Effects of androgenic modulation on the morphophysiology of the adrenal cortex of male gerbils.

The study aimed to investigate the repercussions of androgen modulation on the adrenal cortex of male gerbils, focusing on the morphophysiology, proliferation, and cell death, as well as the expression of hormone receptors and steroidogenic enzymes. Mongolian gerbils (Meriones unguiculatus) were divided into three experimental groups: Control (C), Testosterone (T), animals received injections of testosterone cypionate and Castrated (Ct), animals underwent orchiectomy. The results showed that castration increased the zona fasciculata and promoted cell hypertrophy in all zones. Testosterone supplementation increased cell proliferation and cell death. Androgen modulation promoted an increase in AR, Erα, and ERß. Castration promoted an increase in the CYP19, while decreasing 17ßHSD enzymes. Testosterone supplementation, on the other hand, reduced CYP17 and increased CYP19 and 3ßHSD enzymes. By analyzing the effects of androgen supplementation and deprivation, it can be concluded that testosterone is responsible for tissue remodeling in the cortex, regulating the rate of cell proliferation and death, as well as cell hypertrophy. Testosterone also modulate steroid hormone receptors and steroidogenic enzymes, consequently affecting the regulation, hormone synthesis and homeostasis of this endocrine gland.

Trimethylation profile of histones H3 lysine 4 and 9 in late preantral and early antral caprine follicles grown in vivo versus in vitro in the presence of anethole.

This study assessed the histones methylation profile (H3K4me3 and H3K9me3) in late preantral (PA) and early antral (EA) caprine follicles grown in vivo and in vitro, and the anethole effect during in vitro culture of PA follicles. Uncultured in vivo-grown follicles (PA, n = 64; EA, n = 73) were used as controls to assess the methylation profile and genes' expression related to apoptosis cascade (BAX, proapoptotic; BCL2, antiapoptotic), steroidogenesis (CYP17, CYP19A1), and demethylation (KDM1AX1, KDM1AX2, KDM3A). The isolated PA follicles (n = 174) were cultured in vitro for 6 days in α-MEM+ in either absence (control) or presence of anethole. After culture, EA follicles were evaluated for methylation, mRNA abundance, and morphometry. Follicle diameter increased after culture, regardless of treatment. The methylation profile and the mRNA abundance were similar between in vivo-grown PA and EA follicles. Anethole treatment led to higher H3K4me3 fluorescence intensity in EA follicles. The mRNA abundances of BAX, CYP17, and CYP19A1 were higher, and BCL2 and KDM3A were lower in in vitro-grown EA follicles than in vivo-grown follicles. In conclusion, in vitro follicle culture affected H3K4me3 fluorescence intensity, mRNA abundance of apoptotic genes, and steroidogenic and demethylase enzymes compared with in vivo-grown follicles.

Glioblastoma cells express crucial enzymes involved in androgen synthesis: 3ß-hydroxysteroid dehydrogenase, 17-20α-hydroxylase, 17ß-hydroxysteroid dehydrogenase and 5α-reductase.

Glioblastoma (GB) is the most common and aggressive primary brain tumour in adult humans. Therapeutic resistance and tumour recurrence after surgical removal contribute to poor prognosis for glioblastoma patients. Men are known to be more likely than women to develop an aggressive form of GB, and differences in sex steroids have emerged as a leading explanation for this finding. Studies indicate that the metabolism and proliferation of GB-derived cells are increased by sex steroids, the expression of androgen receptors (ARs) and the synthesis of androgens and oestrogens, suggesting that these hormones have a role in the tumour pathogenesis. The expression of aromatase, the enzyme that converts androgens to oestrogens, has been reported in glial cells and GB cell lines. Thus, it was necessary to test whether the steroidogenic enzymes involved in androgen synthesis are expressed in GB cells. Therefore, here, we investigated the expression of four key enzymes involved in androgen synthesis in human-derived GB cells. U87 cells were cultured in Dulbecco's modified Eagle medium plus foetal bovine serum and antibiotics on slides for immunocytochemistry or immunofluorescence. U87, LN229 and C6 cells were also cultured in multi-well chambers to obtain proteins for Western blotting. We used primary antibodies against 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17α-hydroxilase/17,20-lyase (P450c17), 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and 5α-reductase. Immunocytochemistry, and immunofluorescence results revealed that glioblastoma cells express 3ß-HSD, P450c17, 17ß-HSD and 5α-reductase proteins in their cytoplasm. Moreover, Western blot analyses revealed bands corresponding to the molecular weight of these four enzymes in the three GB cell lines. Thus, glioblastoma cells have the key enzymatic machinery necessary to synthesize androgens, and these enzymes might be useful targets for new therapeutic approaches.

Clinical, Molecular, Functional, and Structural Characterization of CYP17A1 Mutations in Brazilian Patients with 17-Hydroxylase Deficiency.

17-Hydroxylase-deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The aim of the work was to study clinical, biochemical, and the follow up of 17OHD patients and evaluate the function and structure of CYP17A1 mutations. Brazilian patients (three 46, XX and four 46, XY; 17±1.9 years) with combined 17-hydroxylase/17,20-lyase deficiency were evaluated. CYP17A1 gene was sequenced. Functional analysis was performed transfecting COS7 cells, which were exposed to progesterone or 17α-hydroxypregnolone substrates. Hormones were determined by RIA or LC-MS/MS. Three-dimensional structural modeling was performed by Modeller software. All patients presented prepubertal female external genitalia, primary amenorrhea, hypergonadotrophic hypogonadism, hypokalemic hypertension, decreased cortisol, and increased ACTH and corticosterone levels. Five patients presented previously described mutations: p.W406R/p.W406R, IVS2-2A>C/p.P428L, and p.P428L/p.P428L. Two patients presented the compound heterozygous p.G478S/p.I223Nfs*10 mutations, whose CYP17A1 activity and the three dimensional structural modeling are originally studied in this paper. CYP17A1 activity of p.G478S was 13 and 58% against progesterone and 17-hydroxypregnenolone, respectively. The p.I223Nfs*10 caused a truncated inactive protein. Three-dimensional p.G478S structural modeling showed different internal hydrophobic interaction with W313 and created an additional chain side contact with L476 residue. Due to the rarity of 17OHD, the long term follow up (15.3±3.1 years) of our patients will help endocrinologists on the management of patients with 17OHD. The mutation p.G478S/pI223Nfs*10 led to severe 17OHD and impaired CYP17A1 structure and function. The integration of in silico and in vitro analysis showed how the amino acid changes affected the CYP17A1 activity and contributed to clarify the molecular interactions of CYP17A1.

Relationship of Biological Markers of Body Fat Distribution and Corticosteroidogenic Enzyme Activities in Women with Polycystic Ovary Syndrome.

The aim of the study is to determine the impact of different anthropometric measurements of fat distribution on baseline sex-steroid concentrations and corticosteroidogenic enzyme activity in women with polycystic ovary syndrome compared to those with regular menstrual cycles. The current cross-sectional study included 106 normal cycling controls and 268 polycystic ovary syndrome patients. Patients with polycystic ovary syndrome, diagnosed by Rotterdam criteria, were divided in normoandrogenemic (n=91) and hyperandrogenemic (n=177). Anthropometric, biochemical, and hormone parameters were assessed and correlated with corticosteroidogenic enzyme activities in all three groups. Corticosteroidogenic enzyme activities were calculated using product-to-precursor ratios. Regarding sex-steroids individually, anthropometric parameters correlated with the concentrations of several androgens in polycystic ovary syndrome patients, most of them in patients with biochemical hyperandrogenism. The androgen precursors androstenedione, 17-hydroxyprogesterone, and dehydroepiandrosterone were less correlated with anthropometric parameters. The 17,20 lyase activity, in both Δ4 and Δ5 pathways, correlated with several anthropometric measurements in normo- and hyperandrogenemic polycystic ovary syndrome patients. The 17,20 lyase enzyme activity (Δ4 pathway) also correlated with conicity index, visceral adiposity index, and lipid accumulation product in the control group. 17-Hydroxylase activity positively correlated with waist-height ratio in both polycystic ovary syndrome groups. In contrast, 17-hydroxilase negatively correlated with the conicity index. Anthropometric markers of adiposity are associated with androgen levels and their precursors in blood. Body fat distribution correlates with the activities of some steroidogenic enzyme in both normo-and hyperandrogenemic polycystic ovary syndrome phenotypes. The molecular mechanisms involved in these associations are largely unclear and more investigations are required.

Effect of mice Taenia crassiceps WFU cysticerci infection on the ovarian folliculogenesis, enzyme expression, and serum estradiol.

The murine infection with Taenia crassiceps WFU (T. crassiceps WFU) cysticerci has been widely used as an experimental model to better understand human cysticercosis. Several reports have established that the host hormonal environment determines the susceptibility and severity of many parasite infections. Female mice are more susceptible to infection with T. crassiceps cysticerci suggesting that a rich estrogen environment facilitates their reproduction. Ovarian androgens and estrogens are synthesized by key enzymes as P450-aromatase and 17α-hydroxilase/17, 20 lyase (P450C17). The aim of this study was to determine the effect of chronic intraperitoneal infection of T. crassiceps WFU cysticerci on mice ovarian follicular development, ovulation, the expression of ovarian P450-aromatase and P450C17, and serum 17ß-estradiol, key enzymes of the ovarian steroidogenic pathway. To perform this study ovaries and serum were obtained at two, four and six months from T. crassiceps WFU cysticerci infected mice, and compared to those of healthy animals. The ovaries were fixed and processed for histology or lysed in RIPA buffer for Western blot using specific antibodies for P450C17 and P450-aromatase. 17ß-estradiol serum concentration was measured by ELISA. The results showed that the infection with T. crassiceps WFU cysticerci significantly reduced the number of primordial and primary follicles after two months of infection. Through the course of the study, the corpus luteum number began to decrease, whereas atretic follicles increased. The expression of ovarian P450C17 and P450-aromatase as well as serum E2 concentration were significantly increased in the infected group compared to control. These findings show that chronic infection with Taenia crassiceps WFU may alter the reproductive functions of the female mice host.

The Impact of Variants in Genes Associated with Estradiol Synthesis on Hormone Levels and Oocyte Retrieval in Patients Who Underwent Controlled Ovarian Hyperstimulation.

AIMS: To correlate differences in estradiol levels in serum and follicular fluid with genetic variants and to determine if they play a role in the results following assisted reproductive technology (ART). PATIENTS AND METHODS: A cross-sectional study was developed at the Ideia Fértil Institute of Reproductive Health. Two hundred two female patients were selected and underwent controlled ovarian hyperstimulation cycles. Patients for this study were chosen based on their male partners' infertility. Genotypes of selected variants of CYP19A1, CYP17A1, HSD17, and COMT were compared to the estradiol measurements from follicular fluid and serum, as well as to the number and maturation status of the oocytes retrieved. RESULTS: Patients with the variant homozygous genotype AA of CYP19A1 (rs10046) showed increased serum concentrations of estradiol when compared to patients with other genotypes (p = 0.005). The same polymorphism effect was not observed in follicular fluid. This CYP19A1 variant did not affect the number of oocytes recovered nor their maturation level. CONCLUSION: The CYP19A1 variant is associated with an estradiol imbalance in serum. Other pathways, however, may contribute to the formation of the final estradiol metabolite in follicular fluid as well as its impact on the oocyte maturation.

Classic congenital adrenal hyperplasia and its impact on reproduction.

Women with classic congenital adrenal hyperplasia (CAH) can suffer from impaired fertility rates as a result of increased androgen secretion or impaired sex steroid production. In virilizing CAH forms, such as 21-hydroxylase and 11ß-hydroxylase deficiency, the low reported pregnancy rate is mainly secondary to a diminished desire to conceive. Optimal glucocorticoid and/or mineralocorticoid replacement, sufficient to normalize androgen and P levels in the follicular phase, allows natural conception in most cases. The remaining CAH forms exemplified by StAR, P450scc, P450-oxidoreductase, and 17α-hydroxylase/17-20 lyase deficiencies are associated with impaired sex steroid production. Several factors are involved in the true low fertility rate in this group: folliculogenesis arrest, uterine hypoplasia, and inadequate endometrial thickness related to aberrant androgen, estrogen, and P secretion. There are several reports of successful term pregnancies achieved through controlled ovarian hyperstimulation, followed by estrogen replacement and IVF. Progress in female genitalia reconstructive surgery, individualized hormonal therapies, psychosexual evaluation, and assisted reproductive technology have improved fertility and pregnancy outcomes in women with classic CAH. Finally, successful gestational management in CAH patients requires the close coordination of care between endocrinologists and obstetricians.

Effect of glucocorticoids on androgen biosynthesis in the testes of the toad Rhinella arenarum (Amphibia, Anura).

In rat Leydig cells, glucocorticoids (GCs) inhibit testosterone production through the interaction with the glucocorticoid receptor (GR). However, the sensitivity of those cells to GCs is regulated by the enzyme 11ß-hydroxysteroid dehydrogenase Type 1 (11ß-HSD1). In the testes of the toad Rhinella arenarum, the presence of an 11ß-HSD similar to type 2 and a cytosolic GR has also been described. However, there is a lack of information regarding the effects of GCs on amphibian testicular steroidogenesis. In this study, the effects of corticosterone on androgen production, and the activity of two steroidogenic enzymes in toad testes were reported. Corticosterone inhibits androgen production via the GR because the GR antagonist RU486 prevents corticosterone-induced inhibition of testosterone. Corticosterone also reduced the activity of the cytochrome P450 17-hydroxylase, C17,20-lyase (Cyp450 c17 ) without affecting the 3ß-hydroxysteroid dehydrogenase/isomerase activity. This effect on Cyp450 c17 was likewise inhibited by RU486. On the other hand, corticosterone had no effect on the amount of steroidogenic acute regulator protein. These results suggest that GCs inhibit steroidogenesis in toad testes by reducing of Cyp450 c17 activity via a GR-mediated mechanism.

Three new Brazilian cases of 17α-hydroxylase deficiency: clinical, molecular, hormonal, and treatment features.

BACKGROUND: Deficiency of 17α-hydroxylase (17OHD) is a rare form of adrenal hyperplasia. Diagnosis is generally delayed, impairing appropriate treatment. CASE PRESENTATION: Here, we report the clinical, molecular, hormonal, and treatment data of three unrelated 17OHD patients, aged 14-16 years with hypergonadotrophic hypogonadism; uncontrolled hypertension; primary adrenal insufficiency; and high progesterone, low to normal potassium, and low dehydroepiandrosterone, androstenedione, and testosterone levels. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) at baseline and after an adrenocorticotropic hormone test showed low cortisol and cortisone and high deoxycorticosterone (DOC) and corticosterone levels; both DOC/21-deoxycortisol and costicosterone/cortisol ratios were very high. Patient 2 had 46,XX karyotype and patients 1 and 3, had 46,XY. A molecular analysis showed that two of the patients were homozygous for p.W406R mutation and the other patient was compound heterozygous for p.W406R and p.P428L. Hypertension was controlled only after the administration of both prednisone and mineralocorticoid antagonist. CONCLUSIONS: Hypertension in young women must lead to diagnostic suspicion, even in the pre-pubertal period. The basal level of progesterone is an indicator of 17OHD. Mineral and glucocorticoid ratios obtained from LC-MS/MS can reinforce the diagnosis. Hypertension can be controlled using glucocorticoid replacement therapy and mineralocorticoid antagonist.

Leydig cell dysfunction is associated with post-transcriptional deregulation of CYP17A1 in men with Sertoli cell-only syndrome.

STUDY QUESTION: Is the expression of steroidogenic enzyme 17α-Hydroxylase/17,20-Lyase (CYP17A1) down-regulated in Leydig cells (LCs) of men with spermatogenic failure and compensated impairment of LC function, i.e. a low testosterone to LH (T/LH) ratio? SUMMARY ANSWER: Although the transcriptional expression of CYP17A1 is increased, its protein expression is decreased, in isolated LCs of men with spermatogenic failure and reduced serum T/LH. WHAT IS KNOWN ALREADY: Primary spermatogenic defects have been associated with functional and morphological abnormalities of LCs, characterized by decreased serum testosterone (T) levels, decreased T/LH, increased 17ß-estradiol (E2) and E2/T ratio, and larger clusters of LCs (LC hyperplasia). CYP17A1 is a key enzyme in the testosterone pathway and has been implicated in the steroidogenic lesion produced by E2 stimulation. STUDY DESIGN, SIZE, DURATION: We studied 18 azoospermic patients with Sertoli cell-only syndrome (SCOS) and signs of LC dysfunction (cases) and 10 obstructive azoospermic/oligozoospermic men with normal spermatogenesis (controls). The SCOS patients were sub-grouped into 9 cases with T/LH <2 and 9 cases with T/LH ≥2. All of the men underwent testicular biopsy for sperm retrieval at the Reproductive Unit of a University Hospital. PARTICIPANTS/MATERIALS, SETTING, METHODS: The transcriptional expression of CYP17A1 and SF-1 (steroidogenic factor 1) was quantified by SYBR®Green-based qPCR in LCs isolated by laser capture microdissection (LCM), and relative expression to the control pool was assessed. CYP17A1 protein expression was semi-quantified by indirect immunofluorescence (IFI) using Image-Pro Plus v7.0 (Media Cybernetics) in testicular tissue. FSH and LH serum concentrations, and serum and intratesticular T (ITT) and E2 (ITE2) were measured by IRMA and RIA, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Relative CYP17A1 mRNA expression was increased in cases with T/LH <2 compared to cases with T/LH ≥2, by a mean of 3.3-fold (P = 0.002). No corresponding increase in protein expression was found; in fact, CYP17A1 immunostaining intensity assessed by the Integrated Optical Density (IOD) parameter was lower in the cases with T/LH <2 compared to controls (P = 0.008). Relative SF-1 mRNA expression was similar in both case subgroups. CYP17A1 mRNA expression correlated with ITE2 and intratesticular E2/T (r = 0.536; P = 0.026 and r = 0.542; P = 0.016, respectively), while an inverse association was observed for ITE2 and protein level expression (r = -0.421; P = 0.05). LARGE SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: We should interpret the results of the semi-quantification of immunofluorescent staining by Image-Pro Plus software with caution, because it is a semi-quantitative method that may have certain difficulties regarding the disposition of protein in the cells. However, it is not influenced by variations in the number of cells that express the protein, as could be the case of western blot analysis in testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: Dysfunctional LCs of men with SCOS show post-transcriptional deregulation of CYP17A1, with increased mRNA and decreased protein expression, which may be modulated by increased ITE2 levels. In addition, transcriptional expression of CYP17A1 was not associated with changes in SF-1 mRNA expression. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Fund for Scientific and Technological Development (FONDECYT) of Chile to A.C. [grant number 1120176]. The authors declare no conflict of interest.

The role of ARMC5 in human cell cultures from nodules of primary macronodular adrenocortical hyperplasia (PMAH).

The participation of aberrant receptors and intra-adrenal ACTH in hyperplastic tissue are considered mechanisms that regulate hypercortisolism in PMAH. Additionally, germline ARMC5 mutations have been described as the most frequent genetic abnormality found in patients diagnosed with PMAH. Previous functional studies analyzed ARMC5 role using H295R cells. Therefore, we investigated the role of ARMC5 in cell cultures obtained from PMAH nodules containing steroidogenic cells, aberrant receptors and intra-adrenal ACTH. ARMC5 silencing in non-mutated PMAH cell cultures decreased steroidogenesis-related genes and increased CCNE1 mRNA expression and proliferative capacity without affecting cell viability. Additionally, ARMC5 overexpression induced cell death in PMAH mutated cell cultures, thereby decreasing cell viability. We confirmed the role of ARMC5 as an important pro-apoptotic protein involved in PMAH-related steroidogenesis. We also report for the first time the involvement of ARMC5 in controlling proliferation and regulating cell cycle in PMAH cell cultures; these effects need to be explored further.

Steroidogenesis during postnatal testicular development of Galea spixii.

The androgen/estrogen balance is essential for normal sexual development and reproduction in mammals. Studies performed herein investigated the potential for estrogen synthesis in cells of the testes of a hystricomorph rodent, Galea spixii The study characterized the expression of the key enzymes responsible for estrogen and androgen synthesis, cytochromes P450 aromatase (P450arom), 17α-hydroxylase/17,20-lyase (P450c17) respectively, as well as the redox partner NADPH cytochrome P450 oxido-reductase (CPR) required to support electron transfer and catalysis of these P450s, by immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR) analysis, throughout postnatal sexual development. Testes (immature, pre-pubertal, pubertal and post-pubertal) were collected, fixed for IHC (CYP19, CYP17 and CPR) and stored frozen for qPCR for the relevant gene transcripts (Cyp19a1 and Cyp17a1). Expression of P450c17 was significantly elevated at the pre-pubertal and pubertal stages. Based on IHC, P450c17 was expressed only in Leydig cell clusters. The expression of P450arom was detectable at all stages of sexual development of Galea spixii IHC data suggest that estrogen synthesis was not restricted to somatic cells (Leydig cells/Sertoli cells), but that germ cells may also be capable of converting androgens into estrogens, important for testicular function and spermatogenesis.

A time-course regulatory and kinetic expression study of steroid metabolizing enzymes by calcitriol in primary cultured human placental cells.

1,25-dihydroxivitamin D3 (calcitriol), is a secoesteroid involved in several placental functions. In particular, we and others showed that calcitriol regulates peptides, proteins, cytokines and hormones production in human trophoblastic cells. On the other hand, calcitriol modifies the activity and expression of some steroidogenic enzymes, a process that is considered tissue-specific. However, the effects of calcitriol on the expression of enzymes involved in the synthesis of sex steroids in placental tissue have not yet been entirely studied. The aim of the present study was to investigate the effects of calcitriol upon gene expression of several steroid enzymes such as cytochrome P450scc (CYP11A1), type 1 3ß-hydroxysteroid dehydrogenase(3ß-HSDI), 17ß-HSD3, 17α-hydroxylase/17,20 lyase (CYP17A1) and aromatase (CYP19A1) in primary cultures of human placental cells. Cell cultures were performed using placentas obtained immediately after delivery by caesarean section from normotensive healthy women and calcitriol effects were evaluated, at level of transcription, by qPCR. The results showed that: 1) from basal expression values of the five genes studied, 3ß-HSDI was the most expressed gene (P<0.05); 2) basal expression of all enzymes was significantly higher in cultured syncytiotrophoblast than in cytotrophoblasts (P<0.05); 3) the presence of calcitriol in cultured trophoblast cells generally resulted in a stimulatory effect of CYP11A1, CYP19A1 and 17ß-HSD3 gene expression at 3h of treatment whereas 3ß-HSDI was induced at 6h (P<0.05). However, a time-dependent variable was also observed; 4) protein expression of CYP11A1 and 3ß-HSDI were not modified significantly by calcitriol, however that of CYP19A1 was regulated in similar fashion as gene expression. In conclusion, calcitriol affected in a time-dependent manner the expression of steroids metabolizing enzymes in human placental cell cultures.

Expression of androgen-producing enzyme genes and testosterone concentration in Angus and Nellore heifers with high and low ovarian follicle count.

Follicle population is important when animals are used in assisted reproductive programs. Bos indicus animals have more follicles per follicular wave than Bos taurus animals. On the other hand, B taurus animals present better fertility when compared with B indicus animals. Androgens are positively related with the number of antral follicles; moreover, they increase growth factor expression in granulose cells and oocytes. Experimentation was designed to compare testosterone concentration in plasma, and follicular fluid and androgen enzymes mRNA expression (CYP11A1, CYP17A1, 3BHSD, and 17BHSD) in follicles from Angus and Nellore heifers. Heifers were assigned into two groups according to the number of follicles: low and high follicle count groups. Increased testosterone concentration was measured in both plasma and follicular fluid of Angus heifers. However, there was no difference within groups. Expression of CYP11A1 gene was higher in follicles from Angus heifers; however, there was no difference within groups. Expression of CYP17A1, 3BHSD, and 17BHSD genes was higher in follicles from Nellore heifers, and expression of CYP17A1 and 3BHSD genes was also higher in HFC groups from both breeds. It was found that Nellore heifers have more antral follicles than Angus heifers. Testosterone concentration was higher in Angus heifers; this increase could be associated with the increased mRNA expression of CYP11A1. Increased expression of androgen-producing enzyme genes (CYP17A1, 3BHSD, and 17BHSD) was detected in Nellore heifers. It can be suggested that testosterone is acting through different mechanisms to increase follicle development in Nellore and improve fertility in Angus heifers.

Effect of hyperandrogenism on ovarian function.

The objective of this work was to study the ovarian function when follicular development is induced during a hyperandrogenic condition. Female rats were injected with either equine chorionic gonadotropin (eCG group) to induce folliculogenesis or eCG together with DHEA to induce folliculogenesis in a hyperandrogenic condition (eCG+HA group). The control group was injected with vehicle. Ovarian mRNA levels of the peroxisome proliferator-activated receptor gamma (PPARγ) co-activator PGC1α, the PPARγ co-repressor NCoR, the main enzymes involved in the ovarian steroidogenesis (CYP17, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-HSD, and CYP19A), and cyclooxygenase 2 (COX2) were evaluated only by real-time PCR. COX2 was evaluated by both real-time PCR and western blot. Serum steroid hormones and both the oxidative and inflammatory statuses were also quantified. We found that eCG-induced folliculogenesis induced increased mRNA levels of PGC1α and decreased those of NCoR when compared with controls. In addition, we found an increase in serum estradiol (E2) levels and enhanced mRNA expression of CYP19A. A pro-inflammatory status and a pro-oxidant status were also established. When folliculogenesis was induced in a hyperandrogenic condition, the mRNA levels of the PPARγ co-repressor NCoR remained higher than in controls and the pro-inflammatory and pro-oxidant statuses were enhanced. In addition, the enzymes involved in ovarian steroidogenesis were altered leading to the accumulation of testosterone and an unfavorable E2/testosterone ratio. These alterations led to abnormal follicular development.

Steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation.

OBJECTIVE: To analyze steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation. METHODS: Thirty-two virgin adult female rats were randomized to two groups as follows: the control group GI received vehicle and the experimental group GII received melatonin supplementation (10 µg/night per animal) for 60 consecutive days. After the treatment, animals were anesthetized and the collected ovaries were immediately placed in liquid nitrogen for complementary deoxyribonucleic acid microarray analyses. A GeneChip(®) Kit Rat Genome 230 2.0 Affymetrix Array was used for gene analysis and the experiment was repeated three times for each group. The results were normalized with the GeneChip(®) Operating Software program and confirmed through analysis with the secondary deoxyribonucleic acid-Chip Analyzer (dChip) software. The data were confirmed by real-time reverse transcription polymerase chain reaction analysis. Genes related to ovarian function were further confirmed by immunohistochemistry. RESULTS: We found the upregulation of the type 9 adenylate cyclase and inhibin beta B genes and the downregulation of the cyclic adenosine monophosphate response element modulator and cytochrome P450 family 17a1 genes in the ovarian tissue of GII compared to those of the control group. CONCLUSION: Our data suggest that melatonin supplementation decreases gene expression of cyclic adenosine monophosphate, which changes ovarian steroidogenesis.

Comparison of steroidogenic pathways among normoandrogenic and hyperandrogenic polycystic ovary syndrome patients and normal cycling women.

AIM: To compare the corticosteroidogenic enzyme activities between normal cycling non-polycystic ovary syndrome (PCOS), and normoandrogenic PCOS (NA-PCOS) and hyperandrogenic PCOS (HA-PCOS) patients. METHODS: This cohort study was conducted at Julio Muller University Hospital and Tropical Institute of Reproductive Medicine and Menopause, and enrolled 114 non-PCOS women and 355 PCOS patients. The steroidogenic enzyme activities were measured using the serum steroid product/precursor molar ratio. RESULTS: In the Δ5 pathway the 17,20 lyase activity was equally low in the NA-PCOS and HA-PCOS women compared with the non-PCOS women (P < 0.01 and P < 0.001, respectively). In the Δ4 pathway, the 17,20 lyase activity was higher only in the HA-PCOS group (P < 0.001). The 17-hydroxylase activity was the same in PCOS and non-PCOS subjects (P > 0.05). The 3ß-hydroxysteroid dehydrogenase II (3ß-HSDII) activity was higher in the conversion of dehydroepiandrosterone into androstenedione in the HA-PCOS than in the NA-PCOS (P < 0.05) and the non-PCOS patients (P < 0.01). The aromatase activity was lower in the HA-PCOS than in the NA-PCOS (P < 0.05) patients and non-PCOS subjects (P < 0.01). In HA-PCOS subjects, the 17,20 lyase activity was related to insulin, estradiol, total testosterone concentrations and free androgen index in the Δ5 pathway. 3ß-HSDII showed weak correlation with estradiol in the HA-PCOS group. Anthropometric parameters had little impact, if any, on the steroidogenic enzyme activities. CONCLUSION: The NA-PCOS and HA-PCOS patients demonstrated different enzyme activities, and the results provided new directions for future studies including PCOS patients with different phenotypes.

A-ring modified steroidal azoles retaining similar potent and slowly reversible CYP17A1 inhibition as abiraterone.

Abiraterone acetate is a potent inhibitor of human cytochrome P450c17 (CYP17A1, 17α-hydroxylase/17,20-lyase) and is clinically used in combination with prednisone for the treatment of castration-resistant prostate cancer. Although many studies have documented the potency of abiraterone (Abi) in a variety of in vitro and in vivo systems for several species, the exact potency of Abi for human CYP17A1 enzyme has not yet been determined, and the structural requirements for high-potency steroidal azole inhibitors are not established. We synthesized 4 Abi analogs differing in the A-B ring substitution patterns: 3α-hydroxy-Δ(4)-Abi (13), 3-keto-Δ(4)-Abi (11), 3-keto-5α-Abi (6), and 3α-hydroxy-5α-Abi (5). We measured the spectral binding constants (Ks) using purified and modified human CYP17A1 along with the determination constants (Ki) applying a native human CYP17A1 enzyme in yeast microsomes for these compounds as well as for ketoconazole. For Abi, 3-keto-Δ(4)-Abi, 3-keto-5α-Abi, and 3α-hydroxy-5α-Abi, the type 2 spectral changes gave the best fit for a quadratic equation, since in these experiments Ks values were 0.1-2.6nM, much lower than that for ketoconazole and 3α-hydroxy-Δ(4)-Abi (Ks values were 140 and 1660nM, respectively). Inhibition experiments showed mixed inhibition patterns with Ki values of 7-80nM. Abi dissociation from the CYP17A1-Abi complex was incomplete and slow; the t1/2 for dissociation was 1.8h, with 55% of complex remaining after 5h. We conclude that Abi and the 3 related steroidal azoles (3-keto-Δ(4)-Abi, 3-keto-5α-Abi, and 3α-hydroxy-5α-Abi), which also mimic natural substrates, are extraordinarily potent inhibitors of human CYP17A1, whereas the 3α-hydroxy-Δ(4)-Abi is moderately potent and comparable to ketoconazole.

Preventive effect of tert-butylhydroquinone on scrotal heat-induced damage in mouse testes.

To investigate the effect of tert-butylhydroquinone (tBHQ) on scrotal heat-induced damage in mice testes, 8-week-old mice were divided into 6 groups and administered with or without tBHQ through diet (10 mg/g), intraperitoneal injection (100 mg/kg body weight), or intratestis injection (12.5 mg/kg body weight), respectively. After single scrotal heat exposure (42 °C for 25 min), trunk blood and testes were collected 48 h later. The testes from diet and intraperitoneal tBHQ-treated mice showed more compact interstitial cells and less germ cell loss in the seminiferous epithelium compared with their corresponding non-tBHQ groups. However, intratestis tBHQ treatment showed no marked difference relative to the non-treatment group. In addition, pre-treatment of tBHQ caused lower testosterone concentrations and reduced expression of cytochrome P450 17α-hydroxylase/17,20-lyase (CYP 17) compared to the corresponding non-tBHQ groups. The results indicated that scrotal heat-induced structural damage was partly prevented by pre-treatment of tBHQ, which could be used as an effective antioxidant for preventing scrotal heat-mediated male infertility.