CYP19A1 Expression Research Articles

The effects of AdipoRon on cytochrome P450-related gene expression, acute steroidogenic regulatory protein, and structure of ovary in polycystic ovary syndrome model.

One of the most common causes of infertility in adult women is polycystic ovary syndrome (PCOS) which has been identified with symptoms such as chronic hyperandrogenism, anovulation, and polycystic ovaries. Adiponectin modulates steroidogenesis and the expression of ovulation-related genes. Herein, we assessed the effect of AdipoRon (adiponectin agonist) in the PCOS model mice. The PCOS model was induced with letrozole in the adult female mice and the animals received intraperitoneal injection of AdipoRon (5 mg/kg) for 10 days. Expression of CYP11A, CYP17A, and CYP19A genes, StAR protein, and histomorphology of the ovary were evaluated using real-time RT-PCR, western blotting, and histochemistry methods, respectively. Although administration of letrozole caused an increase in the expression of CYP11A, CYP17A, and StAR and a decrease in the CYP19A1 expression, injection of AdipoRon reversed these changes. Moreover, AdipoRon treatment resulted in an improvement of folliculogenesis and a reduction of cysts compared to the letrozole-treated mice. It is likely that AdipoRon has protective effects on the PCOS through modulation of cytochrome P450-related genes and steroidogenesis but needs further study to be sure.

Interleukin-22 promotes proliferation and reverses LPS-induced apoptosis and steroidogenesis attenuation in human ovarian granulosa cells: implications for polycystic ovary syndrome pathogenesis

Objective Interleukin 22 (IL-22) plays a role in inflammatory diseases. However, whether IL-22 affects the function of ovarian granulosa cells (GCs) and its relationship with Polycystic Ovary Syndrome (PCOS)remains unclear. Methods We investigated the level of IL-22 in human follicular fluid using ELISA. The expression and localization of the IL-22 receptor 1 (IL-22R1) in GCs were investigated by RT-PCR and immunofluorescence staining, respectively. The proliferation of KGN cells (human GCs line) was assessed by CCK-8 assay and EdU assay after treatment with recombinant human IL-22 (rhIL-22) and lipopolysaccharide (LPS). Apoptosis was assessed using flow cytometry. Apoptotic proteins and steroidogenic genes were detected by western blotting. Results ELISA's results showed that compared with the control group, PCOS patients showed lower expression of IL-22 in follicular fluid. Immunofluorescence showed that IL-22R1 is expressed and localized in human granulosa cell membranes. IL-22 promoted cell proliferation and reversed LPS-induced inhibition of cell proliferation. IL-22 alone did not affect apoptotic or steroidogenic protein expression, however, it reversed LPS-induced apoptosis via downregulation of Bcl-2, upregulation of Bax and cleaved caspase-3, and restoration of LPS-downregulated StAR, CYP11A1, and CYP19A1 expression. Western blotting confirmed that IL-22 activated the JAK2/STAT3 signaling. Conclusion IL-22 promotes cell proliferation, inhibits apoptosis, and regulates KGN cell steroidogenesis confronted with LPS, and decreased IL-22 may be involved in the development of PCOS.

Sex differences in distribution and identity of aromatase gene expressing cells in the young adult rat brain

BackgroundAromatase catalyzes the synthesis of estrogens from androgens. Knowledge on its regional expression in the brain is of relevance to the behavioral implications of these hormones that might be linked to sex differences in mental health. The present study investigated the distribution of cells expressing the aromatase coding gene (Cyp19a1) in limbic regions of young adult rats of both sexes, and characterized the cell types expressing this gene.MethodsCyp19a1 mRNA was mapped using fluorescent in situ hybridization (FISH). Co-expression with specific cell markers was assessed with double FISH; glutamatergic, gamma-aminobutyric acid (GABA)-ergic, glial, monoaminergic, as well as interneuron markers were tested. Automated quantification of the cells expressing the different genes was performed using CellProfiler. Sex differences in the number of cells expressing Cyp19a1 was tested non-parametrically, with the effect size indicated by the rank-biserial correlation. FDR correction for multiple testing was applied.ResultsIn the male brain, the highest percentage of Cyp19a1+ cells was found in the medial amygdaloid nucleus and the bed nucleus of stria terminalis, followed by the medial preoptic area, the CA2/3 fields of the hippocampus, the cortical amygdaloid nucleus and the amygdalo-hippocampal area. A lower percentage was detected in the caudate putamen, the nucleus accumbens, and the ventromedial hypothalamus. In females, the distribution of Cyp19a1+ cells was similar but at a lower percentage. In most regions, the majority of Cyp19a1+ cells were GABAergic, except for in the cortical-like regions of the amygdala where most were glutamatergic. A smaller fraction of cells co-expressed Slc1a3, suggesting expression of Cyp19a1 in astrocytes; monoaminergic markers were not co-expressed. Moreover, sex differences were detected regarding the identity of Cyp19a1+ cells.ConclusionsFemales show overall a lower number of cells expressing Cyp19a1 in the limbic brain. In both sexes, aromatase is expressed in a region-specific manner in GABAergic and glutamatergic neurons. These findings call for investigations of the relevance of sex-specific and region-dependent expression of Cyp19a1 in the limbic brain to sex differences in behavior and mental health.

DNMT1-mediated DNA methylation contributes to CYP11A1 expression in progesterone synthesis in placental trophoblast syncytialization

DNMT1-mediated DNA methylation contributes to CYP11A1 expression in progesterone synthesis in placental trophoblast syncytialization

FXR Maintains the Intestinal Barrier and Stemness by Regulating CYP11A1-Mediated Corticosterone Synthesis in Biliary Obstruction Diseases.

Biliary obstruction diseases are often complicated by an impaired intestinal barrier, which aggravates liver injury. Treatment of the intestinal barrier is often neglected. To investigate the mechanism by which intestinal bile acid deficiency mediates intestinal barrier dysfunction after biliary obstruction and identify a potential therapeutic modality, we mainly used a bile duct ligation (BDL) mouse model to simulate biliary obstruction and determine the important role of the bile acid receptor FXR in maintaining intestinal barrier function and stemness. Through RNA-seq analysis of BDL and sham mouse crypts and qRT-PCR performed on intestinal epithelial-specific Fxr knockout (FxrΔIEC) and wild-type mouse crypts, we found that FXR might maintain intestinal stemness by regulating CYP11A1 expression. Given the key role of CYP11A1 during glucocorticoid production, we also found that FXR activation could promote intestinal corticosterone (CORT) synthesis by ELISA. Intestinal organoid culture showed that an FXR agonist or corticosterone increased crypt formation and organoid growth. Further animal experiments showed that corticosterone gavage treatment could maintain intestinal barrier function and stemness, decrease LPS translocation, and attenuate liver injury in BDL mice. Our study hopefully provides a new theoretical basis for the prevention of intestinal complications and alleviation of liver injury after biliary obstruction.

Pyridoxamine protects human granulosa cells against advanced glycation end-products-induced steroidogenesis disturbances.

Ovarian advanced glycation end-products (AGEs) accumulation is associated with ovarian granulosa cells (GCs) dysfunction. Vitamin B6 derivatives positively affected reproduction. The current study was conducted to elucidate the AGEs effects on human luteinized mural GCs steroidogenesis in the presence or absence of pyridoxamine (PM). Isolated GCs of 50 healthy women were divided into four parts and treated with media alone (Control), PM alone, or human glycated albumin (HGA) with/without PM. Main steroidogenic enzymes and hormones were assessed by qRT-PCR and ELISA. The AGE receptor (RAGE) protein was also determined using Western blotting. The non-toxic concentration of HGA increased the expression of RAGE, StAR, 3β-HSD, and 17β-HSD (P < 0.0001 for all) but decreased the expression of CYP19A1 at mRNA levels. The increased RAGE protein expression was also confirmed by western blot analysis. These effects resulted in declined estradiol (E2), slightly, and a sharp rise in progesterone (P4) and testosterone (T) levels, respectively. PM, on its own, ameliorated the HGA-altered enzyme expression and, thereby, corrected the aberrant levels of E2, P4, and T. These effects are likely mediated by regulating the RAGE gene and protein expression. This study indicates that hormonal dysfunctions induced by the AGEs-RAGE axis in luteinized GCs are likely rectified by PM treatment. This effect is likely acquired by reduced expression of RAGE. A better understanding of how AGEs and PM interact in ovarian physiology and pathology may lead to more targeted therapy for treating ovarian dysfunction.

ACTH and prolactin synergistically and selectively regulate CYP17 expression and adrenal androgen production in human foetal adrenal organ cultures.

The essential role of ACTH on the growth and function of the human foetal adrenal (HFA) has long been recognized. In addition, many studies have suggested a role of the pituitary hormone prolactin (PRL) in the regulation of the HFA, but the effects of this hormone on steroidogenesis and gene expression are still unknown. Our objective was to investigate the effect of ACTH and PRL on the steroidogenic capacities of the HFA. In vitro/ex vivo experimental study. We used a hanging drop in vitro organ culture system. First trimester HFA samples were cultured for 14 days in basal conditions or treated with ACTH, PRL, or a combination of the 2 (3 to 11 replicates depending on the experiment). Steroids were measured by liquid chromatography/tandem mass spectrometry or immunoassay, gene expression by RT-qPCR, and protein expression by immunoblot. ACTH significantly increased corticosterone, cortisol, and cortisone production, both by itself and when used together with PRL. PRL stimulation by itself had no effect. Combined stimulation with ACTH + PRL synergistically and selectively increased adrenal androgen (DHEAS and Δ4-androstenedione) production and CYP17A1 expression in the HFA, while treatment with each single hormone had no significant effect on those steroids. These results have important implications for our understanding of the hormonal cues regulating adrenal steroidogenesis in the HFA during the first trimester in physiological and pathological conditions and warrant further studies to characterize the molecular mechanisms of converging ACTH and PRL signalling to regulate CYP17A1 expression.

Protein kinase B (Akt) blockade inhibits LH/hCG-mediated 17,20-lyase, but not 17α-hydroxylase activity of Cyp17a1 in mouse Leydig cell steroidogenesis

Androgens are produced by adrenal and gonadal cells thanks to the action of specific enzymes. We investigated the role of protein kinase B (Akt) in the modulation of Δ4 steroidogenic enzymes' activity, in the mouse Leydig tumor cell line mLTC1. Cells were treated for 0–24 h with the 3 × 50% effective concentration of human luteinizing hormone (LH) and choriogonadotropin (hCG), in the presence and in the absence of the specific Akt inhibitor 3CAI. Cell signaling analysis was performed by bioluminescence resonance energy transfer (BRET) and Western blotting, while the expression of key target genes was investigated by real-time PCR. The synthesis of progesterone, 17α-hydroxy (OH)-progesterone and testosterone was measured by immunoassay. Control experiments for cell viability and caspase 3 activation were performed as well. We found that both hormones activated cAMP and downstream effectors, such as extracellularly-regulated kinase 1/2 (Erk1/2) and cAMP response element-binding protein (Creb), as well as Akt, and the transcription of Stard1, Hsd3b1, Cyp17a1 and Hsd17b3 genes, boosting the Δ4 steroidogenic pathway. Interestingly, Akt blockade decreased selectively Cyp17a1 expression levels, inhibiting its 17,20-lyase, but not the 17-hydroxylase activity. This effect is consistent with lower Cyp17a1 affinity to 17α-OH-progesterone than progesterone. As a result, cell treatment with 3CAI resulted in 17α-OH-progesterone accumulation at 16–24 h and decreased testosterone levels after 24 h. In conclusion, in the mouse Leydig cell line mLTC1, we found substantial Akt dependence of the 17,20-lyase activity and testosterone synthesis. Our results indicate that different intracellular pathways modulate selectively the dual activity of Cyp17a1.

Adropin may regulate corpus luteum formation and its function in adult mouse ovary.

Adropin, a unique peptide hormone, has been associated with the regulation of several physiological processes, including glucose homeostasis, fatty acid metabolism, and neovascularization. However, its possible role in ovarian function is not understood. Our objective was to examine the expression of adropin and its putative receptor, GPR19, in the ovaries of mice at various phases of the estrous cycle. Immunohistochemistry and western blot analysis were performed to explore the localization and changes in expression of adropin and GPR19 in the ovaries during different phases of the estrous cycle in mice. Hormonal assays were performed with ELISA. An in vitro study was performed to examine the direct effect of adropin (10, 100 ng/ml) on ovarian function. A western blot study showed that adropin and GPR19 proteins were maximum during the estrus phase of the estrous cycle. Interestingly, adropin and GPR19 displayed intense immunoreactivity in granulosa cells of large antral follicles and corpus luteum. This suggested the possible involvement of adropin in corpus luteum formation. Adropin treatment stimulated progesterone synthesis by increasing GPR19, StAR, CYP11A1, and 3β-HSD expressions, while it decreased estrogen synthesis by inhibiting 17β-HSD and aromatase protein expressions. Moreover, adropin treatment upregulated the cell cycle arrest-CDK inhibitor 1B (p27kip1), pERK1/2, and angiogenic protein (EG VEGF) that are involved in the process of luteinization. Adropin GPR19 signaling promotes the synthesis of progesterone and upregulates the expression of p27kip1, EG VEGF, and erk1/2, resulting in cell cycle arrest and neovascularization, which ultimately leads to corpus luteum formation.

Steroidogenic activity of liposomal methylated resveratrol analog 3,4,5,4′-tetramethoxystilbene (DMU-212) in human luteinized granulosa cells in a primary three-dimensional in vitro model

PurposeSteroid hormone secretion is one of the key functions of granulosa cells (GCs). Resveratrol is a natural polyphenol, known for its beneficial health effects, such as improving reproductive health. However, its application is limited due to poor bioavailability. The methoxy derivative of resveratrol (DMU-212) was demonstrated to be more lipophilic, and therefore of greater bioavailability. However, since the addition of methoxy groups to the stilbene scaffold was found to make the molecule insoluble in water, DMU-212 was loaded into liposomes. This study aimed to evaluate how the liposomal formulation of DMU-212 (lipDMU-212) alters estradiol and progesterone secretion of human ovarian GCs in a primary three-dimensional cell culture model.MethodsDMU-212-loaded liposomes were prepared by thin film hydration followed by extrusion. Cell viability was measured after exposure of GCs spheroids to the liposomal formulation of DMU-212 using CellTiter-Glo® 3D Cell Viability Assay. The secretion of estradiol and progesterone was determined using commercial ELISA kits. RT-qPCR was conducted to analyze the expression of steroidogenesis-related genes. Finally, the western blot technique was used to analyze the effect of lipDMU-212 and FSH treatments on CYP11A1 and HSD3B1 protein levels.ResultslipDMU-212 was found to significantly increase estradiol and progesterone secretion in a dose-dependent manner by enhancing the expression of CYP11A1, HSD3B1, StAR, CYP17A1, CYP19A1, and HSD17B1 genes. We have also shown that lipDMU-212, used alone and in combination with FSH, significantly increased the expression of the HSD3B1 and CYP11A1 proteins in GCs. Furthermore, our study suggests that lipDMU-212 increases FSH activity.ConclusionsThis is the first study to describe the steroidogenic activity of liposomal formulation of DMU-212, possibly through increasing the StAR and CYP19A1 expression. These findings suggest that lipDMU-212 might have a beneficial effect in the treatment of disorders related to estrogen deficiency and hyperandrogenism, such as PCOS.

CYP19A1 mediates severe SARS-CoV-2 disease outcome in males

Male sex represents one of the major risk factors for severe COVID-19 outcome. However, underlying mechanisms that mediate sex-dependent disease outcome are as yet unknown. Here, we identify the CYP19A1 gene encoding for the testosterone-to-estradiol metabolizing enzyme CYP19A1 (also known as aromatase) as a host factor that contributes to worsened disease outcome in SARS-CoV-2-infected males. We analyzed exome sequencing data obtained from a human COVID-19 cohort (n= 2,866) using a machine-learning approach and identify a CYP19A1-activity-increasing mutation to be associated with the development of severe disease in men but not women. We further analyzed human autopsy-derived lungs (n= 86) and detect increased pulmonary CYP19A1 expression at the time point of death in men compared with women. In the golden hamster model, we show that SARS-CoV-2 infection causes increased CYP19A1 expression in the lung that is associated with dysregulated plasma sex hormone levels and reduced long-term pulmonary function in males but not females. Treatment of SARS-CoV-2-infected hamsters with a clinically approved CYP19A1 inhibitor (letrozole) improves impaired lung function and supports recovery of imbalanced sex hormones specifically in males. Our study identifies CYP19A1 as a contributor to sex-specific SARS-CoV-2 disease outcome in males. Furthermore, inhibition of CYP19A1 by the clinically approved drug letrozole may furnish a new therapeutic strategy for individualized patient management and treatment.

CircEpha5 regulates the synthesis and secretion of androgen in mouse preantral follicles by targeting miR-758-5p

Circular RNAs are involved in the pathogenesis of various diseases, although its expression pattern and role in polycystic ovary syndrome (PCOS), characterised by hyperandrogenism, are not very clear. This article assessed the circRNAs expression profile in the ovaries of PCOS mice by circRNAs high-throughput sequencing and explored the role of circEpha5 in hyperandrogenism. The results showed that the overexpression of circEpha5 in mouse preantral follicles could increase the expression of Cyp17a1, an androgen synthesis-related gene, which resulted in a higher serum level of testosterone. Dual-luciferase reporter gene studies identified miR-758-5p as a direct target of circEpha5. Consequently, miR-758-5p expression was downregulated upon circEpha5 overexpression. Ectopically expressed miR-758-5p reversed the stimulation effects of circEpha5 on steroidogenesis-related gene expression and testosterone release. Therefore, circEpha5 could sponge miR-758-5p to regulate the expression of Cyp17a1, thereby promoting the synthesis and secretion of androgen in the preantral follicles. This work is contributed to the understanding of the pathogenesis of hyperandrogenemia and lays the foundation for the development of therapeutic targets of PCOS hyperandrogenism.

Network pharmacology and experimental validation on yangjing zhongyu decoction against diminished ovarian reserve

Ethnopharmacological relevanceDiminished ovarian reserve (DOR) was considered a refractory reproductive endocrine condition that negatively affected female reproductivity. Yangjing Zhongyu Decoction (YJZYD) had effects on treating infertility. However, there were few studies on the mechanisms of YJZYD preserving ovarian reserve. Aim of the studyTo explore the possible mechanisms of YJZYD against DOR by UPLC-ESI-MS/MS, network pharmacology, and experimental validation. MethodsThe chemicals of YJZYD were measured by UPLC-ESI-MS/MS. The correlating targets of YJZYD and DOR were identified by the ETCM database, GeneCards database, and PubMed database. The common targets were employed with the DAVID database and visualized with the PPI network. GO and KEGG enrichment analyses were carried out to explore biological progression and pathways. In vivo experiments, energy production was assessed by ATP, and apoptosis rate was analyzed by TUNEL. The serum FSH, AMH, and E2 levels were evaluated by ELISA. Western blotting and immunohistochemistry were used to measure the expression of SIRT1, PGC1α, NRF1, COX IV, FSHR, CYP19A1, PI3K, p-Akt, Akt, Bcl-2, and Bax. Results132 components in YJZYD were identified by UPLC-ESI-MS/MS. 149 overlapped targets were extracted from YJZYD and DOR, and the top 20 common targets included AKT1 and CYP19A1. ATP binding was involved in GO analysis. In the KEGG enrichment analysis, the metabolic pathway was the top, and the PI3K-Akt signaling pathway was included. In vivo experiments, YJZYD improved ovarian index and histomorphology. After YJZYD treatment, serum FSH, E2, and AMH were well-modulated, and the content of ATP was up-regulated. Besides, the expression of Bax was suppressed in ovarian tissue, while the expressions of SIRT1, PGC1α, NRF1, COX IV, FSHR, CYP19A1, PI3K, Bcl-2, and p-Akt/Akt were enhanced. ConclusionYJZYD could attenuate reproductive endocrine disturbance and ovarian lesions in vivo by mediating steroidogenesis, energy metabolism, and cell apoptosis. This study uncovered the mechanisms of YJZYD against DOR, providing a theoretical basis for further study.

New Target for Minoxidil in the Treatment of Androgenetic Alopecia.

To investigate the mechanism of minoxidil in treating androgenetic alopecia (AGA). The mechanism of action of minoxidil on AGA was first systematically investigated from the viewpoint of network pharmacology, including minoxidil-AGA target prediction, protein-protein interaction (PPI) network analysis, molecular docking and enrichment analysis of targets related to minoxidil and AGA, and dermal papilla cell assays to confirm the viability of prediction. The combined analysis revealed that minoxidil treatment of AGA not only acts on androgenic receptors (AR) but also on 2 new targets, steroid 17-alpha-hydroxylase/17,20 lyase (CYP17A1) and aromatase (CYP19A1). The biological processes linked to these targets were concentrated on several pathways, including enzymes and hormones. Further experiments have revealed that minoxidil suppresses the expression of AR and CYP17A1, boosts the activity of CYP19A1, decreases the formation and binding of dihydrotestosterone, and enhances the production of estradiol. Through these changes, minoxidil acts as a treatment for AGA. Minoxidil may act by altering hormonal and enzymatic pathways. Our study finds two new targets (CYP17A1, CYP19A1) of minoxidil and demonstrates that minoxidil inhibits AR. These targets may provide new ideas for drug research.

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.

Effect of IGF1 and FSH on the function of granulosa cells from prehierarchal follicles in chickens†.

Insulin-like growth factor 1 (IGF1) is an essential regulator of mammalian follicle development and synergizes with follicle-stimulating hormone (FSH) to amplify its effects. In avian preovulatory follicles, IGF1 increases the expression of genes involved in steroidogenesis and progesterone and inhibin A production. The role of IGF1 in prehierarchal follicles has not been well studied in chickens. The aim of this study was to investigate the role of IGF1 in granulosa cells from prehierarchal follicles and to determine whether IGF1 and FSH synergize to promote follicle development. Granulosa cells of 3-5 and 6-8mm prehierarchal follicles were cultured with IGF1 (0, 10, 100ng/mL) in the presence or absence of FSH (0, 10ng/mL). Cell proliferation, expression of genes important in follicle development (FSHR, IGF1R, AMH, STAR, CYP11A1, INHA, and INHBA), and progesterone production were evaluated following treatment. IGF1 treatment alone significantly increased STAR, CYP11A1, and INHBA mRNA expression and cell proliferation in granulosa cells of 6-8mm follicles. IGF1 and FSH synergized to increase STAR mRNA expression in 6-8mm follicles. IGF1 and FSH co-treatment were necessary to increase INHA mRNA expression in 6-8mm follicles. Although IGF1 significantly increased the expression of genes involved in steroidogenesis, progesterone production in granulosa cells of 6-8mm follicles was not affected. IGF1 did not affect AMH mRNA expression, although FSH significantly decreased AMH expression in granulosa cells of 3-5mm follicles. These results suggest that IGF1 may act with FSH to promote follicle selection at the prehierarchal follicle stage.

Investigation of subfertility in the female Nsmf knockout mouse

To study if a pituitary or ovarian defect contributes to subfertility of the female Nsmf knockout (KO) mouse, an animal model of the hypogonadotropic hypogonadism gene NSMF. Analysis of hypothalamic, pituitary and ovarian gene expression at baseline, serum gonadotropin levels before and after gonadotropin-releasing hormone (GnRH) stimulation, ovarian response and implantation after superovulation, gonadotropin effects after ovariectomy, and ovarian NSMF protein expression. University research laboratory. None; mice were used. Gonadotropin-releasing hormone stimulation, superovulation, and ovariectomy in separate experiments. Gene expression in the hypothalamus, pituitary, and ovary; ovarian response and implantation after superovulation; serum gonadotropins after GnRH stimulation and ovariectomy; Western blot to measure ovarian NSMF expression. We found increased hypothalamic Kiss1, Gnrh1, and Jak2 mRNA expression in female Nsmf KO vs. wild type (WT) mice. However, pituitary gonadotropin, and GnRH receptor gene expression was not affected, and serum gonadotropin levels were normal. Gonadotropins increased after ovariectomy for both groups. Baseline Kiss1, Fshr, Prkaca, Prkar1a, and Gdf9 ovarian mRNA expression was increased and Cyp19a1 expression was decreased in Nsmf KO mice, while superovulated Nsmf KO mice had reduced ovarian Kiss1r, Prkar1a, and Fshr mRNA expression, 50% less oocytes, and normal implantation. Western blot demonstrated NSMF protein expression in the ovary of WT mice. Altered hypothalamic and ovarian gene expression was demonstrated in female Nsmf KO mice. It is possible that increased hypothalamic Gnrh1 and Kiss1 mRNA expression could compensate for reduced NSMF enabling a normal pituitary gonadotropin response. Impaired superovulation response, altered ovarian gene expression, and decreased number of oocytes indicate ovarian dysfunction, but a uterine factor cannot be excluded. These findings provide an anatomic basis for future mechanistic studies of subfertility in female Nsmf KO mice.

Porcine-specific expression ofthe three functional CYP19paralogs in early conceptus, placenta, and gonads.

Although three porcine aromatase isoforms have been identified, their gene expression profiles in reproduction are still poorly understood. Here, we identified by Sanger sequencing unique nucleotide signatures for the three paralogous copies of Cyp19 and analyzed by RT-PCR the occurrence of the Cyp19 and Cyp17a1 transcripts at different tissues and stages of conceptus and fetal-placental development. Cyp19a1 and Cyp19a3 expression was detected in conceptuses and gonads, respectively. Cyp19a2 transcripts were identified on both conceptuses and placenta samples. Transcripts for Cyp17a1 were detected predominantly in conceptus and gonads. In the endometrium of Day 21 pregnant, as well as Days 12 and 17 pseudopregnant females, we did not detect expression of Cyp19a1, Cyp19a2 or Cyp19a3. In our study we have demonstrated distinct transcriptional regulation for the three functional Cyp19 paralogs and a potential role for Cyp17a1 in controlling secretion of estrogen from the conceptus and the placenta.

In view of ovarian steroidogenesis and luteal construction to explore the effects of Bushen Huoxue recipe in mice of ovarian hyperstimulation

Ethnopharmacological relevanceBushen Huoxue recipe (BSHXR) is a widely used prescription medicine for treating gynecological diseases. We have previously found that BSHXR can improve the pregnancy outcome of controlled ovarian hyperstimulation (COH) mice by modulating the abnormal high level of progesterone. While the pharmacological mechanism of such therapeutic effect is not clear. Aim of the studyWe aimed to investigate the effects of BSHXR on the ovarian steroidogenesis and luteal function in mice undergoing COH. Materials and methodsA COH mouse model was established via an intraperitoneal injection of 0.4 IU/g pregnant mare serum gonadotropin (PMSG) and 1 IU/g human chorionic gonadotropin (HCG). The histological features of ovaries were observed using hematoxylin-eosin staining. The expression levels of FSHR, LHCGR, and key molecules in ovarian steroidogenesis, including CYP11A1, CYP17A1, CYP19A1, HSD3B1, and StAR, were examined via immunohistochemical staining, western blotting, and RT-qPCR. CD31, VEGFA, and FGF2 levels were assessed to evaluate ovarian vascularization. The protein and mRNA levels of ovarian ERK1/2, p-ERK1/2, MEK1/2, and p-MEK1/2 were also detected using western blotting, RT-qPCR, or immunofluorescence staining. ResultsCOH mice had a significantly increased volume and weight of the ovary and number of corpora lutea. In particular, COH exhibited a long-term influence on ovarian FSHR and LHCGR expression, disrupting the levels of CYP11A1, HSD3B1, and CYP17A1, causing poorer luteal angiogenesis. Compared with normal mice, the expression levels of ovarian VEGFA and FGF2 in COH mice were considerably lower on Day 1 after PMSG. On concomitant HCG treatment, both VEGFA and FGF2 expression surged dramatically on ED1 and then declined on ED4 and ED8. Moreover, the expression pattern of MEK1/2-ERK1/2 was almost consistent with that of VEGFA and FGF2. After treatment, BSHXR increased ovarian LHCGR, FSHR, CYP11A1, HSD3B1, and CYP17A1 levels, boosted luteal vascularization, and restored MEK1/2-ERK1/2 signaling in COH mice. ConclusionBSHXR restored the abnormally high progesterone level by regulating the CYP11A1 and HSD3B1 expression as well as promoted luteal angiogenesis, which was related with LHCGR-MEK1/2-ERK1/2-VEGFA/FGF2 signaling pathway in the ovary. This effect prevented the fluctuation of sex hormones in COH mice and benefited the outcome of pregnancy.

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.