Cytochrome P450 Cholesterol Side-chain Cleavage Research Articles

Unique features of KGN granulosa-like tumour cells in the regulation of steroidogenic and antioxidant genes.

The ovarian KGN granulosa-like tumour cell line is commonly used as a model for human granulosa cells, especially since it produces steroid hormones. To explore this further, we identified genes that were differentially expressed by KGN cells compared to primary human granulosa cells using three public RNA sequence datasets. Of significance, we identified that the expression of the antioxidant gene TXNRD1 (thioredoxin reductase 1) was extremely high in KGN cells. This is ominous since cytochrome P450 enzymes leak electrons and produce reactive oxygen species during the biosynthesis of steroid hormones. Gene Ontology (GO) analysis identified steroid biosynthetic and cholesterol metabolic processes were more active in primary granulosa cells, whilst in KGN cells, DNA processing, chromosome segregation and kinetochore pathways were more prominent. Expression of cytochrome P450 cholesterol side-chain cleavage (CYP11A1) and cytochrome P450 aromatase (CYP19A1), which are important for the biosynthesis of the steroid hormones progesterone and oestrogen, plus their electron transport chain members (FDXR, FDX1, POR) were measured in cultured KGN cells. KGN cells were treated with 1 mM dibutyryl cAMP (dbcAMP) or 10 μM forskolin, with or without siRNA knockdown of TXNRD1. We also examined expression of antioxidant genes, H2O2 production by Amplex Red assay and DNA damage by γH2Ax staining. Significant increases in CYP11A1 and CYP19A1 were observed by either dbcAMP or forskolin treatments. However, no significant changes in H2O2 levels or DNA damage were found. Knockdown of expression of TXNRD1 by siRNA blocked the stimulation of expression of CYP11A1 and CYP19A1 by dbcAMP. Thus, with TXNRD1 playing such a pivotal role in steroidogenesis in the KGN cells and it being so highly overexpressed, we conclude that KGN cells might not be the most appropriate model of primary granulosa cells for studying the interplay between ovarian steroidogenesis, reactive oxygen species and antioxidants.

The expression of cholesterol synthesis and steroidogenic markers in females of the Chinese brown frog (Rana dybowskii) during pre-spawning and pre-hibernation.

The oviduct of the Chinese brown frog (Rana dybowskii) expands in pre-hibernation rather than in pre-spawning, which is one of the physiological phenomena that occur in preparation for hibernation. Steroid hormones are known to regulate oviductal development. Cholesterol synthesis and steroidogenesis may play important role in the expansion of the oviduct before hibernation. In this study, we investigated the expression patterns of the markers that are involved in the de novo steroid synthesis pathway in the oviduct of R. dybowskii during pre-spawning and pre-hibernation. According to histological analysis, the oviduct of R. dybowskii contains epithelial cells, glandular cells, and tubule lumens. During pre-hibernation, oviductal pipe diameter and weight were significantly larger than during pre-spawning. 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), Low-density lipoprotein receptor (LDLR), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) were detected in epithelial cells in pre-hibernation and glandular cells during pre-spawning. HMGCR, LDLR, StAR, and P450scc protein expression levels were higher in pre-hibernation than during pre-spawning, but the SF-1 protein expression level did not significantly differ. HMGCR, LDLR, StAR, P450scc (CYP11A1), and SF-1 (NR5A1) mRNA expression levels were significantly higher in pre-hibernation compared to pre-spawning. The transcriptome results showed that the steroid synthesis pathway was highly expressed during pre-hibernation. Existing results indicate that the oviduct is able to synthesize steroid hormones using cholesterol, and that steroid hormones may affect the oviductal functions of R. dybowskii.

OR13-5 Bone Morphogenetic Protein-4 (BMP4) Inhibits Adrenal Cell Cortisol Synthesis by Blocking CYP17A1 and CYP11B1 Expression.

Abstract Background Cytochrome P450c17 (17α-hydroxylase, encoded by CYP17A1) and cytochrome P450c11 (11β-hydroxylase, encoded by CYP11B1) are essential for production of adrenal cortisol. The expression of CYP17A1 and CYP11B1 in adrenocortical cells is stimulated by adrenocorticotropic hormone (ACTH) through the cAMP and protein kinase A signal transduction pathway. Several growth factors can act as autocrine/paracrine regulators for modulating the steroidogenesis in the adrenal cortex. Among these factors, bone morphogenetic protein-4 (BMP4), a member of the superfamily of transforming growth factor-beta (TGFβ) has been previously reported to have an inhibitory effect on adrenal cell CYP17A1 expression and dehydroepiandrosterone (DHEA) production [1]. The role of BMP4 within the adrenal and its mechanisms regulating steroidogenesis are still unclear. In the present study, we examined human adrenal expression of BMP4 and its chronic effects on the H295R adrenal cell line. Objective To define the autocrine/paracrine role of BMP4 in adrenocortical zonation and adrenal steroidogenesis. Methods BMP4 expression in normal human adrenal glands was studied using microarray analysis with laser-captured adrenal capsule and cortical zones. Adrenal BMP4 protein expression was examined following immunohistochemistry (IHC) with a rabbit polyclonal antibody against human BMP4. In vitro studies were performed using H295R and human adrenal cells treated with or without ACTH (10nM), forskolin (10 µM) and BMP4 (50 ng/mL). The experimental medium was collected every 24 h and analyzed for cortisol. Experiments were terminated at 96 h followed by isolation of RNA and qPCR analysis for PPIA (normalization transcript), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), CYP17A1 and CYP11B1. Results Microarray analysis of adrenal zone RNA demonstrated a BMP4 expression gradient with zona glomerulosa (ZG) expressing 3 and 11-fold higher levels than the zona fasciculata (ZF) and zona reticularis (ZR), respectively. IHC confirmed that BMP4 expression was highest in the ZG and the outer ZF. Administration of BMP4 to cultures of H295R cells caused a significant decrease in basal and forskolin-stimulated cortisol production at 48 h. By 96 h, BMP4 inhibited forskolin-stimulated cortisol by 60%. Cortisol inhibition occurred in conjunction with an inhibition in forskolin-stimulated CYP17A1 (↓75%) and CYP11B1 (↓65%). However, CYP11A1 expression was unaffected. In primary cultures of human adrenal cells BMP4 inhibited the ability of ACTH to increase cortisol production (↓75%), as well as CYP11B1(↓65%) and CYP17A1 (↓57%) expression. Conclusion Our findings indicate that the human adrenal has outer cortex localized expression of BMP4 that could play a paracrine/autocrine role in zone-specific production of aldosterone vs. cortisol through BMP4 selective inhibition of CYP17A1 and CYP11B1. Bibliography: 1. Rege J, Nishimoto HK, Nishimoto K, Rodgers RJ, Auchus RJ, Rainey WE. Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis. Endocrinology 2015; 156: 2530–2540. Presentation: Sunday, June 12, 2022 12:00 p.m. - 12:15 p.m.

Paullinia cupana seed extract ameliorated methotrexate-induced testicular dysfunction through the regulation of antioxidants, inflammatory, apoptosis/anti-apoptosis, and steroidogenesis-associated genes.

Methotrexate (MXT) is a medication used for cancer and rheumatoid treatment with severe organs toxicity as a side effect. Paullinia cupana (Guarana) is a plant with pleiotropic functions used to overcome the side effects of some chemotherapeutic medications. Current study aimed to examine the possible protective effect of guarana against oxidative stress induced by a single dose of MTX in testis. Forty male mice were divided into 4 groups (8weeks old; 30g weight), 1st group is negative control. The 2nd group is positive intoxicated group, received a single dose of MTX intraperitoneally (IP; 20mg/kg BW in saline) on day 7. The 3rd group received guarana seed extract orally (300mg/kg BW daily) for 12days. The protective group was given guarana seed extract orally for 1week, then on day 7 injected with MTX, and continued with guarana for extra 5days. Blood was taken for biochemical measurement (hormones, antioxidants, cytokines, and oxidative stress biomarkers). Testicular tissues were taken for gene quantification (qRT-PCR), testicular oxidative stress activity (malondialdehyde; MDA, and SOD) and comet assay (sperm DNA damage), and histopathological changes at the end of experimental design. MTX intoxication caused a decrease in testicular SOD, GSH, and catalase and an increase in serum and tissue levels of MDA. Biomarkers of oxidative stress were increased by MTX intoxication, and were ameliorated by guarana administration to MTX-intoxicated mice. Guarana prevented the increase in IL-1β and IL-6 levels compared to mice intoxicated with MTX alone. MTX upregulated the expression of caspase-3 and downregulated Bcl-2 expression using qRT-PCR analysis. These negative impacts of MTX were protected by guarana pre-administration. MTX decreased reproductive hormones and altered spermogram parameters (sperm concentration and motility, and percentage of live and dead sperms). In addition, the mRNA expression of steroidogenesis-associated genes, such cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 17β hydroxyl steroid dehydrogenase (17β-HSD) was downregulated in the MTX-treated group, all were prevented by guarana administration. The sperm DNA damage revealed by a comet assay was increased in MTX group and was reversed to control levels by guarana supplementation. Finally, testis histology of MTX-group showed marked spermatocytes vacuolization and a decrease in spermatogenesis. Guarana administration abrogated histopathological changes reported in the Leydig cells and testicular tissues. In conclusion, guarana has the potential as a supplement medication to antagonize testicular oxidative stress induced by methotrexate.

1420P The pharmacokinetics and the pharmacodynamic effect of ODM-208, an inhibitor of cholesterol side-chain cleavage enzyme (CYP11A1)

ODM-208 is a first-in-class, oral, non-steroidal small molecule that selectively and completely inhibits the mitochondrial cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme, the first and rate limiting step in steroidogenesis, leading to deficiency of endogenous steroid hormones. The pharmacokinetics (PK) of ODM-208 and the pharmacodynamics (PD) of ODM-208 by measuring serum testosterone levels were studied in the Cypides phase 1 trial in males with progressive mCRPC, who had received at least one prior line of both 2nd generation AR pathway inhibitor (ARPI) and taxane chemotherapy.

Cadmium-induced preeclampsia-like phenotype in the rat is related to decreased progesterone synthesis in the placenta

Cadmium (Cd) is a toxic element, which is associated with preeclampsia (PE). We treated pregnant rats with cadmium chloride from gestational days (GDs) 9–12 to introduce the PE-like animal model. Maternal systolic blood pressures (SBPs) and body weights were measured on GDs 0, 5, 10, 15, and 20. Foetuses were delivered by Caesarean section on GD20. Then, the dams were sacrificed and the specimens were obtained. The morphological analysis of the placentae was carried out by haematoxylin and eosin staining examination and immunohistochemistry assay. Our study showed that Cd-treated rats developed PE-like phenotypes, such as hypertension, albuminuria, and foetal growth restriction. Moreover, Cd-injected rats displayed abnormal placental angiogenesis and lower progesterone (P4) levels. We further demonstrated that Cd also inhibited the mRNA and protein expressions of steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1), and 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1), which are involved in P4 synthesis in the rat placenta. Our study demonstrates that maternal Cd exposure disrupts the local synthesis of P4 in the placenta, which contributes to the onset of PE in pregnant rats. Supplementing P4 at the early gestational stage may be a promising therapeutic strategy to prevent PE, which requires further investigation.

Triadimefon increases fetal Leydig cell proliferation but inhibits its differentiation of male fetuses after gestational exposure

Triadimefon is a broad-spectrum fungicide widely applied in the agriculture. It is believed to be an endocrine disruptor. Whether triadimefon can inhibit the development of fetal Leydig cells and the underlying mechanisms are unknown. Thirty-two female pregnant Sprague-Dawley rats were randomly assigned into four groups and were dosed via gavage of triadimefon (0, 25, 50, and 100 mg/kg/day) for 9 days from gestational day (GD) 12–20. Triadimefon significantly reduced serum testosterone level in male fetuses at 100 mg/kg. The double immunofluorescence staining of proliferating cell nuclear antigen (PCNA) and cytochrome P450 cholesterol side-chain cleavage (a biomarker for fetal Leydig cells) was used to measure PCNA-labeling in fetal Leydig cells. It markedly increased fetal Leydig cell number primarily via increasing single cell population and elevated the PCNA-labeling of fetal Leydig cells in male fetuses at 100 mg/kg while it induced abnormal aggregation of fetal Leydig cells. The expression levels of fetal Leydig cell genes, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3 and Nr5a1, were determined to explore its effects on fetal Leydig cell development. We found that triadimefon markedly down-regulated the expression of Leydig cell genes, Hsd17b3, Insl3, and Nr5a1 as low as 25 mg/kg and Scarb1 and Cyp11a1 at 100 mg/kg. It did not affect Sertoli cell number but markedly down-regulated the expression of Sertoli cell gene Amh at 50 and 100 mg/kg. Triadimefon significantly down-regulated the expression of antioxidant genes Sod1, Gpx1, and Cat at 25–100 mg/kg, suggesting that it can induce oxidative stress in fetal testis, and it reduced the phosphorylation of ERK1/2 and AKT2 at 100 mg/kg, indicating that it can inhibit the development of fetal Leydig cells. In conclusion, gestational exposure to triadimefon inhibits the development of fetal Leydig cells in male fetuses by inhibiting its differentiation.

Seasonal expressions of SF-1, StAR and P450scc in the scent glands of the muskrats (Ondatra zibethicus)

The steroidogenesis occurs in specific cells and tissues in the mammals which begins with the transfer and intracellular processing of cholesterol converted to pregnenolone. This study investigated the gene and protein expression levels of steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) in the scent glands of the muskrats during the breeding and non-breeding seasons. The immunohistochemical localizations of StAR and P450scc were identified in the glandular cells and epithelial cells while SF-1 was only expressed in glandular cells during the breeding and non-breeding seasons. The gene and protein expression levels of SF-1, StAR and P450scc in the scent glands were remarkedly higher in the breeding season than those of the non-breeding season. The interaction of micro RNAs (miRNAs) and transcriptome results showed that miR-762 and miR-4454 might be the genes encoding (Nr5a1, Star and Cyp11a1) in key biological processes. Taken together, these results suggested that the scent glands of the muskrats potentially owned ability to synthesize steroid hormones de novo, and the steroid hormones might affect the scent glandular functions of the muskrats during the breeding and non-breeding seasons.

Expression and functional role of fibroblast growth factors (FGF) in placenta during different stages of pregnancy in water buffalo (Bubalus bubalis)

The present study documented the expression and functional role of Fibroblast growth factors (FGFs) family and their receptors (Fibroblast growth factor receptor, FGFRs) in placenta (Cotyledon; COT, Caruncle; CAR) during different stages of pregnancy in water buffalo. Samples were collected from Early pregnancy 1 (EP1); Early pregnancy 2 (EP2); Mid pregnancy (MP) and Late pregnancy (LP) while diestrus stage of oestrus cycle (NP) was taken as control. In addition, modulatory role of FGF2 on mRNA expression of von Willebrand factor (vWF), Proliferating cell nuclear antigen (PCNA), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase (3βHSD) and BCL2 Associated X (BAX) were studied in cultured trophoblast cells (TCC), obtained from EP2. Real-time PCR (qPCR), Western blot, and immunohistochemistry were applied to investigate mRNA and protein expressions, and the localization of examined factors whereas, P4 secretion was assessed by RIA. The mRNA and protein expression of FGFs and its receptors were maximum (P < 0.05) during EP (EP1 and EP2) in COT. However, FGFR1 and FGFR4 were upregulated (P < 0.05) during EP2 and MP in COT. Similarly, the mRNA and protein expression of FGFs and its receptors were upregulated (P < 0.05) during all stages of pregnancy in CAR. FGF family members were localized in the cytoplasm of trophoblast cells as well as in fetal blood vessels. At 100 ng/ml dosage, FGF2 stimulated the transcript of vWF maximally (P < 0.05). P4 secretion in trophoblast cells treated with FGF2 was maximum with the highest dose at 72 h. These findings corroborate that FGF acts locally in the trophoblast cells to modulate steroid hormone viz. progesterone synthesis, promote angiogenesis and favors cell survivability indicating that this factor may play an essential role in the regulation of placental formation and function in buffalo.

Sex differences in the brain expression of steroidogenic molecules under basal conditions and after gonadectomy.

The brain is a steroidogenic tissue. It expresses key molecules involved in the synthesis and metabolism of neuroactive steroids, such as steroidogenic acute regulatory protein (StAR), translocator protein 18kDa (TSPO), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenases (3β-HSD), 5α-reductases (5α-R) and 3α-hydroxysteroid oxidoreductases (3α-HSOR). Previous studies have shown that the levels of brain steroids are different in male and female rats under basal conditions and after gonadectomy. In the present study, we assessed gene expression of key neurosteroidogenic molecules in the cerebral cortex and cerebellum of gonadally intact and gonadectomised adult male and female rats. In the cerebellum, the basal mRNA levels of StAR and 3α-HSOR were significantly higher in females than in males. By contrast, the mRNA levels of TSPO and 5α-R were significantly higher in males. In the cerebral cortex, all neurosteroidogenic molecules analysed showed similar mRNA levels in males and females. Gonadectomy increased the expression of 5α-R in the brain of both sexes, although it affected the brain expression of StAR, TSPO, P450scc and 3α-HSOR in females only and with regional differences. Although protein levels were not investigated in the present study, our findings indicate that mRNA expression of steroidogenic molecules in the adult rat brain is sexually dimorphic and presents regional specificity, both under basal conditions and after gonadectomy. Thus, local steroidogenesis may contribute to the reported sex and regional differences in the levels of brain neuroactive steroids and may be involved in the generation of sex differences in the adult brain function.

Inhibition of Luman/CREB3 expression leads to the upregulation of testosterone synthesis in mouse Leydig cells.

Luman, also known as cAMP-response element-binding protein 3, is an endoplasmic reticulum stress-related protein that has been identified as a novel transcriptional coregulator of a variety of nuclear receptors. Herein, immunohistochemistry results showed that Luman was specifically expressed in mouse Leydig cells in an age-dependent increase manner, from prepuberty to sexual maturation. Luman was not detected in Sertoli cells within the seminiferous tubules at any developmental period. The immunofluorescent experiment indicated that Luman was mainly located within the cytoplasm of murine Leydig tumor cells (MLTC-1) and primary Leydig cells (PLCs). To investigate the physiological function of Luman, experiments were conducted to examine the consequences of short hairpin RNA- and small interfering RNA-mediated Luman knock-down in MLTC-1 and PLCs, respectively. Luman knock-down significantly upregulated the expression of steroidogenic acute regulatory, cytochrome P450 cholesterol side-chain cleavage enzymes, 3β-hydroxysteroid dehydrogenase, and 17-α-hydroxylase/C17-20 lyase in MLTC-1 cells and PLCs. Luman knock-down caused an increase in human chorionic gonadotropin-stimulated testosterone production in vitro and in vivo. The nuclear receptors SF-1 and Nur-77 were significantly increased upon Luman knock-down in MLTC-1. By contrast, the level of the nuclear receptor SHP decreased. Luciferase reporter assay results demonstrated that Luman knock-down upregulated the activity of SF-1 and Nur-77 promoters. These data suggested that Luman expressed in mouse Leydig cells in an age-dependent increase manner. Luman knock-down upregulated the activity of SF-1 and Nur-77 promoters, which lead to the increase of testosterone synthesis and steroidogenesis genes expression. In conclusion, these findings provide us with new insights into the role Luman played in male reproduction.

Molecular mechanism of mercuric chloride inhibiting progesterone secretion in ovarian granulosa cells of laying hens.

This study investigated the effect of mercury (Hg) on progesterone secretion in ovarian granulosa cells of laying hens. The gene expressions of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway and intracellular calcium ion (Ca2+ ) were further investigated to uncover the molecular mechanism. Results revealed that the cell viability was gradually decreased after Hg exposure from 0 to 24hr. Besides, progesterone secretion was significantly decreased (p<0.05) as the concentration of Hg increased from 0 to 4μM followed by a plateau in 6μM Hg group at 12-hr time point. Compared with 0μM Hg group, 4 and 6μM Hg for 48hr had significantly decreased progesterone secretion (p<0.05), while Hg exposure for 6 and 24hr had no apparent effect on progesterone secretion. In addition, positive correlations occurred among intracellular progesterone, cAMP, PKA, mRNA expressions of StAR, P450scc and 3β-HSD at 12-h and 24-h time points. On the contrary, intracellular Ca2+ level was negatively related to cAMP level at 6 time point and was negatively correlated with progesterone and PKA level at 48 time point. It could be concluded that Hg dose- and time-dependently inhibited progesterone secretion by means of attenuating cAMP-PKA signal pathway, gene expressions of StAR, P450scc and 3β-HSD and enhancing intracellular Ca2+ in ovarian granulosa cells of laying hens.

The impact of low‐intensity extracorporeal shock wave therapy on testicular function in adult rats

Low-intensity extracorporeal shock wave therapy (Li-ESWT) has been introduced as a treatment for penile diseases. Its impact on testicular function during treatment remains unknown. To clarify whether Li-ESWT impairs testicular function during the treatment of penile diseases by investigating the impact of Li-ESWT on testosterone synthesis and spermatogenesis in adult rats. Twenty-four male Sprague-Dawley rats were equally divided into the following three groups: control group, 1.6 BAR group, and 3.2 BAR group. Rats in the experimental groups were treated with Li-ESWT at different energy levels (300 shocks at 1.6 BAR or 3.2 BAR, 2Hz frequency) three times a week for 3weeks. The control group did not receive any treatment during the same period of time. One day after the last shock wave treatment, serum and testicular tissue testosterone concentrations were measured, and sperm quality was assayed. Histologic examination of the testes and quantitative real-time PCR were performed. Testosterone levels in both the serum and testicular tissue did not change after Li-ESWT exposure. The expression levels of steroidogenic enzymes (steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD)) were not impacted by Li-ESWT. The 3.2 BAR group showed a significantly lower sperm count and lower expression of synaptonemal complex protein 3 (SYCP3) in testicular tissue than the control group. No significant differences in sperm quality or SYCP3 expression were observed between the control group and the 1.6 BAR group. Li-ESWT exposure at 3.2 BAR inhibited spermatogenesis and decreased sperm quality, which indicated that male patients with a desire to preserve fertility should undergo low-energy Li-ESWT or other treatment modalities.

Corticosteroid-binding globulin, induced in testicular Leydig cells by perfluorooctanoic acid, promotes steroid hormone synthesis.

Perfluorooctanoic acid (PFOA) is an abundant perfluoroalkyl substance widely applied in industrial and consumer products. It is a ubiquitous environmental pollutant and suspected endocrine disruptor. Corticosteroid-binding globulin (CBG) is a monomeric glycoprotein that can bind specifically to anti-inflammatory steroids, such as glucocorticoids and progesterone, in circulation. Our previous proteomic profile analysis revealed that CBG levels increased in testes after PFOA treatment. In the present study, we verified its increase in mouse testes following oral exposure to PFOA (0, 1.25 and 5mg/kg/day for 28days) by immunohistochemical analysis and Western blotting. In addition, RNA fluorescence in situ hybridization (FISH) confirmed that testicular CBG was specifically expressed in Leydig cells. Serum CBG levels in all three PFOA groups also increased, accompanied by increased corticosterone in the 5 and 20mg/kg/day groups and decreased adrenocorticotropic hormone in the 20mg/kg/day group. Thus, the influence of PFOA on blood CBG may change free steroid hormone concentrations, thereby serving as an endocrine disruptor. A stimulation effect of PFOA on CBG was also observed in vitro using the Leydig tumor mLTC-1 cell line. Overexpression of CBG in mLTC-1 cells increased progesterone release in culture media. In addition, CBG-induced proteins involved in steroidogenesis in mLTC-1 cells, including steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1), 17α-hydroxylase/17,20 lyase (CYP17A1), and 3β-hydroxysteroid dehydrogenase (3β-HSD), which may be the mechanism behind increased progesterone. Furthermore, the production and release of CBG in mLTC-1 cells were also induced by luteinizing hormone, though this mechanism requires further exploration.

The effects of in utero bisphenol A exposure on ovarian follicle numbers and steroidogenesis in the F1 and F2 generations of mice

Bisphenol A (BPA) is a commonly used plasticizer. Previous studies show that in utero exposure to BPA affects reproductive outcomes in the F1–F3 generations of mice. However, its multigenerational effects on ovarian histology and steroidogenesis over the reproductive lifespan are unknown. Thus, we tested the hypothesis that BPA has multigenerational effects on follicle numbers and steroidogenesis. Mice were exposed in utero to vehicle control or BPA (0.5, 20, and 50μg/kg/day). Ovaries were collected for histological and gene expression analyses and sera were collected for hormone assays. In utero BPA exposure decreased preantral follicle numbers, cytochrome P450 aromatase mRNA levels, and estradiol levels in the F1 generation, whereas it decreased testosterone levels and altered steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage, 3β-hydroxysteroid dehydrogenase 1, and cytochrome P450 aromatase mRNA levels in the F2 generation. These data suggest that BPA has multigenerational effects on the ovary in mice.

Continuous soy isoflavones exposure from weaning to maturity induces downregulation of ovarian steroidogenic factor 1 gene expression and corresponding changes in DNA methylation pattern

Female Wistar rats were treated with orally administered soy isoflavones at concentrations of 0, 25, 50, or 100mg/kg body weight from weaning until sexual maturity (3 mo.), and ovarian steroidogenesis was evaluated. After soy isoflavones were administered, a significant (P<0.05) decrease (44%) in the serum estrodial levels of the high-dose (HD) group were observed. Cultured granulosa cells from the middle- (MD) and HD groups showed significantly (P<0.05) reduced (31%, 45%, respectively) in vitro estradiol secretion, and those from the HD group showed significantly (P<0.05) reduced progesterone (25%) secretion. Compared with the control group, the mRNA expression of the steroidogenic acute regulatory protein (Star), cytochromeP450 cholesterol side chain cleavage (Cyp11a1 and Cyp19a1), and hydroxysteroid dehydrogenase 3b (Hsd3b) genes also decreased. Real-time quantitative PCR and Western blotting revealed a significant (P<0.05) decrease in key transcription factor steroidogenic factor-1 (SF-1) expression in the HD group. The detection of DNA methylation using bisulfitesequencing PCR (BSP) suggested a significantly (P<0.05) increased total methylation rate in the proximal SF-1 promoter in the HD group. Further studies showed that treatment with soy isoflavones can significantly (P<0.05) increase the mRNA expression of DNA methyltransferase (DNMT) 1 and DNMT3a. This study proved that soy isoflavone administration from weaning until sexual maturity could inhibit ovarian steroidogenesis, suggesting that SF-1 might play an important role in this effect. In addition, DNA methylation might play a role in the downregulation of SF-1 gene expression induced by soy isoflavones.

Formaldehyde Inhibits Sexual Behavior and Expression of Steroidogenic Enzymes in the Testes of Mice

BackgroundFormaldehyde, a ubiquitous environmental pollutant, is used extensively and has been proved to impair male reproduction in mammals. However, no trials have explored whether formaldehyde affects sexual function. AimTo evaluate the effect of long-term formaldehyde exposure on sexual behavior and to investigate the potential mechanism. MethodsForty C57BL/6 male mice were randomly allocated to four equally sized groups. Mice were exposed to formaldehyde at a dose of 0 (control), 0.5, 5.0, or 10.0 mg/m3 by inhalation for 60 days. OutcomesSexual behavior, body and reproductive organ weights, testosterone concentration in serum and testicular tissue, expression of steroidogenic enzymes, quality of sperm, and testicular structure were measured. ResultsFormaldehyde inhibited sexual behavior and decreased reproductive organ weights in mice. Serum testosterone levels and intratesticular testosterone concentrations were decreased in the formaldehyde-treated groups. Expression levels of steroidogenic enzymes, including steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase (3β-HSD), also were decreased in the testes of mice exposed to formaldehyde. Moreover, the structure of seminiferous tubules was destroyed and sperm quality decreased after formaldehyde exposure. In addition, the results indicated that the effects of formaldehyde were dose dependent. Clinical ImplicationsEfforts should be undertaken to decrease impairment of sexual function caused by formaldehyde exposure. Strengths and LimitationsThe relatively small sample might have affected the outcomes. Further experiments are needed to study the mechanism of action of formaldehyde. ConclusionExposure to formaldehyde gas inhibited sexual behavior, caused reproductive organ atrophy, and impaired spermatogenesis in male mice, which might have been induced by suppressed expression of steroidogenic enzymes in Leydig cells and decreased testosterone synthesis.Zang Z-J, Fang Y-Q, Ji S-Y, et al. Formaldehyde Inhibits Sexual Behavior and Expression of Steroidogenic Enzymes in the Testes of Mice. J Sex Med 2017;14:1297–1306.

A Potential Role for Endoplasmic Reticulum Stress in Progesterone Deficiency in Obese Women.

Obesity in reproductive-aged women is associated with a shorter luteal phase and lower progesterone levels. Lipid accumulation in follicles of obese women compromises endoplasmic reticulum (ER) function, activating ER stress in granulosa cells. We hypothesized that ER stress activation in granulosa-lutein cells (GLCs) would modulate progesterone production and contribute to obesity-associated progesterone deficiency. Pretreatment with an ER stress inducer, tunicamycin or thapsigargin, inhibited human chorionic gonadotropin (hCG)-stimulated progesterone production in cultured human GLCs. Pretreatment of human GLCs with tunicamycin inhibited hCG-stimulated expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) messenger RNAs (mRNAs) without affecting expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), as determined by real-time quantitative polymerase chain reaction. Pretreatment with tunicamycin also inhibited hCG-stimulated expression of StAR protein and 3β-HSD enzyme activity in cultured human GLCs, as determined by Western blot analysis and an enzyme immunoassay, respectively, but did not affect hCG-induced intracellular 3',5'-cyclic adenosine monophosphate accumulation. Furthermore, tunicamycin attenuated hCG-induced protein kinase A and extracellular signal-regulated kinase activation, as determined by Western blot analysis. In vivo administration of tunicamycin to pregnant mare serum gonadotropin-treated immature mice prior to hCG treatment inhibited the hCG-stimulated increase in serum progesterone levels and hCG-induced expression of StAR and 3β-HSD mRNA in the ovary without affecting serum estradiol levels or the number of corpora lutea. Our findings indicate that ER stress in the follicles of obese women contributes to progesterone deficiency by inhibiting hCG-induced progesterone production in granulosa cells.

Modulatory role of leptin on ovarian functions in water buffalo (Bubalus bubalis)

The aim of the present study was to demonstrate the modulatory role of leptin on bubaline granulosa cells (GCs) and luteal cells (LCs) functions using an in vitro cell culture system and to establish a cross talk between leptin and insulin-like growth factor-1 (IGF-1). GCs were collected from group IV follicles (>13 mm size) and LCs from mid-luteal phase corpus luteum and were grown in serum-containing media supplemented with leptin at three different dose rates (0.1, 1, and 10 ng/mL) and time durations (24, 48, and 72 hours). We evaluated the production and secretion of estradiol (E2) and progesterone (P4) using RIA and the mRNA expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 aromatase (CYP19A1), sterol regulatory element–binding protein 1 (SREBP1), steroidogenic factor-1 (SF1), anti-apoptotic gene PCNA, pro-apoptotic gene caspase 3 and endothelial cell marker, Von Willebrand factor (vWF), using quantitative real-time polymerase chain reaction. The results depicted a direct inhibitory action of leptin on GCs steroidogenesis in a time-dependent manner (P < 0.05), whereas in the presence of IGF-1 the inhibitory effect was reverted. Furthermore, leptin augmented both cellular proliferation (PCNA) and apoptosis (caspase 3). On the other hand, in LCs, leptin alone showed an apparent stimulatory effect on steroidogenesis (P < 0.05); however, in the presence of IGF-1, an antagonistic effect was witnessed. Moreover, leptin had an inhibitory effect on apoptosis while promoted cellular proliferation and angiogenesis. These findings were further strengthened by immunocytochemistry. To conclude, these observations for the first time reported that in buffaloes leptin has a direct dose-, time-, and tissue-dependent effect on ovarian steroidogenesis, angiogenesis, and cytoprotection, and furthermore, it can regulate the effect of systemic factors like IGF-1. Hence, this in vitro study provides an insight into the putative roles of leptin alone and its interactions in vivo.

Immunohistochemical analysis of steroidogenic enzymes in ovarian-type stroma of pancreatic mucinous cystic neoplasms: Comparative study of subepithelial stromal cells in intraductal papillary mucinous neoplasms of the pancreas.

Mucinous cystic neoplasms (MCNs) are generally defined as cyst-forming epithelial neoplasms that arise in the pancreas and harbor characteristic ovarian-type stroma beneath the epithelium. In this study, we compared the immunoreactivity of steroid-related factors in these subepithelial stromal cells in MCNs to those in intraductal papillary mucinous neoplasms (IPMNs) to further characterize this unique MCN ovarian-type stroma through evaluation of sex steroid biosynthesis. Twenty MCNs and twenty IPMNs were examined. Immunoreactivity of steroid hormone receptors, including estrogen receptor (ERα and ERβ), progesterone receptor (PR, PR-A, and PR-B), and androgen receptor (AR), was more frequently detected in MCN ovarian-type stromal cells than in IPMN stromal cells (P < 0.01). The H-scores (mean ± SD) of steroidogenic factor (SF)-1 were also significantly higher in MCNs (112.3 ± 33.1) than in IPMNs (0.9 ± 1.2) (P < 0.01). The steroidogenic enzymes cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), cytochrome P450 17 alpha-hydroxylase (P450c17) and 3β-hydroxysteroid dehydrogenase (3β-HSD) showed immunoreactivity in 9/20 (45.0 %), 15/20 (75.0 %) and 13/20 (65.0 %), respectively, of ovarian-type stroma from MCN cases. These results demonstrate that the ovarian-type stroma of MCNs can express steroidogenic enzymes. Thus, the ovarian-type stroma of MCNs can produce sex steroids that may also act on these cells.