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Endocrine-disrupting chemicals (EDC) are linked to human health and diseases as they mimic or block the normal functioning of endogenous hormones. The present work dealt with a comparative study of the androgenic potential of wastewater treatment plant (WWTP) influents and effluents in Northern region of India, well known for its polluted water. Water samples were screened for their androgenic potential using the Hershberger assay and when they were found positive for androgenicity, we studied their mode of action in intact rats. The data showed a significant change in the weight and structure of sex accessory tissues (SATs) of castrated and intact rats. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis demonstrated a significant change in the expression patterns of the major steroidogenic enzymes in adrenal and testis: cytochrome P450(SCC), cytochrome P450(C17), 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase. This was further supported by increased enzymatic activities measured in vitro spectrophotometrically. Serum hormone profile showed a decreased level of gonadotrophic hormones and increased testosterone level. Further, increase in the serum level of alkaline phosphatase, SGPT and SGOT and histopathological changes in kidney and liver of treated animals, confirmed the toxic effects of contaminating chemicals. Analysis of water samples using HPLC and GC-MS showed the presence of various compounds and from them, four prominent aromatic compounds viz. nonylphenol, hexachlorobenzene and two testosterone equivalents, were identified. Our data suggest that despite rigorous treatment, the final treated effluent from WWTP still has enough androgenic and toxic compounds to affect general health.

Exogenous sex steroids markedly alter sex differentiation in fish. The endocrine and molecular mechanisms involved in these changes remain unclear. To further clarify the mechanism of androgen-induced testicular differentiation, we treated female tilapia Oreochromis niloticus with methyltestosterone (MT at a dose of 50 microg/g diet) and examined the expression of P450 cholesterol-side-chain-cleavage, 3beta-hydroxysteroid dehydrogenase, and cytochrome P450 aromatase (P450arom) in the gonads. MT treatment resulted in 100% masculinization. Untreated fish showed normal ovarian differentiation with strong expression of all three steroidogenic enzymes. In gonads of MT-treated fish, expression of all three steroidogenic enzymes was attenuated within 15 days and completely disappeared within 30 days of treatment. Our results indicate that exogenous androgen treatment suppresses the expression of key steroidogenic enzymes, including P450arom throughout sex differentiation in tilapia, thus masculinizing the animal. Whether the absence of aromatase or the presence of androgens is responsible for testicular differentiation remains to be determined.

Histomorphological changes and steroidogenic potential of the gonads during sexual differentiation and development were studied in Rana curtipes from tadpole stage 25 (Gosner) until maturity. In stage 25 tadpoles of smaller snout-vent length (SVL; 4-5 mm) the gonads were indifferent, containing a few somatic and germ cells, whereas in larger tadpoles (SVL > 7 mm) gonads were differentiated into ovaries with a central lumen. Onset of meiosis was seen in these ovaries. At stage 26, diplotene and first growth phase oocytes were found. With advancement in developmental stage and after metamorphosis the ovaries progressively enlarged due to increase in number and size of oocytes. Vitellogenesis began in the ovary of 4-month-old frogs. Females attained maturity 6 months after metamorphosis. The frogs showed amplexus and one frog spawned. Onset of testicular formation seen at stage 31 was associated with the degeneration of oocytes and infiltration of darkly stained somatic cells in the central region. By stage 35 all oocytes degenerated, leaving behind a large number of somatic and germ cells interspersed with each other. At stage 38, formation of seminiferous tubules enclosing spermatogonia and pre-Sertoli cells was seen. Initiation of meiosis was evident at metamorphic climax. Cysts of elongated spermatids associated with Sertoli cells were seen in 45-day-old frogs. Histochemically, delta(5)-3 beta-hydroxysteroid dehydrogenase activity was localized in the ooplasm, follicular cells, and interstitium of the ovary from stage 28 onward. The enzyme activity in the testis appeared in 45-day-old froglets. In R. curtipes gonadal differentiation is a semidifferentiated type since gonads initially differentiate into ovaries, and later, in the prospective males, the ovaries degenerate and transform into testes. The males attain maturity much earlier than the females. In R. curtipes gonadal sex differentiation precedes the onset of gonadal steroidogenesis., (Copyright 2000 Academic Press.)

Songbirds have emerged as important animal models for understanding how sex steroids influence brain and behavior, particularly how they direct the sexually dimorphic development of the neural circuits controlling song and then activate adult song behavior. Presumably, sex steroids synthesized in the gonads are responsible for these actions on brain. However, experiments do not always reveal a direct relationship between gonadal function, circulating sex steroids, and activation and/or organization of song. Thus, it is critical that we understand more about the sites and mechanisms of sex steroid synthesis in this group of birds. Toward this end, we have established the use in zebra finches of chicken cDNA probes to the principal androgen synthetic enzymes, CYP11A1, 3beta-HSD, and CYP17. On Northern blots, these probes recognized bands of the appropriate size and in tissues similar to those seen in chickens. With these probes, and a probe to CYP19 specific to the zebra finch, we used in situ hybridization to examine the cellular expression of these enzymes in gonads and adrenals of adult and developing zebra finches (1 to 20 days posthatching). In adults, we identified significant expression of CYP11A1 and CYP17 in large ovarian follicles, particularly the thecal cell layer and over the testicular interstitial area. 3beta-HSD was expressed by both theca and granulosa and in testicular interstitial and seminiferous tubular cells. In adrenals, CYP11A1 and 3beta-HSD are abundant with lesser amounts of CYP17. Developmentally, we identified high expression of CYP11A1 and 3beta-HSD in the adrenals, CYP17 in both testes and ovaries, and CYP19 in ovaries only. These results suggest that the ovaries but not the testes may secrete estrogen developmentally and the adrenals may contribute precursors for gonadal steroidogensis., (Copyright 2000 Academic Press.)

The development of the adrenal gland in the lizard Calotes versicolor was studied histologically and histochemically from the day of oviposition (stage 27) to 60 days after hatching. At stage 27, the adrenocortical cells are found in association with the genital ridge (primordial gonad). The separation of adrenocortical cells from the gonad takes place at stage 31. Organization of adrenocortical cells into cords takes place at stage 34. The catecholamine-secreting chromaffin cells can be seen distinctly on the dorsal region of the adrenal at stage 36, indicating the presence of biologically active catecholamines; the noradrenaline-secreting chromaffin cells appear first at stage 36 and the adrenaline-secreting cells appear later at stage 41. The cortico-medullary ratio of 6:1 during early embryonic development decreases with the increase in age and is 3:1 in posthatching lizards. The histochemical localization of Delta(5)-3beta-hydroxysteroid dehydrogenase (3beta-HSD) and glucose-6-phosphate dehydrogenase in the adrenocortical cells as early as at stage 27 (prior to the gonadal differentiation) indicates the capability of these cells to synthesize steroids. The intensity of the enzyme activity is maximum on the day of hatching and remains more or less the same in the posthatching lizards. The localization of 17beta-HSD enzyme activity observed in the adrenocortical cells at stage 34 is suggestive of their ability to synthesize sex steroids during embryonic life. The intense 3beta-HSD activity on the day of hatching in C. versicolor suggests high production of steroids which may be corticoids. The results of the present work also suggest that the onset of steroid secretion occurs prior to catecholamine secretion during embryogenesis of the adrenal gland in C. versicolor. In addition, there is a significant relationship between ontogenic steroidogenesis of the adrenal gland and sexual differentiation of the gonad., (Copyright 2000 Academic Press.)

Ovarian interstitial cells (OICs) are a common feature of mammalian gonads but little is understood concerning their origin or functional significance. This study investigated the development and steroidogenic potential of OIC in feral and colony-reared feline queens. Reproductive tracts, collected from a total of 50 female colony and feral cats, were fixed and analyzed by morphometry. Ovarian sections were also immuno-stained for the expression of the steroidogenic enzymes 17alpha-hydroxylase/17,20 lyase cytochrome P450 (P450c17), 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD), and aromatase. These findings were related to serum estradiol and testosterone concentrations and to the degree of existing cystic endometrial hyperplasia (CEH). Feral cats had three times as many OICs as colony-reared queens (2713 +/- 855 vs 744 +/- 494 cells/mm(2), P < 0.01). These cells were lipid laden and expressed both P450c17 and 3beta-HSD at levels that were higher than those seen in the theca interna of adjacent follicles. Aromatase expression was undetectable. The pattern of enzyme expression was consistent with development of interstitial tissue from atretic follicles and the potential for continued steroid secretion during the anestrum. The incidence of CEH was higher in older (>5 years old; 88.2%) than in younger (2-4 years; 30%) colony queens (P < 0. 01), whereas no such disease was evident in any of the feral cats. Estradiol levels were higher in colony-reared than in feral cats, but testosterone levels were not different. These data are consistent with the transformation of the theca interna of atretic follicles in cats into OICs that retain a similar, or even enhanced, steroidogenic phenotype. Colony-reared cats exhibit a predisposition to CEH compared with feral queens that is associated with elevated serum estradiol concentrations. Whether or not OICs somehow prevent the development of uterine disease or otherwise reflect a gonadal response to reduced negative feedback on the hypothalamic-pituitary axis remains to be determined., (Copyright 1999 Academic Press.)

Recent reports have implicated an important role for arginine vasotocin (AVT) in the socially mediated sexual differentiation of fishes. This study focuses on the plainfin midshipman (Porichthys notatus) which exhibits two male morphs, type I and type II, differing in a suite of behavioral, neurobiological, and endocrine traits. Immunocytochemical techniques were used to detect neurons containing AVT-like peptide in the forebrain of juveniles, adult females, and type I and type II males. AVT immunoreactive (ir) somata were localized to three regions: the terminal nerve ganglion, the preoptic area (POA), and the pineal stalk. The profile area, or size, of AVT-ir POA neurons differed across the four classes of midshipman and was strongly correlated to differences in body size among the groups. By contrast, the number of AVT-ir cells in the POA exhibited no difference across the classes of midshipman. The number of POA cells containing AVT is therefore likely to be set early in development and not to change with the growth of the animal. An analysis of AVT-ir cell number normalized by body mass revealed that the larger morphs, type I males and females, have fewer cells per gram body mass than type II males and juveniles. Therefore, type II males have a juvenile-like AVT POA phenotype with smaller cells and more numerous cells per unit body mass than type I males. Type II males also exhibit more variability in the number of AVT-ir cells found in the POA compared to type I males., (Copyright 1998 Academic Press.)

Steroidogenesis in the gonad of the protandrous sea bass, Lates calcarifer, was examined in vitro in spermiating testis, previtellogenic ovary, and transitional gonads. Gonadal tissues were incubated with tritiated androstenedione. Metabolites were analyzed by thin-layer chromatography, high-performance liquid chromatography, microchemical reactions, and crystallization to constant specific activity. 17 beta-Hydroxysteroid dehydrogenase, 5 beta-reductase, and 3 alpha-hydroxysteroid dehydrogenase activities were found in all of the sex types. On the other hand, 11 beta-hydroxysteroid dehydrogenase and 11 beta-hydroxylase activities were found only when testicular tissue was present, i.e., in testis and early transitional gonad. A low aromatase activity leading to estrone synthesis was detected in the previtellogenic ovary. In late transitional gonads, a major metabolite (metabolite X) was suggestively identified as a 3-ester of 17 beta-estradiol according to its chemical and immunological characteristics. Levels of 17 beta-estradiol (E2), the metabolite X, testosterone (T), and 11-ketotestosterone (11KT) were also measured by radioimmunoassay in plasma, before (January and February) and during (March and April) the sex inversion process. Plasma E2 was virtually undetectable (means below 25 pg/ml), although higher levels of metabolite X were found in transitional fish (485 +/- 432 pg/ml in March). Throughout this period, plasma levels of T and 11KT and the androgens/estrogens ratio were significantly higher in males than in transitional fish, where these levels decreased during the sex inversion period. The level of in vitro synthesis of metabolite X was high in transitional gonads, but their concentrations were very low (0.07 +/- 0.09 ng of equivalent E2/g in transitional gonads against 0.22 +/- 0.37 ng of equivalent E2/g in testes and 2.16 +/- 2.7 ng of equivalent E2/g in ovaries).

Hokkaido brown bears (Ursus arctos yesoensis) are seasonal breeders and the profile of their serum testosterone concentrations undergoes annual changes. However, precise sites of steroidogenesis in the bear testis have not been identified. Therefore, our objective was to localize steroidogenic enzymes by immunocytochemistry using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. The steroidogenic enzymes localized were cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17), and aromatase cytochrome P450 (P450arom) as biosynthetic sites of pregnenolone, progesterone or androstenedione, androgens, and estrogens, respectively. Testes were collected from three adult bears prior to the mating season (April, Hokkaido, Japan) and prepared for immunostaining with primary antibodies, followed by an avidin-biotin-peroxidase method. P450scc was localized in Leydig cells and spermatids. 3 beta-HSD was found only in Leydig cells. P450c17 was identified in Leydig cells and spermatids. Finally, P450arom was found in Leydig cells and in spermatids that stained very intensely. Therefore, Leydig cells appear to be a site of progestin (both delta 4 and delta 5 C21 steroids), androgen, and estrogen production, whereas spermatids appear to be a site of pregnenolone, androgen, and estrogen production. Immunolocalization of steroidogenic enzymes suggests that steroidogenesis may occur not only in Leydig cells, but also in spermatids of the Hokkaido brown bear testis prior to the mating season. We also suggest that Leydig cells and spermatids are the predominant sites of androgen and estrogen synthesis, respectively, in the Hokkaido brown bear testis.

To gauge the relative regulative roles of adrenal, gonadal, and thyroid hormones on uropygial gland of male adult pigeons, morphometric, histological, and histochemical observations have been made on a seasonal basis in normal as well as experimentally manipulated birds. Normal birds showed a parallel adrenal-gonadal-uropygial relationship and inverse adrenal-thyroid, thyroid-gonadal, and thyroid-uropygial relationships. Induced hypocorticalism by dexamethasone in the breeding season and hypercorticalism by ACTH or corticosterone treatment in the nonbreeding season were marked by inhibitory and stimulatory changes respectively in the uropygial gland and testis and by inverse thyroid activity. Further, cyproterone acetate treatment in the breeding season completely suppressed testicular functions and increased thyroid activity without affecting either adrenal or uropygial weight, structure, and functions. Based on the observations it is concluded that adrenal steroids are principally involved in regulating the uropygial gland while the gonadal steroids are involved in qualitative aspects of secretion during the breeding phase and thyroid hormones in maintaining the general metabolic profile.