Immunolocalization of cytochrome P450 17alpha-hydroxylase/c17-20 lyase in the ovary of pregnant pigs and fetal gonads

The study was designed to localize P450 17alpha-hydroxylase/c17-20 lyase (P450c17) in the ovaries of pregnant pigs and fetal gonads. Immunoexpression of P450c17 was investigated in the porcine ovaries (follicles and corpora lutea; CL) collected on days 10, 18, 32, 50, 70 and 90 post coitum (p.c.), and fetal gonads (testes and ovaries) on days 50, 70 and 90 p.c. The presence of P450c17 in ovarian follicles was demonstrated on all examined days of pregnancy but was restricted to theca interna cells. In CL, P450c17 was detected on all examined days of pregnancy but only in small luteal cells. In the female porcine fetuses, P450c17 immunostaining was found in oocyte nests and granulosa cells of primary ovarian follicles, while in the male fetuses in fetal Leydig cells. In conclusion, the immunolocalization of P450c17, detected in the ovaries of pregnant pigs and fetal porcine gonads, indicates the potential sites of androgen synthesis. We suggest that androgens may play a role in the maintenance of pregnancy and in the development of prenatal gonads in pigs.     

Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: Immunohistochemical expression of 17α-hydroxylase/17,20-lyase cytochrome P450

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries.     

Immunolocalization of steroidogenic enzymes in the fetal, neonatal and adult testis of the shiba goat.

The localizations of steroidogenic enzymes (P450scc, 3betaHSD, P450c17 and P450arom) in testes of Shiba goats were investigated by immunohistochemistry. P450scc, 3betaHSD, P450c17 and P450arom were detected in all Leydig cells of adults. P450scc and P450c17 were observed in most Leydig cells in the fetus (90 days) and neonate (15 days). 3betaHSD and P450arom were found in some Leydig cells of the fetus with weak immunostaining but the numbers of immunopositive Leydig cells and intense immunostaining were increased in Leydig cells of the neonate. These results suggest that Shiba goat testes have the ability to synthesize progestin, androgen and estrogen in the fetus, neonate and adult, and synthesis of these steroid hormones showed an age-related rise.     

Adrenarche: postnatal adrenal zonation and hormonal and metabolic regulation

Adrenarche is the direct consequence of the organogenesis of the zona reticularis (ZR). Proliferation of cortical cells could take place in the outermost layers of the adrenal cortex. Cells could then migrate to differentiate the zona glomerulosa (ZG) and zona fasciculata (ZF) during fetal life, and the ZR during postnatal life. After adrenarche, there are detectable increases in circulating DHEA and DHEA-S. Adrenarche could result from an increase in 17,20-lyase activity of P450c17 secondary to high levels of cytochrome b(5) expression, and from a decrease in 3betaHSD2 expression along with an increase in the expression of SULT2A1 in the ZR. The GH-IGF system and insulin, among other factors, might also modulate adrenal androgen production. Furthermore, high concentrations of estradiol enhance basal and ACTH-stimulated DHEA-S production, while aromatase expression was observed in the human adrenal medulla but not in the ZR, suggesting that estrogens produced in the adrenal medulla might be involved in the regulation of androgen production in the ZR. Premature adrenarche might be associated with ovarian hyperandrogenism and polycystic ovarian syndrome in females, as well as with insulin resistance in both sexes. However, many questions remain, transforming adrenal androgens into markers of diseases important for human health.     

Biochemical assessment of limits to estrogen synthesis in porcine follicles

Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b5, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17 alpha-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b5, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b5 expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b5, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b5 appear to regulate P450 activities, but the expression of cytochrome b5 in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.     

Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).

Cytochrome P-45017 alpha catalyzes both 17 alpha-hydroxylation and 17,20-side-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P-45017 alpha in swine testis, ovary, and adrenal, we undertook the simultaneous detection of P-45017 alpha mRNA and protein by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 KB) of porcine testis P-45017 alpha as DNA probe. Immunohistochemical study was performed by employing anti-P-45017 alpha. Hybridization signals were obtained in Leydig cells of the testis, theca interna of the ovarian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the cDNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulosa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P-45017 alpha expression at both the protein and mRNA levels in swine adrenal glands and gonads. This approach of simultaneous immunohistochemistry and in situ hybridization analysis of steroidogenic enzymes can be applied in the future to tissues exhibiting abnormal steroid metabolism and should contribute to a better understanding of steroidogenesis.     

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.     

Assessment of the ability of type 2 cytochrome b5 to modulate 17,20-lyase activity of human P450c17

The 17 alpha-hydroxylase and 17,20-lyase activities of P450c17 lead to the production of 17 alpha-hydroxypregnenolone (17 alpha-OH-Preg) and dehydroepiandrosterone (DHEA), respectively, in different tissues. The mechanisms of differential regulation of these two activities are not yet fully elucidated. It has been previously shown that cytochrome b5 (cyt-b5) could facilitate the 17,20-lyase activity of human P450c17. Recently, a cDNA (type 2 cyt-b5) sharing 45.8% homology with type 1 cyt-b5 has been isolated from human testis. Since high 17,20-lyase activity is required for the production of androgens in the testis, we wanted to determine the importance of this second cDNA in the modulation of P450c17 17,20-lyase activity and hence, its role in the formation of active androgens. We therefore isolated type 2 cyt-b5 from human testis by RT-PCR and analyzed, by transient transfection in transformed human embryonic kidney cells (HEK-293) of various amounts of vectors expressing cyt-b5, P450-reductase and P450c17, its ability to modulate the 17,20-lyase activity of human P450c17. Results show that, in the presence of NADPH cytochrome P450 reductase (P450-red), type 2 cyt-b5 increases 17,20-lyase activity to a level comparable to that of type 1. These results support the idea that types 1 and 2 cyt-b5 could be involved in the differential modulation of 17 alpha-hydroxylase and 17,20-lyase activities of P450c17. Furthermore, the analysis of mRNA expression of types 1 and 2 cyt-b5 by RT-PCR using primers specific to each type showed that both types are present in the liver but also in the adrenal and testis.     

Protein phosphatase 2A and phosphoprotein SET regulate androgen production by P450c17

Cytochrome P450c17 catalyzes 17 alpha-hydroxylation needed for cortisol synthesis and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of P450c17 specifically increases 17,20 lyase activity, but the physiological factors regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin, and cantharidin increased 17,20 lyase activity, suggesting involvement of protein phosphatase 2A (PP2A) or 4 (PP4). PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells, but neither affected 17 alpha-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was concentration-dependent, could be inhibited by okadaic acid, and was restored by endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17, and suppression of PP2A by small interfering RNA increased 17,20 lyase activity. Phosphoprotein SET found in adrenals inhibited PP2A, but not PP4, and fostered 17,20 lyase activity. The identification of PP2A and SET as post-translational regulators of androgen biosynthesis suggests potential additional mechanisms contributing to adrenarche and hyperandrogenic disorders such as polycystic ovary syndrome.     

Down-regulation of steroidogenic cytochrome P450XVII in cryptorchid rat testes

The objective of the present study was to investigate the regulation of a key component of testicular androgen biosynthesis, i.e. the cytochrome P450XVII of the steroid-17 alpha-monooxygenase/C17,20-lyase, after surgical induction of bilateral cryptorchidism in vivo. Seven days after induction of cryptorchidism, P450XVII concentrations are diminished (as compared to sham-operated controls) by 64% in isolated purified Leydig cells but only by 44% in the total Leydig cell compartment of the testis, since the Leydig cell yield from cryptorchid testes is by 53% higher than that from control testes. Using microsomal suspensions prepared from testicular homogenates, P450XVII content per testis equivalent is found to be decreased by 36% seven days after incubation of cryptorchidism, whereas the P450XVII concentration per gram testis is not changed due to testicular involution. Fourteen days after induction of cryptorchidism, the induction of the Leydig cell system appears to superimpose on the down-regulation of P450XVII. The study demonstrates both a strong sensitivity of P450XVII to short-term elevation of testicular temperature and a differentiation between effects of cryptorchidism on total testicular content and specific cellular and subcellular concentration of this steroidogenic protein.     

Immunohistochemical evidence: testicular and scented glandular androgen synthesis in muskrats (Ondatra zibethicus) during the breeding season

In order to elucidate the relationship between androgens and the function of the muskrat (Ondatra zibethicus) scented glands during the breeding season, we investigated immunolocalization of steroidogenic enzymes P450scc, 3βHSD and P450c17 in the muskrat testes and scented glands. Nine adult muskrats were obtained in March (n=3), May (n=3) and July (n=3) 2010. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal P450scc, human placental 3βHSD and porcine testicular P450c17. Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes. Glandular cells, interstitial cells, epithelial cells and excretory tubules were identified in scented glands during the breeding season. P450scc, 3βHSD and P450c17 were only identified in Leydig cells during the breeding season; P450scc and P450c17 were observed in glandular cells of scented glands, however, 3βHSD was not found in scented glands during the breeding season. These novel findings provide the first evidence showing that scented glands of the muskrats are capable of locally synthesizing androgens and androgens acting via an endocrine, autocrine or paracrine manner may play an important role in scented gland function during the breeding season.     

Phosphorylation of Human Cytochrome P450c17 by p38α Selectively Increases 17,20 Lyase Activity and Androgen Biosynthesis

Cytochrome P450c17, a steroidogenic enzyme encoded by the CYP17A1 gene, catalyzes the steroid 17α-hydroxylation needed for glucocorticoid synthesis, which may or may not be followed by 17,20 lyase activity needed for sex steroid synthesis. Whether or not P450c17 catalyzes 17,20 lyase activity is determined by three post-translational mechanisms influencing availability of reducing equivalents donated by P450 oxidoreductase (POR). These are increased amounts of POR, the allosteric action of cytochrome b₅ to promote POR-P450c17 interaction, and Ser/Thr phosphorylation of P450c17, which also appears to promote POR-P450c17 interaction. The kinase(s) that phosphorylates P450c17 is unknown. In a series of kinase inhibition experiments, the pyridinyl imidazole drugs SB202190 and SB203580 inhibited 17,20 lyase but not 17α-hydroxylase activity in human adrenocortical HCI-H295A cells, suggesting an action on p38α or p38β. Co-transfection of non-steroidogenic COS-1 cells with P450c17 and p38 expression vectors showed that p38α, but not p38β, conferred 17,20 lyase activity on P450c17. Antiserum to P450c17 co-immunoprecipitated P450c17 and both p38 isoforms; however, knockdown of p38α, but not knockdown of p38β, inhibited 17,20 lyase activity in NCI-H295A cells. Bacterially expressed human P450c17 was phosphorylated by p38α in vitro at a non-canonical site, conferring increased 17,20 lyase activity. This phosphorylation increased the maximum velocity, but not the Michaelis constant, of the 17,20 lyase reaction. p38α phosphorylates P450c17 in a fashion that confers increased 17,20 lyase activity, implying that the production of adrenal androgens (adrenarche) is a regulated event.     

Changes in Leydig cell gene expression during development in the mouse

Developmental changes in the expression of 18 Leydig cell-specific mRNA species were measured by real-time polymerase chain reaction to partially characterize the developmental phenotype of the cells in the mouse and to identify markers of adult Leydig cell differentiation. Testicular interstitial webs were isolated from mice between birth and adulthood. Five developmental patterns of gene expression were observed. Group 1 contained mRNA species encoding P450 side chain cleavage (P450(scc)), P450(c17), relaxin-like factor (RLF), glutathione S-transferase 5-5 (GST5-5), StAR protein, LH receptor, and epoxide hydrolase (EH); group 2 contained 3beta-hydroxysteroid dehydrogenase (3beta-HSD) VI, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) III, vascular cell adhesion molecule 1, estrogen sulfotransferase, and prostaglandin D (PGD)-synthetase; group 3 contained patched and thrombospondin 2 (TSP2); group 4 contained 5alpha-reductase 1 and 3alpha-hydroxysteroid dehydrogenase; group 5 contained sulfonylurea receptor 2 and 3beta-HSD I. Group 1 contained genes that were expressed in fetal and adult Leydig cells and which increased in expression around puberty toward a maximum in the adult. Group 2 contained genes expressed only in the adult Leydig cell population. Group 3 contained genes with predominant fetal/neonatal expression in the interstitial tissue. Group 4 contained genes with a peak of expression around puberty, whereas genes in group 5 show little developmental change in expression. Highest mRNA levels in descending order were RLF, P450(c17), EH, 17beta-HSD III, PGD-synthetase, GST5-5, and P450(scc). Results identify five genes expressed in the mouse adult Leydig cell population, but not in the fetal population, and one gene (TSP2) that may be expressed only in the fetal Leydig cell population. The developmental pattern of gene expression suggests that three distinct phases of adult Leydig cell differentiation occur.     

Deletion of the mouse P450c17 gene causes early embryonic lethality

Dehydroepiandrosterone (DHEA), a 19-carbon precursor of sex steroids, is abundantly produced in the human but not the mouse adrenal. However, mice produce DHEA and DHEA-sulfate (DHEAS) in the fetal brain. DHEA stimulates axonal growth from specific populations of mouse neocortical neurons in vitro, while DHEAS stimulates dendritic growth from those cells. The synthesis of DHEA and sex steroids, but not mouse glucocorticoids and mineralocorticoids, requires P450c17, which catalyzes both 17 alpha-hydroxylase and 17,20-lyase activities. We hypothesized that P450c17-knockout mice would have disordered sex steroid synthesis and disordered brain DHEA production and thus provide phenotypic clues about the functions of DHEA in mouse brain development. We deleted the mouse P450c17 gene in 127/SvJ mice and obtained several lines of mice from two lines of targeted embryonic stem cells. Heterozygotes were phenotypically and reproductively normal, but in all mouse lines, P450c17(-/-) zygotes died by embryonic day 7, prior to gastrulation. The cause of this early lethality is unknown, as there is no known function of fetal steroids at embryonic day 7. Immunocytochemistry identified P450c17 in embryonic endoderm in E7 wild-type and heterozygous embryos, but its function in these cells is unknown. Enzyme assays of wild-type embryos showed a rapid rise in 17-hydroxylase activity between E6 and E7 and the presence of C(17,20)-lyase activity at E7. Treatment of pregnant females with subcutaneous pellets releasing DHEA or 17-OH pregnenolone at a constant rate failed to rescue P450c17(-/-) fetuses. Treatment of normal pregnant females with pellets releasing pregnenolone or progesterone did not cause fetal demise. These data suggest that steroid products of P450c17 have heretofore-unknown essential functions in early embryonic mouse development.     

Gonadotropin induction of a regulatory mechanism of steroidogenesis in fetal Leydig cell cultures

Studies were conducted to define further the development of the gonadotropin induced, E2 mediated steroidogenic lesion (17-alpha-hydroxylase/17,20-desmolase) in fetal Leydig cell cultures. Analysis of dispersed fetal testes purified by centrifugal elutriation demonstrated a group of cells with sedimentation velocity 12 less than to less than 16.8 mm/h.g containing a small population of adult like "transitional" Leydig cells and homogeneous "fetal" Leydig cell population collected at greater than 19.3 mm/h.g. After cells were cultured for 3 days with addition of 1 microgram oLH at 3 day intervals, the transitional cells showed testosterone accumulation comparable to the fetal cells. In contrast, transitional cells had 10-fold higher basal and hCG-stimulated aromatase activity than fetal cells, and a lack of testosterone response to acute (3 h) hCG stimulation. At day 6, transitional cells steroidogenic ability declined markedly. The fetal population maintained in culture with LH additions every 3 days, showed typical immature Leydig cell response, with enhancement of acute testosterone response to hCG at 3 day (1-fold) and at 6 day of culture (5-fold). Higher doses of LH (5 micrograms/day) or daily treatment of 1 microgram to fetal cultures, induced a lesion of 17 alpha-hydroxylase/17,20-desmolase with reduction of enzymatic activities (P less than 0.01) and impaired testosterone production (P less than 0.01) in response to acute hCG stimulation. Also aromatase was stimulated by hCG + 140% and 50% and E2 receptors were increased by 100 and 180% at 3 days and 6 days of cultures with daily or high dose LH addition, findings consistent with the observation of the E2-mediated lesion during LH action. In conclusion, the cultured fetal Leydig cell provides a useful model to elucidate molecular mechanisms involved in the development of gonadotropin-induced estradiol-mediated desensitization. Treatment of fetal Leydig cell cultures with multiple or frequent doses of LH elevate aromatase activity to necessary levels for the induction of desensitization. We have isolated small population of transitional Leydig cells with morphological characteristics of cells found in 15 day post-natal testis but functional capabilities of adult cells. We have also demonstrated the emergence of a functional adult-like population from the fetal Leydig cell.