RESUMEN
Casein kinase 1α (CK1α), acting as one member of the ß-catenin degradation complex, negatively regulates the Wnt/ß-catenin signaling pathway. CK1α knockout usually causes both Wnt/ß-catenin and p53 activation. Our results demonstrated that conditional disruption of CK1α in spermatogonia impaired spermatogenesis and resulted in male mouse infertility. The progenitor cell population was dramatically decreased in CK1α conditional knockout (cKO) mice, while the proliferation of spermatogonial stem cells (SSCs) was not affected. Furthermore, our molecular analyses identified that CK1α loss was accompanied by nuclear stability of p53 protein in mouse spermatogonia, and dual-luciferase reporter and chromatin immunoprecipitation assays revealed that p53 directly targeted the Sox3 gene. In addition, the p53 inhibitor pifithrin α (PFTα) partially rescued the phenotype observed in cKO mice. Collectively, our data suggest that CK1α regulates spermatogenesis and male fertility through p53-Sox3 signaling, and they deepen our understanding of the regulatory mechanism underlying the male reproductive system.
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Caseína Quinasa Ialfa , Animales , Caseína Quinasa Ialfa/metabolismo , Masculino , Ratones , Factores de Transcripción SOXB1/metabolismo , Espermatogénesis/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
Macrophage polarization contributes to obesity-induced insulin resistance. Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone specialized for folding and quality control of secreted and membrane proteins. To determine the role of GRP94 in macrophage polarization and insulin resistance, macrophage-specific GRP94 conditional knockout (KO) mice were challenged with a high-fat diet (HFD). Glucose tolerance, insulin sensitivity, and macrophage composition were compared with control mice. KO mice showed better glucose tolerance and increased insulin sensitivity. Adipose tissues from HFD-KO mice contained lower numbers of M1 macrophages, with lower expression of M1 macrophage markers, than wild-type (WT) mice. In vitro, WT adipocytes cocultured with KO macrophages retained insulin sensitivity, whereas those cultured with WT macrophages did not. In addition, compared with WT bone marrow-derived macrophages (BMDMs), BMDMs from GRP94 KO mice exhibited lower expression of M1 macrophage marker genes following stimulation with LPS or IFN-γ, and exhibited partially increased expression of M2 macrophage marker genes following stimulation with interleukin-4. These findings identify GRP94 as a novel regulator of M1 macrophage polarization and insulin resistance and inflammation.
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Dieta Alta en Grasa , Resistencia a la Insulina/genética , Activación de Macrófagos/genética , Macrófagos/inmunología , Glicoproteínas de Membrana/genética , Obesidad/inmunología , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Técnicas de Cocultivo , Citocinas/genética , Citocinas/inmunología , Prueba de Tolerancia a la Glucosa , Inflamación/genética , Inflamación/inmunología , Resistencia a la Insulina/inmunología , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Noqueados , Obesidad/genética , Obesidad/metabolismo , ARN Mensajero/metabolismoRESUMEN
We previously demonstrated the efficient production of trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) in Lactococcus lactis by ectopically expressing a Propionibacterium acnes isomerase (pai) gene and also mentioned that a recombinant strain was unable to accumulate t10c12-CLA product, despite the normal transcription. Here, the molecular analysis indicated that this mutated strain harbors a pai gene with a single-nucleotide mutation converting GC50A to GTA, leading to a corresponding change of Alanine residue into Valine. The expression of the reverse mutation resulted in the recovery for enzyme activity. Site-directed mutagenesis indicated that the codon usage of Val17 was not responsible for the enzyme inactivation in the Ala17Val mutation. Western blot analysis revealed that the recombinant PAI protein was not detectable in the His tag-marked Ala17Val mutant. It is, therefore, reasonable to assume that Ala17 residue is critical for PAI functionality.Abbreviations: pai: propionibacterium acnes isomerase; CLA: conjugated linoleic acid; t10c12-CLA: trans 10, cis 12-CLA; LA: linoleic acid (18:2n-6); FAD: flavin adenine dinucleotide.
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Sustitución de Aminoácidos , Flavina-Adenina Dinucleótido/metabolismo , Isomerasas/antagonistas & inhibidores , Propionibacterium acnes/enzimología , Western Blotting , Codón , Cristalografía por Rayos X , Isomerasas/química , Isomerasas/genética , Isomerasas/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , Mutación con Pérdida de Función , Propionibacterium acnes/genética , Conformación ProteicaRESUMEN
MicroRNAs, including microRNA-7 (miR-7), are important modulators of numerous gene expressions and the related biological processes. Melatonin is a key hormone regulating daily and seasonal rhythms, in which a variety of positive and negative regulatory factors, such as norepinephrine (NE) and leptin, are involved. However, the interactions among these factors and the mechanisms remain to be elucidated. The aims of the present study were to identify the functions and the related mechanisms of miR-7 in regulating melatonin synthesis and secretion through in vitro and in vivo experiments in pineal gland of pigs, which is an important animal model for agricultural and biomedical studies. Our results firstly show that miR-7 is specifically expressed in porcine pinealocytes and negatively regulates melatonin synthesis. The further functional studies show that the dynamic expression levels of miR-7 are contrary to the melatonin levels throughout the day, and the forced inhibition of endogenous miR-7 in porcine pinealocytes sharply increases arylalkylamine N-acetyltransferase (AANAT) expression by 80.0% (P = 0.0031) and melatonin levels by 81.0% (P = 0.0421), whereas miR-7 over-expression down-regulates AANAT expression by 38.6% (P = 0.0004) and melatonin levels by 37.6% (P = 0.0212). In addition, the miR-7 expression is up-regulated by leptin through the JAK/STAT3 signaling pathway, and the in vivo intracerebroventricular injection of leptin increases miR-7 expression by 80.0% (P = 0.0044) in porcine pineal glands and reduces melatonin levels by 57.1% (P = 0.0060) compared with the controls. This functional inhibition of melatonin synthesis by miR-7 is accomplished by its binding to the 3'-UTR of Raf1. Further, our results demonstrate that the RAF1/MEK/ERK signaling pathway mediates NE-induced AANAT expression, whereas leptin attenuates NE's function through miR-7. Taken together, the results demonstrated that leptin activates the JAK/STAT3 signaling pathway to increase the expression of miR-7, which acts as a negative regulatory molecule inhibiting NE-activated RAF1/MEK/ERK signaling pathway by targeting Raf1, resulting in decreased AANAT expression and melatonin synthesis. These findings suggest that miR-7 is a novel negative regulator of melatonin synthesis and links leptin- and NE-mediated signaling pathways in porcine pineal glands, which will contribute to our understanding in the establishment of the biological rhythms resulting from melatonin.
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Regulación de la Expresión Génica/fisiología , Leptina/metabolismo , Melatonina/biosíntesis , MicroARNs/metabolismo , Norepinefrina/metabolismo , Glándula Pineal/metabolismo , Animales , Masculino , Transducción de Señal/fisiología , PorcinosRESUMEN
Melatonin is a key hormone that regulates circadian rhythms, metabolism, and reproduction. However, the mechanisms of melatonin synthesis and secretion have not been fully defined. The purpose of this study was to investigate the functions of the LIM homeobox transcription factor Isl1 in regulating melatonin synthesis and secretion in porcine pineal gland. We found that Isl1 is highly expressed in the melatonin-producing cells in the porcine pineal gland. Further functional studies demonstrate that Isl1 knockdown in cultured primary porcine pinealocytes results in the decline of melatonin and arylalkylamine N-acetyltransferase (AANAT) mRNA levels by 29.2% and 72.2%, respectively, whereas Isl1 overexpression raised by 1.3-fold and 2.7-fold. In addition, the enhancing effect of norepinephrine (NE) on melatonin synthesis was abolished by Isl1 knockdown. The in vivo intracerebroventricular NE injections upregulate Isl1 mRNA and protein levels by about threefold and 4.5-fold in the porcine pineal gland. We then examined the changes in Isl1 expression in the pineal gland and global melatonin levels throughout the day. The results show that Isl1 protein level at 24:00 is 2.5-fold higher than that at 12:00, which is parallel to melatonin levels. We further found that Isl1 increases the activity of AANAT promoter, and the effect of NE on Isl1 expression was blocked by an ERK inhibitor. Collectively, the results presented here demonstrate that Isl1 positively modulates melatonin synthesis by targeting AANAT, via the ERK signaling pathway of NE. These suggest that Isl1 plays important roles in maintaining the daily circadian rhythm.
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Proteínas con Homeodominio LIM/metabolismo , Melatonina/metabolismo , Glándula Pineal/metabolismo , Animales , N-Acetiltransferasa de Arilalquilamina/genética , N-Acetiltransferasa de Arilalquilamina/metabolismo , Western Blotting , Inmunohistoquímica , Proteínas con Homeodominio LIM/genética , Masculino , Norepinefrina/farmacología , Radioinmunoensayo , Reacción en Cadena en Tiempo Real de la Polimerasa , PorcinosRESUMEN
CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase.
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Ligasas de Carbono-Nitrógeno/metabolismo , Núcleo Celular/enzimología , Citoplasma/enzimología , Células 3T3 , Animales , Línea Celular , Citoesqueleto/enzimología , Glutamina/análogos & derivados , Glutamina/deficiencia , Glutamina/metabolismo , Células HEK293 , Células HeLa , Humanos , RatonesRESUMEN
As a positional and geometrical isomer of linoleic acid, trans 10, cis 12 conjugated linoleic acid (t10c12-CLA) reduces white fat by reducing food intake, modulating lipid metabolism, and stimulating energy expenditure. However, the t10c12-CLA products are mostly mixtures, making it difficult to obtain accurate results. Studies are needed to investigate the effects of pure t10c12-CLA on animals and humans. In this study, we used the biallelic transgenic (tg) mice, which could produce t10c12-CLA itself, to investigate the effects of pure t10c12-CLA on female reproductive ability. The results showed that the body and relative ovary weights had no significant difference between tg and wild-type (wt) littermates at ages 3 or 10 weeks. While the fecundity test found that tg mice had a significantly longer first litter time (32.0 ± 4.70 days vs. 21.3 ± 2.31 days, P<0.05), and a significantly lower number of litters (4.75 ± 2.75 vs. 6.67 ± 0.57, P<0.05) when compared with wt mice during continuous mating within seven months. Hormone profiles showed that serum estradiol levels did not change in tg mice; however, significantly (P<0.05) decreased progesterone and increased prostaglandin E2 levels were observed in tg mice compared with those of wt mice. Hematoxylin-eosin staining showed no pathological characteristics in tg ovaries, except for the increased atresia follicles (P<0.05). Moreover, the tg mice had a significantly more extended diestrus period than the wt mice (48.4 ± 6.38% vs. 39.6 ± 3.81%, P<0.05). In summary, t10c12-CLA could affect serum progesterone and prostaglandin E2 levels, lead to a disordered estrus cycle, and impact the reproductive performance of female mice. This study provided theoretical and biosafety recommendations for applying t10c12-CLA in female mammals.
RESUMEN
Dietary trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) is a potential candidate in anti-obesity trials. A transgenic mouse was previously successfully established to determine the anti-obesity properties of t10c12-CLA in male mice that could produce endogenous t10c12-CLA. To test whether there is a different impact of t10c12-CLA on lipid metabolism in both sexes, this study investigated the adiposity and metabolic profiles of female Pai mice that exhibited a dose-dependent expression of foreign Pai gene and a shift of t10c12-CLA content in tested tissues. Compared to their gender-match wild-type littermates, Pai mice had no fat reduction but exhibited enhanced lipolysis and thermogenesis by phosphorylated hormone-sensitive lipase and up-regulating uncoupling proteins in brown adipose tissue. Simultaneously, Pai mice showed hepatic steatosis and hypertriglyceridemia by decreasing gene expression involved in lipid and glucose metabolism. Further investigations revealed that t10c10-CLA induced excessive prostaglandin E2, adrenaline, corticosterone, glucagon and inflammatory factors in a dose-dependent manner, resulting in less heat release and oxygen consumption in Pai mice. Moreover, fibroblast growth factor 21 overproduction only in monoallelic Pai/wt mice indicates that it was sensitive to low doses of t10c12-CLA. These results suggest that chronic t10c12-CLA has system-wide effects on female health via synergistic actions of various hormones.
Asunto(s)
Corticosterona , Dinoprostona , Epinefrina , Factores de Crecimiento de Fibroblastos , Glucagón , Ácidos Linoleicos Conjugados , Ratones Transgénicos , Animales , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Ratones , Ácidos Linoleicos Conjugados/farmacología , Ácidos Linoleicos Conjugados/metabolismo , Corticosterona/metabolismo , Dinoprostona/metabolismo , Glucagón/metabolismo , Epinefrina/metabolismo , Termogénesis/efectos de los fármacos , Termogénesis/genética , Masculino , Metabolismo de los Lípidos/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/efectos de los fármacos , Hígado Graso/metabolismo , Hígado Graso/genética , Lipólisis/efectos de los fármacos , Hipertrigliceridemia/metabolismo , Hipertrigliceridemia/genética , Adiposidad/efectos de los fármacosRESUMEN
Little is known about how foreign DNA is randomly integrated into chromosomes in transgenic animals. In the current study, the insertion sites of 36 transgenic mice were mapped by thermal asymmetric interlaced PCR, and 38 junction sequences were obtained from 30 samples. Analysis of the 38 sequences revealed that 44.7 % of integration events occurred within host gene regions, including 13.2 % (5/38) in exonic regions and 31.6 % (12/38) in intronic regions. The results also revealed that all non-end side integrations of foreign DNA were mediated by short sequence homologies (microhomologies) and that the end side integrations occurred in the presence or absence of microhomologies. In addition, microhomology-mediated mechanisms were also confirmed in four transgenic Arabidopsis thaliana lines. The results indicate that foreign DNA is easily integrated into host gene regions. These results also suggest that the integration of both ends of foreign DNA follows the above-mentioned mechanism in many transgenic/transformed organisms.
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Arabidopsis/genética , Ratones Transgénicos/genética , Plantas Modificadas Genéticamente/genética , Transformación Genética/genética , Transgenes/genética , Animales , Secuencia de Bases , Southern Blotting , Cartilla de ADN/genética , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) from ruminant-derived foodstuffs can induce body fat loss after oral administration. In the current study, a transgenic mouse that produced t10c12-CLA had been generated by inserting the Propionibacterium acnes isomerase (Pai) expression cassette into the Rosa26 locus, and its male offspring were used to elucidate the enduring influence of t10c12-CLA on overall health. Compared to their wild-type (wt) C57BL/6J littermates, both biallelic Pai/Pai and monoallelic Pai/wt mice exhibited reduced plasma triglycerides levels, and Pai/wt mice exclusively showed increased serum fibroblast growth factor 21. Further analysis of Pai/Pai mice found a decrease in white fat and an increase in brown fat, with more heat release and less physical activity. Analysis of Pai/Pai brown adipose tissues revealed that hyperthermia was associated with the over-expression of carnitine palmitoyltransferase 1B, uncoupling proteins 1 and 2. These findings suggest that the systemic and long-term impact of t10c12-CLA on obesity might be mediated through the pathway of fibroblast growth factor 21 when low doses are administered or through enhanced thermogenesis of brown adipose tissues when high doses are employed.
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Adiposidad , Ácidos Linoleicos Conjugados , Masculino , Ratones , Animales , Ratones Transgénicos , Ácidos Linoleicos Conjugados/farmacología , Ratones Endogámicos C57BL , ObesidadRESUMEN
We investigated the etiology and molecular mechanisms of bladder outlet obstruction (BOO). Transgenic (Tg) male mice overexpressing aromatase (Cyp19a1) under the ubiquitin C promoter in the estrogen-susceptible C57Bl/6J genetic background (AROM+/6J) developed inguinal hernia by 2 months and severe BOO by 9 to 10 months, with 100% penetrance. These mice gradually developed uremia, renal failure, renal retention, and finally died. The BOO bladders were threefold larger than in age-matched wild-type (WT) males and were filled with urine on necropsy. Hypotrophic smooth muscle cells formed the thin detrusor urinae muscle, and collagen III accumulation contributed to the reduced compliance of the bladder. p-AKT and ERα expression were up-regulated and Pten expression was down-regulated in the BOO bladder urothelium. Expression of only ERα in the intradetrusor fibroblasts suggests a specific role of this estrogen receptor form in urothelial proliferation. Inactivation of Pten, which in turn activated the p-AKT pathway, was strictly related to the activation of the ERα pathway in the BOO bladders. Human relevance for these findings was provided by increased expression of p-AKT, PCNA, and ERα and decreased expression of PTEN in severe human BOO samples, compared with subnormal to mild samples. These findings clarify the involvement of estrogen excess and/or imbalance of the androgen/estrogen ratio in the molecular pathogenetic mechanisms of BOO and provide a novel lead into potential treatment strategies for BOO.
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Aromatasa/metabolismo , Obstrucción del Cuello de la Vejiga Urinaria/enzimología , Obstrucción del Cuello de la Vejiga Urinaria/patología , Anciano , Animales , Aromatasa/genética , Receptor alfa de Estrógeno/metabolismo , Estrógenos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Fenotipo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/enzimología , Vejiga Urinaria/patologíaRESUMEN
Background: The v-raf-leukemia viral oncogene 1 (RAF1) plays an essential physiological role in reproduction and development through the mediation of steroid hormone synthesis. Follicle-stimulating hormone (FSH) signaling pathway was not involved in the majority of RAF1 studies, whether RAF1 takes part in the signaling events of gonadotropic hormones such as FSH in ovarian tissue is unknown. Methods: The process is blocked by treating granulosa cells (GCs) with the RAF1 inhibitor, RAF709. Inhibition of RAF1 activity by RAF709 decreased extracellular regulated protein kinases (ERK) phosphorylation and suppressed the expression of the cytochrome P450 subfamily 19 member 1 (CYP19A1), which is a major rate-limiting enzyme that participates in the last step of E2 biosynthesis. Results: We found that RAF1, acting as a downstream molecule, mediates FSH signalling to stimulate estradiol (E2) synthesis and secretion in mouse ovarian GCs. Gene expression of RAF1 was induced by FSH and the secretion of E2 increased into the bloodstream of mice and into the supernatant of primary GCs. Our in vitro and in vivo studies clearly illustrate RAF1 plays an important medium adjusting role in the FSH signaling pathway, and RAF1 acting as a downstream molecule to trigger ERK phosphorylation to stimulate GC E2 synthesis and secretion. Conclusions: RAF1 plays a pivotal mediating role in the FSH signaling pathway by inducing the phosphorylation of ERK and promoting E2 synthesis.
RESUMEN
Prolactin (PRL) is a pituitary hormone that regulates multiple physiological processes. However, the mechanisms of PRL synthesis have not been fully elucidated. The aims of the present study were to study the functions and the related mechanisms of miR-375 regulating PRL synthesis. We initially found that miR-375 mainly expressed in the lactotrophs of mouse pituitary gland. To identify the function of miR-375 in the pituitary gland, the miR-375 knockout mice were generated by using Crispr/Cas9 technique. The results showed that miR-375 knockout resulted in the decline of pituitary PRL mRNA and protein levels by 75.7 and 60.4%, respectively, and the serum PRL level reduced about 46.1%, but had no significant effect on FSH, LH and TSH. Further, we identified that Estrogen receptor 1 (alpha) (Esr1) was a downstream molecule of miR-375. The real-time PCR and Western blot results showed that ESR1 mRNA and protein levels markedly decreased by 40.9 and 42.9% in the miR-375 knockout mouse pituitary, and these were subsequently confirmed by the in vitro study using transfections of miR-375 mimics and inhibitors in pituitary lactotroph GH4 cells. Further, Rasd1 was predicted by bioinformatic tools and proved to be the direct target of miR-375 in lactotrophs using the dual-luciferase reporter assay. Rasd1-siRNA transfection results revealed the negative effect of Rasd1 in regulating ESR1. Collectively, the results presented here demonstrate that miR-375 positively modulates PRL synthesis through Rasd1 and Esr1, which are crucial for understanding the regulating mechanisms of pituitary hormone synthesis.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Lactotrofos/metabolismo , MicroARNs/metabolismo , Prolactina/biosíntesis , Proteínas ras/metabolismo , Animales , Femenino , Ratones Endogámicos ICR , Ratones Noqueados , HipófisisRESUMEN
BACKGROUND: It is not clear how foreign DNA molecules insert into the host genome. Recently, we have produced transgenic mice to investigate the role of the fad2 gene in the conversion of oleic acid to linoleic acid. Here we describe an integration mechanism of fad2 transgene by homologous illegitimate random integration. RESULTS: We confirmed that one fad2 line had a sole integration site on the X chromosome according to the inheritance patterns. Mapping of insertion sequences with thermal asymmetric interlaced and conventional PCR revealed that the foreign DNA was inserted into the XC1 region of the X chromosome by a homologous illegitimate replacement of an entire 45,556-bp endogenous genomic region, including the ovarian granulosa cell tumourigenesis-4 allele. For 5' and 3' junction sequences, there were very short (3-7 bp) common sequences in the AT-rich domains, which may mediate the recognition of the homologous arms between the transgene and the host genome. In addition, analysis of gene transcription indicated that the transgene was expressed in all tested fad2 tissues and that its transcription level in homozygous female tissues was about twice as high as in the heterozygous female (p < 0.05). CONCLUSIONS: Taken together, the results indicated that the foreign fad2 behaved like an X-linked gene and that foreign DNA molecules were inserted into the eukaryotic genome through a homologous illegitimate random integration.
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ADN/genética , Genes Ligados a X , Ratones Transgénicos/genética , Recombinación Genética , Transgenes , Cromosoma X/genética , Animales , Secuencia de Bases , Ácido Graso Desaturasas/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodosRESUMEN
The effect of human SCD1 heterologous expression on cellular fatty acid synthesis was investigated in the current study. The SCD1 gene expression cassette and PGK-neomycin-selectable marker cassette were co-introduced into HEK 293 cells by electroporation, and subsequently, SCD1 expression was evaluated by fatty acid analysis. RT-PCR analysis indicated that the foreign SCD1 gene could be expressed in transformed cell lines. Total lipid analysis of the transformed cells fed with vaccenic acid (t11-18:1) as a substrate showed that SCD1 expression resulted in an increase in c9t11-CLA from 0.73-1.03% to 2.69-2.86% (p<0.05) and that the conversion efficiency was elevated from 5.11-6.88% to 16.49-20.06% (p<0.05). Surprisingly, the concentration of t10c12-CLA was also increased, from 0.10-0.41% to 1.35-1.69% in SCD1 cells (p<0.05). SCD1 expression also resulted in a significant (p<0.05) increase in palmitoleic acid (16:1 n-7) from 1.56-2.26% to 3.47-4.04% and cis-vaccenic acid (18:1 n-7) from 2.42-3.97% to 6.20-7.22%, and the corresponding conversion ratio of n-7 fatty acid was elevated from 12.01-16.70% to 22.62-24.13% (p<0.05). This study demonstrates that the foreign SCD1 gene was expressed with high efficiency and induced elevated c9t11-CLA, t10c12-CLA, and n-7 fatty acid levels in mammalian cells.
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Ácidos Grasos Insaturados/biosíntesis , Ácidos Linoleicos Conjugados/biosíntesis , Estearoil-CoA Desaturasa/biosíntesis , Línea Celular , Humanos , Estearoil-CoA Desaturasa/genéticaRESUMEN
The possibility of elevating the omega-3 fatty acid contents in mammalian cells using the sdd17 gene from Saprolegnia diclina was investigated in the current study. The nucleotide sequence of the sdd17 gene was optimized and the pSDD17-IRES-GFP plasmid was introduced into murine 3T3 fibroblast cells by electroporation, following which its heterologous expression was evaluated by fatty acid analysis. Evaluation of GFP co-expression and RT-PCR analysis indicated that sdd17 could be expressed at very high levels in mammalian cells. Total cellular lipid analysis of transformed cells fed with arachidonic acid (20:4 n-6) as a substrate showed that the sdd17 expression resulted in an 82-155% (p<0.05) increase in eicosapentaenoic acid (20:5 n-3) compared with the control. This expression also reduced the arachidonic acid/(eicosapentaenoic+docosapentaenoic+docosahexaenoic acid) ratio from approximately 4:1 in control cells to 1.5:1 in sdd17-transformed cells (p<0.05). This study demonstrated that the foreign sdd17 gene from EPA-rich fungus was expressed at a high efficiency and caused the omega-3 fatty acid contents in mammalian cells to be elevated. It also provided a basis for potential applications of this gene in animal transgenesis.
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Ácido Araquidónico/metabolismo , Ácido Eicosapentaenoico/biosíntesis , Ácido Graso Desaturasas/metabolismo , Saprolegnia/enzimología , Células 3T3 , Animales , Ácido Graso Desaturasas/genética , Ratones , Saprolegnia/genética , Transformación GenéticaRESUMEN
Previous studies demonstrated that depression is more prevalent in women with polycystic ovary syndrome (PCOS). In this study, we aimed to determine whether amitriptyline (AMT), an antidepressant drug, plays a role in preventing PCOS. The results showed that AMT modified ovarian morphology improved the ovarian functions and estrus cycle in estradiol valerate (EV)-induced polycystic ovary (PCO). AMT restored the levels of estradiol (E2), testosterone (T) and progesterone (P4) to normal, and elevated the level of luteinizing hormone (LH) in EV-induced PCO. No significant changes in follicle stimulating hormone (FSH) levels were observed in rats with EV or AMT treatment. The restoration of norepinephrine (NE) level was detected in rats with EV-induced PCO. AMT also altered the expression levels of steroidogenesis genes and beta2-adrenoceptor (beta2-AR) in EV-induced PCO. Our data revealed that AMT improves the ovarian morphology and modifies ovarian expression of beta2-AR and steroidogenesis genes in rats with EV-induced rat PCO. Our data provide support for the hypothesis that AMT is considered as a candidate drug for preventing and treating PCOS along with depression.
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Amitriptilina/farmacología , Antidepresivos/farmacología , Ovario/efectos de los fármacos , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Animales , Estradiol , Femenino , Ovario/metabolismo , Ovario/patología , Síndrome del Ovario Poliquístico/inducido químicamente , Síndrome del Ovario Poliquístico/patología , Ratas , Ratas Wistar , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Espermatogénesis/efectos de los fármacos , Espermatogénesis/genéticaRESUMEN
Smooth muscle myosin heavy chain (SM-MHC) is exclusively expresses in smooth muscle, which takes part in smooth muscle cell contraction. Here, we used an insertional mutation mouse whose heavy polypeptide 11 (Myh11) gene has been disrupted and no SM-MHC protein has been detected. Compared to the wild-type and SM-MHC+/- mice, the SM-MHC-/- neonates had large round bellies, thin-walled giant bladders, and large stomachs with huge gas bubbles. Most of it died within 10 h and the rest within 20 h after birth. Further analysis of the developing foetuses from 16.5 days postcoitum (dpc) stage to newborn showed no significant (P<0.05) difference in the ratio of Mendelian inheritance and average body weight among SM-MHC+/+ , SM-MHC+/- and SM-MHC-/- mice, whereas the abnormal exterior appearance was observed in each SM-MHC-/- bladders from 16.5 dpc. Histological analysis showed no difference in stomach tissues but evidently thin-walled smooth muscle layer and a giant cavity in bladders of SM-MHC-/- foetuses at various stages from 15.5 dpc to newborn. The results indicated that the defect of SM-MHC lead to the bladder developing lesions initially at 15.5 dpc stage in mouse and also implied that the SM-MHC loss might result in the gas bubbles in stomach. The study should facilitate further detailed analyses of the potential role of SM-MHC in bladder and stomach development.
Asunto(s)
Desarrollo Fetal/genética , Mucosa Gástrica/metabolismo , Músculo Liso/metabolismo , Cadenas Pesadas de Miosina/genética , Vejiga Urinaria/metabolismo , Animales , Animales Recién Nacidos , Secuencia de Bases , Femenino , Masculino , Ratones , Ratones Noqueados , Músculo Liso/embriología , Cadenas Pesadas de Miosina/deficiencia , Estómago/embriología , Factores de Tiempo , Vejiga Urinaria/embriologíaRESUMEN
Zearalenone (ZEA) acts as an environmental endocrine disruptor (EED) to cause health detriments. miRNAs were reported to influence the synthesis and secretion of pituitary hormones. However, the interactions between ZEA and miRNAs and related mechanisms remain unclear. The aims of this study were to determine whether and how miR-7 affects animal reproduction by its interactions with ZEA in the pig pituitary, which is sensitive to ZEA and has been used as an important animal model in medical research. Expressions of miRNA were detected by real-time PCR, in situ hybridization, and immunohistochemistry. The effects of ZEA, miR-7, and their interactions in the pituitary gland were identified by using an ovariectomized pig model, transfecting miR-7 mimics and inhibitor, radioimmunoassay, luciferase reporter assay, and Western blotting. The ZEA dosage was 7.5 mg/kg body weight in vivo and 1 µM in vitro. Our results demonstrate miR-7 acts to regulate gonadotropin synthesis and secretion. Furthermore, we found that ZEA leads to reproductive defects by enhancing miR-7 expression, which subsequently inhibits FSH synthesis and secretion. In vitro and in vivo experiments revealed that the effects of ZEA rely on G protein-coupled estrogen receptor 1, and miR-7 functions by mediating ZEA signaling pathway and targeting the Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog (FOS) gene. These findings show that miRNAs are key intrinsic factors regulating pituitary gonadotropins by mediating EED signaling in pituitary glands, and the actions of miRNAs and EEDs should be seriously considered in related studies about medical practice and animal production.
Asunto(s)
Estrógenos no Esteroides/farmacología , Hormona Folículo Estimulante/metabolismo , Genes fos/efectos de los fármacos , MicroARNs/farmacología , Hipófisis/efectos de los fármacos , Zearalenona/farmacología , Animales , Western Blotting , Disruptores Endocrinos/farmacología , Femenino , Genes fos/genética , Inmunohistoquímica , Hibridación in Situ , Técnicas In Vitro , Ovariectomía , Hipófisis/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Sus scrofa , PorcinosRESUMEN
The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the forced inhibition of endogenous MAP3K8 by using MAP3K8 small interfering RNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly block the P(4) synthesis and neutralize the enhancing effect of E(2) on P(4) production in the CL. In addition, our results here demonstrate that the stimulating effect of E(2) on P(4) synthesis relies on the estrogen no-classical protein-coupled receptor 30, and MAP3K8 is involved in mediating the protein-coupled receptor 30signaling of E(2) affecting P(4) synthesis via stimulating ERK phosphorylation. These novel findings are critical for our understanding the ovary physiology and pathological mechanism.