P450 Cholesterol Side-chain Cleavage Enzyme Research Articles

Effect of Notch Signal Pathway on Steroid Synthesis Enzymes in TM3 Cells.

Studies have indicated that the conservative Notch pathway contributes to steroid hormone synthesis in the ovaries; however, its role in hormone synthesis of the testis remains unclear. We have previously reported Notch 1, 2, and 3 to be expressed in murine Leydig cells and that inhibition of Notch signaling caused G0/G1 arrest in TM3 Leydig cells. In this study, we have further explored the effect of different Notch signal pathways on key steroidogenic enzymes in murine Leydig cells. TM3 cells were treated with Notch signaling pathway inhibitor MK-0752, and different Notch receptors were also overexpressed in TM3 cells. We evaluated the expression of key enzymes of steroid synthesis, including p450 cholesterol side-chain cleavage enzyme (P450Scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and steroidogenic acute regulatory protein (StAR), and key transcriptional factors for steroid synthesis, including steroidogenic factor 1 (SF1), GATA-binding protein 4 (GATA4) and GATA6. We found the level of P450Scc, 3β-HSD, StAR and SF1 to be decreased after treatment with MK-0752, while overexpression of Notch1 up-regulated the expression of 3β-HSD, P450Scc, StAR and SF1. MK-0752 and overexpression of different Notch members had no influence on the expression of GATA4 and GATA6. In conclusion, Notch1 signaling may contribute to the steroid synthesis in Leydig cells through regulating SF1 and downstream steroidogenic enzymes (3β-HSD, StAR and P450Scc).

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.

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.

Arsenic exposure diminishes ovarian follicular reserve and induces abnormal steroidogenesis by DNA methylation

Arsenic contamination is a worldwide public health problem, and the effect of arsenic on male reproduction has been extensively studied; however, data on the biotoxicity of arsenic in terms of female reproduction are more scarce. In this study, a human-cell-animal translational strategy was applied to explore the effect of arsenic exposure on ovarian steroidogenesis and its potential mechanism. We conducted a 1:1 propensity score matched case–control study involving 127 diminished ovarian reserve (DOR) cases and 127 healthy controls. The ovarian follicular fluid levels of 21 metal elements, including arsenic, were measured. The results showed that there were significant differences in follicular fluid metal profiles between DOR patients and controls and that arsenic, molybdenum, and strontium played important roles in DOR progression [OR (95 % CI): 2.203 (1.385, 3.503), 2.308 (1.490, 3.575) and 2.922 (1.864, 4.580), respectively]. In the primary ovarian granulosa cell culture model, we found that treatment with 8 μM arsenic for 24 and 48 h induced a decrease in human granulosa cell viability. The estradiol (E2) level was significantly decreased after arsenic exposure (P < 0.05), which was dependent on significant alterations (P < 0.05) in key enzymes in steroidogenesis. In addition, a model for sodium arsenite exposure through water in rats from weaning to sexual maturity was established. We evaluated ovarian development by monitoring the estrous cycle, observing ovarian pathology, and calculating the follicular proportion. RT–qPCR, Western blotting, and bisulfite-sequencing PCR were used to investigate the effect of arsenic exposure on ovarian steroidogenesis and its possible mechanism. The results indicated that steroidogenic factor-1 (SF-1) was an important target of the steroidogenesis disorder induced by arsenic exposure. Arsenic significantly increased the DNA methylation level (P < 0.05) in the promoter region of SF-1 to reduce its expression, subsequently decreasing the levels of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (CYP11A1), and aromatase (CYP19A1) (P < 0.05), leading to premature depletion of ovarian follicles.

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.

Human HAND1 inhibits the conversion of cholesterol to steroids in trophoblasts

Steroidogenesis from cholesterol in placental trophoblasts is fundamentally involved in the establishment and maintenance of pregnancy. The transcription factor gene heart and neural crest derivatives expressed 1 (Hand1) promotes differentiation of mouse trophoblast giant cells. However, the role of HAND1 in human trophoblasts remains unknown. Here, we report that HAND1 inhibits human trophoblastic progesterone (P4) and estradiol (E2) from cholesterol through downregulation of the expression of steroidogenic enzymes, including aromatase, P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1). Mechanically, although HAND1 inhibits transcription of aromatase by directly binding to aromatase gene promoter, it restrains transcription of P450scc by upregulation of the methylation status of P450scc gene promoter through its binding to ALKBH1, a demethylase. Unlike aromatase and P450scc, HAND1 decreases 3β-HSD1 mRNA levels by the reduction of its RNA stability through binding to and subsequent destabilizing protein HuR. Finally, HAND1 suppresses circulating P4 and E2 levels derived from JEG-3 xenograft and attenuates uterine response to P4 and E2. Thus, our results uncover a hitherto uncharacterized role of HAND1 in the regulation of cholesterol metabolism in human trophoblasts, which may help pinpoint the underlying mechanisms involved in supporting the development and physiological function of the human placenta.

Reproductive performance is associated with seasonal plasma reproductive hormone levels, steroidogenic enzymes and sex hormone receptor expression levels in cultured Asian yellow pond turtles (Mauremys mutica).

In order to understand the endocrine mechanism associated with fecundity of seasonally breeding animals, we investigated the plasma reproductive hormones levels and detected the differences in steroidogenic enzymes and sex hormone receptor mRNA levels in female Mauremys mutica. These turtles were divided into higher fecundity (HF) group than those in lower fecundity (LF) group based on paternity identification in our previous research. The plasma estrogen (E2), testosterone (T) and progesterone (P4) levels were significantly higher in pre-breeding season (PBS) than those in non-breeding season (NBS) and were markedly higher in the HF group than those in LF group. In the hypothalamus, there was significantly higher mRNA abundance of P450-cholesterol side-chain cleavage enzyme (P450Scc) encoded by Cyp11α1, aromatase (Cyp19α1) and 5-reductase (5α-R), but significantly lower mRNA levels of follicular stimulating hormone receptor (FSHR) and progesterone receptor (PR) detected in PBS than those in NBS. The pituitary steroidogenic acute regulatory protein (StAR), cytochrome P450-17alpha-hydroxylase (Cyp17α1), 3-hydroxy-steroid dehydrogenase (3βHSD), 17-hydroxy-steroid dehydrogenase 3 (17βHSD3), Cyp19α1, 5α-R, FSHR, estrogen receptor 1 (ESR1), androgen receptor (AR) and PR transcriptional levels in HF group were up-regulated significantly compared with the LF group. In the ovary, Cyp17α1 and 17βHSD3 transcriptional levels were markedly higher in PBS than those in NBS. We detected significantly increased expression levels of all steroidogenic enzymes, but notably lower mRNA levels of FSHR and PR in uterus during the PBS, and the HF group has significantly higher expression levels of StAR, Cyp17α1, 5α-R and AR than LF group. Our work reveals seasonal variations in hormone regulation as well as gene regulation in turtles, providing reliable information to understand the mechanisms underlying the different reproductive capacity of reptiles.

Oyster peptide prevents the occurrence of exercise-hypogonadal male condition by improving the function of pituitary gonadal axis in male rats.

This study evaluates the protective role of oyster peptide (OP) on the occurrence of Exercise-Hypogonadal Male Condition. Male rats were given heavy-load swimming training and / or OP was supplemented for 6 consecutive weeks. After heavy-load training, sperm count, sperm viability and sperm motility in epididymis, testosterone in serum and testis, glutathione peroxidase (GSH-px) and androgen receptor (AR) in testis and mating times were remarkably decreased, malondialdehyde (MDA), capture latency and mating latency were significantly increased, mRNA expression of steroidogenic acute regulatory (StAR) and P450 cholesterol side-chain cleavage enzyme (P450scc) were obviously down-regulated, but serum follicle-stimulating hormone (FSH) and luteinising hormone (LH) were not statistically changed. Conversely, when OP was supplemented at heavy-load training, sperm count, sperm viability and sperm motility in epididymis, serum FSH, LH, testosterone, GSH-px, superoxide dismutase (SOD), testosterone, AR in testis and mating times were dramatically increased, while testicular MDA, capture latency and mating latency were significantly decreased, and mRNA expression of StAR, StARD7, P450scc and 3β-hydroxysteroid dehydrogenase (3β-HSD) were significantly up-regulated. In conclusion, heavy-load training causes testicular spermatogenic and steroidogenic disorders by enhancing the generation of reactive oxygen species (ROS), which can be protected by the co-administration of OP by enhancing the function of pituitary gonad axis and lowering ROS generation.

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.

Platelet-derived growth factor B restores vascular barrier integrity and diminishes permeability in ovarian hyperstimulation syndrome.

Although advances in the prediction and management of ovarian hyperstimulation syndrome (OHSS) have been introduced, complete prevention is not yet possible. Previously, we and other authors have shown that vascular endothelial growth factor, angiopoietins (ANGPTs) and sphingosine-1-phosphate are involved in OHSS etiology. In addition, we have demonstrated that ovarian protein levels of platelet-derived growth factor (PDGF) ligands -B and -D decrease in an OHSS rat model, whilst PDGFR-β and ANGPT2 remain unchanged. In the present work, we investigated the role of PDGF-B in OHSS by evaluating ligand protein levels in follicular fluid (FF) from women at risk of developing OHSS and by using an immature rat model of OHSS. We demonstrated that PDGF-B and PDGF-D are lower in FF from women at risk of developing OHSS compared to control patients (P < 0.05). In the OHSS rat model, PDGF-B (0.5 µg/ovary) administration decreased ovarian weight (P < 0.05), reduced serum progesterone (P < 0.05) and lowered the percentage of cysts (P < 0.05), compared to untreated OHSS rats, but had no effect on the proportion of follicles or corpora lutea (CL). PDGF-B treatment also restored the expression of steroidogenic acute regulatory protein (P < 0.05) and P450 cholesterol side-chain cleavage enzyme (P < 0.01) to control levels. In addition, PDGF-B increased the peri-endothelial cell area in CL and cystic structures, and reduced vascular permeability compared to untreated OHSS ovaries. Lastly, PDGF-B increased the levels of junction proteins claudin-5 (P < 0.05), occludin (P < 0.05) and β-catenin (P < 0.05), while boosting the extracellular deposition of collagen IV surrounding the ovarian vasculature (PP < 0.01), compared to OHSS alone. In conclusion, our findings indicate that PDGF-B could be another crucial mediator in the onset and development of OHSS, which may lead to the development of novel prediction markers and therapeutic strategies.

Cadmium exposure during prenatal development causes progesterone disruptors in multiple generations via steroidogenic enzymes in rat ovarian granulosa cells.

Exposure to the heavy metal cadmium (Cd) in the environment is linked to adverse health. To fully understand the adverse effects of this important endocrine-disrupting compound (EDC) requires studies that address multigenerational effects and epigenetic mechanisms. The present study orally dosed pregnant SD rats with Cd from gestation day 1 until birth. First filial generation (F1) female rats were mated with untreated males to generate the secondary filial generation (F2). Ovarian granulosa cells (OGCs) were collected at postnatal day (PND) 56 from both generations after prenatal Cd exposure, and hormone secretion examinations showed a progesterone disorder. Significant decreases in steroidogenic enzymes (steroidogenic acute regulatory protein (StAR) and P450 cholesterol side-chain cleavage enzyme (CYP11A1)) were observed in F1 and F2 rats. However, F1 and F2 rats had different patterns of mRNA and protein expression of steroidogenic factor 1 (SF-1). We also found that microRNAs were significantly changed using a microarray, and miR-10b-5p and miR-27a-3p were upregulated in F1 and F2 rats. The COV434cell line microRNA-knockdown model showed that these two important microRNAs regulated the StAR-induced Cd effect on progesterone secretion. Overall, the results of this study indicate that prenatal Cd exposure causes cytotoxicity problems, progesterone disorder and microRNAs expression changed in a multigenerational manner. And progesterone disorder may interfere with the steroidogenic enzymes in offspring. The present study also revealed that environmental pollution produces multigenerational effects.

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.

Prolyl oligopeptidase regulates progesterone secretion via the ERK signaling pathway in murine luteal cells.

Prolyl oligopeptidase (POP), one of the most widely distributed serine endopeptidases, is highly expressed in the ovaries. However, the physiological role of POP in the ovaries is not clear. In this study, we investigated the significance of POP in the corpus luteum. Murine luteal cells were cultured in vitro and treated with a POP selective inhibitor, (2S)-1[[(2 S)-1-(1-oxo-4-phenylbutyl)-2-pyrrolidinyl carbonyl]-2-pyrrolidinecarbonitrile (KYP-2047). We found that KYP-2047 treatment decreased progesterone secretion. In contrast, POP overexpression increased progesterone secretion. Three essential steroidogenic enzymes, including p450 cholesterol side-chain cleavage enzyme (CYP11A), 3β-hydroxysteroid dehydrogenase (3β-HSD), and the steroidogenic acute regulatory protein (StAR), were regulated by POP. Further studies showed that POP overexpression increased ERK1/2 phosphorylation and increased the expression of steroidogenic factor 1 (SF1), while KYP-2047 treatment decreased ERK1/2 phosphorylation and SF1 expression. To clarify the role of ERK1/2 signaling in POP-regulated progesterone synthesis, U0126-EtOH, an inhibitor of the ERK signaling pathway, was used to treat luteal cells. We found that U0126-EtOH decreased progesterone production and the expression of steroidogenic enzymes and SF1. POP overexpression did not reverse the effects of U0126-EtOH. Overall, POP regulates progesterone secretion by stimulating the expression of CYP11A, 3β-HSD, and StAR in luteal cells. ERK signaling and downstream SF1 expression contribute to this process.

Notch signalling regulates steroidogenesis in mouse ovarian granulosa cells.

The Notch signalling pathway in the mammalian ovary regulates granulosa cell proliferation. However, the effects of Notch signalling on steroidogenesis are unclear. In this study we cultured mouse ovarian granulosa cells from preantral follicles invitro and observed the effect of Notch signalling on steroidogenesis through overexpression, knockdown and inhibition of Notch signalling. Activation of Notch signalling decreased progesterone and oestrogen secretion. In contrast, inhibition of Notch signalling increased the production of progesterone and oestrogen. Expression of the genes for steroidogenic-related enzymes, including 3β-hydroxysteroid dehydrogenase, p450 cholesterol side-chain cleavage enzyme and aromatase, was repressed after stimulation of Notch signalling. The expression of upstream transcription factors, including steroidogenic factor 1 (SF1), Wilms' tumour 1 (Wt1), GATA-binding protein 4 (Gata4) and Gata6, was also inhibited after stimulation of Notch signalling. Production of interleukin (IL)-6 was positively correlated with Notch signalling and negatively correlated with the expression of these transcription factors and enzymes. In conclusion, Notch signalling regulated progesterone and oestrogen secretion by affecting the expression of upstream transcription factors SF1, Wt1, Gata4 and Gata6, as well as downstream steroidogenic-related enzymes. IL-6, which may be regulated directly by Notch signalling, may contribute to this process. Our findings add to the understanding of the diverse functions of Notch signalling in the mammalian ovary.

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.

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.

Dietary exposure to di-isobutyl phthalate increases urinary 5-methyl-2′-deoxycytidine level and affects reproductive function in adult male mice

Phthalates are a large family of ubiquitous environmental pollutants suspected of being endocrine disruptors. Epidemiological studies have associated phthalate metabolites with decreased reproductive parameters and linked phthalate exposure with the level of urinary 5-methyl-2′-deoxycytidine (5mdC, a product of methylated DNA). In this study, adult male mice were exposed to 450mg di-isobutyl phthalate (DiBP)/(kg·day) via dietary exposure for 28days. Mono-isobutyl phthalate (MiBP, the urinary metabolite) and reproductive function parameters were determined. The levels of 5mdC and 5-hydroxymethyl-2′-deoxycytidine (5hmdC) were measured in urine to evaluate if their contents were also altered by DiBP exposure in this animal model. Results showed that DiBP exposure led to a significant increase in the urinary 5mdC level and significant decreases in sperm concentration and motility in the epididymis, accompanied with reduced testosterone levels and down-regulation of the P450 cholesterol side-chain cleavage enzyme (P450scc) gene in the mice testes. Our findings indicated that exposure to DiBP increased the urinary 5mdC levels, which supported our recent epidemiological study about the associations of urinary 5mdC with phthalate exposure in the male human population. In addition, DiBP exposure impaired male reproductive function, possibly by disturbing testosterone levels; P450scc might be a major steroidogenic enzyme targeted by DiBP or other phthalates.

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.

Testicular gene expression of steroidogenesis-related factors in prepubertal, postpubertal, and aging dogs

Developmental and aging changes in testicular factors related to steroidogenesis are unknown in dogs. Using reverse transcription quantitative real-time PCR, this study examined testicular mRNA levels of CYP11A1 (P450 cholesterol side-chain cleavage enzyme, P450scc), CYP17A1 (P450 17α-hydroxylase/C17–20 lyase, P450c17), HSD3B2 (3β-hydroxysteroid dehydrogenase, 3β-HSD), CYP19A (P450 aromatase, P450arom), STAR (steroidogenic acute regulatory protein, StAR), cyclooxygenase (COX) -1 and COX-2 in prepubertal (4–6 months of age), postpubertal (1 year of age), and aging (2–18 years of age) dogs. Testicular mRNA levels for P450scc, 3β-HSD, StAR, COX-1, and COX-2 did not change from prepubertal to postpubertal stages, whereas that for P450arom markedly and abruptly increased and that for P450c17 gradually decreased. In postpubertal and aging dogs, a negative correlation was found between aging and testicular P450arom mRNA levels. Based on the rapid testicular growth observed during puberty, these results suggested that total testis gene expression for steroidogenesis-related factors, in particular for P450arom, increases during puberty in dogs. In addition, the decline in P450arom gene expression during aging may affect the ability to synthesize steroids in canine testes.