The dynamic assessment of toxicity and pathological process of DEHP in germ cells of male Sprague Dawley rats.

Di-(2-ethylhexyl) phthalate is representative of Phthalate esters (PAEs), which is one of the most widely used plasticizer and known to act as a reproductive toxicant. However, little is known about the toxicity and pathological process of DEHP exposure in male reproductive system in terms of different concentrations and time points. In this study, peripubertal male Sprague Dawley rats were continually exposed to different DEHP doses (100 mg/kg, 500 mg/kg, and 900 mg/kg) and periods (7 days, 14 days, 21 days, 28 days, and 35 days) during critical periods for sexual maturity. The reproductive parameters have been investigated, including testicular morphology, serum testosterone level, and testicular P450scc, 3ß-HSD, and PCYP17 levels. We observed disarrangement of testicular spermatogenic epithelium coupled with decrease of serum testosterone, testicular P450scc, 3ß-HSD, and PCYP17 levels, and these changes were more obvious with increase of both the exposure time and dosage. Then trend of the time-dose response to DEHP exposure and the pathological process in germ cells were estimated. The results of this study suggested that DEHP exposure could affect the male reproductive system and the degree of adverse effect depended on the dose and extent of exposure.

Paraquat exposure delays stem/progenitor Leydig cell regeneration in the adult rat testis.

Paraquat, a widely used nonselective herbicide, is a serious hazard to human health. However, the effects of paraquat on the male reproductive system remain unclear. In this study, adult male Sprague Dawley rats were intraperitoneally injected ethane dimethane sulfonate (EDS, 75 mg/kg) to initiate a regeneration of Leydig cells. EDS-treated rats were orally exposed to paraquat (0.5, 2, 8 mg/kg/day) from post-EDS day 17 to day 28 and effects of paraquat on Leydig and Sertoli cell functions on post-EDS day 35 and day 56 were investigated. Paraquat significantly decreased serum testosterone levels at 2 and 8 mg/kg. Paraquat lowered Leydig cell Hsd17b3, Srd5a1, and Hsd11b1 mRNA levels but increased Hsd3b1 on post-EDS day 35. Paraquat lowered Cyp11a1, Cyp17a1, and Hsd11b1 but increased Srd5a1 on post-EDS day 56. However, paraquat did not alter Leydig cell number and PCNA labeling index. Epididymal staining showed that few sperms were observed in paraquat-treated rats. Primary culture of adult Leydig cells showed that paraquat diminished testosterone output and induced reactive oxygen species generation at 1 and 10 µM and apoptosis rate at 10 µM. In conclusion, a short-term exposure to paraquat delays Leydig cell regeneration from stem/progenitor Leydig cells, causing low production of testosterone and an arrest of spermatogenesis.

Diethylstilbestrol administration inhibits theca cell androgen and granulosa cell estrogen production in immature rat ovary.

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

Transcriptome Analysis of WHV/c-myc Transgenic Mice Implicates Cytochrome P450 Enzyme 17A1 as a Promising Biomarker for Hepatocellular Carcinoma.

Early detection of hepatocellular carcinoma (HCC) is critical for successful treatment and favorable prognosis. To identify novel HCC biomarkers, we used the WHV/c-myc transgenic (Tg) mice, an animal model of hepatocarcinogenesis. By analyzing their gene expression profiling, we investigated differentially expressed genes in livers of wild-type and Tg mice. The cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1), a hepatic P450 enzyme, was revealed to be overexpressed in the liver tissues of Tg mice at both preneoplastic and neoplastic stages. Mouse-to-human validation demonstrated that CYP17A1 mRNA and protein were also significantly increased in human HCC tissues compared with paired nontumor tissues (P = 0.00041 and 0.00011, respectively). Immunohistochemical studies showed that CYP17A1 was overexpressed in 67% (58 of 87) of HCC, and strong staining of CYP17A1 was observed in well-differentiated HCCs. Consistent with this, the median serum levels of CYP17A1 were also significantly higher in patients with HCC (140.2 ng/mL, n = 776) compared with healthy controls (31.4 ng/mL, n = 366) and to those with hepatitis B virus (57.5 ng/mL, n = 160), cirrhosis (46.1 ng/mL, n = 147), lung cancer (27.4 ng/mL, n = 109), and prostate cancer (42.1 ng/mL, n = 130; all P < 0.001). Notably, the elevations were seen in most AFP-negative HCC cases. Altogether, through mouse-to-human search and validation, we found that CYP17A1 is overexpressed in HCCs and it has great potentiality as a noninvasive marker for HCC detection. These results provide a rationale for the future development and clinical application of CYP17A1 measurement to diagnose HCC more precisely. Cancer Prev Res; 9(9); 739-49. ©2016 AACR.

Correlations among antral follicular echotexture, apoptosis and expression of key steroidogenic enzymes in sheep.

Nineteen cycling ewes underwent transrectal ultrasonography of ovaries followed by ovariectomies during the growth phase of the first follicular wave of the interovulatory interval or the proestrus/estrus phase of the cycle. Quantitative ultrasonographic characteristics of the antrum and follicular wall in a total of forty-three ovine antral follicles were examined for correlations with the protein expression of three steroidogenic enzymes (cytochrome P450 17α-hydroxylase, CYP17; cytochrome P450 aromatase, CYP19; and 3ß-hydroxysteroid dehydrogenase, 3ß-HSD) determined by densitometric analysis of immunohistochemical slides, follicular dimensions, granulosa layer thickness and the percentage of apoptotic granulosa cells. Significant correlations were found between echotextural attributes of ovine antral follicles and the percentage of apoptotic granulosa cells, CYP17 expression (theca), CYP19 expression (granulosa) and 3ß-HSD expression (theca cells). Computer-aided analyses of ultrasonographic images can be beneficial to the development of assisted reproductive technologies and diagnosis of hormonal imbalances without the need for ovarian biopsies or hormone assays.

An innovative LC-MS/MS-based method for determining CYP 17 and CYP 19 activity in the adipose tissue of pre- and postmenopausal and ovariectomized women using 13C-labeled steroid substrates.

CONTEXT: Does adipose tissue produce steroid hormones like an endocrine organ? OBJECT: To clarify whether adipose tissue produces sex steroid hormone like an endocrine organ, we estimated several key steroid hormone levels, as well as CYP17 and CYP19 activity, in ovariectomized, pre- and postmenopausal women by liquid chromatography-tandem mass spectrometry (LC-MS/MS). SUBJECTS AND METHODS: The subjects were 19 premenopausal (n = 12), postmenopausal (n = 4), and ovariectomized women (n = 3) aged 27-68 years. Serum, visceral adipose and sc adipose samples were taken from these subjects and stored at -70°C. The levels of cortisol, cortisone, progesterone (Prog), androstenedione, dehydroepiandrosterone, estrone, estradiol (E2), and T in serum and adipose tissue were estimated simultaneously by LC-MS/MS. CYP17 and CYP19 activity in tissues were assayed with the use of (13)C-labeled steroid precursors and LC-MS/MS-based estimation of the metabolites. RESULTS: E2 and Prog levels in the sera of postmenopausal or ovariectomized women were less than 10% of those in premenopausal women. No marked variations were seen in other hormones. Estrone, androstenedione, dehydroepiandrosterone, and Prog levels in the visceral and sc tissues of postmenopausal and ovariectomized women were 9-60 times higher than those in serum, whereas E2 and T levels were 3- to 7-fold higher than those in serum, and cortisol and cortisone levels were 20% of those found for serum. CYP17 in adipose tissue was found to have 17-hydroxylase and 20,17-lyase activity, with each catalytic activity being essentially equal. Therefore, CYP17 in adipose tissue is of the testicular/ovarian type but not adrenal type, which has 17-hydroxylase activity dominant. The presence of CYP19 activity in adipose tissue was approximately 3% of CYP17. CONCLUSION: Our findings suggest that adipose tissue acts as an endocrine organ, with CYP17 and CYP19 activity playing an essential role in sex steroid hormone biosynthesis.

Cytochrome P450 17α-hydroxylase/C(17,20)-lyase immunoreactivity and molecular expression in the cerebellar nuclei of adult male rats.

Several probes have been developed to identify steroidogenic activity in the brain of vertebrates. However, the presence of the cytochrome P450 17α-hydroxylase/C(17,20)-lyase (P450C(17)), an enzyme that converts pregnenolone and progesterone into dehydroepiandrosterone and androstenedione, in specific areas of the cerebellum such as the deep cerebellar nuclei, remains virtually unexplored. Using Western blot analysis and immunohistochemistry, we found molecular expression of P450C(17) in the lateral, interposed and medial deep cerebellar nuclei. Moreover, double immunofluorescence procedures enabled localization of P450C(17) mainly in neurons, axons and glutamatergic synapses. Taken together, these data demonstrate the occurrence of P450C(17) in the deep cerebellar nuclei, and enable the chemical characterization of the cells that express the cytochrome.

Modulation of estrogen synthesis through activation of protein kinase A in H295R cells by extracts of estuary sediments.

Sediments from two estuaries within Liaodong Bay, China, were examined for the effects on steroidogenesis using H295R human adrenocortical carcinoma cells. Total extracts (TE) isolated from sediments by Soxhlet extraction were separated into three fractions (F1, F2, and F3) using Florisil columns. After exposing H295R cells to each TE and fractions, the expressions of six steroidogenic genes (cytochrome P450 cholesterol side-chain cleavage [CYP11A], 3ß-hydroxysteroid dehydrogenase type 1 [3ß-HSD1], 3ß-hydroxysteroid dehydrogenase type 2 [3ß-HSD2], cytochrome P450 17-hydroxylase/17-20 lyase [CYP17], cytochrome P450 aromatase [CYP19], 17ß-hydroxysteroid dehydrogenase [17ß-HSD]), and the production of six steroid hormones (progesterone [PGT], 17-hydroxyprogesterone [17-HPT], testosterone [TTR], androstenedione [ADD], estrone [E1], and 17ß-estradiol [17ß-E2]) were measured. The gene expressions of CYP11A, CYP17, 3ß-HSD2, and CYP19, and hormone productions of PGT, 17-HPT, TTR, ADD, E1, and 17ß-E2 were significantly increased after exposure to F3 extracts from the Daliao River. In particular, greater concentrations of E1 (up to 48-fold) and 17ß-E2 (up to 20-fold), as well as up-regulation of CYP19 gene expression (up to tenfold), were caused by exposure to the F3 fraction from the Daliao River, but not from the Daling River. Insight into the mechanism of action was obtained by use of principal component analysis (PCA), the results of which were consistent with unidentified constituents in F3 from the Daliao River activating the protein kinase A (PKA) pathway. This hypothesis was confirmed by reversal of the effects caused by F3 through a co-exposure of a PKA inhibitor (H89) and F3 extract. The H89 down-regulated CYP19 messenger RNA (mRNA) expression with concomitant lesser production of E1 and 17ß-E2 in the co-exposure group, indicating unidentified constituents that could modulate estrogen synthesis, primarily through a mechanism of PKA activation.

The Bidder's organ of the toad Rhinella arenarum (Amphibia, Anura). Presence of steroidogenic enzymes.

The Bidder's organ (BO) of male true toads of Bufonidae family is located in the anterior pole of the testis and it has been compared to a rudimentary ovary because of the presence of previtellogenic follicles. In some species, BO remains in both sexes, while in others only adult males preserve the structure. Several studies suggest that the development of BO is inhibited by the differentiation of the corresponding gonad. The purpose of this study is to describe morphological and histological variability of the BO of Rhinella arenarum and also analyze its steroidogenic capacity. Observations indicate that although most bidderian follicles are in pre vitellogenesis, there are others in early or late vitellogenesis. Moreover, we found that BOs weight was significantly lower in males during the pre-reproductive period and that there is no significant correlation between the weights of BO and the adjacent testis. We also analyzed the presence of steroidogenic enzymes using immunohistochemistry. Results indicate that all the follicles were immunoreactive with the antibody against aromatase, while only few of them were positive for the cytochrome P450 side-chain cleavage. Furthermore, activities of 3ß-hydroxysteroid dehydrogenase/isomerase, cytochrome P450 17-hydroxylase, C17,20-lyase and aromatase were detected by the transformation of radioactive substrates into products. Taken together, these results confirm the steroidogenic capacity of the BO in adult males of R. arenarum.

An insight into testis and gubernaculum dynamics of INSL3-RXFP2 signalling during testicular descent in the dog.

Insulin-like 3 (INSL3) plays a prominent role in male development and is supposed to induce the growth of the gubernaculum testis (g.t.), thus being directly involved in testicular descent in humans and rodents. This happens through activation of the RXFP2 receptor (GREAT or LGR8). The INSL3-RXFP2 complex is reputed to play an additional paracrine role in the testis, possibly acting as part of an autocrine feedback loop. The present work provides evidence of the immunolocalisation of INSL3 in the Leydig cells of canine fetuses and of the expression of RXFP2 receptor in different tissues of the g.t. of the same specimens. RXFP2 was localised at the cell membrane of g.t. muscle and connective cells, as well as in the epithelial cells of the developing excurrent ducts. Notably, RXFP2 immunoreactivity of the g.t. was limited to fetuses at ~35-45 days of gestation, which is also the fetal period when the endocrine compartment of the dog testis is active endocrinologically, as confirmed by the anti-P450c17 and anti-INSL3 immunoreactivities of the fetal Leydig cells, and by anti-Müllerian hormone immunoreactivity of the Sertoli cells. The same immunoreactivities were also evaluated in the testes of cryptorchid dogs of different ages. RXFP2 immunoreactivity was absent from genital tracts of cryptorchid testes and g.t. remnants.