StAR Gene Expression Research Articles

Leptin fragment modulates the stimulatory effects of chorionic gonadotropin on testicular steroidogenesis in a model of diet-induced obesity in rats

Leptin, secreted by adipose tissue, indirectly stimulates the activity of GnRH-producing neurons of the hypothalamus and thus regulates the functional activity of the hypothalamic-pituitary-testicular (HPT) axis. As is known, the obesity is accompanied by systemic hyperleptinemia and impaired leptin transport in the central nervous system, which limits the use of full-length leptin as a drug. It was previously shown that intranasally administered leptin fragment MA-[D-Leu4]-OB3 (LF) enhances the steroidogenic effect of human chorionic gonadotropin (hCG) in rats fed a standard diet. An even more urgent task is to assess its effect on testicular steroidogenesis in conditions of obesity, which reduces reproductive functions in men. The aim of the work was to study the ability of LF (200 μg/kg, intranasally, 3 days) to modulate the effect of hCG (10 IU/rat, subcutaneously, once) on testicular steroidogenesis in rats with obesity induced by a high-fat/high-carbohydrate diet (HFHCD), and also to evaluate influence of the GnRH receptor antagonist cetrorelix (ANT, 75 µg/kg, subcutaneously, 3 days) on the effects of LF. Male Wistar rats were used for the study and received HFHCD for 20 weeks. In obese rats, the level of the luteinizing hormone (LH) receptor in the testes was reduced and the expression of the Cyp11a1 gene, encoding the steroidogenic enzyme cytochrome P450scc, was compensatory increased. LF administration enhanced the effect of hCG on the testosterone level in the blood and the expression of the Star gene, encoding the cholesterol transport protein StAR, which indicates the ability of LF to positively modulate the activity of the HPT axis in obesity. Co-administration of ANT and LF, on the contrary, reduced the stimulating effect of hCG on testosterone levels and Star gene expression, which may be due to the testicular effects of LF. Our data indicate the ability of LF to influence various components of the male gonadal axis under conditions of diet-induced obesity.

Nano Spirulina platensis countered cisplatin-induced repro-toxicity by reversing the expression of altered steroid hormones and downregulation of the StAR gene.

Cisplatin is a commonly utilized chemotherapy medication for treating different sarcomas and carcinomas. Its ability interferes with cancer cells' DNA repair pathways and postpones unfavorable outcomes in cancer patients. The current investigation's goal was to ascertain if nano Spirulina platensis (NSP) might shield rat testicles from cisplatin damage by assessing the expression of the StAR and SOD genes, sex hormones, 17ß-hydroxysteroid dehydrogenase(17ß-HSD), sperm profile picture, oxidative condition of testes, testicular histology, and DNA damage. Four equal and random groups of 28 adult male Wistar rats were created; the control group was given saline for 8weeks. An extraction of NSP at a concentration of 2500mg/kg body weight was administered orally for 8weeks to the NSP group. For the first 4weeks, the cisplatin group was intraperitoneally injected with 2mg/kg/body weight of cisplatin, and for the next 4weeks, they were given a dosage of 4mg/kg/body weight. The cisplatin + NSP group was given both NSP and cisplatin. The results of the experiment showed that intake of NSP and cisplatin improved sperm profile; re-established the balance of oxidizing agents and antioxidant state; enhanced testicular histology; promoted the histometric parameters of seminiferous tubules including epithelial height, their diameter, and Johnsen's score, decreasing DNA breakage in testicular tissue; increased testosterone level; decreased 17ß-HSD concentration; and upregulated both the StAR and SOD gene expression in testicles compared to rats exposed to cisplatin alone. These results demonstrate that NSP is a promising agent for improving cisplatin-induced testicular injury and infertility.

Alterations in the Hypothalamic-Pituitary-Adrenal Axis as a Response to Experimental Autoimmune Encephalomyelitis in Dark Agouti Rats of Both Sexes.

Multiple sclerosis (MS) is a chronic inflammatory disease that affects the central nervous system, usually diagnosed during the reproductive period. Both MS and its commonly used animal model, experimental autoimmune encephalomyelitis (EAE), exhibit sex-specific features regarding disease progression and disturbances in the neuroendocrine and endocrine systems. This study investigates the hypothalamic-pituitary-adrenal (HPA) axis response of male and female Dark Agouti rats during EAE. At the onset of EAE, Crh expression in the hypothalamus of both sexes is decreased, while males show reduced plasma adrenocorticotropic hormone levels. Adrenal gland activity is increased during EAE in both males and females, as evidenced by enlarged adrenal glands and increased StAR gene and protein expression. However, only male rats show increased serum and adrenal corticosterone levels, and an increased volume of the adrenal cortex. Adrenal 3β-HSD protein and progesterone levels are elevated in males only. Serum progesterone levels of male rats are also increased, although testicular progesterone levels are decreased during the disease, implying that the adrenal gland is the source of elevated serum progesterone levels in males. Our results demonstrate a sex difference in the response of the HPA axis at the adrenal level, with male rats showing a more pronounced induction during EAE.

The mollifying effect of Sambucus nigra extract on StAR gene expression, oxidative stress, and apoptosis induced by fenpropathrin in male rats

Fenpropathrin (FNP) is a man-made insecticide of to the pyrethroid class, commonly employed in agricultural and horticultural practices. However, it has a prolonged persistence in the environment. Sambucus nigra, also referred to as SN, is a botanical species recognized for its notable antioxidant characteristics. The objective of this study was to examine if SN extract could mitigate the reproductive toxicity induced by FNP in rats. A total of thirty rats were categorized into six distinct groups: a control group with no treatment, two groups getting SN extract at varying doses, a group receiving FNP, and two groups receiving both FNP and SN extract. The exposure to FNP led to a decline in the number and movement of sperm, lowered levels of testosterone, and reduced the activity of the StAR gene in the FNP group compared to the control group (p < 0.05). In addition, FNP resulted in a significant increase in malondialdehyde levels with a significant drop in GSH content compared to the control group (p < 0.05). Also, a significant increase in the expression of caspase 3. Nevertheless, the administration of SN extract alleviated these effects and reinstated spermatogenesis, thereby bringing the parameters closer to those observed in the control group. The data indicate that FNP can induce testicular harm and infertility, but SN extract can mitigate these detrimental consequences.

The influence of indole propionic acid on molecular markers of steroidogenesis, ER stress, and apoptosis in rat granulosa cells exposed to high glucose conditions

Hyperglycemia is known as one of the main causes of infertility in human societies. Indole propionic acid (IPA) is produced by intestinal microbiota and has antioxidant and anti-inflammatory properties. This study aims to investigate the effects of IPA on molecular indices of steroidogenesis, ER stress, and apoptosis induced by high glucose (HG) in granulosa cells. Primary GCs, isolated from ovarian follicles of Rats were cultured in 5 mM (control) and 30 mM (HG) of glucose and in the presence of 10 and 20 µM of IPA for 24 h. The cell viability was assessed by MTT. The gene expression of P450SCC, 3βHSD, CYP19A, BAX, BCL2, and STAR was evaluated by Real-Time PCR. Protein expression of ATF6, PERK, GRP78, and CHOP determined by western blot. Progesterone, estradiol, IL-1β, and TNF-α were measured by ELISA. HG decreased the viability, and expression of P450SCC, 3βHSD, CYP19A, BCL2, STAR, and increased BAX. 10 and 20 µM of IPA increased cell viability, expression of P450SCC, 3βHSD, CYP19A, BCL2 and STAR and decreased BAX compared to the HG group. The expression of ATF6, PERK, GRP78, and CHOP proteins increased by HG and IPA decreased the expression of these proteins compared to the HG group. Also, HG decreased progesterone and estradiol levels and increased IL-1β and TNF-α. IPA significantly increased progesterone and estradiol and decreased IL-1β and TNF-α compared to the HG group. IPA can improve the side effects of HG in GCs of rats, as responsible cells for fertility, by improving steroidogenesis, regulation of ER-stress pathway, suppression of inflammation, and apoptosis.

The effect of adipose tissue StAR gene expression on ICSI among Polycystic Ovarian Syndrome cases and matched controls: a case-control study

The effect of adipose tissue StAR gene expression on ICSI among Polycystic Ovarian Syndrome cases and matched controls: a case-control study

Fertility protection: a novel approach using pretreatment with mesenchymal stem cell exosomes to prevent chemotherapy–induced ovarian damage in a mouse model

BackgroundPrimary ovarian insufficiency (POI) refers to the loss of ovarian functionbefore the age of 40 years and results in amenorrhea and infertility. POI has diverse causes, but a common cause is exposure to gonadotoxic chemotherapy for cancer treatment. Due to the risk of developing POI, patients who want to preserve their fertility may consider various procedures for fertility preservation. However, current fertility preservation options are highly invasive, carry substantial risks, and have uncertain success rates. Recent studies from our group and others reported that mesenchymal stem cells (MSCs) and MSC-derived exosomes can restore ovarian functions in preclinical models of POI by restoring damaged cells and inhibiting apoptosis. Although the restorative effect of MSC-derived exosomes has been well reported in previous studies, the potential of MSC-derived exosomes for preventing ovarian damage has not been fully elucidated. ObjectiveWe hypothesized that the antiapoptotic potential of MSC-derived exosomes may protect ovarian tissue from chemotherapy-induced damage. Study designIn this study, we delivered MSC-derived exosomes directly into mouse ovaries before chemotherapy. Sixty mice were divided into three groups (20 per group), which were labeled the control, CTx (chemotherapy), and FP (fertility protection) groups. Only the FP group mice received exosomes, while the control and CTx group mice received saline. After exosome injection, the CTx and FP groups of mice were subjected to chemotherapy treatment to induce ovarian damage. After chemotherapy, we evaluated the protective effects of exosome treatment on ovarian function, such as estrus cyclicity, serum hormone levels, and the fertility rate, by comparing these outcomes between the CTx and FP groups. These outcomes were also compared with those of the control group for comparison with outcomes under healthy conditions. ResultsAfter intraovarian injection of exosomes before chemotherapy, the mice were able to maintain their estrus cycle (4–5-day cyclicity), serum AMH level (66.06±26.40 ng/ml, not significantly different from that of the healthy controls), folliculogenesis (32.2±11.3 in the CTx group/46.4±14.1 in the FP group, p<0.05), steroidogenesis marker StAR gene expression (0.44±0.11-fold in the CTx group and 0.88±0.31-fold in the FP group, p<0.05), and fertility (2 of 8 in the CTx group and 5 of 8 in the FP group), preventing chemotherapy-induced damage. We found that exosome treatment before chemotherapy can preserve ovarian function and protect fertility through the overexpression of ABC transporters, such as ABCB1b (10.17±17.75-fold in the CTx group and 44.14±33.25-fold in the FP group; p<0.05), and ABCC10 (3.25±0.59-fold in the CTx group and 5.36±1.86-fold in the FP group; p<0.05). ConclusionIn this study, we present a novel fertility protection method using MSC-derived exosomes. We conclude that MSC-derived exosomes are a promising simple treatment option for fertility protection in reproductive-age patients receiving gonadotoxic chemotherapy.

The relationship between CYP19A1 gene expression in luteinized granulosa cells and follicular estradiol output in women with endometriosis

Endometriosis was claimed to negatively affect the intrafollicular environment, hindering oocyte competence. Previous studies evaluated expression levels of cytochrome P450 aromatase (CYP19A) in granulosa and cumulus oophorus cells collected from endometriosis women, but results are controversial. To further investigate the intrafollicular environment whose alteration may potentially disturb ovarian steroidogenesis in endometriosis, gene expression of CYP19A and of its upstream enzymes, StAR and 3βHSD was assessed in luteinized granulosa cells isolated from follicular fluids (FF) collected during Assisted Reproduction Technology (ART) procedures in women with stage III-IV disease and from subjects without the condition. In a subgroup of patients, cumulus oophorus cells (COCs) were also assessed for CYP19A, StAR and 3βHSD gene expression. No difference in mRNA expression of CYP19A1, StAR and 3βHSD in both granulosa cells and COCs was observed between the two groups of patients. No significant difference was also found between estradiol FF levels detected in endometriosis patients (median=873, IQR=522–1221 ng/ml)) and control patients (median=878, IQR=609–1137 ng/ml). To gain more insight into the intrafollicular regulation of CYP19A in patients with endometriosis, associations between expression of the analyzed genes, systemic and follicular 17β-estradiol levels and ART outcomes were assessed. While in the control group, levels of CYP19A1, StAR and 3βHSD transcripts significantly correlated with follicular estradiol levels (adjusted R² of 0.60), no significant association was detected in affected women (adjusted R² of 0.23). After stratification of the populations based on the presence of the disease, CYP19A1 expression was shown to correlate with the number of oocytes retrieved [β:− 1.214;95%CI: − 2.085 - (−0.343); p = 0.007] in the control group while this association was not present in patients with endometriosis [β:− 0.003; 95%CI:− 0.468–0.461; p = 0.988)]. These results do not support data from the literature indicating a reduced aromatase expression in granulosa cells of affected women, but they highlight a potential subtle mechanism affecting the ovulation process in these women.

Blumea balsamifera Leaf Extract Maintain Testosterone Levels in Hypercholesterolemic Rats Through Antioxidant Mechanism and Upregulation of StAR Gene Expression

High cholesterol levels can increase lipid peroxidation in tissues that are potentially toxic to reproductive organ cells, especially the Leydig cells that produce the testosterone hormone. Blumea balsamifera leaf extract (BBLE) has the main content in the form of flavonoid compounds with antihypercholesterolemic activity. This study aimed to determine the effect of BBLE administration on MDA levels, StAR mRNA expression, Leydig cell counts, and testosterone levels in hypercholesterolemic rats. A posttest-only control group design was utilized in this research. For 50 days, 36 male Wistar rats had been separated into two groups: 1) the control group (HCD + 1 ml/day sterile aquadest) and 2) the BBLE group (HCD + 4 mg/bb rats per day). After treatment, MDA testicular tissue levels, StAR mRNA expression, Leydig cell count, and testosterone levels were measured in both groups. The data collected were statistically examined using the Independent T-test and path analysis. The results indicated that the MDA level was lower in the BBLE group, though StAR gene expression, Leydig cell count, and testosterone levels were significantly greater (p0.05). StAR mRNA expression had a significant direct effect on testosterone levels. Administration of BBLE had been shown to improve testosterone hormone secretion in hypercholesterolemic rats by preventing oxidative stress in testicular tissue with the signs of lower MDA levels, up-regulation of the StAR mRNA, and Leydig cell regeneration.

Oral follicle-stimulating hormone receptor agonist affects granulosa cells differently than recombinant human FSH

To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing invitro fertilization. Basic science research with a preclinical allosteric FSHR agonist. University hospital. Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS. Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle. Estradiol (E2) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells. TOP5300 consistently stimulated E2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS. TOP5300 appears to stimulate E2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E2 production.

Liraglutide ameliorates diabetic-induced testicular dysfunction in male rats: role of GLP-1/Kiss1/GnRH and TGF-β/Smad signaling pathways.

Introduction: Glucagon-like peptide -1 (GLP-1) is released by intestinal cells to stimulate glucose-dependent insulin release from the pancreas. GLP-1 has been linked to ameliorating obesity and/or diabetic complications as well as controlling reproductive function. Liraglutide is a GLP-1 receptor agonist (GLP-1RA) with 97% homology with GLP-1. The main objective of this study was to investigate the ameliorative role of liraglutide in diabetic-induced reproductive dysfunction in male rats. Methods: Rats were randomly allocated into 3 groups; a control group, a diabetic group, and a liraglutide-treated diabetic group. Results: In the diabetic group, a significant increase in BMI, FBG, HbA1c, HOMA-IR, TC, TAG, LDL, IL6, TNFα, and MDA, as well as decreased serum insulin, HDL, GSH, total testosterone, LH, and FSH, were shown compared to the control group. Furthermore, A significant downregulation in relative hypothalamic gene expression of GLP-1R, PPAR-α, PGC-1α, kiss, kiss1R, leptin, leptin R, GnRH GLP-1R, testicular PGC-1α, PPARα, kiss1, kiss1R, STAR, CYP17A1, HSD17B3, CYP19A, CYP11A1, and Smad7, as well as upregulation in hypothalamic GnIH and testicular TGF- β and Smad2 expression, were noticed compared to the control group. Liraglutide treatment significantly improved such functional and structural reproductive disturbance in diabetic rats. Conclusion: GLP-1RAs ameliorated the deleterious effects of diabetes on reproductive function by targeting GLP-1/leptin/kiss1/GnRH, steroidogenesis, and TGF- β/Smad pathways.

Rat Ovarian Function Is Impaired during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease affecting the CNS and occurring far more prevalently in women than in men. In both MS and its animal models, sex hormones play important immunomodulatory roles. We have previously shown that experimental autoimmune encephalomyelitis (EAE) affects the hypothalamic-pituitary-gonadal axis in rats of both sexes and induces an arrest in the estrous cycle in females. To investigate the gonadal status in female rats with EAE, we explored ovarian morphometric parameters, circulating and intraovarian sex steroid levels, and the expression of steroidogenic machinery components in the ovarian tissue. A prolonged state of diestrus was recorded during the peak of EAE, with maintenance of the corpora lutea, elevated intraovarian progesterone levels, and increased gene and protein expression of StAR, similar to the state of pseudopregnancy. The decrease in CYP17A1 protein expression was followed by a decrease in ovarian testosterone and estradiol levels. On the contrary, serum testosterone levels were slightly increased. With unchanged serum estradiol levels, these results point at extra-gonadal sites of sex steroid biosynthesis and catabolism as important regulators of their circulating levels. Our study suggests alterations in the function of the female reproductive system during central autoimmunity and highlights the bidirectional relationships between hormonal status and EAE.

Ameliorative effects of nano Moringa on fluoride-induced testicular damage via down regulation of the StAR gene and altered steroid hormones

Fluoride is a common environmental contaminant that has harmful effects on human health when it is present in high concentrations. Fluoride enters the bloodstream after being absorbed by the gastrointestinal system when fluoride-contaminated groundwater is consumed by people. The aim of the present study was to determine whether polyphenol-rich nano Moringa oleifera (NMO) could protect rat testicles from sodium fluoride (NaF) damage by evaluating sperm quality, sex hormones, testicular oxidative status, histopathology, and StAR gene expression. Twenty-eight adult Wistar rats were divided equally and randomly into four groups: group one received distilled water; group two received NMO at a dosage of 250mg/kg/body weight; group three received NaF at a dosage of 10mg/kg/body weight; and group four received NaF and NMO. The rats were orally administrated daily for a duration of eight weeks. The study's findings demonstrated that, in comparison to rats exposed to NaF alone, co-administration of NMO and NaF enhanced sperm motility and viability, decreased sperm morphological changes, restored the balance between oxidant and antioxidant status, improved testosterone and dehydroepiandrosterone, improved testicular histology, raised the Johnson score, and upregulated the StAR gene in testicular tissue. These findings show that NMO is promise as a prophylactic medication against sodium fluoride-induced testicular damage because administration of NMO had no adverse effects and enhanced reproductive health.

Sinapic and ferulic acid phenethyl esters increase the expression of steroidogenic genes in MA-10 tumor Leydig cells

During aging, the production of androgens by the testis Leydig cells gradually decreases. Phenolic compounds can improve testosterone biosynthesis and delay the onset of hypogonadal symptoms in males. In this study, sinapic acid phenethyl ester was evaluated for its ability to regulate gene expression and steroid production in Leydig cells. Specifically, the effects of this ester on the transcriptome of MA-10 Leydig cells were investigated by RNA-Seq. To better establish a structure-function relationship of the hydroxy-methoxyphenyl moiety of sinapic and phenethyl ester, its influences on gene expression were compared to those of its ferulic acid phenethyl ester analogue. According to the transcriptomic analysis, most genes encoding enzymes related to cholesterol biosynthesis are increased in response to sinapic acid phenethyl ester treatment of MA-10 Leydig cells. Interestingly, treatments with 10 μM of ferulic acid phenethyl ester increased cAMP-dependent Star promoter activation, gene expression and protein levels. In addition, treatments of MA-10 Leydig cells with 10 μM of sinapic or ferulic acid phenethyl ester resulted in increased progesterone production. Thus, our results indicate that sinapic and ferulic acid phenethyl esters can improve cholesterol and steroid biosynthesis in testicular Leydig cells.

A Short Promoter Region Containing Conserved Regulatory Motifs Is Required for Steroidogenic Acute Regulatory Protein (Star) Gene Expression in the Mouse Testis.

In the testis, Leydig cells produce steroid hormones that are needed to masculinize typical genetic males during fetal development and to initiate and maintain spermatogenesis at puberty and adulthood, respectively. Steroidogenesis is initiated by the transfer of cholesterol from the outer to the inner mitochondrial membrane through the action of steroidogenic acute regulatory protein (STAR). Given its importance for the steroidogenic process, the regulation of STAR gene expression has been the subject of numerous studies. These studies have involved the characterization of key promoter sequences through the identification of relevant transcription factors and the nucleotide motifs (regulatory elements) that they bind. This work has traditionally relied on in vitro studies carried out in cell cultures along with reconstructed promoter sequences. While this approach has been useful for developing models of how a gene might be transcriptionally regulated, one must ultimately validate that these modes of regulation occur in an endogenous context. We have used CRISPR/Cas9 genome editing to modify a short region of the mouse Star promoter (containing a subset of regulatory elements, including conserved CRE, C/EBP, AP1, and GATA motifs) that has been proposed to be critical for Star transcription. Analysis of the resultant mutant mice showed that this short promoter region is indeed required for maximal STAR mRNA and protein levels in the testis. Analysis also showed that both basal and hormone-activated testosterone production in mature mice was unaffected despite significant changes in Star expression. Our results therefore provide the first in vivo validation of regulatory sequences required for Star gene expression.

P-300 Evaluation of CYP19A1 gene expression in luteinized granulosa cells of women affected by endometriosis undergoing assisted reproductive technology (ART) treatments

Abstract Study question Does endometriosis affect the expression of the aromatase gene CYP19A1 in the cumulus oophorous (COCs) and mural lutein-granulosa cells (GCs) influencing ART procedures? Summary answer Endometriosis does not impair CYP19A1 gene expression. However, the correlation between the aromatase expression and the number of oocytes retrieved is lost in endometriosis patients. What is known already Endometriosis-related infertility could be associated with a dysregulation of oocytes development. Indeed, endometriosis seems to have a negative effect on the intrafollicular environment, hindering oocyte maturation. A dysregulated synthesis of steroid hormones by GCs in the ovaries of affected women may be at the basis of an inadequate folliculogenesis. In line, some studies have investigated the expression levels of aromatase p450 ( CYP19A1 ) -the key enzyme involved in 17β-estradiol (E2) synthesis - in GCs and COCs collected from endometriosis women, reporting controversial results. Study design, size, duration In order to identify novel prognostic factors of ART outcomes in affected women, we set the evaluation of CYP19A1 expression in GCs samples isolated from endometriosis patients undergoing ART in comparison to control women. In a subgroup of patients, COCs were also collected. CYP19A1, StAR and 3βHSD gene expression was evaluated in both cell types. Finally, we evaluated the association between the expression of the analyzed genes and E2 levels with the clinical ART outcomes Participants/materials, setting, methods GCs were isolated from follicular fluids(FF) of n = 68 women with stage III-IV endometriosis and of n = 69 control patients. CYP19A1 gene expression was quantified by qPCR. 17β-estradiol levels in FF were assessed using an ELISA kit. In addition to CYP19A1 gene expression, mRNA levels of StAR and 3βHSD both in GCs and COCs (n = 20 endometriosis;n=21 controls) were evaluated in both cell types using qPCR. Differences between the two patients’ groups were estimated using linear regression models. Main results and the role of chance qPCR results showed no differences in mRNA expression of CYP19A1, StAR and 3βHSD in both GCs and COCs between the two groups of ART patients. These results were supported by the presence of the same concentration of E2 in the FF of controls (median: 877.7 ng/mL) and endometriosis patients (median: 878.3 ng/mL) (p-value=0.87). Linear regression model including as input variables gene expression values and ART outcomes showed that the blastulation rate was the only ART outcome associated with the expression levels of CYP19A1 (p-value=0.043, 95% CI: 0.001-0.061). In particular, a decrease of aromatase levels was associated with an increase in blastulation rate. After stratification of the population based on the presence of the disease, it emerged that, in the control group, the CYP19A1 expression correlated with the number of oocytes retrieved [β:-1.214;95%CI: -2.085 - (-0.343); p-value=0.007], while in the group of patients with endometriosis this association was no more present [β:-0.003;95%CI:-0.468 - 0.461; p-value=0.988)]. These results do not support data from the literature indicating that aromatase expression is reduced in GCs of affected women, but they highlight a potential disease-related mechanism affecting the ovulation process in these women Limitations, reasons for caution These findings need to be validated in a different cohort of samples. An RNA-seq approach is needed in order to validate our results and to obtain the overall transcriptome profiles of GCs and COCs in endometriosis patients. Wider implications of the findings Our data do not confirm previous evidence supporting a reduced expression/activity of aromatase in GCs in endometriosis. However, they suggest that aromatase may have a complex and sophisticated regulation of its expression in this cell type, which is not maintained in presence of endometriosis. Trial registration number not applicable

Parental High-Fat High-Sugar Diet Intake Programming Inflammatory and Oxidative Parameters of Reproductive Health in Male Offspring.

Parental nutrition can impact the health of future generations, programming the offspring for the development of diseases. The developing germ cells of the offspring could be damaged by the maternal or the paternal environment. The germ cells in development and their function could be affected by nutritional adversity and therefore, harm the health of subsequent generations. The paternal or maternal intake of high-fat diets has been shown to affect the reproductive health of male offspring, leading to imbalance in hypothalamic-pituitary-gonadal axis, testicular oxidative stress, low testosterone production, and changes in sperm count, viability, motility, and morphology. There is a need for studies that address the combined effects of diets with a high-fat and high-sugar (H) content by both progenitors on male reproduction. In this context, our study evaluated epigenetic parameters and the inflammatory response that could be associated to oxidative stress in testis and epididymis of adult offspring. 90 days-old male rats were divided according to the combination of the parental diet: CD (control paternal and maternal diet), HP (H paternal diet and control maternal diet), HM (H maternal diet and control paternal diet) and HPM (H paternal and maternal diet).We evaluated serum levels of testosterone and FSH; testicular gene expression of steroidogenic enzymes Star and Hsd17b3 and epigenetic markers Dnmt1, Dnmt3a, Dnmt3b, and Mecp2; testicular and epididymal levels of TNF-α, IL-6, IL-10, and IL-1β; testicular and epididymal activity of SOD, CAT, and GST; the oxidative markers MDA and CP; the daily sperm production, sperm transit time, and sperm morphology. Testicular epigenetic parameter, inflammatory response, oxidative balance, and daily sperm production of the offspring were affected by the maternal diet; paternal diet influenced serum testosterone levels, and lower daily sperm production was exacerbated by the interaction effect of both parental intake of high-fat high-sugar diet in the testis. There was isolated maternal and paternal effect in the antioxidant enzyme activity in the cauda epididymis, and an interaction effect of both parents in protein oxidative marker. Maternal effect could also be observed in cytokine production of cauda epididymis, and no morphological effects were observed in the sperm. The potential programming effects of isolated or combined intake of a high-fat high-sugar diet by the progenitors could be observed at a molecular level in the reproductive health of male offspring in early adulthood.

Perfluorooctanoic Acid Disrupts Ovarian Steroidogenesis and Folliculogenesis in Adult Mice.

Perfluorooctanoic acid (PFOA) is a synthetic fluorosurfactant used in the manufacturing of fluorotelomers. Although PFOA is no longer produced in the United States, it is environmentally persistent and found in imported food packaging, cookware, and textiles. Previous studies have identified developmental toxicity of PFOA, but little is known about the effects of PFOA on the adult ovary. Thus, this study examined the effects of PFOA on hormone levels, ovarian steroidogenic gene expression, and folliculogenesis in mice in vitro and in vivo. For the in vitro studies, antral follicles from adult female mice were cultured with vehicle control or 1, 10, or 100 μg/ml PFOA for 96 h. For the in vivo studies, adult CD-1 female mice were orally dosed with vehicle control or 1, 5, 10, or 20 mg/kg/day PFOA for 10 days. Gene expression of steroidogenic enzymes, levels of sex steroid hormones, and follicle counts were analyzed. In vitro, PFOA (100 μg/ml) significantly decreased follicle growth, estradiol and estrone levels, and gene expression of StaR, Cyp11a1, and Hsd3b1 compared with controls. In vivo, exposure to PFOA significantly decreased progesterone and pregnenolone levels (5 mg/kg), increased testosterone levels (1 mg/kg), and increased gene expression of Cyp19a1 (1 mg/kg) compared with controls. Exposure to PFOA also significantly altered follicle counts by decreasing primordial follicles and increasing preantral and antral follicles (5 and 10 mg/kg) compared with controls. Collectively, these data show that PFOA disrupts adult ovarian function in a nonmonotonic matter and may pose a risk for premature ovarian failure.

Effects of Supplementation of Moringa Oleifera Leaf Powder on Some Reproductive Performance in Laying Hens

This study aimed to evaluate the effects of adding Moringa oleifera leaf powder (MOLP) to the basal diets of chickens at late laying period on clutch trait, reproductive organs, serum reproductive hormones and reproduction-related genes expression. A total of 350 hens (37 weeks old) with similar laying rate and clutch trait were randomly allocated into five groups, and fed 0 (Control, CON), 2.5 (MOLP2.5), 5, 7.5 and 10% MOLP supplemented diets for 6 weeks. The hens supplemented with 2.5% MLOP had the average clutch length, clutches and clutch intensity close to those in CON. The numbers and weight of hierarchal follicle were significantly increased at the supplementation 2.5% MOLP group. The estrogen concentration was highest in MOLP2.5 group and lowest in MOLP10 group. Expression levels of steroidogenesis-related genes of StAR and Cyp19a1 were higher in MOLP2.5, MOLP5 and MOLP7.5 groups compared with that in the control. These findings suggested that dietary supplementation with 2.5% MOLP effectively increased hierarchal follicle numbers, estrogen level and gene expression of StAR and Cyp19a1, which has a potential beneficial effect on laying performance in laying chicken.

Growth Hormone-induced STAT5B Regulates Star Gene Expression Through a Cooperation With cJUN in Mouse MA-10 Leydig Cells.

Leydig cells produce androgens that are essential for male sex differentiation and reproductive function. Leydig cell function is regulated by several hormones and signaling molecules, including growth hormone (GH). Although GH is known to upregulate Star gene expression in Leydig cells, its molecular mechanism of action remains unknown. The STAT5B transcription factor is a downstream effector of GH signaling in other systems. While STAT5B is present in both primary and Leydig cell lines, its function in these cells has yet to be ascertained. Here we report that treatment of MA-10 Leydig cells with GH or overexpression of STAT5B induces Star messenger RNA levels and increases steroid hormone output. The mouse Star promoter contains a consensus STAT5B element (TTCnnnGAA) at -756 bp to which STAT5B binds in vitro (electrophoretic mobility shift assay and supershift) and in vivo (chromatin immunoprecipitation) in a GH-induced manner. In functional promoter assays, STAT5B was found to activate a -980 bp mouse Star reporter. Mutating the -756 bp element prevented STAT5B binding but did not abrogate STAT5B-responsiveness. STAT5B was found to functionally cooperate with DNA-bound cJUN. The STAT5B/cJUN cooperation was only observed in Leydig cells and not in Sertoli or fibroblast cells, indicating that additional Leydig cell-enriched transcription factors are required. The STAT5B/cJUN cooperation was lost only when both STAT5B and cJUN elements were mutated. In addition to identifying the Star gene as a novel target for STAT5B in Leydig cells, our data provide important new insights into the mechanism of GH and STAT5B action in the regulation of Leydig cell function.