism?

Δ⁵-3β-Ηydroxysteroid dehydrogenase (Δ⁵-3β-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3β-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Δ⁵-3β-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Δ⁵-3β-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed. Received: 20 January 1999 / Accepted: 5 May 1999     

Expression,Purification, and Characterization of a Human Recombinant 17β-Hydroxysteroid Dehydrogenase Type 1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reaction of estrone with NADPH to form estradiol and NADP⁺, thereby regulating the biological activity of sex steroid hormones in a variety of tissues. Here, we present an efficient method for expressing and purifying human 17β-HSD1 from Escherichia coli. The expression vector pET28a/17β-HSD1 was constructed and transformed into Escherichia coli BL21(DE3) cells. We found that the active enzyme can be obtained by inducing 17β-HSD1 expression at 0.25 mM IPTG, 13°C for overnight. The protein is purified by single step Ni–NTA affinity chromatography and yields 2.8 mg/L of culture. The kinetic study shows V/E _t of (1.21 ± 0.05) × 10⁻²/s and K _estradiol of 0.8 μM in the oxidation of estradiol with NADP⁺ as cofactor at pH 9.3. The new bacterial expression system for recombinant 17β-HSD1 is useful for the easy purification of large amounts and will facilitate the functional study of this enzyme.     

Immunolocalization of steroidogenic enzymes in gonads of European eel, Anguilla anguilla (L.)

The localization of cytochrome P450 cholesterol side-chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase (P450arom) was investigated using polyclonal antibodies during gonad development in wild European eels, Anguilla anguilla (L.), from the River Po Delta (Ferrara, Italy). The first steroidogenic cells, observed in undifferentiated gonads of 14–16 cm yellow eels, showed no P450scc, 3β-HSD or P450arom activity, but positive regions appeared in head kidney insulae from this stage until the silver eel stage. In undifferentiated gonads of 16–20 cm yellow eels the steroidogenic cells were positive to all enzymes. Pre-Leydig steroidogenic cells, identified in Syrski organs of yellow eels of 22–26 cm evolving into testes, were positive to 3β-HSD and P450scc, but negative to P450arom. However, steroidogenic cells in Syrski organs evolving towards ovaries and in small but fully differentiated ovaries were positive to all enzymes. Immature testes of yellow and silver eels had Leydig cells positive to P450scc and 3β-HSD; the same reactions were also observed in some Sertoli cells of silver eel testes containing meiotic cells. Sex differentiation in A. anguilla apparently occurs through an initial female stage controlled by P450arom activity. Leydig and Sertoli cells appear involved in different steps of hormonal control of spermatogenesis: Leydig cells begin their steroidogenic activity before meiosis, while Sertoli cells begin their activity during meiosis.     

Specific neural phase relation of serotonin and dopamine modulate the testicular activity in Japanese quail

Specific phase relation of serotonin and dopamine modulate the hypothalamo–hypophyseal–gonadal axis as well as photosexual responses in Japanese quail, but the effect of these specific phase relations on testicular activity and steroidogenesis is not yet been investigated. We hypothesized that temporal phase relation induced alteration in local testicular gonadotropin-releasing hormone (GnRH)–Gonadotropin-inhibitory hormone (GnIH) and their receptor system may modulate the testicular activity and steroidogenesis through local (paracrine and autocrine) action. To validate this hypothesis, we have checked the alterations in the expression of gonadotropin-releasing hormone receptor (GnRH-R), gonadotropin-inhibitory hormone receptor (GnIH-R) messenger RNA (mRNA), growth hormone receptor (GH-R), proliferating cell nuclear antigen (PCNA), cell communication and gap junctional proteins (14-3-3 and connexin-43 [Cnx-43]), steroidogenic factor-1 (SF-1), steroidogenic acute regulatory (StAR) protein, steroidogenic enzyme (3β-hydroxysteroid dehydrogenase [3β-HSD]) in testis as well as androgen receptor (AR) in testis and epididymis of control, 8-, and 12-hr quail. Experimental findings clearly indicate the increased expression of GnIH-R mRNA and suppression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3β-HSD in testis as well as AR in testis and epididymis in 8-hr quail, while 12-hr quail exhibited the opposite results that is significantly decreased expression of GnIH-R mRNA and increased expression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3β-HSD in testis as well as AR in testis and epididymis. The significantly increased intratesticular testosterone has been observed in the 12-hr quail while, 8-hr quail showed opposite result. Hence, it can be concluded that 12-hr quail showed significantly increased testicular activity and steroidogenesis while opposite pattern was observed in 8-hr quail.     

Histochemical localization of 3 - and 17 -hydroxysteroid dehydrogenases in the male reprodutive tract of the domestic fowl (Gallus domesticus)

Synopsis 3- and 17-hydroxysteroid dehydrogenase activities were studied histochemically in the male reproductive tract of the domestic fowl. 3-hydroxysteroid dehydrogenase was NAD⁺-linked and was capable of metabolizing the three substrates used, namely, pregnenolone, 17-hydroxypregnenolone and dehydroepiandrosterone. 17-hydroxysteroid dehydrogenase oxidized testosterone in the presence of NAD⁺ or NADP⁺. The pattern of distribution of formazan granules was essentially the same with all the substrates used, and they were located in the Leydig cells, seminiferous tubules and the lining epithelia of the entire excurrent duct system of the testis except the rete testis. The activity of both enzymes appeared to be highest in the ductuli efferentes and decreased distally along the tract. The evidence suggests that steroid synthesis may occur in the epithelial lining of the excurrent ducts as well as in the cells of the testis.     

Rapid estrogen regulation of DHEA metabolism in the male and female songbird brain

In the songbird brain, dehydroepiandrosterone (DHEA) is metabolized to the active and aromatizable androgen androstenedione (AE) by 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD). Thus, brain 3β-HSD plays a key role in regulating the steroidal milieu of the nervous system. Previous studies have shown that stress rapidly regulates brain 3β-HSD activity in a sex-specific manner. To elucidate endocrine regulation of brain 3β-HSD, we asked whether 17β-estradiol (E₂) regulates DHEA metabolism in adult zebra finch ( Taeniopygia guttata ) and whether there are sex-specific effects. Brain tissue was homogenized and centrifuged to obtain supernatant lacking whole cells and cell nuclei. Supernatant was incubated with [³H]DHEA and radioinert E₂ in vitro . Within only 10 min, E₂ significantly reduced 3β-HSD activity in both male and female brain. Interestingly, the rapid effects of E₂ were more pronounced in females than males. These are the first data to show a rapid effect of estrogens on the songbird brain and suggest that rapid estrogen effects differ between male and female brains.     

THE OCCURRENCE OF HISTOCHEMICAL ACTIVITY OF 3β-HYDROXYSTEROID DEHYDROGENASE IN THE DEVELOPING TESTES OF POECILIA RETICULATA

In the mature testes of the guppy, Poecilia reticulata , some groups of cells, distributed sparsely in the interspace between the peripheral germ cell layer and the hilar duct system, show evident histochemical response for Δ⁵-3β-hydroxysteroid dehydrogenase (3β-HSD). In the testis of newly delivered guppies, somatic cells are present in the testicular hilus as a compact mass without revealing any structural differentiation. In the testis of juvenile fish of the 8mm stage about 7 days after birth, interstitial cells resembling histologically those of adult testes become differentiated from the somatic cell mass and, though only in some specimens, coincidentally begin to display weak but obvious histochemical response for 3β-HSD. Thereafter the occurrence of enzyme activity becomes increasingly regular in the developing testes, and attains the adult pattern of distribution in testes of all specimens after the 11 13mm stage or 17 ∽ 20 days of age.
The appearance and enhancement of 3β-HSD activity in the testis is concurrent with the differentiation and development of the testicular duct system. Treatments of newly delivered fish with methyltestosterone (30 ∽ 50 μg/g diet) distinctly stimulate the development of the duct system, which suggests a possible role of androgen secretion occurring in the early phase of the testicular development in the control of testicular organogenesis in the guppy.     

A study on the 3β- and 17β-hydroxysteroid dehydrogenase activities in the gonads of Monopterus albus (Pisces: Teleostei) at various sexual phases during natural sex reversal

Activities of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in Monopterus gonads were studied at different sexual phases during natural sex reversal. Before sexual transformation, positive reactions for 3β-HSD in the follicular epithelium were found in the granulosa cells of some large, maturing follicles in some females during the breeding season. Weak reaction for this enzyme was also detected in some scattered interstitial cells found occasionally in some ovaries. At the intersexual and the male phases, intense 3β-HSD activities were demonstrated exclusively in the interstitial Leydig cells. No 17β-HSD activities were observable in the gonads at any stage of development. The reaction intensity of 3β-HSD in the interstitial cells exhibited a marked increase during the process of sex change from female to the intersexual and the male phases and there is a definite correlation with the density and nuclear size of these cells. It is concluded that in Monopterus , the granulosa cells in the ovary and the interstitial cells of the intersexual and male gonads are the major sites for the biosynthesis of oestrogens and androgens, respectively, and that the intensive development of interstitial tissue with increasing steroidogenic enzyme activities at the intersexual and male phases was directly related to the increase in androgen production in vitro reported previously. The occasional presence of some 3β-HSD positive interstitial cells in the ovary suggests that interstitial cell development might precede testicular lobule formation during natural sex reversal.     

The Pineal Gland,but Not Melatonin,Is Associated with the Termination of Seasonal Testicular Activity in an Annual Reproductive Cycle in Roseringed Parakeet Psittacula krameri

The role of the pineal gland and its hormone melatonin in the regulation of annual testicular events was investigated for the first time in a psittacine bird, the roseringed parakeet (Psittacula krameri). Accordingly, the testicular responsiveness of the birds was evaluated following surgical pinealectomy with or without the exogenous administration of melatonin and the experimental manipulations of the endogenous levels of melatonin through exposing the birds to continuous illumination. An identical schedule was followed during the four reproductive phases, each characterizing a distinct testicular status in the annual cycle, namely, the phases of gametogenic quiescence (preparatory phase), seasonal recovery of gametogenesis (progressive phase), seasonal initiation of sperm formation (pre‐breeding phase), and peak gametogenic activity (breeding phase). In each reproductive phase, the birds were subjected to various experimental conditions, and the effects were studied comparing the testicular conditions in the respective control birds. The study included germ cell profiles of the seminiferous tubules, the activities of steroidogenic enzymes 17β‐hydroxysteroid dehydrogenase (17β‐HSD), and Δ⁵3β‐hydroxysteroid dehydrogenase (Δ⁵3β‐ HSD) in the testis, and the serum levels of testosterone and melatonin. An analysis of the data reveals that the pineal gland and its hormone melatonin may play an inhibitory role in the development of the testis until the attainment of the seasonal peak in the annual reproductive cycle. However, in all probability, the termination of the seasonal activity of the testis or the initiation of testicular regression in the annual reproductive cycle appears to be the function of the pineal gland, but not of melatonin.     

Developmental pattern of Δ5 3β-hydroxysteroid dehydrogenase activity in isolated rat Leydig cells

A direct method for determination of Δ⁵ 3β-hydroxysteroid dehydrogenase (3β-HSD) activity was employed in isolated Leydig cells (LC) derived from rats on fetal day 19 (F₁₉) and postnatal (N) days 1,12,24, 34 and 45 and adults. The activity of 3β-HSD in the adult LC was 1.15 ± 0.02 (μmole/μg DNA/hr, mean ± SEM, n = 73). Activities in the other groups, expressed as a percentage of the respective adult control, were: F₁₉-38%; N₁-39%; N₁₂-8%; N₂₄-89%; N₃₄-166%; and N₄₅-118%. A good correlation was found between histochemical staining for 3β-HSD and the quantitive method employed. Using (³H)-DHA as a substrate, LC isolated from F₁₉, n₁ and N₁₂ produced testosterone in appreciable amounts (41%, 55% and 20% of the toal products respectively) whereas at advanced stages of development (N₂₄ to adulthood) the major product was androstenedione (93 ± 1%). These findings may be explained by the observed decrease in 17β-hydroxysteroid dehydrogenase (17β-HSD) activity, due to an insufficient supply of NADPH, in the older vs. earlier stages of development. This study indicates the presence of steroidogenic enzymatic activity in LC throughout development in the rat. It also provides a relatively simple in vitro model for studies of testicular regulation during development.     

Immunocytochemical localization of 17β-hydroxysteroid dehydrogenase in porcine testis

Summary The immunocytochemical localization of 17-hydroxysteroid dehydrogenase (17-HSD) in porcine testes was examined by applying an indirect-immunofluorescence method using an antiporcine testicular 17-HSD antibody. Only the Leydig cells located in the interstitial tissue exhibited a positive immunoreaction for 17-HSD: the germ cells and Sertoli cells located in the seminiferous tubules were entirely negative. These results suggest that, in porcine testis, the biosynthesis of testicular testosterone, the final step of which is the conversion of androstenedione to testosterone, takes place in the Leydig cells.Supported by grants from the Ministry of Education, Science, and Culture, Japan     

11β-HSD1, Inflammation,Metabolic Disease and Age-related Cognitive (dys)Function

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an intracellular amplifier of glucocorticoid action. By converting intrinsically inert glucocorticoids (cortisone, 11-dehydrocorticosterone) into their active forms (cortisol, corticosterone), 11β-HSD1 increases glucocorticoid access to receptors. Glucocorticoid hormones modulate diverse physiological processes, linking circadian rhythms to food seeking, motivational and cognitive behaviours, as well as intermediary metabolism and immune responses. They are a key component of pathways that buffer the organism against stressful challenges. Here we review the part played in these processes by 11β-HSD1, and discuss the promise of inhibitors of 11β-HSD1 in alleviating disorders associated with cumulative stress. Special issue article in honor of George Fink.     

The role of phosphoenolpyruvate carboxykinase in neuronal steroidogenesis under acute inflammation

Phosphoenolpyruvate carboxykinase (PEPCK) is a key gluconeogenic enzyme found in many tissues throughout the body including brain. In the present study, we have investigated the effect of bacterial lipopolysaccharide (LPS) on PEPCK and its role in neuronal steroidogenesis. Adult female albino rats were administered LPS (5 mg/kg body weight) to induce acute inflammation. LPS administration resulted in a significant increase of PEPCK mRNA expression with concomitant increase in mRNA levels of steroidogenic acute regulatory (StAR) protein and other steroidogenic enzymes including 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and aromatase in brain tissue. Further, the inhibition of PEPCK expression by glipizide significantly decreased the mRNA expression of steroidogenic proteins and concurrently increased the mRNA levels of proinflammatory cytokines under LPS administration. The results of this study suggest a novel finding that PEPCK may have an important role in neuronal steroidogenesis; which serves as an adaptive response under inflammation.     

Altered testicular microsomal steroidogenic enzyme activities in rats with lifetime exposure to soy isoflavones

Androgen production in the testis is carried out by the Leydig cells, which convert cholesterol into androgens. Previously, isoflavones have been shown to affect serum androgen levels and steroidogenic enzyme activities. In this study, the effects of lifelong exposure to dietary soy isoflavones on testicular microsomal steroidogenic enzyme activities were examined in the rat. F1 male rats were obtained from a multi-generational study where the parental generation was fed diets containing alcohol-washed soy protein supplemented with increasing amounts of Novasoy, a commercially available isoflavone supplement. A control group was maintained on a soy-free casein protein-based diet (AIN93G). The diets were designed to approximate human consumption levels and ranged from 0 to 1046.6 mg isoflavones/kg pelleted feed, encompassing exposures representative of North American and Asian diets as well as infant fed soy-based formula. Activities of testicular 3β-hydroxysteroid dehydrogenase (3β-HSD), P450c17 (CYP17), 17β-hydroxysteroid dehydrogenase (17β-HSD) were assayed on post natal day (PND) 28, 70, 120, 240 and 360 while 5-reducatase was assayed on PND 28. At PND 28, 3β-HSD activity was elevated by approximately 50% in rats receiving 1046.6 mg total isoflavones/kg feed compared to those on the casein only diet. A similar increase in activity was observed for CYP17 in rats receiving 235.6 mg total isoflavones/kg feed, a level representative of infant exposure through formula, compared to those receiving 0 mg isoflavones from the casein diet. These results demonstrate that rats fed a mixture of dietary soy isoflavones showed significantly altered enzyme activity profiles during development at PND 28 as a result of early exposure to isoflavones at levels obtainable by humans.     

Estradiol Secretion by Tadpole Ovaries Masculinized by Implanted Capsules of Cyanoketone

Female tadpoles of Rana catesbeiana were laparotomized at metamorphic stages XI-XIII and an empty capsule or one containing cyanoketone (CK), which is an inhibitor of Δ⁵-3β-hydroxysteroid dehydrogenase (Δ⁵-3β-HSD), was implanted intraperitoneally. Ovarian activity of Δ⁵-3β-HSD was examined histochemically 2 months later, estradiol-17β (E₂) secretion by the ovaries was measured by RIA 4 months later and histological changes of the ovaries were examined 6 months later. The Δ⁵-3β-HSD activity of the CK-treated ovaries was much lower than that of controls. E₂ secretion per froglet by CK-treated ovaries was about one third that of controls (p<0.001). Histological examination showed various degrees of masculinization of the ovaries, about 28% of which were totally transformed into testis-like structures.
As a result of suppressed Δ⁵-3β-HSD activity, dehydroepiandrosterone would have accumulated, resulting in deficient E₂ secretion and, therefore, ovarian masculinization. In tadpoles, this effect does not depend on the pituitary, whereas interrenal hyperplasia and hyperactivity do, indicating that interrenal function is not essential for ovarian masculinization. From these findings and our previous results, we suggest that disturbance of steroidogenesis by CK in the ovaries results in their masculinization.