RESUMO
In teleost fish, sex steroids are involved in sex determination, sex differentiation, and fertility. Cyp17a1 (Cytochrome P450 family 17 subfamily A member 1) is thought to play essential roles in fish steroidogenesis. Therefore, to further understand its roles in steroidogenesis, sex determination, and fertility in fish, we constructed a cyp17a1 gene mutant in Nile tilapia (Oreochromis niloticus). In XX fish, mutation of the cyp17a1 gene led to a female-to-male sex reversal with a significant decline in 17ß-estradiol (E2) and testosterone (T) production, and ectopic expression of male-biased markers (Dmrt1 and Gsdf) in gonads from the critical window of sex determination. Sex reversal was successfully rescued via T or E2 administration, and ovarian characteristics were maintained after termination of E2 supplementation in the absence of endogenous estrogen production in cyp17a1-/- XX fish. Likewise, deficiencies in T and 11-ketotestosterone (11-KT) production in both cyp17a1-/- XX sex-reversed males and cyp17a1-/- XY mutants resulted in meiotic initiation delays, vas deferens obstruction and sterility due to excessive apoptosis and abnormal mitochondrial morphology. However, 11-KT treatment successfully rescued the dysspermia to produce normal sperm in cyp17a1-/- male fish. Significant increases in gonadotropic hormone (gth) and gth receptors in cyp17a1-/- mutants may excessively upregulate steroidogenic gene expression in Leydig cells through a feedback loop. Taken together, our findings demonstrate that Cyp17a1 is indispensable for E2 production, which is fundamental for female sex determination and differentiation in XX tilapia. Additionally, Cyp17a1 is essential for T and 11-KT production, which further promotes spermatogenesis and fertility in XY males.
Assuntos
Ciclídeos/fisiologia , Família 17 do Citocromo P450/fisiologia , Hormônios Esteroides Gonadais/biossíntese , Infertilidade Masculina/genética , Processos de Determinação Sexual/genética , Animais , Animais Geneticamente Modificados , Ciclídeos/genética , Ciclídeos/metabolismo , Família 17 do Citocromo P450/genética , Feminino , Fertilidade/genética , Peixes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Infertilidade Masculina/veterinária , Masculino , Redes e Vias Metabólicas/genéticaRESUMO
Polycystic ovary syndrome (PCOS) results from functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Cultured theca cells from polycystic ovaries of women with the most common form of PCOS overexpress most androgen producing enzymes, particularly CYP450c17. In this study, a murine model was used of PCOS induced by chronic feeding with a high-fat diet that exhibits the reproductive, hyperandrogenic, and metabolic constellation of PCOS symptoms seen in women. Oral administration of KDT501, a hops-derived bitter taste receptor (Tas2R 108) isohumulone ligand resulted in resolution of PCOS-associated endocrine and metabolic disturbances and restored reproductive function. Pioglitazone, a PPARγ agonist, also improved metabolic and reproductive function, though not to the same degree as KDT501. Specifically, treatment of the murine PCOS model with KDT501 resulted in reduced testosterone and androstenedione levels in the absence of significant changes in LH or FSH, improved glucose tolerance and lipid metabolism, and reduced hepatic lipid infiltration and adiposity. There was significant improvement in estrous cyclicity and an increase in the number of ovarian corpora lutea, indicative of improved reproductive function after exposure to KDT501. Finally, ex vivo exposure of murine ovaries to KDT501 attenuated androgen production and ovarian expression of CYP450c17. Interestingly, the ovaries expressed Tas2R 108, suggesting a potential regulation of ovarian steroidogenesis through this chemosensory receptor family. In summary, a therapeutic strategy for PCOS possibly could include direct influences on ovarian steroidogenesis that are independent of gonadotrophic hormone regulation.
Assuntos
Extratos Vegetais/administração & dosagem , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Adiposidade/efeitos dos fármacos , Androstenodiona/metabolismo , Animais , Família 17 do Citocromo P450/genética , Família 17 do Citocromo P450/metabolismo , Modelos Animais de Doenças , Ciclo Estral/efeitos dos fármacos , Feminino , Humanos , Humulus/química , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Extratos Vegetais/química , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/fisiopatologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Testosterona/metabolismoRESUMO
BACKGROUND: Metformin influences insulin receptor signaling, which might interfere with the proliferation of ovarian follicular structures and steroidogenesis. We hypothesize that reductions in glucose and insulin levels might interfere with CYP-17 expression and histomorphological changes in an androgenized rat model. The aim of this study was to analyze the effect of metformin on CYP-17 expression, follicular dynamics, and proliferative parameters in neonatally androgenized female rats. METHODS: Thirty-six newborn rats were randomly allocated to the following three groups on the third day of life: control (CG, n = 12), androgenized (GA, n = 12), and androgenized + metformin (GAmet, n = 12). The GA and GAmet animals were administered 0.1 mL of testosterone propionate (1.25 mg/animal) diluted in castor oil (vehicle) in a single dose; the CG rats received a subcutaneous injection of the vehicle in the dorsum. After 90 days, gavage treatment was initiated, distilled water was administered to the CG and GA rats, and metformin (150 mg/kg) was administered to the GAmet animals. The treatment was administered daily for six weeks. Following anesthesia, blood was drawn for biochemical measurements, and the ovaries were removed for histological and immunohistochemical analyses of Ki67, VEGFA and CYP17 expression. The glucose and insulin levels were also measured. RESULTS: The comparison of the GA and GAmet animals revealed that metformin decreased the weight as well as the glucose and insulin levels, slowed the proliferation of the theca interna and interstitial cells, as evidenced by Ki-67 and VEGF-A expression, and diminished CYP17 expression in the analyzed ovarian structures. In addition, metformin reduced the number of degenerating follicles and interstitial cells and improved angiogenesis. CONCLUSION: Metformin improves the carbohydrate metabolism, reduces proliferation, and decreases CYP-17 expression in the follicular structures of androgenized rats.
Assuntos
Família 17 do Citocromo P450/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Metformina/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Células Tecais/efeitos dos fármacos , Células Tecais/metabolismo , Animais , Biomarcadores , Proliferação de Células/efeitos dos fármacos , Família 17 do Citocromo P450/metabolismo , Modelos Animais de Doenças , Feminino , Glucose/metabolismo , Imuno-Histoquímica , Insulina/metabolismo , Síndrome do Ovário Policístico/etiologia , Síndrome do Ovário Policístico/metabolismo , RatosRESUMO
The aim of this study was to determine the steroidogenic endocrine disrupting effect of three widely used serotonin-noradrenaline reuptake inhibitors duloxetine, venlafaxine and tramadol, using two in vitro models, the H295R assay and a recombinant CYP17 enzyme assay. Steroid hormones were quantified using LC-MS/MS. Duloxetine showed endocrine disrupting effects at 5-20µM with CYP17 being the main target. Venlafaxine also affected the steroidogenesis, mainly by affecting the CYP17 lyase reaction, although at much higher concentrations i.e. 100µM. Tramadol only exerted minor effects on the steroidogenesis with the lowest observed effect at 314µM. Based on the H295R results, the inhibition of CYP17 by duloxetine and venlafaxine was investigated in a recombinant CYP17 assay with the use of the 4 major CYP17 substrates pregnenolone, progesterone, 17α-hydroxypregnenolone and 17α-hydroxyprogesterone. Both duloxetine and venlafaxine inhibited CYP17 enzyme activity, but duloxetine was most potent. IC50-values were in the range 5.3-21µM for duloxetine and 1318-2750µM for venlafaxine. Overall, results from the recombinant CYP17 assay confirmed the results from the H295R cell assay. Using testosterone as end point, the margin of safety (defined as NOAEL/Cmax) for duloxetine was 1.6 indicating that duloxetine may have endocrine disrupting effects. In contrast, venlafaxine and tramadol showed higher margins of safety (venlafaxine: 24; tramadol: 157) indicating a lower potential to disrupt the human steroidogenesis.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Família 17 do Citocromo P450/antagonistas & inibidores , Cloridrato de Duloxetina/efeitos adversos , Disruptores Endócrinos/efeitos adversos , Inibidores da Recaptação de Serotonina e Norepinefrina/efeitos adversos , Tramadol/efeitos adversos , Cloridrato de Venlafaxina/efeitos adversos , Córtex Suprarrenal/metabolismo , Corticosteroides/biossíntese , Corticosteroides/química , Corticosteroides/metabolismo , Analgésicos Opioides/efeitos adversos , Antidepressivos/efeitos adversos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450/efeitos adversos , Família 17 do Citocromo P450/genética , Família 17 do Citocromo P450/metabolismo , Humanos , Limite de Detecção , Estrutura Molecular , Nível de Efeito Adverso não Observado , Concentração Osmolar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos TestesRESUMO
Although age-related ovarian failure in female mammals cannot be reversed, recent strategies have focused on improving reproductive capacity with age, and rapamycin is one such intervention that has shown a potential for preserving the ovarian follicle pool and preventing premature ovarian failure. However, the application is limited because of its detrimental effects on follicular development and ovulation during long-term treatment. Herein, we shortened the rapamycin administration to 2 weeks and applied the protocol to both young (8 weeks) and middle-aged (8 months) mouse models. Results showed disturbances in ovarian function during and shortly after treatment; however, all the treated animals returned to normal fertility 2 months later. Following natural mating, we observed prolongation of ovarian lifespan in both mouse models, with the most prominent effect occurring in mice older than 12 months. The effects of transient rapamycin treatment on ovarian lifespan were reflected in the preservation of primordial follicles, increases in oocyte quality, and improvement in the ovarian microenvironment. These data indicate that short-term rapamycin treatment exhibits persistent effects on prolonging ovarian lifespan no matter the age at initiation of treatment. In order not to disturb fertility in young adults, investigators should in the future consider applying the protocol later in life so as to delay menopause in women, and at the same time increase ovarian lifespan.
Assuntos
Envelhecimento/efeitos dos fármacos , Fertilidade/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Reprodução/genética , Sirolimo/farmacologia , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Biomarcadores/metabolismo , Microambiente Celular/efeitos dos fármacos , Família 17 do Citocromo P450/genética , Família 17 do Citocromo P450/metabolismo , Família 19 do Citocromo P450/genética , Família 19 do Citocromo P450/metabolismo , Ciclo Estral/efeitos dos fármacos , Ciclo Estral/genética , Feminino , Fertilidade/genética , Expressão Gênica , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Oócitos/citologia , Oócitos/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fatores de TempoRESUMO
Using the domestic cat as a non-rodent, larger animal model, the objective was to determine the impact of a brief incubation in a hypertonic microenvironment on (1) ovarian follicle and oocyte growth in vitro, (2) developmental capacity of the resident oocyte, and (3) expression of aquaporin (AQP) genes in parallel with genes involved in regulation of folliculogenesis. In Study 1: Secondary or early antral follicles encapsulated in 0.5% alginate were allocated to one of three treatment groups: 1) culture in standard medium at 290 mOsm for 15 d (Control); 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for 15 d (Hypertonic-1h); or 3) incubation in 350 mOsm medium for 24 h followed by incubation in standard medium for additional 14 d (Hypertonic-24h). After measuring follicle and oocyte diameters on Day 15, in vitro-grown oocytes were incubated for 24 h before assessing nuclear status. In Study 2: secondary or early antral follicles were subjected to one of the three treatments: 1) culture in standard medium at 290 mOsm for 48 h; 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for additional 47 h; or 3) incubation in 350 mOsm medium for 24 h followed by culture in standard medium for additional 24 h. At the end of the culture period, all follicles were assessed for mRNA level of Cyp17a1, Cyp19a1, Star, Aqp1, 3, 5, 7 and 8 as well as Fshr using qPCR. Freshly collected follicles also were subjected to gene expression analysis and served as the 'Non-cultured control'. Hypertonic-24h follicles grew larger (P < 0.05) than the control, whereas those in Hypertonic-1h group exhibited intermediate growth, especially when the culture started at the early antral stage. Oocytes in the Hypertonic-24h group were larger and resumed meiosis at a higher rate than in the other treatments. In vitro culture affected (P < 0.05) mRNA expression of Cyp19a1, Star, Aqp1, and Aqp7 in both the secondary and early antral stage while Fshr was only affected in the former compared to the non-cultured control. Pre-incubating follicles in 350 mOsm medium for 24 h enhanced (P < 0.05) Star and Aqp7 while decreasing (P < 0.05) Aqp1 expression compared to the control in secondary follicles, but not in the early antral stage. In summary, short-term hypertonic exposure promoted cat follicle development in vitro (including the meiotic competence of the enclosed oocyte) possibly through a mechanism that does not involve water transport genes.
Assuntos
Aquaporinas/metabolismo , Aromatase/metabolismo , Família 17 do Citocromo P450/metabolismo , Soluções Hipertônicas/farmacologia , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Receptores do FSH/metabolismo , Animais , Aquaporinas/genética , Aromatase/genética , Gatos , Técnicas de Cultura de Células/veterinária , Família 17 do Citocromo P450/genética , Feminino , Expressão Gênica , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , RNA Mensageiro/metabolismo , Receptores do FSH/genéticaRESUMO
Myeloid cell leukemia-1 (MCL1), an anti-apoptotic member of the BCL2 family, is expressed abundantly in the testis. Previous characterization revealed that MCL1 is expressed exclusively in the Leydig cells in the mouse testis, yet what it does in these cells remains unknown. We therefore analyzed testosterone biosynthesis in isolated primary Leydig cells and the MA-10 cell line, in which MCL1 was knocked down using an siRNA strategy. The mRNA abundance of the steroidogenic genes Star, Cyp11a1, Cyp17a1, Hsd3b1, Srd5a, and the luteinizing hormone/choriogonadotropin receptor Lhcgr were significantly reduced following MCL1 knockdown. Of the two enzymes required for testosterone biosynthesis, STAR and P450 SCC (encoded by Cyp11a1) enzyme abundance was also reduced following Mcl1 siRNA treatment, possibly leading to the reduced production of sex steroid precursors, and testosterone in these knockdown cells. Despite its classification as an anti-apoptosis protein, Mcl1 siRNA treatment did not affect cell survival. Collectively, our findings indicate that MCL1 plays a pivotal role in Leydig-cell steroidogenesis, and might provide novel insights into metabolic regulation in this cell. Mol. Reprod. Dev. 83: 226-235, 2016. © 2016 Wiley Periodicals, Inc.