RESUMEN
Kisspeptin and its receptor KISS1R have been proven as pivotal regulators on controlling the hypothalamus-pituitary-gonad axis. Inactivating mutations in one of them cause idiopathic hypogonadotropic hypogonadism in human as well as rodent models. Notably, gonadotropin insensitivity, failure in hCG response, was presented in the male patients with loss-function-mutations in KISS1R gene; this reveals the essential role of KISS1R signaling in regulating testosterone production beyond the hypothalamic functions of kisspeptin. In this study, we hypothesized that the autocrine action of kisspeptin on Leydig cells may modulate steroidogenesis. Based on the mouse cell model, we first demonstrated that the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) signaling pathway mediated gonadotropin-induced kisspeptin expression. By using siRNA interfering technique, knockdown of Kiss1r in MA-10 cells, a mouse Leydig tumor cell line, significantly reduced progesterone productions in both basal and hCG-treated conditions. Integrating the results from both quantitative real-time PCR and steroidogenic enzyme-activity assay, we found that this steroidogenic defect was associated with decreased luteinizing hormone/choriogonadotropin receptor (Lhcgr) and StAR protein (Star) expressions. Furthermore, exogenous expression of human LHCGR completely rescued hCG-stimulated progesterone production in the KISS1R-deficient cells. In conclusion, we proposed that the reproductive functions of KISS1R signaling in Leydig cell include modulating Lhcgr and steroidogenic gene expressions, which may shed the light on the pathophysiology of gonadotropin insensitivity.
Asunto(s)
Gonadotropina Coriónica/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Hormona Luteinizante/farmacología , Progesterona/biosíntesis , Receptores de Kisspeptina-1/metabolismo , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Receptores de Kisspeptina-1/genética , Sustancias para el Control de la Reproducción/farmacología , Transducción de SeñalRESUMEN
Androgens, especially testosterone produced in Leydig cells, play an essential role in development of the male reproductive phenotype and fertility. However, testicular oxidative stress may cause a decline in testosterone production. Many antioxidants have been used as reactive oxygen species (ROS) scavengers to eliminate oxidative stress to protect steroidogenesis. Astaxanthin (AST), a natural extract from algae and plants ubiquitous in the marine environment, has been shown to have antioxidant activity in many previous studies. In this study, we treated primary mouse Leydig cells or MA-10 cells with hydrogen peroxide (H2O2) to cause oxidative stress. Testosterone and progesterone production was suppressed and the expression of the mature (30 kDa) form of StAR protein was down-regulated in MA-10 cells by H2O2 and cAMP co-treatment. However, progesterone production and expression of mature StAR protein were restored in MA-10 cells by a one-hour pretreatment with AST. AST also reduced ROS levels in cells so that they were lower than the levels in untreated controls. These results provide additional evidence of the potential health benefits of AST as a potential food additive to ease oxidative stress.
Asunto(s)
Antioxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/aislamiento & purificación , Línea Celular , Peróxido de Hidrógeno/toxicidad , Células Intersticiales del Testículo , Masculino , Ratones , Progesterona/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Testosterona/metabolismo , Xantófilas/aislamiento & purificación , Xantófilas/farmacologíaRESUMEN
Cellular cholesterol is known to be under homeostatic control in nonsteroidogenic cells, and this intrigued us to understand how such control works in steroidogenic cells that additionally use cholesterol for steroid hormone synthesis. We employed primary culture of rat ovarian granulosa cells to study how steroidogenic cells adapt to acquire sufficient cholesterol to meet the demand of active steroidogenesis under the stimulation of gonadotropin follicle-stimulating hormone (FSH) and cytokine transforming growth factor (TGF)ß1. We found that TGFß1 potentiated FSH to upregulate scavenger receptor class B member I (SR-BI) and LDL receptor (LDLR), both functional in uptaking cholesterol as hHDL(3) and hLDL supplementation enhanced progesterone production, and the effect of each lipoprotein was completely or partially blocked by SR-BI selective inhibitor BLT-1. Uptaken cholesterol could also be stored in lipid droplets. Importantly, LDLR and SR-BI responded to sterol with different sensitivity. Giving cells lipoproteins or 25-hydroxycholesterol downregulated Ldlr but not Scarb1; Scarb1 was ultimately downregulated by excessive sterol accumulation under 25-hydroxycholesterol and aminoglutethimide (inhibitor of steroidogenesis) cotreatment. Furthermore, transcription factors sterol regulatory element-binding protein (SREBP)-2 and liver receptor homolog (LRH)-1 crucially mediated Ldlr and Scarb1 differential response to sterol challenge. This study reveals that ovarian granulosa cells retain the cholesterol homeostatic control machinery like nonsteroidogenic cells, although during active steroidogenesis, they utilize SR-BI to evade such feedback control.
Asunto(s)
Antígenos CD36/metabolismo , Colesterol/metabolismo , Células de la Granulosa/metabolismo , Homeostasis , Esteroides/biosíntesis , Animales , Femenino , Hormona Folículo Estimulante/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de LDL/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
In vitro and in vivo studies implicate that follicle-stimulating hormone (FSH) and transforming growth factor ß1 (TGFß1) play crucial physiological roles in regulating ovarian granulosa cell function essential to fertility control in females. FSH induces cAMP and calcium signaling, thereby activating transcription factor CREB to upregulate steroidogenic gene expression, and TGFß1 greatly enhances FSH-stimulated steroidogenesis. A CREB coactivator CRTC2/TORC2 was identified to function as a cAMP and calcium-sensitive coincidence sensor. This led us to explore the role of CRTC2 and its regulator calcineurin in FSH and TGFß1-stimulated steroidogenesis. Primary culture of granulosa cells from gonadotropin-primed immature rats was used. Immunoblotting analysis shows that FSH rapidly and transiently induced dephosphorylation/activation of CRTC2, and FSH + TGFß1 additionally induced late-phase CRTC2 dephosphorylation. Immunofluorescence analysis further confirms FSH ± TGFß1 promoted CRTC2 nuclear translocation. Using selective inhibitors, we demonstrate that FSH activated CRTC2 in a PKA- and calcineurin-dependent manner, and TGFß1 acting through its type I receptor (TGFßRI)-modulated FSH action in a calcineurin-mediated and PKA-independent fashion. Next, we investigated the involvement of calcineurin and CRTC2 in FSH and TGFß1-stimulated steroidogenesis. Calcineurin and TGFßRI inhibitor dramatically reduced the FSH ± TGFß1-increased progesterone synthesis and protein levels of StAR, P450scc, and 3ß-HSD enzyme. Furthermore, chromatin-immunoprecipitation and immunoprecipitation analyses demonstrate that FSH ± TGFß1 differentially increased CRTC2, CREB, and CBP binding to these steroidogenic genes, and CREB nuclear association with CRTC2 and CBP. In all, this study reveals for the first time that CRTC2 and calcineurin are critical signaling mediators in FSH and TGFß1-stimulated steroidogenesis in ovarian granulosa cells.
Asunto(s)
Calcineurina/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Hormona Folículo Estimulante/metabolismo , Células de la Granulosa/metabolismo , Progesterona/biosíntesis , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Transporte Activo de Núcleo Celular , Animales , Western Blotting , Proteína de Unión a CREB/metabolismo , Inhibidores de la Calcineurina , Células Cultivadas , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Inmunoprecipitación de Cromatina , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Células de la Granulosa/efectos de los fármacos , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Progesterona Reductasa/genética , Progesterona Reductasa/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Ovinos , Transducción de Señal , Factores de TiempoRESUMEN
Consumption of ponderosa pine needles causes late-term abortions in cattle and is a serious poisonous plant problem in foothill and mountain rangelands. Isocupressic acid (IA) is the component of pine needles responsible for the abortifacient effect, its abortifacient effect may be due to inhibition of steroidogenesis. To investigate the more detail molecular mechanism, we used MA-10 cell, which is wild used to investigate molecular mechanism of steroidogenesis, to characterize the molecular mechanisms underlying the actions of IA in more detail. In this report, we focus on the function of IA on important steroidogenic genes, including steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). We found that IA does not affect enzyme activities of these genes but inhibits transcription of P450scc and translation of StAR and P450scc through attenuating cAMP-PKA signaling. Thus, steroid productions of cells were suppressed.
RESUMEN
BACKGROUND: Kisspeptin (KISS1) and kisspeptin receptor (KISS1R) are essential gatekeepers of the reproductive system. The functions of KISS1 and KISS1R in corpus luteal cells remain ambiguous. The objective was to observe normal physiologic functions of corpus luteal cells in vivo and clarify the functions of KISS1 in vitro. METHODS: We conducted an in vivo observation of cellular patterns as well as the levels of steroidogenic enzymes and KISS1/KISS1R in corpus luteal cells obtained from female crossbred Taiwan native goats in the estrous cycle; the observation was performed using hematoxylin and eosin and immunohistochemistry staining. Subsequently, we used kisspeptin-10 (Kp-10) to stimulate temperature sensitive-caprine luteal cell line (ts-CLC-D) cells to investigate the progesterone (P4) levels, steroidogenic messenger RNA (mRNA)/protein levels, cell survival rate, intracellular Ca2+ concentration, and cell proliferation-related mRNA/protein levels in the mitogen-activated protein kinase pathway in vitro by applying immunofluorescence staining, Western blotting, 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, and real-time polymerase chain reaction. RESULTS: We observed the presence of proteins and mRNAs for STAR, CYP11A1, HSD3B, KISS1, and KISS1R in the corpus luteal cells from goats in vivo. In vitro, the addition of Kp-10 reduced the P4 levels (p < 0.01) and increased cell proliferation (p < 0.05) of the ts-CLC-D cells. Furthermore, we found that the levels of proteins and mRNA for STAR, CYP11A1, and HSD3B decreased significantly when Kp-10 was added (p < 0.05). However, adding Kp-10 did not affect the mRNA levels for PLCG2, DAG1, PRKCA, KRAS, RAF1, MAP2K1, MAP2K2, MAPK3, MAPK1, and MAPK14. CONCLUSION: We determined that KISS1 could affect the P4 levels, steroidogenesis, and cell proliferation in luteal cells. However, further research is required to clarify how KISS1 regulates proliferation and steroid production in luteal cells.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Kisspeptinas/farmacología , Células Lúteas/efectos de los fármacos , Animales , Supervivencia Celular , Femenino , Expresión Génica/genética , Cabras , Reacción en Cadena de la Polimerasa , ARN Mensajero , TaiwánRESUMEN
BACKGROUND: Kisspeptin and its receptor KISS1R have been found to be essential regulators of reproductive function. Previous data have revealed the presence of Kiss1 and Kiss1r mRNAs in the hypothalamus and the testis of humans and rodents. However, the precise location and possible physiological role of the kisspeptin/KISS1R system in the testis remain ambiguous. METHODS: We first produced an anti-KISS1R immunoglobulin Y antibody for KISS1R identification. To detect the exact sites of KISS1R and kisspeptin expression in the testis, we conducted immunohistochemistry assays on sections of testes. We used real-time polymerase chain reactions to identify Kiss1r in mice and to determine the expression levels of testicular genes. Finally, to verify the upstream regulation on the Kisspeptin/KISS1 receptor system, we treated primary mouse Leydig cells and MA-10 cells with luteinizing hormone (LH) and Br-cAMP, respectively, and examined Kiss1 and Kiss1r mRNA expression. RESULTS: Immunohistochemistry assays revealed that kisspeptin was expressed in Leydig cells and KISS1R was localized in the seminiferous tubules. With real-time polymerase chain reactions, we found Kiss1r mRNA was constitutively expressed in the mouse testis from birth until the postnatal fourth week. Furthermore, mRNA expression of Kiss1 was synchronized with that of Insl3 and Cyp19a. However, the expression of the LH receptor-encoding gene increased 1 week earlier than did Kiss1 expression. This indicated that the kisspeptin/KISS1R system in the testis may be controlled by LH and cAMP signaling pathways. Finally, we confirmed that Kiss1 mRNA expression was increased in both LH-treated primary Leydig cells and Br-cAMP-treated MA-10 cells (p < 0.05). On the other hand, cotreatment of both cell lines with Br-cAMP and a protein kinase A inhibitor RP-cAMP significantly suppressed 50% of Br-cAMP-induced Kiss1 expression (p < 0.05). CONCLUSION: We discovered that Kiss1 expression in mouse Leydig cells was induced by LH through the cAMP/PKA pathway. Based on the presence of kisspeptin receptors on spermatids, we inferred that kisspeptin- and development-related factors have synergistic effects on spermatogenesis. Nevertheless, more studies are required to elaborate the role of the kisspeptin/KISS1R system in testicular development.
Asunto(s)
Receptores de Kisspeptina-1/metabolismo , Testículo/crecimiento & desarrollo , Animales , Humanos , Masculino , RatonesRESUMEN
Obese men have lower circulating testosterone than men with an optimal body mass index. Elevated fatty acids (FAs) caused by obesity have been reported to suppress the steroidogenesis of Leydig cells. Recent studies have demonstrated that autophagy regulates steroidogenesis in endocrine cells; however, few studies have investigated the molecular mechanisms of FA-impaired steroidogenesis. To study FA regulation in the steroidogenesis of Leydig cells, MA-10 cells were treated with an FA mixture and co-treated with 8-Br-cAMP to stimulate the steroidogenesis capacity. We showed that FAs led to cellular lipid accumulation and decreased steroidogenesis of MA-10 cells, and FA-suppressed steroidogenesis was largely recovered by P5 treatment but not by 22R-OHC treatment, suggesting the primary defect was the deficiency of CYP11A1. To examine the involvement of autophagy in the steroidogenesis of Leydig cells, we treated MA-10 cells with autophagy regulators, including rapamycin, bafilomycin, and chloroquine. Inhibition of late-stage autophagy including FA-upregulated Rubicon suppressed the steroidogenesis of MA-10 cells. More interestingly, Rubicon played a novel regulatory role in the steroidogenesis of MA-10 cells, independent of inhibitors of late-stage autophagy. Collectively, this study provides novel targets to investigate the interaction between FAs and steroidogenesis in steroidogenic cells.
Asunto(s)
Autofagia/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Ácidos Grasos/metabolismo , Esteroides/metabolismo , Animales , Autofagia/efectos de los fármacos , Cloroquina/farmacología , Células Endocrinas/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Lípidos/genética , Macrólidos/farmacología , Masculino , Ratones , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Sirolimus/farmacología , Esteroides/biosíntesisRESUMEN
The usefulness of acupuncture (AP) as a complementary and/or alternative therapy in animals is well established but more research is needed on its clinical efficacy relative to conventional therapy, and on the underlying mechanisms of the effects of AP. Cardiac magnetic resonance imaging (CMRI), an important tool in monitoring cardiovascular diseases, provides a reliable method to monitor the effects of AP on the cardiovascular system. This controlled experiment monitored the effect electro-acupuncture (EA) at bilateral acupoint Neiguan (PC6) on recovery time after ketamine/xylazine cocktail anesthesia in healthy cats. The CMRI data established the basic feline cardiac function index (CFI), including cardiac output and major vessel velocity. To evaluate the effect of EA on the functions of the autonomic nervous and cardiovascular systems, heart rate, respiration rate, electrocardiogram and pulse rate were also measured. Ketamine/xylazine cocktail anesthesia caused a transient hypertension in the cats; EA inhibited this anesthetic-induced hypertension and shortened the post-anesthesia recovery time. Our data support existing knowledge on the cardiovascular benefits of EA at PC6, and also provide strong evidence for the combination of anesthesia and EA to shorten post-anesthesia recovery time and counter the negative effects of anesthetics on cardiac physiology.
RESUMEN
BACKGROUND: Kisspeptin and its receptor KISS1R have been found to be essential regulators of reproductive function. Previous data have revealed the presence of Kiss1 and Kiss1r mRNAs in the hypothalamus and the testis of humans and rodents. However, the precise location and possible physiological role of the kisspeptin/KISS1R system in the testis remain ambiguous. METHODS: We first produced an anti-KISS1R immunoglobulin Y antibody for KISS1R identification. To detect the exact sites of KISS1R and kisspeptin expression in the testis, we conducted immunohistochemistry assays on sections of testes. We used real-time polymerase chain reactions (PCR) to identify Kiss1r in mice and to determine the expression levels of testicular genes. Finally, to verify the upstream regulation on the Kisspeptin/KISS1 receptor system, we treated primary mouse Leydig cells and MA-10 cells with luteinizing hormone (LH) and Br-cAMP, respectively and examined Kiss1 and Kiss1r mRNA expression. RESULTS: Immunohistochemistry assays revealed that kisspeptin was expressed in Leydig cells and KISS1R was localized in the seminiferous tubules. With real-time PCR, we found Kiss1r mRNA was constitutively expressed in the mouse testis from birth until the postnatal fourth week. Furthermore, mRNA expression of Kiss1 was synchronized with that of Insl3 and Cyp19a. However, the expression of the LH receptor-encoding gene increased 1 week earlier than did Kiss1 expression. This indicated that the kisspeptin/KISS1R system in the testis may be controlled by LH and cAMP signaling pathways. Finally, we confirmed that Kiss1 mRNA expression was increased in both LH-treated primary Leydig cells and Br-cAMP-treated MA-10 cells (p < 0.05). On the other hand, cotreatment of both cell lines with Br-cAMP and a protein kinase A inhibitor RP-cAMP significantly suppressed 50% of Br-cAMP-induced Kiss1 expression (p < 0.05). CONCLUSION: We discovered that Kiss1 expression in mouse Leydig cells was induced by LH through the cAMP/PKA pathway. Based on the presence of kisspeptin receptors on spermatids, we inferred that kisspeptin and development-related factors have synergistic effects on spermatogenesis. Nevertheless, more studies are required to elaborate the role of the kisspeptin/KISS1R system in testicular development.
RESUMEN
Patients with lung cancer harboring activating epidermal growth factor (EGFR) mutations and pre-existing diabetes have been demonstrated to exhibit poor responses to first-line EGFR-tyrosine kinase inhibitor (TKI) therapy. Strategies for the management of acquired resistance to EGFR-TKIs in patients with advanced non-small cell lung cancer (NSCLC) are urgently required. Only a limited number of studies have been published to date on the effects of insulin on EGFR-TKI resistance in NSCLC. Hence, the aim of the present study was to investigate the roles of hyperinsulinemia and hyperglycemia in mediating gefitinib resistance in NSCLC cells with activating EGFR mutations. In the present study, the HCC4006 cell line, which harbors EGFR mutations, was co-treated with gefitinib and long-acting insulin glargine. Whether hyperinsulinemia is able to mediate EGFR-TKI resistance in the NSCLC cell line harboring activating EGFR mutations was also investigated, and the possible underlying mechanisms responsible for these actions were explored. The inhibition of cell proliferation, and the potential mechanism of gefitinib resistance, were examined using an MTS proliferation assay and western blot analysis, and through the transfection of siRNAs. Whether the inhibition of AKT is able to overcome EGFR-TKI resistance induced by long-acting insulin was also investigated. The results obtained suggested that hyperinsulinemia induced by glargine upregulated NSCLC cell proliferation and survival, and induced gefitinib resistance. By contrast, the morphology and proliferation of the cells in a medium containing a 2-fold concentration of glucose were not significantly affected. Gefitinib resistance induced by hyperinsulinemia may have been mediated via the phosphoinositide 3-kinase (PI3K)/AKT pathway rather than the mitogen-activated protein kinase extracellular signal regulated kinase (MAPK/ERK) pathway. AKT serine/threonine kinase 1 knockdown by siRNA rescued the gefitinib resistance that was induced by hyperinsulinemia. In conclusion, hyperinsulinemia, but not hyperglycemia, was identified to cause the development of gefitinib resistance in NSCLC cells with activating EGFR mutations. However, additional studies are required to investigate strategies, such as co targeting hyperinsulinemia and the PI3K/AKT pathway, for overcoming EGFR-TKI resistance in patients with NSCLC.
RESUMEN
Previous studies have demonstrated the important role of kisspeptin in impaired glucose-stimulated insulin secretion (GSIS). In addition, it was reported that the activation of autophagy in pancreatic ß-cells decreases insulin secretion by selectively degrading insulin granules. However, it is currently unknown whether kisspeptin suppresses GSIS in ß-cells by activating autophagy. To investigate the involvement of autophagy in kisspeptin-regulated insulin secretion, we overexpressed Kiss1 in NIT-1 cells to mimic the long-term exposure of pancreatic ß-cells to kisspeptin during type 2 diabetes (T2D). Interestingly, our data showed that although kisspeptin potently decreases the intracellular proinsulin and insulin ((pro)insulin) content and insulin secretion of NIT-1 cells, autophagy inhibition using bafilomycin A1 and Atg5 siRNAs only rescues basal insulin secretion, not kisspeptin-impaired GSIS. We also generated a novel in vivo model to investigate the long-term exposure of kisspeptin by osmotic pump. The in vivo data demonstrated that kisspeptin lowers GSIS and (pro)insulin levels and also activated pancreatic autophagy in mice. Collectively, our data demonstrated that kisspeptin suppresses both GSIS and non-glucose-stimulated insulin secretion of pancreatic ß-cells, but only non-glucose-stimulated insulin secretion depends on activated autophagic degradation of (pro)insulin. Our study provides novel insights for the development of impaired insulin secretion during T2D progression.
Asunto(s)
Autofagia/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Kisspeptinas/fisiología , Animales , Línea Celular , Diabetes Mellitus Tipo 2/fisiopatología , Genes Reporteros , Glucosa/farmacología , Kisspeptinas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Proinsulina/metabolismo , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The biosynthesis of steroids from steroidogenic cells are catalyzed by the two major enzymes, P450 side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). This article describes the construction of two novel polyclonal antibodies against conserved recombinant protein and the validation of these antibodies on fixed tissue sections of bovine corpus luteum. The polyclonal antibodies were used successfully in Western blots and specifically reacted with P450scc and 3beta-HSD protein in bovine luteal cell extracts. Thus, P450scc and 3beta-HSD are two specific polyclonal antibodies that are integral products in the investigation of the biological function and regulatory mechanism involved in steroidogenesis.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/inmunología , Anticuerpos/inmunología , Cuerpo Lúteo/inmunología , Sistema Enzimático del Citocromo P-450/inmunología , 3-Hidroxiesteroide Deshidrogenasas/genética , Animales , Anticuerpos/química , Western Blotting/métodos , Bovinos , Mezclas Complejas/química , Mezclas Complejas/inmunología , Cuerpo Lúteo/química , Cuerpo Lúteo/enzimología , Sistema Enzimático del Citocromo P-450/genética , Femenino , Hormonas Esteroides Gonadales/inmunología , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
We report production of a polyclonal antibody against the StAR (steroidogenic acute regulatory) protein of steroidogenic cells and immunohistochemistry (IHC) staining of bovine adrenal gland tissue available in paraffin block. The epitope-specific polyclonal antibody was produced in a rabbit immunized against a synthetic 26 amino acid peptide (82AMQRALGILKDQEGWKKESRANGDE107) derived from the coding sequences reported for the bovine StAR gene (Gene Bank Accession No. Q28918). Western blots were developed using the StAR-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The antiserum was found to be highly specific for StAR, which exhibited an estimated molecular weight of about 30 KDa for all species analyzed. Finally, the peptide antiserum was successfully employed to localize StAR protein by immunohistochemical staining of thin sections prepared from bovine adrenal gland tissue. This study is the first to report a polyclonal peptide antiserum that apparently recognizes native StAR protein, regardless of the species of origin. The successful production of the antibody has provided a useful tool for studying regulation of StAR protein.
Asunto(s)
Sueros Inmunes/inmunología , Fosfoproteínas/análisis , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Glándulas Suprarrenales/química , Hormona Adrenocorticotrópica/farmacología , Secuencia de Aminoácidos , Animales , Bovinos , Hidrocortisona/metabolismo , Inmunohistoquímica , Datos de Secuencia Molecular , Fosfoproteínas/inmunología , Conejos , RatasRESUMEN
A caprine luteal cell line (tsCLC-D) that synthesizes progesterone (P4) was established following by transformation with a temperature-sensitive A209 (tsA209) mutant of simian virus 40 (SV40). The transformed cells have temperature-sensitive for morphology, cell propagation and progesterone steroidogenesis. At the permissive temperature of 34 degrees C, these cells were spindle-shaped and grew with a similar rapidity as tumor cells. However, at the nonpermissive temperature of 40 degrees C, the cells have exhibited a round shape and ceased to proliferate because the gene for maintenance of transformation was not expressed. The tsCLC-D cell line responds to 8-Br-cyclic AMP, 22-hydroxycholesterol and pregnenolone treatment with an increase in progesterone biosynthesis. This cell line still express StAR protein, 3beta-HSD and P450scc enzyme of three kinds of steroidogenic protein and enzymes, this characteristic is similar to normal luteal cell. However, the addition of any doses oLH did not increase progesterone secretion. We speculate that tsCLC-D might lose the responsiveness to gonadotropins during the immortalization process, while retaining steroidogenic enzyme activity and progesterone production. To our knowledge, this is the first report of a stable cell line derived from corpus luteum of ruminant. The tsCLC-D retains steroidogenic capacity, which will make this cell line useful for the studies of regulation of steroidogenesis.
Asunto(s)
Transformación Celular Viral , Cabras , Células Lúteas/patología , Células Lúteas/virología , Virus 40 de los Simios/fisiología , Esteroides/biosíntesis , Temperatura , Animales , Línea Celular Transformada , Separación Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Femenino , Hormonas/biosíntesis , Células Lúteas/efectos de los fármacos , Células Lúteas/metabolismo , Virus 40 de los Simios/patogenicidadRESUMEN
Expression of nuptial color is usually energetically costly, and is therefore regarded as an 'honest signal' to reflect mate quality. In order to choose a mate with high quality, both sexes may benefit from the ability to precisely evaluate their mates through optimizing visual systems which is in turn partially regulated by opsin gene modification. However, how terrestrial vertebrates regulate their color vision sensitivity is poorly studied. The green-spotted grass lizard Takydromus viridipunctatus is a sexually dimorphic lizard in which males exhibit prominent green lateral colors in the breeding season. In order to clarify relationships among male coloration, female preference, and chromatic visual sensitivity, we conducted testosterone manipulation with mate choice experiments, and evaluated the change of opsin gene expression from different testosterone treatments and different seasons. The results indicated that males with testosterone supplementation showed a significant increase in nuptial color coverage, and were preferred by females in mate choice experiments. By using quantitative PCR (qPCR), we also found that higher levels of testosterone may lead to an increase in rhodopsin-like 2 (rh2) and a decrease in long-wavelength sensitive (lws) gene expression in males, a pattern which was also observed in wild males undergoing maturation as they approached the breeding season. In contrast, females showed the opposite pattern, with increased lws and decreased rh2 expression in the breeding season. We suggest this alteration may facilitate the ability of male lizards to more effectively evaluate color cues, and also may provide females with the ability to more effectively evaluate the brightness of potential mates. Our findings suggest that both sexes of this chromatically dimorphic lizard regulate their opsin expression seasonally, which might play an important role in the evolution of nuptial coloration.
Asunto(s)
Lagartos/genética , Opsinas/genética , Caracteres Sexuales , Testosterona/genética , Animales , Visión de Colores/genética , Femenino , Regulación de la Expresión Génica/genética , Lagartos/fisiología , Masculino , Preferencia en el Apareamiento Animal , Pigmentación/genética , Reproducción/genética , Estaciones del Año , Conducta Sexual/fisiologíaRESUMEN
Previous studies have demonstrated that saturated fatty acids (SFAs) are more lipotoxic than unsaturated fatty acids (UFAs) in inhibiting hepatic autophagy and promoting non-alcoholic steatohepatitis (NASH). However, there have been few studies have investigated the effects of carbon chain length on SFA-induced autophagy impairment and lipotoxicity. To investigate whether SFAs with shorter carbon chain lengths have differential effects on hepatic autophagy and NASH development, we partially replaced lard with coconut oil to elevate the ratio of medium-chain fatty acids (MCFAs) to long-chain fatty acids (LCFAs) in a mouse high-fat diet (HFD) and fed mice for 16 weeks. In addition, we treated HepG2 cells with different combinations of fatty acids to study the mechanisms of MCFAs-mediated hepatic protections. Our results showed that increasing dietary MCFA/LCFA ratio mitigated HFD-induced Type 2 diabetes and NASH in mice. Importantly, we demonstrated that increased MCFA ratio exerted its protective effects by restoring Rubicon-suppressed autophagy. Our study suggests that the relative amount of LCFAs and MCFAs in the diet, in addition to the amount of SFAs, can significantly contribute to autophagy impairment and hepatic lipotoxicity. Collectively, we propose that increasing dietary MCFAs could be an alternative therapeutic and prevention strategy for Type 2 diabetes and NASH.
Asunto(s)
Autofagia , Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 2/prevención & control , Dieta Alta en Grasa/efectos adversos , Grasas de la Dieta/farmacología , Ácidos Grasos/farmacología , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Animales , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
BACKGROUND: Kisspeptin, encoded by the Kiss1 gene, has many forms including kisspeptin54, kisspeptin14, kisspeptin13, and kisspeptin10, and all these peptides have the same affinity to their receptor KISS1R encoded by the Kiss1r gene. The KISS1-KISS1R system was discovered in neurons, and many reports stress on their function in the brain. However, recent studies have shown that Kiss1 and Kiss1r are expressed in the testes. The goal of this study was to demonstrate the roles of Kiss1 and Kiss1r in testicular function, especially their steroidogenic activity. METHODS: Kisspeptin10 and the kisspeptin10 antagonist peptide234 were used to determine their effect on testosterone production. Moreover, expression of steroidogenic genes in mouse testes and their gonadosomatic index (weight of the testes divided by the total body weight) and also serum testosterone level were studied between the ages of 2 weeks and 15 weeks. RESULTS: Kisspeptin10 and peptide234 did not affect testosterone production in primary Leydig cells from adult mice. Kiss1 and Esr1 expression also increased during puberty. The peak gonadosomatic index occurred at 4 weeks of age, and serum testosterone levels plateaued after the age of 4 weeks. CONCLUSION: Our results suggest that kisspeptin10 does not affect steroidogenesis in adult Leydig cells, but its pattern of expression follows the stages of testicular development. Future studies should determine if kisspeptin regulates testicular development during puberty.
Asunto(s)
Kisspeptinas/fisiología , Células Intersticiales del Testículo/fisiología , 3-Hidroxiesteroide Deshidrogenasas/genética , Factores de Edad , Animales , Células Cultivadas , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Receptor alfa de Estrógeno/genética , Masculino , Ratones , Ratones Endogámicos ICR , Receptores Acoplados a Proteínas G/fisiología , Receptores de Kisspeptina-1 , Testosterona/sangreRESUMEN
A plasma progesterone profile obtained from three consecutive blood samples with an interval of 7 days was evaluated for usefulness as the basis for the diagnosis and treatment of anestrous gilts and sows. Four reproductive statuses were categorized based on the plasma progesterone levels and pathological examination of the reproductive organs from 25 gilts and 12 sows with anestrus. Category 1: fluctuating (at least one sample <2.5 ng/ml and one >10 ng/ml) with normal ovary; Category 2: sustained low (<2.5 ng/ml) with inactive ovary; Category 3: persistent high (>5 ng/ml) with normal sized or cystic corpora lutea; and Category 4: animals not included in the categories mentioned, such as pigs with luteinized cysts and follicular cysts. Using the plasma progesterone profiles and this categorization, the reproductive status of 54 gilts and 38 sows with anestrus was predicted. Hormonal treatments were performed with moderate to high success. Results from this study indicate that plasma progesterone profiles can be useful for the determination of estrus status, for the diagnosis of the causes of anestrus, and for the prediction of the next estrus for an appropriate hormonal treatment in anestrous gilts and sows.
Asunto(s)
Anestro/sangre , Progesterona/sangre , Reproducción , Porcinos/sangre , Animales , Gonadotropina Coriónica/administración & dosificación , Cuerpo Lúteo/anatomía & histología , Dinoprost/administración & dosificación , Estro/sangre , Detección del Estro , Femenino , Gonadotropinas Equinas/administración & dosificación , Valores de ReferenciaRESUMEN
Betel quid (BQ) is a favorite chewing item among many communities in different parts of Asia where it is popular by different names. BQ is a unique combination of nut or fruit from the Areca catechu Linn. (AN) tree, leaf from the Piper betle Linn. (BL) vine, slaked lime, paste of bark from the Acacia catechu tree and other spices. AN has been used successfully in various traditional medicines by different civilizations over several ages. Initially condemned by the medical communities for its health hazards, identification and application of potent pharmacologically bioactive compounds from different constituents of BQ have rekindled growing interest in related investigations. Curious about the stimulating role of BQ, we investigated the potential steroidogenic activity of hot water extract from BQ and its constituents and arecoline on testosterone producing ability in an in vitro experiment. Enzyme dissociated interstitial cells from adult mouse testes (ICR strain) were cultured with/without different doses of the extracts and the level of testosterone produced was assayed by an enzyme immunoassay (EIA) technique. It was found that at lower doses of arecoline, AN and BL extracts had significantly stimulated testosterone production over the basal level (p < 0.05). BQ extract, on the other hand, did not show any significant effect on testosterone production. Combinations of arecoline at low doses with 10 ng/ml ovine leutinizing hormone (oLH) showed increases in testosterone produced, while cyclic adenosine monophosphate (cAMP) co-culture showed dose-related inhibition. Our current finding hints at the possible dose-dependent dualistic role of AN and BL extracts and arecoline for testosterone production employing possible non-cAMP-dependent pathway of steroidogenesis. However, the identity of the active compounds besides arecoline and the exact mechanism involved remains to be further investigated.