RESUMEN

Fish sex is largely influenced by steroid hormones, especially sex hormones. Here, we established a steroid hormone-free genetic model by mutation of cyp11a1 in Nile tilapia, which was confirmed by EIA assay. Gonadal phenotype and transcriptome analyses showed that the XX mutants displayed sex reversal from female to male but with defective spermatogenesis. Despite the sex reversal, the aromatase encoding gene cyp19a1a was continuously expressed in the gonads of the XX mutants, which might be caused by androgen deficiency. Whole-mount fluorescence in situ hybridization and transcriptome analysis showed that the gonads of the XX mutants firstly developed towards ovary but shifted to testis between 10 to 15 days after hatching. Detailed expression analysis of key sex differentiation pathway genes foxl3 and dmrt1 combined with apoptosis analysis revealed transdifferentiation of germ cells from female to male during sex reversal. Rescue experiments showed that both P5 and E2 treatment rescued the sex reversal of cyp11a1 mutant XX fish. Overall, our results revealed a transient ovary-like stage and transdifferentiation of germ cells from female to male in the early gonads of the steroid hormone-deprived cyp11a1 mutant XX fish.

Asunto(s)

Mutación , Ovario , Diferenciación Sexual , Animales , Femenino , Ovario/metabolismo , Masculino , Diferenciación Sexual/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Cíclidos/genética , Cíclidos/metabolismo , Trastornos del Desarrollo Sexual/genética , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Tilapia/genética , Tilapia/metabolismo , Procesos de Determinación del Sexo/genética

RESUMEN

The ovarian KGN granulosa-like tumour cell line is commonly used as a model for human granulosa cells, especially since it produces steroid hormones. To explore this further, we identified genes that were differentially expressed by KGN cells compared to primary human granulosa cells using three public RNA sequence datasets. Of significance, we identified that the expression of the antioxidant gene TXNRD1 (thioredoxin reductase 1) was extremely high in KGN cells. This is ominous since cytochrome P450 enzymes leak electrons and produce reactive oxygen species during the biosynthesis of steroid hormones. Gene Ontology (GO) analysis identified steroid biosynthetic and cholesterol metabolic processes were more active in primary granulosa cells, whilst in KGN cells, DNA processing, chromosome segregation and kinetochore pathways were more prominent. Expression of cytochrome P450 cholesterol side-chain cleavage (CYP11A1) and cytochrome P450 aromatase (CYP19A1), which are important for the biosynthesis of the steroid hormones progesterone and oestrogen, plus their electron transport chain members (FDXR, FDX1, POR) were measured in cultured KGN cells. KGN cells were treated with 1 mM dibutyryl cAMP (dbcAMP) or 10 µM forskolin, with or without siRNA knockdown of TXNRD1. We also examined expression of antioxidant genes, H2O2 production by Amplex Red assay and DNA damage by γH2Ax staining. Significant increases in CYP11A1 and CYP19A1 were observed by either dbcAMP or forskolin treatments. However, no significant changes in H2O2 levels or DNA damage were found. Knockdown of expression of TXNRD1 by siRNA blocked the stimulation of expression of CYP11A1 and CYP19A1 by dbcAMP. Thus, with TXNRD1 playing such a pivotal role in steroidogenesis in the KGN cells and it being so highly overexpressed, we conclude that KGN cells might not be the most appropriate model of primary granulosa cells for studying the interplay between ovarian steroidogenesis, reactive oxygen species and antioxidants.

Asunto(s)

Antioxidantes , Aromatasa , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Células de la Granulosa , Humanos , Femenino , Antioxidantes/metabolismo , Aromatasa/genética , Aromatasa/metabolismo , Línea Celular Tumoral , Células de la Granulosa/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Tiorredoxina Reductasa 1/metabolismo , Tiorredoxina Reductasa 1/genética , Regulación Neoplásica de la Expresión Génica , Tumor de Células de la Granulosa/genética , Tumor de Células de la Granulosa/metabolismo , Tumor de Células de la Granulosa/patología , Esteroides/biosíntesis , Progesterona/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología

RESUMEN

Steroidogenesis occurs locally in peripheral tissues and via adrenal and gonadal glands' biosynthesis. The C2C12 mouse myoblast cell line and rat skeletal muscles harbor a local steroidogenesis pathway for glucocorticoids, and corticosterone is biosynthesized from skeletal muscle cells. However, Cyp11a1 and StAR protein expressions are not observed in C2C12 cells or rat muscular tissues. In this context, this study investigated the relationship between DNA methylation and key steroidogenic genes. Bioinformatics analysis of methylated DNA immune precipitation showed that C2C12 myoblasts and myotubes did not have remarkable DNA methylated regions in the gene-body of Cyp11a1. However, a highly methylated region in the CpG island was detected in the intronic enhancer of Ad4BP/SF-1, known as the transcriptional factor for steroidogenic genes. After C2C12 myoblasts treatment with 5-aza-2-deoxycytidine, the gene expressions of Ad4BP/SF-1, Cyp11a1, and StAR were significantly time- and concentration-dependent upregulated. To clarify the contribution of Ad4BP/SF-1 on Cyp11a1 and StAR transcripts, we silenced Ad4BP/SF-1 during the 5-aza-2-deoxycytidine treatment in C2C12 myoblasts, resulting in significant suppression of both Cyp11a1 and StAR. Additionally, pregnenolone levels in the supernatants of C2C12 cells were enhanced by 5-aza-2-deoxycytidine treatment, whereas pregnenolone production by C2C12 myoblasts was significantly suppressed by Ad4BP/SF-1 knockdown. These results indicate that DNA methylation of Ad4BP/SF-1 might be involved in the downregulation of steroidogenic genes, such as Cyp11a1 and StAR in C2C12 myoblasts.

Asunto(s)

Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Islas de CpG , Metilación de ADN , Mioblastos , Fosfoproteínas , Animales , Metilación de ADN/genética , Metilación de ADN/efectos de los fármacos , Ratones , Mioblastos/metabolismo , Mioblastos/efectos de los fármacos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Línea Celular , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Islas de CpG/genética , Decitabina/farmacología , Ratas , Azacitidina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

Cholesterol and Helicobacter pylori (H. pylori) are both risk factors for gastric cancer (GC). However, the relationship between cholesterol and H. pylori and their function in the progression of GC are controversial. In this study, we addressed that H. pylori could induce mitochondrial cholesterol accumulation and promote GC proliferation and protect GC cells against apoptosis via cholesterol. Metabolomic and transcriptomic sequencing were used to identify CYP11A1 responsible for H. pylori-induced cholesterol accumulation. In vitro and in vivo function experiments revealed that cholesterol could promote the proliferation of GC and inhibit apoptosis. Mechanically, the interaction of Cytotoxin-associated gene A (CagA) and CYP11A1 redistributed mitochondrial CYP11A1 outside the mitochondria and subsequently caused mitochondrial cholesterol accumulation. The CYP11A1-knockdown upregulated cholesterol accumulation and reproduced the effect of cholesterol on GC in a cholesterol-dependent manner. Moreover, CYP11A1-knockdown or H. pylori infection inhibited mitophagy and maintained the mitochondria homeostasis. H. pylori could contribute to the progression of GC through the CagA/CYP11A1-mitoCHO axis. This study demonstrates that H. pylori can contribute to the progression of GC via cholesterol, and eradicating H. pylori is still prognostically beneficial to GC patients.

Asunto(s)

Colesterol , Helicobacter pylori , Mitocondrias , Neoplasias Gástricas , Helicobacter pylori/metabolismo , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Colesterol/metabolismo , Humanos , Mitocondrias/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Animales , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Línea Celular Tumoral , Ratones , Apoptosis , Masculino , Proliferación Celular

RESUMEN

Alterations in steroid hormone regulation have been implicated in the etiology and progression of autism spectrum disorders (ASD), with the enzyme cytochrome P450 family 11 subfamily A member 1 (CYP11A1)-a key catalyst in cholesterol side-chain cleavage, prominently expressed in the adrenal glands, ovaries, testes, and placenta-standing at the forefront of these investigations. The potential link between aberrations in placental Cyp11a1 expression and the resultant neurodevelopmental disorders, along with the mechanisms underpinning such associations, remains inadequately delineated. In this study, we employed a placental trophoblast-specific Cyp11a1 Hipp11 (H11) knock-in murine model to dissect the phenotypic manifestations within the placenta and progeny, thereby elucidating the underlying mechanistic pathways. Behavioral analyses revealed a diminution in social interaction capabilities alongside an augmented anxiety phenotype, as evidenced by open field and elevated plus maze assessments; both phenotypes were ameliorated after vitamin D3 supplementation. Electrophysiological assays underscored the augmented inhibition of paired-pulse facilitation, indicating impaired neuroplasticity in Cyp11a1 H11-modified mice. An elevation in progesterone concentrations was noted, alongside a significant upregulation of Th1-related cytokines (IL-6 and TNFα) across the plasma, placental, and frontal cortex-a pathological state mitigable through vitamin D3 intervention. Western blotting revealed a vitamin D-mediated rectification of vitamin D receptor and PGC-1α expression dysregulations. Immunofluorescence assays revealed microglial activation in the knock-in model, which was reversible upon vitamin D3 treatment. In conclusion, Cyp11a1 overexpression in the placenta recapitulated an autism-like phenotype in murine models, and vitamin D3 administration effectively ameliorated the resultant neurobehavioral and neuroinflammatory derangements. This study substantiates the application of Cyp11a1 as a biomarker in prenatal diagnostics and posits that prenatal vitamin D3 supplementation is a viable prophylactic measure against perturbations in steroid hormone metabolism associated with ASD pathogenesis.

Asunto(s)

Trastorno del Espectro Autista , Encéfalo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Modelos Animales de Enfermedad , Placenta , Animales , Femenino , Embarazo , Placenta/metabolismo , Ratones , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Encéfalo/metabolismo , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/genética , Vitamina D/metabolismo , Masculino , Trastorno Autístico/metabolismo , Trastorno Autístico/genética , Efectos Tardíos de la Exposición Prenatal/metabolismo , Progesterona/metabolismo , Técnicas de Sustitución del Gen

RESUMEN

Abstract: Reactive oxygen species (ROS) are a by-product of the activity of cytochrome P450 steroidogenic enzymes. Antioxidant enzymes protect against ROS damage. To identify if any particular antioxidant enzyme is used to protect against ROS produced by granulosa cells as follicles enlarge and produce oestradiol, we measured in the bovine granulosa cells the expression of two steroidogenic enzymes (CYP11A1, CYP19A1), important for progesterone and oestradiol production. We also measured the expression of the members (FDXR, FDX1, POR) of their electron transport chains (ETC). We measured antioxidant enzymes (GPXs 1-8, CAT, SODs 1 and 2, PRDXs 1-6, GSR, TXN, TXNRDs 1-3). Since selenium is an active component of GPXs, the selenium-uptake receptors (LRPs 2 and 8) were measured. Only the selenium-dependent GPX1 showed the same increase in expression as the steroidogenic enzymes did with increasing follicle size. GPX4 and PRDX2/6 decreased with follicle size, whereas SOD1/2, CAT, GSR, and TXNRD3 were lowest at the intermediate sizes. The other antioxidant enzymes were unchanged or expressed at low levels. The expression of the selenium-uptake receptor LRP8 also increased significantly with follicle size. Correlation analysis revealed statistically significant and strongly positive correlations of the steroidogenic enzymes and their ETCs with both GPX1 and LRP8. These results demonstrate a relationship between the expression of genes involved in steroidogenesis and selenium-containing antioxidant defence mechanisms. They suggest that during the late stages of folliculogenesis, granulosa cells are dependent on sufficient expression of GPX1 and the selenium transporter LRP8 to counteract increasing ROS levels caused by the production of steroid hormones. Lay summary: In the ovary, eggs are housed in follicles which contain the cells that produce oestrogen in the days leading up to ovulation of the egg. Oestrogen is produced by the action of enzymes. However, some of these enzymes also produce by-products called reactive oxygen species (ROS). These are harmful to eggs. Fortunately, cells have protective antioxidant enzymes that can neutralise ROS. This study was interested in which particular antioxidant enzyme(s) might be involved in neutralising the ROS in follicle cells. It was found that only one antioxidant enzyme, GPX1, appeared to be co-regulated with the enzymes that produce oestrogen and progesterone in the follicular cells. GPX1 contains the essential mineral selenium. In summary, this study has identified which antioxidant appears to be involved in neutralising ROS in the days leading to ovulation. It highlights the importance of selenium in the diet.

Asunto(s)

Glutatión Peroxidasa GPX1 , Glutatión Peroxidasa , Células de la Granulosa , Femenino , Células de la Granulosa/metabolismo , Animales , Bovinos , Glutatión Peroxidasa/metabolismo , Glutatión Peroxidasa/genética , Selenio/metabolismo , Antioxidantes/metabolismo , Aromatasa/metabolismo , Aromatasa/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Progesterona/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estradiol/metabolismo , Folículo Ovárico/metabolismo

RESUMEN

OBJECTIVE: The objective of this study was to investigate the allele frequencies of polymorphisms in genes CYP11A1 rs4886595 and CYP11A1 rs4887139 that are responsible for the steroidogenesis mechanism in polycystic ovary syndrome patients and control females. METHODS: Samples were obtained from the Department of Obstetrics and Gynecology in the Near East University Hospital from September 2019 to December 2019. Only the nonobese patients between the ages of 18-40 years were included in this study following informed consent. Obese patients and patients more than 40 years of age were excluded from the study. Nonobese women and normal ovulation were included in the control group. DNA was isolated from blood samples. Real-time polymerase chain reaction (PCR) was used to analyze single nucleotide polymorphisms (SNPs) in various genes linked to polycystic ovary syndrome. The studies were carried out using the samples obtained from 120 women, of whom 55 were nonobese and had normal ovulation, and 65 were polycystic ovary syndrome patients. The allelic frequencies of SNPs in genes linked to polycystic ovary syndrome were calculated using real-time PCR outcomes. RESULTS: The variation of the CYP11A1 rs4887139 G>A did not show any significance, while the variation of CYP11A1 rs4886595 C>A showed significant differences between the patient and the control groups (p=0.01), respectively. CONCLUSION: Future research ought to focus on elucidating the susceptible causes of polycystic ovary syndrome with a wide range of SNPs and more sample size. The genome-wide association studies in polycystic ovary syndrome patients of different origin will be important to identify candidate genes as well as proteins that are implied in polycystic ovary syndrome risk.

Asunto(s)

Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Frecuencia de los Genes , Síndrome del Ovario Poliquístico , Polimorfismo de Nucleótido Simple , Humanos , Síndrome del Ovario Poliquístico/genética , Femenino , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Adulto , Frecuencia de los Genes/genética , Adulto Joven , Estudios de Casos y Controles , Adolescente , Predisposición Genética a la Enfermedad/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Genotipo

RESUMEN

Polycystic ovary syndrome (PCOS), a prevalent reproductive disorder in women of reproductive age, features androgen excess, ovulatory dysfunction, and polycystic ovaries. Despite its high prevalence, specific pharmacologic intervention for PCOS is challenging. In this study, we identified artemisinins as anti-PCOS agents. Our finding demonstrated the efficacy of artemisinin derivatives in alleviating PCOS symptoms in both rodent models and human patients, curbing hyperandrogenemia through suppression of ovarian androgen synthesis. Artemisinins promoted cytochrome P450 family 11 subfamily A member 1 (CYP11A1) protein degradation to block androgen overproduction. Mechanistically, artemisinins directly targeted lon peptidase 1 (LONP1), enhanced LONP1-CYP11A1 interaction, and facilitated LONP1-catalyzed CYP11A1 degradation. Overexpression of LONP1 replicated the androgen-lowering effect of artemisinins. Our data suggest that artemisinin application is a promising approach for treating PCOS and highlight the crucial role of the LONP1-CYP11A1 interaction in controlling hyperandrogenism and PCOS occurrence.

Asunto(s)

Proteasas ATP-Dependientes , Artemisininas , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Proteínas Mitocondriales , Síndrome del Ovario Poliquístico , Animales , Femenino , Humanos , Ratones , Ratas , Andrógenos/metabolismo , Artemisininas/uso terapéutico , Artemisininas/farmacología , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Modelos Animales de Enfermedad , Hiperandrogenismo/tratamiento farmacológico , Hiperandrogenismo/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Ovario/efectos de los fármacos , Ovario/metabolismo , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Proteolisis , Ratones Endogámicos C57BL , Adulto Joven , Adulto , Ratas Sprague-Dawley , Proteasas ATP-Dependientes/genética , Proteasas ATP-Dependientes/metabolismo

RESUMEN

Acorus gramineus has a number of beneficial effects, including protective effects against age-related disorders. In this study, the effects of A. gramineus on testosterone production and andropause symptoms were evaluated. We first treated TM3 mouse Leydig cells, responsible for testosterone production, with A. gramineus aqueous extract at different concentrations. In TM3 cells, the testosterone concentration increased in a concentration-dependent manner compared with those in the control. In addition, at 400 µg/mL extract, the mRNA expression level of the steroidogenic enzyme CYP11A1 was increased. Subsequently, 23-week-old Sprague-Dawley (SD) rats exhibiting an age-related reduction in serum testosterone (approximately 80% lower than that in 7-week-old SD rats) were administered A. gramineus aqueous extract for 8 weeks. Serum total testosterone and free testosterone levels were higher and serum estradiol, prostate-specific antigen levels, and total cholesterol levels were lower in the AG50 group (A. gramineus aqueous extract 50 mg/kg of body weight/day) than in the OLD (control group). The AG50 group also showed significant elevations in sperm count, grip strength, and mRNA expression of StAR, CYP11A1, 17ß-HSD, and CYP17A1 compared with those in the OLD group. In conclusion, A. gramineus aqueous extract facilitated steroidogenesis in Leydig cells, elevated testosterone levels, lowered serum estradiol and total cholesterol levels, and increased muscle strength and sperm count, thus alleviating the symptoms of andropause. These findings suggest that A. gramineus aqueous extract is a potentially effective therapeutic agent against various symptoms associated with andropause.

Asunto(s)

Acorus , Andropausia , Células Intersticiales del Testículo , Extractos Vegetales , Ratas Sprague-Dawley , Testosterona , Animales , Masculino , Testosterona/sangre , Ratones , Extractos Vegetales/farmacología , Ratas , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Acorus/química , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Humanos

RESUMEN

The development of ovarian follicles in poultry is a key factor affecting the performance of egg production. Ovarian follicle development is regulated via the Wnt/ß-catenin signaling pathway, and ß-catenin, encoded by CTNNB1, is a core component of this pathway. In this study, using ovary GCs from laying hens, we investigated the regulatory role of CTNNB1 in steroid synthesis. We found that CTNNB1 significantly regulates the expression of StAR and CYP11A1 (key genes related to progesterone synthesis) and the secretion of progesterone (P4). Furthermore, simultaneous overexpression of CTNNB1 and SF1 resulted in significantly higher levels of CYP11A1 and secretion of P4 than in cells overexpressing CTNNB1 or SF1 alone. We also found that in GCs overexpressing SF1, levels of CYP11A1 and secreted P4 were significantly greater than in controls. Silencing of CYP11A1 resulted in the inhibition of P4 secretion while overexpression of SF1 in CYP11A1-silenced cells restored P4 secretion to normal levels. Together, these results indicate that synergistic cooperation between the ß-catenin and SF1 regulates progesterone synthesis in laying hen ovarian hierarchical granulosa cells to promote CYP11A1 expression.

Asunto(s)

Pollos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Células de la Granulosa , Progesterona , beta Catenina , Animales , Femenino , Progesterona/biosíntesis , Progesterona/metabolismo , beta Catenina/metabolismo , beta Catenina/genética , Células de la Granulosa/metabolismo , Pollos/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Factor Esteroidogénico 1/genética , Factor Esteroidogénico 1/metabolismo , Regulación de la Expresión Génica/fisiología

RESUMEN

Prenatal exposure to diethylhexyl phthalate (DEHP) has been linked with a decline in testosterone levels in adult male rats, but the underlying mechanism remains unclear. We investigated the potential epigenetic regulation, particularly focusing on N6-methyladenosine (m6A) modification, as a possible mechanism. Dams were gavaged with DEHP (0, 10, 100, and 750 mg/kg/day) from gestational day 14 to day 21. The male offspring were examined at the age of 56 days. Prenatal DEHP administration at 750 mg/kg/day caused a decline in testosterone concentrations, an elevation in follicle-stimulating hormone, a downregulated expression of CYP11A1 HSD3B2, without affecting Leydig cell numbers. Interestingly, Methyltransferase Like 4 (METTL4), an m6A methyltransferase, was downregulated, while there were no changes in METTL3 and METTL14. Moreover, CYP11A1 showed m6A reduction in response to prenatal DEHP exposure. Additionally, METTL4 expression increased postnatally, peaking in adulthood. Knockdown of METTL4 resulted in the downregulation of CYP11A1 and HSD3B2 and an increase in SCARB1 expression. Furthermore, the increase in autophagy protection in adult Leydig cells induced by prenatal DEHP exposure was not affected by 3-methyladenosine (3MA) treatment, indicating a potential protective role of autophagy in response to DEHP exposure. In conclusion, prenatal DEHP exposure reduces testosterone by downregulating CYP11A1 and HSD3B2 via m6A epigenetic regulation and induction of autophagy protection in adult Leydig cells as a response to DEHP exposure.

Asunto(s)

Dietilhexil Ftalato , Regulación hacia Abajo , Epigénesis Genética , Células Intersticiales del Testículo , Metiltransferasas , Efectos Tardíos de la Exposición Prenatal , Testosterona , Animales , Femenino , Masculino , Embarazo , Ratas , Adenosina/análogos & derivados , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Dietilhexil Ftalato/toxicidad , Dietilhexil Ftalato/análogos & derivados , Regulación hacia Abajo/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Células Intersticiales del Testículo/efectos de los fármacos , Metiltransferasas/genética , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Ratas Sprague-Dawley , Testosterona/sangre

RESUMEN

At 50 wk of age, broiler breeder roosters exhibit a significant decline of fertility. Therefore, the aim of this study was to assess the impact of incorporating barley sprout (BS) powder, D-aspartic acid (DA), or their combination into the diet on fertility, hatchability, semen quality, and the relative expression of StAR and P450SCC genes in aging broiler roosters. Aging (50 wk) male broiler breeders (n=32) were randomly assigned to one of four dietary treatments (2 × 2 factorial) with 2 levels of BS (0 or 2% basal diet) and DA (0 or 200 mg/kg/BW) for 12 wk. Roosters were individually housed under a 14-h light and 10-h dark cycle, with 150 g/d feed allocation and free access to fresh water, then euthanized. Throughout the study, the body weight of the broiler breeders was measured, along with various parameters related to semen quality, on a weekly basis. Additionally, artificial insemination was performed during the last 2 wk to evaluate reproductive endpoints. The results revealed that both BS and DA decreased (P < 0.01) body weight. Interestingly, the inclusion of BS, either alone or in combination with DA, resulted in a significant increase in total and forward sperm motility. Furthermore, it was demonstrated that the seminal concentration of malondialdehyde, a marker of oxidative stress, was significantly decreased by more than 20% in all groups compared to the control. The combination of both BS and DA led to the highest levels of circulating testosterone, as well as the functionality and membrane integrity of sperms. Additionally, it resulted in increased sperm concentrations, production, and penetration, ultimately leading to improved fertility rate and hatchability percentage. Moreover, a positive association between total motility and fertility was observed (P < 0.01). Furthermore, the combined supplementation of BS and DA up-regulated the relative mRNA expression of P450scc and StAR (P < 0.01). To summarize, dietary inclusion of BS, DA, or their combination have a potential to improve various aspects of reproductive performance in aging roosters.

Asunto(s)

Alimentación Animal , Proteínas Aviares , Pollos , Ácido D-Aspártico , Dieta , Suplementos Dietéticos , Fertilidad , Hordeum , Análisis de Semen , Animales , Masculino , Pollos/fisiología , Pollos/genética , Hordeum/química , Suplementos Dietéticos/análisis , Análisis de Semen/veterinaria , Alimentación Animal/análisis , Dieta/veterinaria , Fertilidad/efectos de los fármacos , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Ácido D-Aspártico/administración & dosificación , Ácido D-Aspártico/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Distribución Aleatoria , Regulación hacia Arriba/efectos de los fármacos , Expresión Génica/efectos de los fármacos

RESUMEN

This study investigated the effect of hCG or GnRH on structural changes of the corpora lutea (CL) and the regulation of the expression of steroidogenic enzymes involved in P4 secretion in post-ovulatory (po-CL) and accessory CL (acc-CL). Sixty-four ewes were assigned to three groups receiving: 300 IU of hCG (hCG) or 4 µg Buserelin (GnRH) or 1 mL of saline solution (Control) on Day (d) 4 post artificial insemination (FTAI). Laparoscopic ovarian were performed on d 4, 14 and, 21 post-FTAI to determine the numbers of CL. Blood samples were collected for serum LH and P4 analysis. On d 14 post-FTAI, both CL were removed from the ovary to determine large luteal cell (LLC) number and to evaluate the expression of steroidogenic enzymes (HSD3B1, STAR, CYP11A1). Only hCG and GnRH treated ewes generated acc-CL. The LLC in both po- and acc-CL were significantly greater in the hCG group compared to GnRH and Control groups (P<0.05). Overall, hCG group showed the greatest immunodetection of HSD3B1and STAR in both po- and acc-CL (P<0.05). rnRNA expression of HSD3B1, STAR and CYP11A1 in the acc-CL tended to be greater in hCG group than in GnRH group (P<0.1). The LH concentration was increased in GnRH group (P<0.05) and P4 concentration was greater in hCG group compared to the other groups (P<0.05). In conclusion, administration of hCG has a notably impact on acc-CL development and the expression of steroidogenic enzymes compared to GnRH treatment in ewes. This leads to elevated P4 concentration and improved luteal function.

Asunto(s)

Gonadotropina Coriónica , Cuerpo Lúteo , Hormona Liberadora de Gonadotropina , Fase Luteínica , Progesterona , Animales , Femenino , Ovinos/fisiología , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/metabolismo , Progesterona/sangre , Progesterona/metabolismo , Gonadotropina Coriónica/farmacología , Gonadotropina Coriónica/administración & dosificación , Hormona Liberadora de Gonadotropina/farmacología , Hormona Liberadora de Gonadotropina/metabolismo , Fase Luteínica/efectos de los fármacos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Hormona Luteinizante/metabolismo , Fosfoproteínas

RESUMEN

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.

Asunto(s)

Fitosteroles , Progesterona , Bovinos , Animales , Pregnenolona/metabolismo , Esteroles , Esteroides , Colesterol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Mamíferos/metabolismo , Éteres

RESUMEN

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.

Asunto(s)

Células Lúteas , Femenino , Animales , Progesterona/metabolismo , Kisspeptinas/genética , Kisspeptinas/farmacología , Kisspeptinas/metabolismo , Regulación hacia Arriba , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Sistema de Señalización de MAP Quinasas , Cuerpo Lúteo/fisiología , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo

RESUMEN

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.

Asunto(s)

Sistema Hipotálamo-Hipofisario , Microbiota , Masculino , Ratones , Animales , Sistema Hipotálamo-Hipofisario/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Espectrometría de Masas en Tándem , Sistema Hipófiso-Suprarrenal/metabolismo , Esteroides/metabolismo , Corticosterona/metabolismo

RESUMEN

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.

Asunto(s)

Pollos , Progesterona , Femenino , Animales , Progesterona/metabolismo , Progesterona/farmacología , Pollos/genética , Quercetina/farmacología , Quercetina/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/farmacología , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Células de la Granulosa/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

The involvement of the molecular clock in regulating cell physiological processes on a specific time scale is a recognized concept, yet its specific impact on optimizing androgen production in Leydig cells has been unclear. This study aimed to confirm the role of the REVERBA (NR1D1) gene in controlling the transcription of key genes related to Leydig cell steroid production. We investigated daily variations by collecting Leydig cells from rats at various times within a 24-h period. Chromatin immunoprecipitation study showed a time-dependent pattern for genes linked to steroid production (Nur77, Star, Cyp11a1, and Cyp17a1), which closely matched the 24-h REVERBA levels in Leydig cells, peaking between zeitgeber time (ZT) 7-11. To understand the physiological significance of REVERBA's interaction with promoters of steroidogenesis-related genes, Leydig cells from rats at two different times (ZT7 and ZT16; chosen based on REVERBA expression levels), were treated with either an agonist (GSK4112) or an antagonist (SR8278). The results revealed that the REVERBA agonist stimulated gene transcription, while the antagonist inhibited it, but only when REVERBA was sufficiently present, indicating a reliance on REVERBA's circadian fluctuation. Moreover, this REVERBA-dependent stimulation had a clear impact on testosterone production in the culture medium, underscoring REVERBA's involvement in the circadian regulation of testosterone. This study indicates that REVERBA, in addition to being a core component of the cellular clock, plays a key role in regulating androgen production in Leydig cells by influencing the transcription of critical steroidogenesis-related genes.

Asunto(s)

Relojes Circadianos , Células Intersticiales del Testículo , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares , Animales , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Ratas , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Relojes Circadianos/genética , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Testosterona/biosíntesis , Testosterona/metabolismo , Esteroides/biosíntesis , Esteroides/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Ratas Sprague-Dawley

RESUMEN

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.

Asunto(s)

Hidrogenasas , Células Intersticiales del Testículo , Masculino , Animales , Células Intersticiales del Testículo/química , Células Intersticiales del Testículo/metabolismo , Cadmio/metabolismo , Testosterona , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Hidroxiesteroides/metabolismo , Hidroxiesteroides/farmacología , Hidrogenasas/metabolismo , Hidrogenasas/farmacología , Espermatogénesis , Colesterol/metabolismo , Colesterol/farmacología

RESUMEN

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.

Asunto(s)

Proteína alfa Potenciadora de Unión a CCAAT , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , ADN (Citosina-5-)-Metiltransferasa 1 , Placenta , Femenino , Humanos , Embarazo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Metilación de ADN , Placenta/metabolismo , Progesterona/metabolismo , Trofoblastos/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo