Steroidogenic Acute Regulatory Protein Research Articles

Protective Effects of Ginger Extract on Oxidative Stress and Steroidogenesis-related Genes in The Ovary of Streptozotocin-induced Diabetic Rats

Background and Aims: Ginger has anti-oxidant and anti-diabetic properties, but its beneficial effects have not been fully understood on ovarian disorders in diabetic conditions. In the current project, the impact of ginger extract was investigated on oxidative stress and steroidogenesis-related genes in streptozotocin (STZ)-induced female rats. 
 Materials and Methods: The STZ-induced rats were utilized as a diabetic model and received 200 or 400 mg/kg/day ginger extract for eight weeks. The biochemical factors were measured by standard procedures in this study. Serum levels of insulin and sex hormones were assessed by the enzyme-linked immunosorbent assay (ELISA) technique, and the mRNA expression of target genes was assayed by real-time polymerase chain reaction.
 Results: An increase in the levels of glucose, testosterone, and malondialdehyde (MDA) as well as a decrease in the levels of insulin, glutathione peroxidase (GPx), 3β-hydroxysteroid dehydrogenase (3βHSD), steroidogenic acute regulatory protein (StAR), 17β-estradiol, and progesterone was observed in the diabetic rats. Ginger (200 mg/kg) exhibited a significant amelioration in the levels of glucose, testosterone, and MDA. Treatment with 200 mg/kg ginger enhanced the levels of GPx, StAR, and 17β-estradiol. Administration with 400 mg/kg ginger extract also ameliorated the level of glucose, testosterone, MDA, and elevated the level of insulin, GPx, 3βHSD, StAR, 17β-estradiol, and progesterone in the diabetic rats.
 Conclusion: The current study suggests ginger extract protects against ovarian damage in STZ-diabetic rats.

Bioactive metabolites reveal the therapeutic consistency of epimedii folium from multi-plant sources for the treatment of kidney-yang deficiency

Ethnopharmacological relevanceEpimedii Folium (Yin-yang-huo in Chinese), a traditional and commonly used herbal medicine (HM), is a representative of multi-plant sources. To date, little is known about the reasons for similar therapeutic effects of this HM from multi-plant sources. Aim of the studyTo investigate the underlying reasons for the similar pharmacological effects of Epimedii Folium from two botanical sources (Epimedium koreanum Nakai and Epimedium wushanense T. S. Ying). Materials and methodsFirstly, the phytochemicals of the extracts of E. koreanum and E. wushanense were systematically analyzed. Meanwhile, their pharmacological effects on kidney-yang deficiency (KYD) syndrome were evaluated in rats induced by hydrocortisone. Subsequently, we proposed a combined effect index (CEI) to assess the effects of two plants on the secretion of testosterone by combing the system exposure of twelve active components in vivo with their regulation activities of testosterone production in vitro. Moreover, the label-free proteomics and Western blot analysis were conducted to evaluate the possible mechanism of Epimedii Folium from two botanical sources. ResultsE. koreanum and E. wushanense exhibited similar pharmacological effects on KYD syndrome with promoting the mating behaviors and testosterone levels of rats, although there is a certain difference in the main components between two plants. The CEI analysis showed that there was no difference (P > 0.05) in the sum of CEIs of two Epimedium, indicating that their similar therapeutic effects are attributed to bioactive metabolites in vivo. Furthermore, Epimedii Folium can regulate testosterone production in rat Leydig cell via reversing expressions of key steroidogenic enzymes, such as steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD). ConclusionOur results supply critical evidence for the similar pharmacological effects of two Epimedium species, acting by consistent bioactive components directly exposing in vivo, not chemical compositions presenting in herbs. It provides a reasonable scientific basis for understanding of the HMs originated from multi-plant sources for the same clinical application.

Ameliorative effect of hesperidin against high dose sildenafil-induced liver and testicular oxidative stress and altered gene expression in male rats

BackgroundThe clinical use of sildenafil citrate (Viagra), a drug used to treat erectile dysfunction, is limited because of its many side effects on tissues. In this context, we aimed to investigate the protective effects of hesperidin, a citrus flavonoid, on hepatic and testicular damage induced by a high dose of sildenafil citrate in male rats. Rats were randomly divided into four groups. The first group was used as the control group. The second group was orally administered sildenafil citrate at a high dose of 75 mg/kg thrice a week. In the third group, hesperidin was administered orally at a dose of 50 mg/kg/day. The fourth group was administered 75 mg/kg sildenafil citrate three times a week with 50 mg/kg hesperidin daily. The experiment lasted for 28 days.ResultsIn the sildenafil-treated groups, blood indices were altered, liver function tests were deranged, and serum testosterone levels were reduced. In the liver and testicular tissue, sildenafil citrate treatment resulted in significant reductions in catalase and total antioxidant capacity; as well as increased malondialdehyde, reactive oxygen species, and nitrous oxide levels. In addition, sildenafil citrate treatment caused abnormal histopathological patterns in both the liver and the testes. Liver vascular endothelial growth factor and testicular steroidogenic acute regulatory protein gene expression were upregulated.ConclusionsHesperidin attenuated the harmful effects of intensive sildenafil citrate treatment on liver and testicular functions, alleviated oxidative stress and normalized blood indices. Therefore, hesperidin could be protective against sildenafil citrate-induced oxidative damage that may develop over the long term.

CRISPR/Cas9-mediated activation of NR5A1 steers female human embryonic stem cell-derived bipotential gonadal-like cells towards a steroidogenic cell fate

The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms’ tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.

Impacts of elevated temperature on morphology, oxidative stress levels, and testosterone synthesis in ex vivo cultured porcine testicular tissue

Heat stress has been recognized as a major environmental factor affecting reproductive performance in livestock. However, the underlying mechanisms through which high temperature impairs testicular function remain elusive. This study aimed to investigate the effects of high temperature on morphology, oxidative stress levels, and testosterone synthesis in porcine testicular tissue in vitro. Testicular tissue samples from boars were subjected to different temperature conditions: control (37 °C) and heat stress (39 °C) for 4 h. The morphology of the testicular tissue was assessed using histological analysis, while oxidative stress levels were evaluated by measuring reactive oxygen species (ROS) production and antioxidant enzyme activities. Additionally, the expression of key enzymes involved in testosterone synthesis was examined using quantitative real-time polymerase chain reaction (qRT-PCR). Our results revealed that exposure to high temperatures significantly altered testicular tissue morphology. Histological analysis demonstrated degeneration and disorganization of seminiferous tubules, reduction in germ cell populations, and disruption of the blood-testis barrier. Moreover, high-temperature exposure significantly increased ROS production and decreased the activities of antioxidant enzymes (p < 0.05), indicating elevated oxidative stress levels in the testicular tissue. Furthermore, qRT-PCR analysis showed that high-temperature exposure suppressed the expression of key enzymes involved in testosterone synthesis, including steroidogenic acute regulatory protein and cytochrome P450 family 11 subfamilies A member 1. These findings suggest high temperature impairs testicular function by disrupting testicular morphology, inducing oxidative stress, and inhibiting testosterone synthesis. In conclusion, our study demonstrates that high-temperature exposure adversely affects morphology, oxidative stress levels, and testosterone synthesis in porcine testicular tissue. These findings provide insights into the potential mechanisms underlying heat-induced reproductive dysfunction in male pigs and highlight the importance of heat stress management in swine production to maintain optimal reproductive performance. Further investigations are warranted to elucidate the precise molecular pathways involved in the heat-induced testicular impairments observed in this study.

Seasonal expressions of the translocator protein (18 kDa), voltage-dependent anion channel, and steroidogenic acute regulatory protein in the scent glands of muskrats (Ondatra zibethicus)

Steroidogenesis machinery involves the steroidogenic acute regulatory protein (StAR), which regulates cholesterol transfer within the mitochondria, and the transport of cholesterol via a channel composed of 18-kDa translocator protein (TSPO), the voltage-dependent anion channel (VDAC) plus some accessory proteins. In this study, we investigated the immunolocalizations and expressions of StAR, TSPO, VDAC and cytochrome P450 side chain cleavage enzyme (P450scc, CYP11A1) in the scent glands of muskrats (Ondatra zibethicus) during the breeding and non-breeding periods. StAR, TSPO, VDAC and CYP11A1 were immunolocalized in the scent glandular, interstitial and epithelial cells in both breeding and non-breeding seasons with stronger immunostaining in the breeding season. The mRNA expression levels of StAR, TSPO, VDAC and CYP11A1 were higher in the scent glands of the breeding season than those of the non-breeding season. The circulating follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) as well as scent glandular T and dihydrotestosterone (DHT) concentrations were also significantly higher in the breeding season. Additionally, the transcriptomic study in the scent glands identified that differentially expressed genes might be related to the lipid metabolic process, integral component of membrane, and steroid hormone receptor activity and hormone activity using GO analysis. Further in vitro study verified that StAR, TSPO, VDAC and CYP11A1 expression levels increased significantly after human chorionic gonadotropin, hCG/FSH treatment compared with the control group. The KEGG pathway enriched by differentially expressed genes detected to be involved in endocrine system or amino acid metabolism. These findings suggested that the scent glands of the muskrats have ability to synthesize steroids de novo, and that the steroid hormones may have an important regulatory role in the scent glandular function via an autocrine/paracrine pathway.

Expression and regulation of Noggin4 gene in chicken ovarian follicles and its role in the proliferation and differentiation of granulosa cells

As a member of Noggin family, Noggin4 is reported to play an important role in the formation of head structure during the embryo development of Xenopus laevis and chicken. We previously detected an increase of Noggin4 transcript in the granulosa cells of chicken hierarchal follicles (Post-GCs) compared to pre-hierarchal follicles (Pre-GCs) by ONT transcriptome sequencing. To further clarify the role of Noggin4 in chicken follicle selection, in this study, we investigated its expression, regulation and function in follicles and granulosa cells. The mRNA expression of chicken Noggin4 exhibited dynamic changes during follicle development. It was significantly higher in the small yellow follicles than in the small white, F6, F5 and F4 follicles, and also increased in Post-GCs than in Pre-GCs. The Noggin4 mRNA could be stimulated by follicle stimulating hormone (FSH) and bone morphogenetic protein 4 (BMP4) in both Pre-GCs and Post-GCs. However, the estrogen and progesterone could exert opposing transcriptional regulations on Noggin 4 mRNA in both Pre- and Post-GCs. In chicken Post-GCs, knockdown of Noggin4 by siRNA reduced the mRNA expression of steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), but increased that of Wnt family member 4 (Wnt4), while overexpression of Noggin4 significantly decreased the mRNA expression of Wnt4 but had no marked effects on that of STAR and CYP11A1. Moreover, Noggin4 significantly decreased the mRNA expression of BMP4 in both Pre-GCs and Post-GCs. Overexpression of Noggin4 inhibited the proliferation of both Pre-GCs and Post-GCs. These data collectively suggest an important role of Noggin4 in chicken follicle selection, especially on the proliferation of granulosa cells.

Knockdown of ZnT4 Induced Apoptosis, Inhibited Proliferation and testosterone synthesis of TM3 cells.

Zinc deficiency has a huge impact on male reproduction. The zinc transporter (ZnT) family is involved in the maintenance of zinc homeostasis and testosterone synthesis. However, the underlying mechanisms remain to be investigated. Therefore, in this study, we aimed to determine the effect of zinc transporter 4 (ZnT4) on testosterone synthesis in male Kunming mice and mouse Leydig cells. The results of this study showed that compared with the zinc normal diet group (Con group), the zinc-deficient diet group (ZnD group) had decreased zinc content and increased ZnT4 expression in testicular tissues, and decreased serum testosterone levels, suggesting that ZnT4 may be involved in Leydig cell injury resulting from a zinc-deficient diet. Subsequently, mouse Leydig cell line TM3 cells were used to analyze the effect of ZnT4 downregulation on TM3 cell proliferation and apoptosis, on testosterone synthesis, and its underlying mechanisms. Here, we show that knockdown of ZnT4 can induce the accumulation of zinc, inhibit the viability, and induce apoptosis in TM3 cells. In addition, knockdown of ZnT4 downregulated testosterone concentration and expression of testosterone synthesis-related proteins steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase/D5-D4 isomerase (3β-HSD) in TM3 cells, while hCG could rescue their levels. We show that it is ZnT4 that plays a role in testosterone production through a mediated PI3K/Akt/mTOR autophagy pathway, whereas mTORC1 complex inhibitor (Rapa) blocks the decrease in testosterone levels caused by ZnT4 downregulation. In conclusion, the above results indicate that ZnT4 plays an important role in regulating testosterone synthesis.

Studying the effect of hyperoside on recovery from cyclophosphamide induced oligoasthenozoospermia

Oligoasthenozoospermia is becoming a serious problem, but effective prevention or treatment is lacking. Hyperoside, one of the main active ingredients in traditional Chinese medicine, may be effective in the treatment of oligoasthenozoospermia. In this study, we used cyclophosphamide (CTX: 50 mg/kg) to establish a mouse model of Oligoasthenozoospermia to investigate the therapeutic effect of hyperoside (30 mg/kg) on CTX-induced oligoasthenozoospermia. All mice were divided into four groups: blank control group (Control), treatment control group (Hyp), disease group (CTX) and treatment group (CTX + H). Mice body weight, testicular weight, sperm parameters and testicular histology were used to assess the reproductive capacity of mice and to explore the underlying mechanism of hyperoside in the treatment of oligoasthenozoospermia by assessing hormone levels, protein levels of molecules related to hormone synthesis and transcript levels of important genes related to spermatogenesis. Treatment with hyperoside significantly improved sperm density, sperm viability and testicular function compared to untreated oligoasthenozoospermia mice. In mechanism, treatment with hyperoside resulted in significant improvement in pathological changes in spermatogenic tubules, with an increase in testosterone production, and upregulations of Protein Kinase CAMP-Activated Catalytic Subunit Beta (PRKACB), Steroidogenic Acute Regulatory Protein (STAR), and Cytochrome P450 Family 17 Subfamily A Member 1 (CYP17A1) for testosterone production. Hyperoside also promoted the cell cycle of germ cells and up-regulated meiosis and spermatogenesis-related genes, including DNA Meiotic Recombinase 1 (Dmc1), Ataxia telangiectasia mutated (Atm) and RAD21 Cohesin Complex Component (Rad21). In conclusion, hyperoside exerted protective effects on oligoasthenozoospermia mice by regulating testosterone production, meiosis and sperm maturation of germ cells.

Effects of exposure to the explosive and environmental pollutant 2,4,6-trinitrotoluene on ovarian follicle development in rats.

Although 2,4,6-trinitrotoluene (TNT) is a dangerous carcinogen in environmental pollution, information on the reproductive effects of TNT explosive contamination is limited. To explore the possible ovarian effects, TNT explosive-exposed rat models were established, and Wistar female rats were exposed to low and high TNT (40 g and 80 g, air and internal) explosives. After a month of exposure, the estrous cycle, ovarian histopathology, and follicle counting were conducted. Serum hormones follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-Müllerian hormone (AMH), progesterone, testosterone, and estradiol were detected, and the mRNA and protein expression of steroidogenic enzymes were measured. The results showed that the diestrus phase duration was significantly (P < 0.05) increased in the high TNT-exposed groups. In addition, the proportions of preantral follicles were significantly (P < 0.05) decreased in the high TNT-exposed groups, as well as the proportions of atretic follicles. The serum estradiol levels were significantly (P < 0.05) increased, and the follicle-stimulating hormone and luteinizing hormone levels were significantly (P < 0.05) decreased in the high TNT-exposed groups. The mRNA levels of steroidogenic acute regulatory protein (Star), cytochrome P450 cholesterol side chain cleavage (Cyp11a1, Cyp17a1 and Cyp19a1), hydroxysteroid dehydrogenase 3b (Hsd3b) and steroidogenic factor-1 (SF-1) were significantly (P < 0.05) increased in the TNT-exposed groups. The protein levels of Star, Cyp11a1 and Hsd3b were increased (P < 0.05) in the TNT-exposed groups. These results indicate that the exposure of rats to TNT explosive can subsequently affect ovarian follicle development, suggesting that the mechanism may involve disrupting steroidogenesis.

Morphological and molecular changes in the Harderian gland of streptozotocin-induced diabetic rats.

Using a rat model of type 1 diabetes (T1D) obtained by treatment with streptozotocin, an antibiotic that destroys pancreatic β-cells, we evaluated the influence of subsequent hyperglycemia on the morphology and physiology of the Harderian gland (HG). HG is located in the medial corner of the orbit of many terrestrial vertebrates and, in rodents, is characterized by the presence of porphyrins, which being involved in the phototransduction, through photo-oxidation, produce reactive oxygen species activating the autophagy pathway. The study focused on the expression of some morphological markers involved in cell junction formation (occludin, connexin-43, and α-tubulin) and mast cell number (MCN), as well as autophagic and apoptotic pathways. The expression of enzymes involved in steroidogenesis [steroidogenic acute regulatory protein (StAR), and 3β-hydroxysteroid dehydrogenase (3β-HSD)] and the level of lipid peroxidation by thiobarbituric acid reactive species assay were also evaluated. The results strongly indicate, for the first time, that T1D has a negative impact on the pathophysiology of rat HG, as evidenced by increased oxidative stress, morphological and biochemical alterations, hyperproduction and secretion of porphyrins, increased MCN, reduced protein levels of StAR and 3β-HSD, and, finally, induced autophagy and apoptosis. All the combined data support the use of the rat HG as a suitable experimental model to elucidate the molecular damage/survival pathways elicited by stress conditions.

High Expression of Adrenal Cortisol Synthases Is Acquired After Intrauterine Inflammation in Periviable Sheep Fetuses.

Intrauterine inflammation, a representative stressor for the fetus, has been shown to alter the hypothalamus-pituitary-adrenal (HPA) axis reactivity in preterm fetuses and increase postnatal cortisol production. However, the mechanism of this alteration has not yet been elucidated. We aimed to clarify the effects of endotoxin-induced intrauterine inflammation on the HPA axis of periviable sheep fetuses. Fetal sheep (0.63 term) were divided into 2 groups: (1) the endotoxin group, in which the endotoxin was injected into the amniotic fluid; and (2) the control group, in which the saline solution was injected instead. A corticotropin-releasing hormone (CRH) challenge test was performed on the third day after injection to evaluate the cortisol-producing capacity of each group. Gene expression levels in the fetal adrenal glands of each group were analyzed by RNA-seq. The cortisol levels were significantly higher in the endotoxin group than in the control group after CRH challenge (P = .02). There were no significant differences in the responsiveness of adrenocorticotropin and cortisone between the 2 groups. Gene expression levels of the following enzymes involved in cortisol synthesis were significantly elevated in the endotoxin group: cytochrome P450 family (CYP) 11 subfamily A member 1 (log2FC 1.75), CYP 17 subfamily A member 1 (log2FC 3.41), 3β-hydroxysteroid dehydrogenase type I (log2FC 1.13), steroidogenic acute regulatory protein (log2FC 1.09), and CYP 21 (log2FC 0.89). Periviable fetuses exposed to inflammation in utero have altered the responsiveness of the HPA axis with increased expression of enzymes involved in cortisol synthesis in the adrenal gland.

α-Linolenic acid-regulated testosterone biosynthesis via activation of the JNK-SF-1 signaling pathway in primary rooster Leydig cells

As a functional fatty acid, α-linolenic acid (ALA) is essential in promoting animal testosterone biosynthesis. This study investigated the effects of ALA on testosterone biosynthesis and the possible mechanism underlying the signaling pathway in primary Leydig cells of the rooster. Methods: Primary rooster Leydig cells were treated with ALA (0, 20, 40, or 80 μmol/L) or pretreated with a p38 inhibitor (50 μmol/L), a c-Jun NH2-terminal kinase (JNK) inhibitor (20 μmol/L), or an extracellular signal-regulated kinase (ERK) inhibitor (20 μmol/L) before ALA treatment. Testosterone content in the conditioned culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The expression of steroidogenic enzymes and JNK-SF-1 signaling pathway factors was detected using real-time fluorescence quantitative PCR (qRT-PCR). Results: Supplementation with ALA significantly increased testosterone secretion within culture media (P < 0.05), and the optimized dose was 40 μmol/L. Compared with the control group, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression significantly increased (P < 0.05) in the 40 μmol/L ALA group; 17-hydroxylase/c17-20 lyase (P450c17) and p38 mRNA expressions were not significantly different in the 40 μmol/L ALA group; ERK and JNK mRNA expressions were significantly upregulated (P < 0.05) in 40 μmol/L ALA group. In the inhibitor group, testosterone levels were significantly downregulated (P < 0.05). Compared with the 40 μmol/L ALA group, StAR, P450scc, and P450c17 mRNA expressions were significantly decreased (P < 0.05), and 3β-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3β-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3β-HSD, and P450c17 mRNA expressions were significantly decreased (P < 0.05) in JNK inhibitor group. Additionally, the increased steroidogenic factor 1 (SF-1) gene expression levels induced by ALA were reversed when the cells were pre-incubated with JNK and ERK inhibitors. The levels in the JNK inhibitor group were significantly lower than those in the control group (P < 0.05). Conclusion: ALA may promote testosterone biosynthesis by activating the JNK-SF-1 signaling pathway to upregulate StAR, P450scc, 3β-HSD, and P450c17 expression in primary rooster Leydig cells.

P-081 In utero exposure to diisopentyl phthalate (DiPeP) disrupts the expression of steroidogenesis-related genes and induces multinucleated gonocytes in the fetal rat testis

Abstract Study question Can the plasticizer diisopentyl phthalate (DiPeP) disrupt testosterone biosynthesis and alter fetal rat testis histology similarly to other common toxic phthalates? Summary answer DiPeP can dose-dependently suppress mRNA levels of steroidogenic genes and induce multinucleated gonocytes in the fetal rat testis, resembling the effects of other toxic phthalates. What is known already DiPeP is an uncommon phthalate, whose metabolites have been ubiquitously detected in urinary samples from Brazilian pregnant women and children. These results, which contrast with the undetected levels of DiPeP metabolites in other human biomonitoring studies worldwide, led us to investigate DiPeP reproductive and developmental toxicity in rats. Our preliminary data indicate that DiPeP is a potent antiandrogenic phthalate, but no studies have been conducted to assess the impact of DiPeP exposures on the incidence of multinucleated gonocytes in the rat fetal testis, a common feature of the rat phthalate syndrome. Study design, size, duration Pregnant Wistar rats (n = 7-9 dams/group) were treated orally (gavage) with DiPeP 0 (control), 11, 33, and 100 mg/kg/day between gestation days 14-20. Dose levels were based on prior rat phthalate toxicity studies and canola oil was used as a vehicle. On gestation day 20, dams were euthanized and up to three male fetuses from each litter were removed for the collection of fetal testes and the assessment of the study endpoints. Participants/materials, setting, methods Fetal rat testes were used for enzyme immunoassay quantification of testosterone production following ex vivo incubation of testes in culture media, analysis of mRNA levels of steroidogenic genes and insulin-like factor 3 (Insl3) by quantitative polymerase chain reaction (qPCR), and histological assessment of multinucleated gonocytes by optical microscopy. Main results and the role of chance No signs of systemic toxicity were observed as indicated by the lack of alterations in maternal body weights and the unchanged number, viability, and weight of fetuses. There were no significant changes in the ex vivo testosterone production at any DiPeP dose. On the other hand, DiPeP induced significant reductions in the mRNA expression of key steroidogenic genes, including suppressed mRNA levels of the steroidogenic acute regulatory protein (Star) and cytochrome P450 family 11 subfamily A member 1 (Cyp11a1) at 100 mg/kg/day and of cytochrome P450 family 17 subfamily A member 1 (Cyp17a1) at 33 and 100 mg/kg/day. Gene expression of Insl3, which is essential for the process of testis descent, was reduced at the highest dose by 58% in relation to control, but this change was not significant (p = 0.07). The number of multinucleated gonocytes, corrected for the number of testicular cord sections or the total area of analyzed testicular cords was significantly increased in the groups exposed to 33 and 100 mg/kg/day. Chance is an unlikely explanation for our results considering the observed dose-dependent responses and the consistency of our findings with prior phthalate reproductive toxicity data. Limitations, reasons for caution The tested doses are higher than the expected human exposure levels and animal-to-human extrapolation should be done with caution. Wider implications of the findings DiPeP suppresses the expression of steroidogenic genes and induces multinucleated gonocytes in the fetal rat testis. These endpoints seem more sensitive than inhibition of ex vivo testosterone production. Although human exposure levels are lower than the doses tested here, DiPeP can potentially act cumulatively with chemicals that share similar mechanisms. Trial registration number not applicable

Resveratrol stimulates StAR expression and progesterone production by GPER-mediated downregulation of Snail expression in human granulosa cells.

Steroidogenic acute regulatory protein (StAR) plays a critical role in the regulation of progesterone (P4) production. Resveratrol (RSV), a natural polyphenol, has beneficial effects on reproductive function. However, its effects on StAR expression and P4 production in human granulosa cells remain undetermined. In this study, we showed that treatment of RSV upregulated StAR expression in human granulosa cells. G protein-coupled estrogen receptor (GPER) and ERK1/2 signaling were involved in RSV-stimulated StAR expression and P4 production. In addition, the expression of a transcriptional repressor, Snail, was downregulated by RSV, which contributed to the RSV-induced inductions of StAR expression and P4 production.

Inhibition of phosphodiesterase PDE8B reduces activation of primordial follicles in mouse ovaries.

In the ovaries, cyclic adenosine 3',5'-monophosphate (cAMP) is a second messenger supporting the generation of steroids. Phosphodiesterases (PDEs) are regulators of intracellular cAMP, and therefore, potential regulators of ovarian function. Interestingly, the family of PDE genes are differentially expressed in human oocytes and granulosa cells from primordial and primary follicles, suggesting diverse roles. In this study, we addressed the functions of PDE3B and PDE8B in primordial follicle regulation using inhibitors of PDE3B and PDE8B in murine ovary primary in vitro cultures. Inhibition of PDE8B in ovarian cultures prevented primordial follicle activation, while inhibition of PDE3B had no effect on follicle distribution in the ovary, under the tested conditions. As cAMP levels may increase steroid levels, we assessed the protein levels of the steroidogenic acute regulatory protein (StAR) and aromatase enzymes, and found that inhibition of PDE3B reduced StAR protein levels, whereas inhibition of PDE8 did not alter StAR expression in our murine ovary culture system conditions. Our results showed that ketotifen-induced inhibition of PDE8B can decrease primordial follicle activation, whereas we observed no effect of follicle distribution, when PDE3B was inhibited. Expression of the StaR enzyme was not altered when PDE8B was inhibited, which might reflect not sufficient inhibition by ketotifen to induce StAR alterations, or redundant mechanisms.

Different types of bisphenols alter ovarian steroidogenesis: Special attention to BPA

Endocrine disruptors such as bisphenol A (BPA) and some of its analogues, including BPS, BPAF, and BPE, are used extensively in the manufacture of plastics. These synthetic chemicals could seriously alter the functionality of the female reproductive system. Although the number of studies conducted on other types of bisphenols is smaller than the number of studies on BPA, the purpose of this review study was to evaluate the effects of bisphenol compounds, particularly BPA, on hormone production and on genes involved in ovarian steroidogenesis in both in vitro (human and animal cell lines) and in vivo (animal models) studies.The current data show that exposure to bisphenol compounds has adverse effects on ovarian steroidogenesis. For example, BPA, BPS, and BPAF can alter the normal function of the hypothalamic-pituitary-gonadal (HPG) axis by targeting kisspeptin neurons involved in steroid feedback signals to gonadotropin-releasing hormone (GnRH) cells, resulting in abnormal production of LH and FSH. Exposure to BPA, BPS, BPF, and BPB had adverse effects on the release of some hormones, namely 17-β-estradiol (E2), progesterone (P4), and testosterone (T). BPA, BPE, BPS, BPF, and BPAF are also capable of negatively altering the transcription of a number of genes involved in ovarian steroidogenesis, such as the steroidogenic acute regulatory protein (StAR, involved in the transfer of cholesterol from the outer to the inner mitochondrial membrane, where the steroidogenesis process begins), cytochrome P450 family 17 subfamily A member 1 (Cyp17a1, which is involved in the biosynthesis of androgens such as testosterone), 3 beta-hydroxysteroid dehydrogenase enzyme (3β-HSD, involved in the biosynthesis of P4), and cytochrome P450 family 19 subfamily A member 1 (Cyp19a1, involved in the biosynthesis of E2). Exposure to BPA, BPB, BPF, and BPS at prenatal or prepubertal stages could decrease the number of antral follicles by activating apoptosis and autophagy pathways, resulting in decreased production of E2 and P4 by granulosa cells (GCs) and theca cells (TCs), respectively. BPA and BPS impair ovarian steroidogenesis by reducing the function of some important cell receptors such as estrogens (ERs, including ERα and ERβ), progesterone (PgR), the orphan estrogen receptor gamma (ERRγ), the androgen receptor (AR), the G protein-coupled estrogen receptor (GPER), the FSHR (follicle-stimulating hormone receptor), and the LHCGR (luteinizing hormone/choriogonadotropin receptor). In animal models, the effects of bisphenol compounds depend on the type of animals, their age, and the duration and dose of bisphenols, while in cell line studies the duration and doses of bisphenols are the matter.

The involvement of let-7 in hCG-induced progesterone synthesis via regulating p27Kip1 and p21Cip1 expression

Progesterone is essential in females to maintain a regular menstrual cycle and pregnancy. The luteinizing hormone (LH) surge induces the luteinization of granulosa cells and thecal cells to form the corpus luteum, which is responsible for progesterone synthesis. However, the specific mechanism of how hCG, the analog of LH, regulates progesterone synthesis has yet to be fully discovered. In this study, we found that progesterone level was increased in adult wild-type pregnant mice 2 and 7 days post-coitum, along with a decrease in let-7 expression compared with the estrus stage. Besides, the let-7 expression was negatively correlated with progesterone level in post-delivery day 23 wild-type female mice after being injected with PMSG and hCG. Then, using let-7 transgenic mice and a human granulosa cell line, we found that overexpression of let-7 antagonized progesterone level via targeting p27Kip1 and p21Cip1 and steroidogenic acute regulatory protein (StAR) expression, which is a rate-limiting enzyme in progesterone synthesis. Furthermore, hCG suppressed let-7 expression by stimulating the MAPK pathway. This study elucidated the role of microRNA let-7 in regulating hCG-induced progesterone production and provided new insights into its role in clinical application.

Downregulation of StAR driven neurosteroid biosynthesis as a distinctive feature in the brains of Alzheimer's disease patients

• The steroidogenic acute regulatory (StAR) protein mediates neurosteroidogenesis that progressively deteriorates during human senescence. • We observed that postmortem brains of Alzheimer's disease (AD) patients exhibit suppression of StAR, free cholesterol (FC), and pregnenolone levels. • Attenuation of StAR expression, concomitant with neurosteroid biosynthesis, in AD brains, was inversely correlated with Aβ and pTau accumulation. • Our findings unequivocally demonstrate that downregulation of StAR governed neurosteroidogenesis correlated with aggregation of APP/Aβ and pTau in AD.

Identification and functional analyses of a novel FOXL2 pathogenic variant causing blepharophimosis, ptosis, and epicanthus inversus syndrome.

To discover the molecular pathogenic basis of the blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), and to predict the clinical subtype according to in vitro experiments, which is significant to the prognosis. A 3-year-old sporadic female patient with typical clinical manifestations of BPES was enrolled. The coding region of forkhead box L2 (FOXL2) gene was sequenced, and the functional assays were performed in vitro by Western blotting, subcellular localization experiment, luciferase reporter assay, and quantitative real-time polymerase chain reaction. A novel FOXL2 point pathogenic variant (c.274G>T) was detected, resulting in a truncated protein (p.E92*). Functional studies demonstrated that the FOXL2 pathogenic variant induced the subcellular mislocalization and the abnormal transcriptional activity on promoters of the steroidogenic acute regulatory protein (StAR or STARD1) gene and the odd-skipped related 2 transcription factor (OSR2) gene. A novel pathogenic variant is identified to expand the spectrum of the known FOXL2 mutations. The in vitro experiments provide reference data and more insights to the molecular pathogenesis of BPES. The predicted high risk of ovarian insufficiency makes it significant for the patient enrolled to have further follow-up and therapy concerning female endocrinology.