Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system.

BACKGROUND: Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. RESULTS: Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. CONCLUSION: The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Enhancement of progesterone biosynthesis via kisspeptin stimulation: Upregulation of steroidogenic transcripts and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) expression in the buffalo luteal cells.

The presence of Kisspeptin (Kp) and its receptors in the corpus luteum (CL) of buffalo has recently been demonstrated. In this study, we investigated the role of Kp in the modulation of progesterone (P4) synthesis in vitro. The primary culture of bubaline luteal cells (LCs) was treated with 10, 50, and 100 nM of Kp and Kp antagonist (KpA) alongside a vehicle control. The combined effect of Kp and KpA was assessed at 100 nM concentration. Intracellular response to Kp treatment in the LCs was assessed by examining transcript profiles (LHR, STAR, CYP11A1, HSD3B1, and ERK1/2) using quantitative polymerase chain reaction (qPCR). In addition, the immunolocalization of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the LCs was studied using immunocytochemistry. Accumulation of P4 from the culture supernatant was determined using enzyme-linked immunosorbent assay (ELISA). The results indicated that LCs had a greater p-ERK1/2 expression in the Kp treatment groups. A significant increase in the P4 concentration was recorded at 50 nM and 100 nM Kp, while KpA did not affect the basal concentration of P4. However, the addition of KpA to the Kp-treated group at 100 nM concentration suppressed the Kp-induced P4 accumulation into a concentration similar to the control. There was significant upregulation of ERK1/2 and CYP11A1 expressions in the Kp-treated LCs at 100 nM (18.1 and 37fold, respectively, p < 0.01). However, the addition of KpA to Kp-treated LCs modulated ERK1/2, LHR, STAR, CYP11A1, and HSD3B1 at 100 nM concentration. It can be concluded that Kp at 100 nM stimulated P4 production, while the addition of KpA suppressed Kp-induced P4 production in the buffalo LCs culture. Furthermore, an increment in p-ERK1/2 expression in the LCs indicated activation of the Kp signaling pathway was associated with luteal steroidogenesis.

De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis.

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.

Microbiota modulates the steroid response to acute immune stress in male mice.

Microbiota plays a role in shaping the HPA-axis response to psychological stressors. To examine the role of microbiota in response to acute immune stressor, we stimulated the adaptive immune system by anti-CD3 antibody injection and investigated the expression of adrenal steroidogenic enzymes and profiling of plasma corticosteroids and their metabolites in specific pathogen-free (SPF) and germ-free (GF) mice. Using UHPLC-MS/MS, we showed that 4 hours after immune challenge the plasma levels of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone (CORT), 11-dehydroCORT and their 3α/ß-, 5α-, and 20α-reduced metabolites were increased in SPF mice, but in their GF counterparts, only CORT was increased. Neither immune stress nor microbiota changed the mRNA and protein levels of enzymes of adrenal steroidogenesis. In contrast, immune stress resulted in downregulated expression of steroidogenic genes (Star, Cyp11a1, Hsd3b1, Hsd3b6) and upregulated expression of genes of the 3α-hydroxysteroid oxidoreductase pathway (Akr1c21, Dhrs9) in the testes of SPF mice. In the liver, immune stress downregulated the expression of genes encoding enzymes with 3ß-hydroxysteroid dehydrogenase (HSD) (Hsd3b2, Hsd3b3, Hsd3b4, Hsd3b5), 3α-HSD (Akr1c14), 20α-HSD (Akr1c6, Hsd17b1, Hsd17b2) and 5α-reductase (Srd5a1) activities, except for Dhrs9, which was upregulated. In the colon, microbiota downregulated Cyp11a1 and modulated the response of Hsd11b1 and Hsd11b2 expression to immune stress. These data underline the role of microbiota in shaping the response to immune stressor. Microbiota modulates the stress-induced increase in C21 steroids, including those that are neuroactive that could play a role in alteration of HPA axis response to stress in GF animals.

BMAL1 positively correlates with genes regulating steroidogenesis in human luteinized granulosa cells.

In brief: In this study, we examined the relationship between BMAL1 expression and the genes regulating steroid biosynthesis in human luteinized granulosa cells. BMAL1 function is crucial for steroid production and proper ovarian function, highlighting the importance of circadian clock regulation in female reproductive health. Abstract: Human luteinized granulosa cells were collected to analyze circadian clock gene expression and its effect on the genes regulating steroid biosynthesis. We used siRNA to knock down the expression of BMAL1 in KGN cells. We measured the expression levels of genes regulating steroid biosynthesis and circadian clock RT-qPCR. We demonstrated that BMAL1 expression positively correlates with genes regulating steroid biosynthesis (CYP11A1, CYP19A1, STAR, and ESR2). The knockdown of BMAL1 in KGN cells revealed a significant decrease in steroid synthase expression. In contrast, when BMAL1 was overexpressed in KGN and HGL5 cells, we observed a significant increase in the expression of steroid synthases, such as CYP11A1 and CYP19A1. These results indicated that BMAL1 positively controls 17ß-estradiol (E2) secretion in granulosa cells. We also demonstrated that dexamethasone synchronization in KGN cells enhanced the rhythmic alterations in circadian clock genes. Our study suggests that BMAL1 plays a critical role in steroid biosynthesis in human luteinized granulosa cells, thereby emphasizing the importance of BMAL1 in the regulation of reproductive physiology.

miR-6881-3p contributes to diminished ovarian reserve by regulating granulosa cell apoptosis by targeting SMAD4.

BACKGROUND: In our previous investigation, we revealed a significant increase in the expression of microRNA-6881-3p (miR-6881-3p) in follicular fluid granulosa cells (GCs) from women with diminished ovarian reserve (DOR) compared to those with normal ovarian reserve (NOR). However, the role of miR-6881-3p in the development of DOR remains poorly understood. OBJECTIVE: This study aimed to elucidate the involvement of miR-6881-3p in the regulation of granulosa cells (GCs) function and the pathogenesis of DOR. MATERIALS AND METHODS: Initially, we assessed the expression levels of miR-6881-3p in GCs obtained from human follicular fluid in both NOR and DOR cases and explored the correlation between miR-6881-3p expression and clinical outcomes in assisted reproduction technology (ART). Bioinformatic predictions and dual-luciferase reporter assays were employed to identify the target gene of miR-6881-3p. Manipulation of miR-6881-3p expression was achieved through the transfection of KGN cells with miR-6881-3p mimics, inhibitor, and miRNA negative control (NC). Following transfection, we assessed granulosa cell apoptosis and cell cycle progression via flow cytometry and quantified target gene expression through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Finally, we examined the correlation between target gene expression levels in GCs from NOR and DOR patients and their association with ART outcomes. RESULTS: Our findings revealed elevated miR-6881-3p levels in GCs from DOR patients, which negatively correlated with ovarian reserve function and ART outcomes. We identified a direct binding interaction between miR-6881-3p and the 3'-untranslated region of the SMAD4. Transfection with miR-6881-3p mimics induced apoptosis in KGN cell. Furthermore, miR-6881-3p expression negatively correlated with both mRNA and protein levels of the SMAD4. The mRNA and protein levels of SMAD4 were notably reduced in GCs from DOR patients, and SMAD4 mRNA expression positively correlated with ART outcomes. In addition, the mRNA levels of FSHR, CYP11A1 were notably reduced after transfection with miR-6881-3p mimics in KGN cell, while LHCGR notably increased. The mRNA and protein levels of FSHR, CYP11A1 were notably reduced in GCs from DOR patients, while LHCGR notably increased. CONCLUSION: This study underscores the role of miR-6881-3p in directly targeting SMAD4 mRNA, subsequently diminishing granulosa cell viability and promoting apoptosis, and may affect steroid hormone regulation and gonadotropin signal reception in GCs. These findings contribute to our understanding of the pathogenesis of DOR.

Perfluorooctanoic acid inhibits androgen biosynthesis in rat immature Leydig cells.

Perfluorooctanoic acid (PFOA) is a commonly used short-chain synthetic perfluoroalkyl agent. Immature Leydig cells (ILCs) are localized in the testis and responsible for androgen biosynthesis and metabolism. Although PFOA shows toxicity in the reproductive system, it is not clear if it disrupts the function of ILCs. In the present study, primary ILCs were isolated from 35-day-old rats and exposed to a range of PFOA concentrations (0, 0.01, 0.1, or 1 µM). It was determined that 0.1 or 1 µM PFOA reduced total androgen biosynthesis in ILCs. Specifically, 22R-hydroxycholesterol (22R), and pregnenolone (P5) mediated androgen biosynthesis were reduced by 0.1 µM PFOA. PFOA also selectively downregulated mRNA and protein expressions of steroidogenic enzymes including LHCGR, CYP11A1, 3ß-HSD1, and NR5A1 at 0.01, 0.1, or 1 µM. Further analysis revealed that 0.1 µM PFOA inhibited CYP11A1 and 3ß-HSD1 enzyme activities. However, PFOA did not significantly affect androgen metabolism and turnover under any of the conditions tested. And PFOA gavaging to 35-day-old rats at 5 or 10 mg/kg for 7 or 14 days also reduced serum androgen levels secreted by ILCs. Moreover, PFOA gavaging also downregulated the mRNA and protein expression levels of LHCGR, CYP11A1, 3ß-HSD1, and NR5A1 in vivo. Taken together, these findings suggest that PFOA inhibits androgen biosynthesis in ILCs by selectively targeting key enzymes in the synthesis pathway.

The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive.

Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)2 cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and koff rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH)2 cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d3-Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step.

Effect of quercetin on granulosa cells development from hierarchical follicles in chicken.

1. The bioflavonoid quercetin is a biologically active component, but its functional regulation of granulosa cells (GCs) during chicken follicular development is little studied. To investigate the effect of quercetin on follicular development in laying hens, an in vitro study was conducted on granulosa cells from hierarchical follicles treated with quercetin.2. The effect of quercetin on cell activity, proliferation and apoptosis of granulosa cells was detected by CCK-8, EdU and apoptosis assays. The effect on progesterone secretion from granulosa cells was investigated by enzyme-linked immunosorbent assay (ELISA). Expression of proliferating cell nuclear antigen (PCNA) mRNA and oestrogen receptors (ERs), as well as the expression of steroid acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA during progesterone synthesis, were measured by real-time quantitative polymerase chain reaction (RT-qPCR). PCNA, StAR and CYP11A1 protein expression levels were detected using Western blotting (WB).3. The results showed that treatment with quercetin in granulosa cells significantly enhanced cell vitality and proliferation, reduced apoptosis and promoted the expression of gene and protein levels of PCNA. The levels of progesterone secretion increased significantly following quercetin treatment, as did the expression levels of StAR and CYP11A1 using the Western Blot (WB) method.4. The mRNA expression levels of ERα were significantly upregulated in the 100 ng/ml and 1000 ng/ml quercetin-treated groups, while there was no significant difference in expression levels of ERß mRNA.

Effect of cadmium on the regulatory mechanism of steroidogenic pathway of Leydig cells during spermatogenesis.

Cadmium is a male reproductive toxicant that interacts with a variety of pathogenetic mechanisms. However, the effect of cadmium on the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis is still ambiguous. Light microscopy, Western blot, immunohistochemistry, immunofluorescence, and quantitative polymerase chain reaction were performed to study the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis. The results indicated that in the control group, Leydig cells showed dynamic immunoreactivity and immunosignaling action with a strong positive significant secretion of 3ß-hydroxysteroid hydrogenase (3ß-HSD) in the interstitial compartment of the testis. Leydig cells showed a high active regulator mechanism of the steroidogenic pathway with increased the proteins and genes expression level of steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol (CYP11A1), cytochrome P450 cholesterol (CYP17A1), 3ß-hydroxysteroid hydrogenase (3ß-HSD) 17ß-hydroxysteroid hydrogenase (17ß-HSD), and androgen receptor (AR) that maintained the healthy and vigorous progressive motile spermatozoa. However, on treatment with cadmium, Leydig cells were irregularly dispersed in the interstitial compartment of the testis. Leydig cells showed reduced immunoreactivity and immunosignaling of 3ß-HSD protein. Meanwhile, cadmium impaired the regulatory mechanism of the steroidogenic process of the Leydig cells with reduced protein and gene expression levels of STAR, CYP11A1, CYP17A1, 3ß-HSD, 17ß-HSD, and AR in the testis. Additionally, treatment with cadmium impaired the serum LH, FSH, and testosterone levels in blood as compared to control. This study explores the hazardous effect of cadmium on the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis.

Resveratrol protects against high glucose-induced steroidogenesis and apoptosis in murine granulosa cells.

Resveratrol (Res) is a polyphenolic compound that exhibits a diverse array of biological effects. Herein, we detected the ability of Res on murine granulosa cells (GCs) against impaired steroidogenesis and apoptotic death in response to high glucose levels. Ovarian GCs were harvested from C57BL/6 mice and cultured in steroidogenic media supplemented with follicle-stimulating hormone (FSH, 30 ng/mL), Res (50 µmol/L), and low or high glucose concentrations (5 mM or 30 mM). After culture for 24 h, cell supernatants were harvested and the levels of progesterone and estradiol therein were measured. Also, caspase-3 activity and the expression of genes associated with apoptosis and steroidogenesis were assessed. High-glucose treatment suppressed steroidogenesis in this assay system, resulting in the impaired expression of steroidogenesis-related genes including Cyp11a1, Cyp19a1, 3ßHSD, and StAR and a concomitant decrease in progesterone and estradiol production. Cells exposed to high glucose also exhibited apoptotic phenotypes characterized by Bax upregulation, Bcl-2 downregulation, and increased caspase-3 activity levels. However, Res treatment was sufficient to reverse this high glucose level-induced apoptotic and steroidogenic phenotypes with improving progesterone and estradiol production, and these maybe related the effects of Res on Cyp11a1, Cyp19a1, 3ßHSD, and StAR expressions. These data suggested that Res is well suited to overcoming the negative effects of hyperglycemia of GC functionality.

Melatonin Inhibits Testosterone Synthesis in Rooster Leydig Cells by Targeting CXCL14 through miR-7481-3p.

Melatonin has been proved to be involved in testosterone synthesis, but whether melatonin participates in testosterone synthesis by regulating miRNA in Leydig cells is still unclear. The purpose of this study is to clarify the mechanism of melatonin on Leydig cells testosterone synthesis from the perspective of miRNA. Our results showed that melatonin could significantly inhibit testosterone synthesis in rooster Leydig cells. miR-7481-3p and CXCL14 were selected as the target of melatonin based on RNA-seq and miRNA sequencing. The results of dual-luciferase reporter assays showed that miR-7481-3p targeted the 3'-UTR of CXCL14. The overexpression of miR-7481-3p significantly inhibited the expression of CXCL14 and restored the inhibitory role of melatonin testosterone synthesis and the expression of StAR, CYP11A1, and 3ß-HSD in rooster Leydig cells. Similarly, interference with CXCL14 could reverse the inhibitory effect of melatonin on the level of testosterone synthesis and the expression of StAR, CYP11A1, and 3ß-HSD in rooster Leydig cells. The RNA-seq results showed that melatonin could activate the PI3K/AKT signal pathway. Interference with CXCL14 significantly inhibited the phosphorylation level of PI3K and AKT, and the inhibited PI3K/AKT signal pathway could reverse the inhibitory effect of CXCL14 on testosterone synthesis and the expression of StAR, CYP11A1 and 3ß-HSD in rooster Leydig cells. Our results indicated that melatonin inhibits testosterone synthesis by targeting miR-7481-3p/CXCL14 and inhibiting the PI3K/AKT pathway.

The Role of DNMT1 and C/EBPα in the Regulation of CYP11A1 Expression During Syncytialization of Human Placental Trophoblasts.

Progesterone synthesized in the placenta is essential for pregnancy maintenance. CYP11A1 is a key enzyme in progesterone synthesis, and its expression increases greatly during trophoblast syncytialization. However, the underlying mechanism remains elusive. Here, we demonstrated that passive demethylation of CYP11A1 promoter accounted for the upregulation of CYP11A1 expression during syncytialization with the participation of the transcription factor C/EBPα. We found that the methylation rate of a CpG locus in the CYP11A1 promoter was significantly reduced along with decreased DNA methyltransferase 1 (DNMT1) expression and its enrichment at the CYP11A1 promoter during syncytialization. DNMT1 overexpression not only increased the methylation of this CpG locus in the CYP11A1 promoter, but also decreased CYP11A1 expression and progesterone production. In silico analysis disclosed multiple C/EBPα binding sites in both CYP11A1 and DNMT1 promoters. C/EBPα expression and its enrichments at both the DNMT1 and CYP11A1 promoters were significantly increased during syncytialization. Knocking-down C/EBPα expression increased DNMT1 while it decreased CYP11A1 expression during syncytialization. Conclusively, C/EBPα plays a dual role in the regulation of CYP11A1 during syncytialization. C/EBPα not only drives CYP11A1 expression directly, but also indirectly through downregulation of DNMT1, which leads to decreased methylation in the CpG locus of the CYP11A1 promoter, resulting in increased progesterone production during syncytialization.

Effects of insulin-like growth factor-1 on the mRNA expression of estradiol receptors, steroidogenic enzymes, and steroid production in bovine follicles.

Insulin-like growth factor-1 (IGF-1) plays a crucial role in follicular growth and stimulates steroid hormone production in bovine follicles. Steroid hormones are synthesized through the actions of steroidogenic enzymes, specifically STAR, CYP11A1, HSD3B, and CYP19A1 in both theca cells (TCs) and granulosa cells (GCs), under the influence of gonadotropins. Particularly, estradiol 17ß (E2) assumes a central role in follicular development and selection by activating estrogen receptors ß (ESR2) in GCs. We assessed ESR2 mRNA expression in GCs of developing follicles and investigated the impact of IGF-1 on the mRNA expression of ESR2, CYP19A1, FSHR, and LHCGR, STAR, CYP11A1, and HSD17B in cultured GCs and TCs, respectively. Additionally, we assessed the influence of IGF-1 on androstenedione (A4), progesterone (P4), and testosterone (T) production in TCs. Small-sized follicles (< 6 mm) exhibited the highest levels of ESR2 mRNA expression, whereas medium-sized follicles (7-8 mm) displayed higher levels than large-sized follicles (≥ 9 mm) (P < 0.05). IGF-1 increased the mRNA expression of ESR2, CYP19A1, and FSHR in GCs of follicles of both sizes, except for FSHR mRNA in medium-sized follicles (P < 0.05). IGF-1 significantly elevated mRNA expression of LHCGR, STAR, CYP11A1, and CYP17B in TCs of small- and medium-sized follicles (P < 0.05). Moreover, IGF-1 augmented the production of A4 and P4 but had no impact on T production in TCs of small- and medium-sized follicles. Taken together, our findings indicate that IGF-1 upregulates steroidogenic enzymes and steroid hormone production, underscoring the crucial role of IGF-1 in follicle development and selection.

Expression of cholesterol synthesis and steroidogenic markers in females of the Chinese brown frog (Rana dybowskii) during prespawning and prehibernation.

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

Perfluorooctanoic acid (PFOA) inhibits steroidogenesis and mitochondrial function in bovine granulosa cells in vitro.

Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant. Due to the ubiquitous presence of PFOA in the environment, the impacts of PFOA exposure not only affect human reproductive health but may also affect livestock reproductive health. The focus of this study was to determine the effects of PFOA on the physiological functions of bovine granulosa cells in vitro. Primary bovine granulosa cells were exposed to 0, 4, and 40 µM PFOA for 48 and 96 h followed by analysis of granulosa cell function including cell viability, steroidogenesis, and mitochondrial activity. Results revealed that PFOA inhibited steroid hormone secretion and altered the expression of key enzymes required for steroidogenesis. Gene expression analysis revealed decreases in mRNA transcripts for CYP11A1, HSD3B, and CYP19A1 and an increase in STAR expression after PFOA exposure. Similarly, PFOA decreased levels of CYP11A1 and CYP19A1 protein. PFOA did not impact live cell number, alter the cell cycle, or induce apoptosis, although it reduced metabolic activity, indicative of mitochondrial dysfunction. We observed that PFOA treatment caused a loss of mitochondrial membrane potential and increases in PINK protein expression, suggestive of mitophagy and mitochondrial damage. Further analysis revealed that these changes were associated with increased levels of reactive oxygen species. Expression of autophagy related proteins phosphoULK1 and LAMP2 were increased after PFOA exposure, in addition to an increased abundance of lysosomes, characteristic of increased autophagy. Taken together, these findings suggest that PFOA can negatively impact granulosa cell steroidogenesis via mitochondrial dysfunction.

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.

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.

Ontogeny of adropin and its receptor expression during postnatal development and its pro-gonadal role in the ovary of pre-pubertal mouse.

Adropin, a highly conserved multifunctional peptide hormone, has a beneficial effect on the maintenance of gluco-lipid homeostasis, endothelial and cardiovascular functions. However, the expression and potential role of adropin in ovarian function are not fully elucidated. The present study aimed to investigate the expression of adropin and GPR19 in the mice ovary during various stages of postnatal development. This study also explored whether the treatment of adropin can modulate the timing of puberty, for which pre-pubertal mice were treated with adropin. The result showed the intense immunoreactivity of adropin in TICs, while GPR19 immunoreactivity was noted in GCs in infantile, pre-pubertal, and pubertal mice ovary. Also, adropin and GPR19 are highly expressed in the CL of the ovary of reproductively active mice. The fact that adropin expression in the ovary at different stages of postnatal development positively correlated with circulating progesterone and estradiol indicated that it has a role in the production of steroid hormones. Furthermore, the results of in vivo studies in pre-pubertal mice showed that adropin promotes early folliculogenesis by enhancing the proliferation (PCNA) of GCs of cortical ovarian follicles and promotes estradiol production by enhancing the expression of GPR19, StAR, CYP11A1 and aromatase proteins. Also, adropin treatment increases the Bax/Bcl2 ratio and expression of cleaved caspase-3 and ERα proteins, which may result in increased apoptosis of medullary follicles leading to the formation of a well-developed interstitium with interstitial glandular cells. Collectively, these findings indicate that adropin may be a factor that accelerates pubertal development in the ovary and could be utilized as a therapeutic approach for treating pubertal delay.

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.

PURPOSE: Steroid 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. METHODS: DMU-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. RESULTS: lipDMU-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. CONCLUSIONS: This 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.