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.

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.

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.

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.

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.

Exploring the impact of CYP11A1's missense SNPs on the interaction between CYP11A1 and cholesterol: A comprehensive structural analysis and MD simulation study.

The process of steroidogenesis plays a vital role in human physiology as it governs the biosynthesis of mineralocorticoids, glucocorticoids, and androgens. These three classes of steroid hormones are primarily produced in the adrenal and gonadal glands through steroidogenesis pathways. Initiated by the side chain cleavage of cholesterol (CLR), this process leads to the conversion of cholesterol into pregnenolone and isocaproic aldehyde. The enzyme CYP11A1, encoded by the CYP11A1 gene, plays a key role in catalyzing the side chain cleavage of CLR. Several single nucleotide polymorphisms (SNPs) have been identified in the CYP11A1 gene, which may predispose carriers to disorders associated with abnormal steroidogenesis. Specifically, missense SNPs in the CYP11A1 gene have the potential to negatively impact the interaction between CYP11A1 and CLR, thus affecting the overall metabolome of steroid hormones. In this computational study, we focused on a specific set of missense SNPs reported in the CYP11A1 gene, aiming to identify variants that directly impact the interaction between CYP11A1 and CLR. The three-dimensional structure of the CYP11A1-CLR complex was obtained from the RCSB Protein Data Bank, while missense SNPs in the CYP11A1 gene were retrieved from Ensembl. To predict the most deleterious variants, we utilized the ConSurf server, SIFT, and PolyPhen. Furthermore, we assessed the impact of induced amino acid (AA) substitutions on the CYP11A1-CLR interaction using the PRODIGY server, PyMol, and Ligplot programs. Additionally, molecular dynamics (MD) simulations were conducted to analyze the effects of deleterious variants on the structural dynamics of the CYP11A1-CLR complex. Among the 8096 retrieved variants, we identified ten missense SNPs (E91K, W147G, R151W, R151Q, S391C, V392M, Q395K, Q416E, R460W, and R460Q) as deleterious for the interaction between CYP11A1 and CLR. MD simulations of the CYP11A1-CLR complexes carrying these deleterious AA substitutions revealed that Q416E, W147G, R460Q, and R460W had the most pronounced impacts on the structural dynamics of the complex. Consequently, these missense SNPs were considered the most deleterious ones. Further functional tests are recommended to assess the impact of these four missense SNPs on the enzymatic activity of CYP11A1. Moreover, Genome-Wide Association Studies (GWAS) should be conducted to determine the significance of their association with abnormal steroidogenesis diseases in various patient groups.

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

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

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

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

Heat enhances the inhibitory effect of lipopolysaccharide on duck granulosa cell proliferation and steroid biosynthesis in vitro.

Lipopolysaccharide (LPS) reduces the reproductive performance of laying ducks, especially during the hot summer months. To study the underlying mechanisms, we investigated the effects of different LPS concentrations and heat on duck granulosa cell (GC) proliferation and steroid biosynthesis in vitro. We investigated GC proliferation, secretion, and activation of the MAPK pathway. The cell cycle results showed that LPS treatment alone did not significantly affect cell proliferation, whereas the mRNA expression levels of IGF2, IGFBP2, and CyclinD1 were downregulated and p27kip1 was significantly upregulated after 2000 ng/mL LPS treatment when compared to untreated cells. In steroid hormone synthesis, although LPS increased the expression of most steroid biosynthesis genes, it inhibited the expression of CYP11A1 at high LPS concentrations. High temperatures enhanced the inhibitory effect of LPS on the expression of proliferation-promoting genes. Heat significantly reduced CYP11A1 and CYP19A1 expression. In addition, the phosphorylation of P38 was significantly upregulated by high temperatures combined with LPS, whereas the phosphorylation of ERK1/2 and JNK was downregulated. The relative protein expression of Bax/BCL-2 was upregulated at high temperatures in combination with LPS. Heat treatment enhanced the inhibitory effects of LPS on the proliferation and hormone biosynthesis of duck GCs in vitro.

Mitochondrial cytochrome P450 1B1 is involved in pregnenolone synthesis in human brain cells.

Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The neurosteroid and major steroid precursor pregnenolone has therapeutical potential in various diseases, such as psychiatric and pain disorders, and may play important roles in myelination, neuroinflammation, neurotransmission, and neuroplasticity. Although pregnenolone is synthesized by CYP11A1 in peripheral steroidogenic organs, our recent study showed that pregnenolone must be synthesized by another mitochondrial cytochrome P450 (CYP450) enzyme other than CYP11A1 in human glial cells. Therefore, we sought to identify the CYP450 responsible for pregnenolone production in the human brain. Upon screening for CYP450s expressed in the human brain that have mitochondrial localization, we identified three enzyme candidates: CYP27A1, CYP1A1, and CYP1B1. We found that inhibition of CYP27A1 through inhibitors and siRNA knockdown did not negatively affect pregnenolone synthesis in human glial cells. Meanwhile, treatment of human glial cells with CYP1A1/CYP1B1 inhibitors significantly reduced pregnenolone production in the presence of 22(R)-hydroxycholesterol. We performed siRNA knockdown of CYP1A1 or CYP1B1 in human glial cells and found that only CYP1B1 knockdown significantly decreased pregnenolone production. Furthermore, overexpression of mitochondria-targeted CYP1B1 significantly increased pregnenolone production under basal conditions and in the presence of hydroxycholesterols and low-density lipoprotein. Inhibition of CYP1A1 and/or CYP1B1 via inhibitors or siRNA knockdown did not significantly reduce pregnenolone synthesis in human adrenal cortical cells, implying that CYP1B1 is not a major pregnenolone-producing enzyme in the periphery. These data suggest that mitochondrial CYP1B1 is involved in pregnenolone synthesis in human glial cells.

A cytochrome P450 CYP87A4 imparts sterol side-chain cleavage in digoxin biosynthesis.

Digoxin extracted from the foxglove plant is a widely prescribed natural product for treating heart failure. It is listed as an essential medicine by the World Health Organization. However, how the foxglove plant synthesizes digoxin is mostly unknown, especially the cytochrome P450 sterol side chain cleaving enzyme (P450scc), which catalyzes the first and rate-limiting step. Here we identify the long-speculated foxglove P450scc through differential transcriptomic analysis. This enzyme converts cholesterol and campesterol to pregnenolone, suggesting that digoxin biosynthesis starts from both sterols, unlike previously reported. Phylogenetic analysis indicates that this enzyme arises from a duplicated cytochrome P450 CYP87A gene and is distinct from the well-characterized mammalian P450scc. Protein structural analysis reveals two amino acids in the active site critical for the foxglove P450scc's sterol cleavage ability. Identifying the foxglove P450scc is a crucial step toward completely elucidating digoxin biosynthesis and expanding the therapeutic applications of digoxin analogs in future work.

Perfluorooctane sulfonic acid modulates expression of placental steroidogenesis-associated genes and hormone levels in pregnant rats.

Perfluorooctane sulfonate (PFOS) is a widespread and persistent chemical in the environment. Reports show that PFOS is a potential endocrine disruptor; however, the possible effects of PFOS on placental endocrine function are unclear. This study aimed to investigate the endocrine-disrupting effects of PFOS on the placenta in pregnant rats and its potential mechanism. Pregnant rats from gestational days 4-20 were exposed to 0, 10, and 50 µg/mL PFOS through drinking water followed by analysis of various biochemical parameters. PFOS dose-dependently decreased fetal and placental weight in both sexes, with a specific decrease in weight of labyrinth but not junctional layer. Plasma progesterone (↑166%), aldosterone (↑201%), corticosterone (↑205%), testosterone (↑45%), luteinizing hormone (↑49%) levels were significantly increased, while estradiol (↓27%), prolactin (↓28%) and hCG (↓62%) levels were reduced in groups exposed to higher doses of PFOS. Real-time quantitative reverse transcriptase-polymerase chain reaction analysis revealed a significant increase in mRNA levels of placental steroid biosynthesis enzymes, including Cyp11A1 and 3ß-HSD1 in male placenta and StAR, Cyp11A1, 17ß-HSD1 and 17ß-HSD3 in female placenta of PFOS dams. Cyp19A1 expression in ovaries was significantly decreased in PFOS dams. mRNA levels for placental steroid metabolism enzyme UGT1A1 increased in male but not in female placenta of PFOS dams. These results suggest that the placenta is a target tissue of PFOS and PFOS-induced dysregulation in steroid hormone production might be related to the altered expression of hormone biosynthesis and metabolism enzyme genes in the placenta. This hormone disruption might affect maternal health and fetal growth.

Spheroid formation and luteinization of granulosa cells of felids in a long-term 3D culture.

In the present study, granulosa cells (GCs) from domestic cats and Persian leopard were cultured and characterized from selected days. The culture period was divided into two phases: maintenance, which lasted for 7 days, and luteinization, which followed for up to 11 days. Luteinization was performed on ultra-low attachment plates, supporting the formation of spheroids in a medium supplemented with insulin, forskolin, and luteinizing hormone (LH). GCs of domestic cat produced estradiol (E2) and progesterone (P4) during the maintenance phase. The gene expressions of some proteins involved in steroidogenesis were stable (STAR, HSD3B1) or decreased over time (CYP11A1, HSD17B1, CYP17A1, and CYP19A1), which was similar to the expressions of gonatropin receptors (LHCGR and FSHR). During the luteinization phase, P4 concentration significantly increased (P < 0.05), and E2, in contrast to the proliferation phase, was below detection range. The expression of genes of proteins involved in steroidogenesis (STAR, CYP11A1, HSD3B1, HSD17B1, CYP17A1, and CYP19A1) and of gonadotropin receptors (LHCGR and FSHR) significantly increased during the luteinization period, but some expressions exhibited a decrease at the end of the phase (LHCGR, FSHR, HSD17B1, CYP19A1). The morphology of the luteinized GCs of domestic cat resembled large luteal cells and had numerous vacuole-like structures. Also, the GCs of Persian leopard underwent luteinization, shown by increasing P4 production and HSD3B1 expression. This study confirms that GCs from felids can be luteinized in a 3D spheroid system which can be a basis for further studies on luteal cell function of felids. Additionally, we could show that the domestic cat can serve as a model species for establishing cell culture methods which can be transferred to other felids.

Comparative biochemical characterization of mammalian-derived CYP11A1s with cholesterol side-chain cleavage activities.

Steroid drugs, the second largest class of pharmaceuticals after antibiotics, have shown significant anti-inflammatory, anti-allergic, and endocrine-regulating effects. A group of cytochrome P450 enzymes, namely, CYP11A1 isoenzymes from different organisms are capable of converting cholesterol into pregnenolone, which is a pivotal reaction in both steroid metabolism and (bio)synthetic network of steroid products. However, the low activity of CYP11A1s greatly restricts the industrial application of these cholesterol side-chain cleavage enzymes. Herein, we investigate ten CYP11A1 enzymes of different origins and in vitro characterize two CYP11A1s with a relatively higher expression level from Capra hircus and Sus scrofa, together with the CYP11A1s from Homo sapiens and Bos taurus as references. Towards five selected sterol substrates with different side chain structures, S. scrofa CYP11A1 displays relatively higher activities. Through redox partners combination screening, we reveal the optimal redox partner pair of S. scrofa adrenodoxin and C. hircus adrenodoxin reductase. Moreover, the semi-rational mutagenesis for the active sites and substrate entrance channels of human and bovine CYP11A1s is performed based on comparative analysis of their crystal structures. The mutant mBtCYP11A1-Q377A derived from mature B. taurus CYP11A1 shows a 1.46 times higher activity than the wild type enzyme. These results not only demonstrate the tunability of the highly conserved CYP11A1 isoenzymes, but also lay a foundation for the following engineering efforts on these industrially relevant P450 enzymes.

Differential tissue expression of sex steroid-synthesizing enzyme CYP11A1 in male Tibetan sheep (Ovis aries).

Steroid metabolism is a fundament to testicular development and function. The cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) is a key rate-limiting enzyme for catalyzing the conversion of cholesterol to pregnenolone. However, despite its importance, what expression and roles of CYP11A1 possesses and how it regulates the testicular development and spermatogenesis in Tibetan sheep remains largely unknown. Based on this, we evaluated the expression and localization patterns of CYP11A1 in testes and epididymides of Tibetan sheep at three developmental stages (three-month-old, pre-puberty; one-year-old, sexual maturity and three-year-old, adult) by quantitative real-time PCR (qPCR), western blot and immunofluorescence. The results showed that CYP11A1 mRNA and protein were expressed in testes and epididymides throughout the development stages and obviously more intense in one- and three-year-old groups than three-month-old group (except for the caput epididymidis). Immunofluorescence assay showed that the CYP11A1 protein was mainly located in Leydig cells and epididymal epithelial cells. In addition, positive signals of CYP11A1 protein were observed in germ cells, epididymal connective tissue and sperms stored in the epididymal lumen. Collectively, these results suggested that the CYP11A1 gene might be mainly involved in regulating spermatogenesis and androgen synthesis in developmental Tibetan sheep testis and epididymis.