RESUMO
BACKGROUND: Gynecological malignancies, such as endometrial cancer (EC) and uterine cancer are prevalent. Increased Acyl-CoA synthetase long-chain family member 1 (ACSL1) activity may contribute to aberrant lipid metabolism, which is a potential factor that contributes to the pathogenesis of endometrial cancer. This study aimed to elucidate the potential molecular mechanisms by which ACSL1 is involved in lipid metabolism in endometrial cancer, providing valuable insights for targeted therapeutic strategies. METHODS: Xenograft mouse models were used to assess the effect of ACSL1 on the regulation of endometrial cancer progression. ACSL1 protein levels were assessed via immunohistochemistry and immunoblotting analysis. To assess the migratory potential of Ishikawa cells, wound-healing and Transwell invasion assays were performed. Changes in lipids in serum samples from mice with endometrial cancer xenotransplants were examined in an untargeted lipidomic study that combined multivariate statistical methods with liquid chromatographyâmass spectrometry (LC/MS). RESULTS: Patient sample and tissue microarray data suggested that higher ACSL1 expression is strongly associated with the malignant progression of EC. Overexpression of ACSL1 enhances fatty acid ß-oxidation and 5'-adenylate triphosphate (ATP) generation in EC cells, promoting cell proliferation and migration. Lipidomic analysis revealed that significant changes were induced by ACSL1, including changes to 28 subclasses of lipids and a total of 24,332 distinct lipids that were detected in both positive and negative ion modes. Moreover, pathway analysis revealed the predominant association of these lipid modifications with the AMPK/CPT1C/ATP pathway and fatty acid ß-oxidation. CONCLUSIONS: This study indicates that ACSL1 regulates the AMPK/CPT1C/ATP pathway, which induces fatty acid ß-oxidation, promotes proliferation and migration, and then leads to the malignant progression of EC.
Assuntos
Neoplasias do Endométrio , Ácidos Graxos , Humanos , Camundongos , Animais , Feminino , Ácidos Graxos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo dos Lipídeos , Neoplasias do Endométrio/genética , Trifosfato de Adenosina/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismoRESUMO
Different development stages of porcine embryos have different tolerance to low temperature. Therefore, we took the porcine embryos after parthenogenetic activation (PA) as the model, to explore the optimal development stage for vitrification during morula (D4), early blastocyst (D5), and expanded blastocyst (D6) after PA (D0). Embryos were observed with microscope and analyzed by different staining after cryo-recovery for 24 hours. The quality of embryos was damaged after vitrification, including embryonic nuclei, DNA, cytoskeleton, and organelles. The re-expansion rate at 24 hours of D5 embryos was significantly higher than those of D4 and D6 embryos (D5 vs. D4 vs. D6, 27.620 ± 0.041 vs. 7.809 ± 0.027 vs. 13.970 ± 0.032, p < 0.05). Therefore, D5 embryos were selected as research objects to explore the effect of vitrification on lipid in vitrified embryos. The results showed that the expression levels of perilipin PLIN3 messenger RNA (mRNA) and triacylglycerol synthesis-related genes AGPAT1 and DGAT mRNA are significantly reduced (p < 0.05). Vitrification affected lipid synthesis, which might have an irreversible impact on embryonic development. In conclusion, our data demonstrated that the optimal stage of vitrification was D5 for early blastocysts.
Assuntos
Criopreservação , Vitrificação , Animais , Blastocisto/fisiologia , Criopreservação/métodos , Criopreservação/veterinária , Desenvolvimento Embrionário/fisiologia , Feminino , Lipídeos , Gravidez , RNA Mensageiro , SuínosRESUMO
BACKGROUND: Multiple common variants identified by genome-wide association studies have shown limited evidence of the risk of breast cancer in Chinese individuals. In this study, we aimed to uncover the relationship between estrogen levels and the genetic polymorphism of estrogen metabolism-related enzymes in breast cancer (BC) and establish a risk prediction model composed of estrogen-metabolizing enzyme genes and GWAS-identified breast cancer-related genes based on a polygenic risk score. METHODS: Unrelated BC patients and healthy subjects were recruited for analysis of estrogen levels and single nucleotide polymorphisms (SNPs) in genes encoding estrogen metabolism-related enzymes. The polygenic risk score (PRS) was used to explore the combined effect of multiple genes, which was calculated using a Bayesian approach. An independent sample t-test was used to evaluate the differences between PRS scores of BC and healthy subjects. The discriminatory accuracy of the models was compared using the area under the receiver operating characteristic (ROC) curve. RESULTS: The estrogen homeostasis profile was disturbed in BC patients, with parent estrogens (E1, E2) and carcinogenic catechol estrogens (2/4-OHE1, 2-OHE2, 4-OHE2) significantly accumulating in the serum of BC patients. We then established a PRS model to evaluate the role of SNPs in multiple genes. PRS model 1 (M1) was established from SNPs in 6 GWAS-identified high risk genes. On the basis of M1, we added SNPs from 7 estrogen metabolism enzyme genes to establish PRS model 2 (M2). The independent sample t-test results showed that there was no difference between BC and healthy subjects in M1 (P = 0.17); however, there was a significant difference between BC and healthy subjects in M2 (P = 4.9*10- 5). The ROC curve results showed that the accuracy of M2 (AUC = 62.18%) in breast cancer risk identification was better than that of M1 (AUC = 54.56%). CONCLUSION: Estrogen and related metabolic enzyme gene polymorphisms are closely related to BC. The model constructed by adding estrogen metabolic enzyme gene SNPs has a good predictive ability for breast cancer risk, and the accuracy is greatly improved compared with that of the PRS model that only includes GWAS-identified gene SNPs.
Assuntos
Neoplasias da Mama/genética , Estrogênios/metabolismo , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Adulto , Teorema de Bayes , Neoplasias da Mama/etiologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Feminino , Estudo de Associação Genômica Ampla , Humanos , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: Type 2 diabetes mellitus (T2DM) is thought to be a risk factor for endometrial hyperplasia, but potential links between the two diseases are unknown. This study aims to evaluate the role of T2DM in the progression of endometrial hyperplasia. METHODS: Female Sprague-Dawley rats were randomly divided into normal (N) group, endometrial hyperplasia (NH) group, T2DM (T) group, and endometrial hyperplasia with T2DM (TH) group. Proteomics analysis was performed to determine the protein profile of endometrial tissues. Proliferation, migration, and invasion of cells with/without GLANT2-knockdown were assessed. Immunohistochemical staining and ELISA were used to examine the expression of GALNT2 in endometrial tissues and serum of clinical samples. RESULTS: The highest uterus index and endometrial thickness were observed in TH group, with the expression of proliferation marker PCNA increased significantly, indicating that T2DM facilitates the progress of endometrial hyperplasia. Proteomics analysis showed that there were significant differences in protein profiles among groups and differential proteins were mainly enriched in metabolic pathways. Further verification by molecular biology analysis indicated that GALNT2 is the key target for T2DM facilitating endometrial hyperplasia. The expression of GALNT2 was significantly decreased in high glucose environment. T2DM could synergize the proliferative function of GALNT2 aberration by activating EGFR/AKT/ERK pathway. The decreased expressions of GALNT2 in clinical samples were associated with worse subtypes of endometrial hyperplasia. CONCLUSION: T2DM promoted the progression of endometrial hyperplasia by regulating the GALNT2-mediated phosphorylation of EGFR and enhancing cell proliferation. GALNT2 has the potential to be a novel biomarker in the treatment of endometrial hyperplasia.
Assuntos
Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Hiperplasia Endometrial/etiologia , Mucinas/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Proliferação de Células/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Progressão da Doença , Hiperplasia Endometrial/fisiopatologia , Feminino , Técnicas de Silenciamento de Genes , Glicosilação , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Cryopreservation of oocytes/embryos is an important technique for genetic resources; however, the success of vitrification in pig oocytes remained at a relatively lower level due to the high content of lipid droplets (LDs). Considering the positive effect of L-carnitine on the function of LDs, the present study was designed to investigate the effect of the addition of L-carnitine on the vitrification of porcine cumulus cells of complexes (cumulus/oocyte complexes [COCs]). First, COCs were randomly divided into two groups: one group of COCs were commonly in vitro maturation (IVM) for 42-46 hours (nonvitrification [NV]), while another group of COCs were IVM with 10 mM L-carnitine (NVL [nonvitrification with L-carnitine addition in IVM]). In addition, random parts of COCs with L-carnitine addition were vitrified (VL [vitrification with L-carnitine addition in IVM]), while vitrification was performed on COCs without L-carnitine used as control group (V). Results showed that the maturation rate of pig oocytes reduced significantly when the vitrification was performed at 16 hours during IVM (VL vs. NVL, 40.09 ± 2.85 vs. 90.76 ± 1.16; V vs. NV, 34.41 ± 2.55 vs. 89.71 ± 1.33, p < 0.01). With the addition of L-carnitine, intracellular LDs were decreased significantly (p < 0.01). However, no difference was observed on the efficiency of vitrification in pig oocytes (VL vs. V, 40.09 ± 2.85 vs. 34.41 ± 2.55, p > 0.05). In addition, not only the reactive oxygen species (ROS) level in pig oocytes with the L-carnitine addition group reduced significantly (p < 0.01), but also the expression of SOD1 gene was improved (p < 0.05). In conclusion, results demonstrated that although no difference could be observed on pig COC vitrification, the LDs and ROS level in pig oocytes could be modified by the addition of L-carnitine, which might be helpful for further development.
Assuntos
Carnitina/farmacologia , Células do Cúmulo/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Vitrificação/efeitos dos fármacos , Animais , Criopreservação/métodos , Células do Cúmulo/fisiologia , Feminino , Técnicas de Maturação in Vitro de Oócitos , Oócitos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , SuínosRESUMO
BACKGROUND: To investigate the correlation between the level of circulating vitamin D and the development of colorectal cancer (CRC) and to clarify the effect and mechanism of vitamin D on the development of CRC. METHODS: Serum samples from 63 patients with CRC (CRC group) and 61 healthy volunteers (normal group) were collected. Azoxymethane + dextran sodium sulfate-induced CRC mouse model and dietary models with different doses of vitamin D were established to verify whether vitamin D supplementation could reverse the occurrence and development of CRC at the overall animal level. Intestinal barrier integrity and microbial defense response were evaluated by detection of intestinal flora and expression of related genes. RESULTS: In the clinical serum samples, compared with the normal group, the level of 25 (OH) D3 in the CRC group was relatively low (P < 0.01), which was consistent with the clinical situation in mice. Vitamin D deficiency aggravated the deterioration of enteritis and intestinal cancer in CRC mice, whereas the overall condition of CRC mice improved after vitamin D supplementation. Vitamin D has a significant regulatory effect on the homeostasis of the intestinal flora, particularly in the regulation of intestinal probiotics, Akkermansia muciniphila-mediated colon barrier integrity. CONCLUSIONS: Vitamin D deficiency is closely related to the high incidence of CRC, and vitamin D supplementation can inhibit the occurrence and development of CRC. Vitamin D plays a role in the reversal of CRC mainly through the regulation of intestinal flora, especially the regulation of A. muciniphila-mediated colon barrier integrity.
Assuntos
Neoplasias Colorretais/etiologia , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/prevenção & controle , Microbioma Gastrointestinal/efeitos dos fármacos , Deficiência de Vitamina D/complicações , Vitamina D/administração & dosagem , Vitamina D/farmacologia , Akkermansia , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos Endogâmicos C57BL , VerrucomicrobiaRESUMO
Ovarian follicular development is a complex process that requires codevelopment of the perifollicular vascular network, which is closely regulated by angiogenic factors, gonadotropins, sex steroids, and their metabolites. To detect the effects of vascular endothelial growth factor 120 (VEGF120), follicle-stimulating hormone (FSH), and 2-methoxyestradiol (2ME2) on follicular angiogenesis during development and atresia, we treated sexually immature and mature female mice with VEGF120, FSH, 2ME2, and FSH receptor (FSHR) antagonist singly or in combination via intraperitoneal injection. The number of follicles and their perifollicular angiogenesis and atresia rates at different developmental stages were examined in paraffin sections after hematoxylin and eosin staining. The results showed that the exogenous factors have specific and precise effects on developmental, angiogenesis, and atresia processes in follicles of different sizes in mature and immature mice. Perifollicular angiogenesis was regulated by VEGFA and closely related to follicular development and atresia. 2ME2 affected angiogenesis through VEGFA and might regulate atresia directly. FSH might control VEGFA function via both transcriptional and posttranscriptional mechanisms because FSHR was required for achieving VEGFA functions at all the follicular development stages. The present study presents insights into the mechanisms of FSH, 2ME2, and VEGFA in follicular development and disorders and provides a foundation for the development of new therapeutic strategies.
Assuntos
2-Metoxiestradiol/farmacologia , Hormônio Foliculoestimulante/farmacologia , Atresia Folicular , Neovascularização Fisiológica/efeitos dos fármacos , Folículo Ovariano , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Feminino , Atresia Folicular/efeitos dos fármacos , Atresia Folicular/metabolismo , Camundongos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismoRESUMO
Zearalenone (ZEA) and imprinted long noncoding RNAs (lncRNAs) are both closely related to reproduction and development. However, whether they have connections in regulating reproduction and development is not clear yet. The aim of this research is to investigate their relationship. lncRNA microarray was performed to analyze differentially expressed genes, and real-time quantitative polymerase chain reaction (PCR) was used to verify the accuracy of microarray analysis. Meanwhile, the technologies of rapid amplification of cDNA ends, RNA fluorescence in situ hybridization and bioinformatics were adopted to characterize the selected lncRNA. Analysis of lncRNA microarray showed lncRNAs and messenger RNAs related to reproduction and development were significantly differently expressed, and Dio3os was probably the target lncRNA. Then, the experiment of real-time quantitative PCR verified the accuracy of microarray data. Characterization of Dio3os showed Dio3os, an antisense lncRNA with 2312 bp and 15 open reading frames, was enriched in the cytoplasm. Our findings suggest ZEA probably exerts toxic effects on reproduction and development by mediating Dio3os.
Assuntos
Endométrio/metabolismo , RNA Antissenso/biossíntese , RNA Longo não Codificante/biossíntese , Reprodução/efeitos dos fármacos , Zearalenona/toxicidade , Animais , Endométrio/patologia , Feminino , Camundongos , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
MicroRNAs (miRNAs) are short, noncoding RNAs that posttranscriptionally regulate gene expression. In the past decade, studies on miRNAs in ovaries have revealed the key roles of miRNAs in ovarian development and function. In this review, we first introduce the development of follicular atresia research and then summarize genome-wide studies on the ovarian miRNA profiles of different mammalian species. Differentially expressed miRNA profiles during atresia and other biological processes are herein compared. In addition, current knowledge on confirmed functional miRNAs during the follicular atresia process, which is mostly indicated by granulosa cell (GC) apoptosis, is presented. The main miRNA families and clusters, including the let-7 family, miR-23-27-24 cluster, miR-183-96-182 cluster and miR-17-92 cluster, and related pathways that are involved in follicular atresia are thoroughly summarized. A deep understanding of the roles of miRNA networks will not only help elucidate the mechanisms of GC apoptosis, follicular development, atresia and their disorders but also offer new diagnostic and treatment strategies for infertility and other ovarian dysfunctions.
Assuntos
Apoptose/genética , Atresia Folicular/genética , Perfilação da Expressão Gênica , Células da Granulosa/metabolismo , MicroRNAs/genética , Folículo Ovariano/metabolismo , Animais , Feminino , Humanos , Infertilidade Feminina/diagnóstico , Infertilidade Feminina/genéticaRESUMO
Because zearalenone (ZEA) causes harmful influence to animals and widely exists in the world, the researches on ZEA have never stopped. However, the mechanisms of ZEA on proliferation, cycle, and apoptosis in endometrial stromal cells (ESCs) remain poorly defined. Therefore, the purpose of our study was to explore the effects of ZEA to ESCs and demonstrate them by transcriptomic analysis. The results showed that after ZEA treatment, ESCs appeared numerous adverse reactions, and the phenomena of cell viability decrease, DNA replication block and apoptosis were detected by flow cytometry, Annexin V-FITC/PI double-staining method, TUNEL assay, and so on. Then, RNA-seq approach was adopted to prove the validity of above experiments, as expected, the results from different expression genes, gene ontology terms, and KEGG pathway were all consistent with those. Overall, the results suggested that ZEA could cause a series of reactions by cytotoxicity to mouse ESCs, meanwhile there must be some substances and mechanisms protect cells against cytotoxicity damage.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Perfilação da Expressão Gênica , Zearalenona/toxicidade , Animais , Ciclo Celular/efeitos dos fármacos , Endométrio/citologia , Endométrio/metabolismo , Endométrio/fisiologia , Feminino , Citometria de Fluxo , Camundongos , Análise de Sequência de RNA , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/fisiologiaRESUMO
Deoxynivalenol (DON) is a type B trichothecene mycotoxin which has toxic effects on humans and animals. Although DON has been studied in various cell types for its cytotoxicity, there is litter information about the effects of DON on mouse endometrial stromal cells (ESCs). Thus, in this study, we investigated the toxic effects of DON on mouse ESCs and its possible mechanisms. DON inhibited the cell viability in a dose- and time-dependent manner. TUNEL assay results showed that DON caused apoptosis and TUNEL-positive cells increased with increasing DON concentrations in mouse ESCs. Western blot showed that DON significantly increased the expression levels of apoptosis-related protein including Caspase-9, Caspase-3, poly (ADP-ribose) polymerase (PARP) and the ratio of Bax/Bcl-2. After DON treatment, the expression levels of cell cycle-related protein including p38/p-p38, Cdc25C/p-Cdc25C, Cdc2/p-Cdc2 and cyclinB1 were significantly decreased and immunoprecipitation analysis showed that cyclinB1-Cdc2 complex was significantly decreased. However, the combination of SB203580 (p38 specific inhibitor) and DON treatment significantly reversed the depression of Cdc25C/p-Cdc25C, Cdc2/p-Cdc2, cyclinB1 and cyclinB1-Cdc2 complex. Collectively, these data suggest that DON causes apoptosis via mitochondria apoptosis pathway and induces G2 arrest via p38 MAPK signaling pathway in mouse ESCs.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases , Células Estromais/efeitos dos fármacos , Tricotecenos/toxicidade , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Sobrevivência Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Fase G2 , Camundongos , Mitocôndrias/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Stathmin 1 (STMN1) is a neuronal growth-associated protein that is involved in microtubule dynamics and plays an important role in synaptic outgrowth and plasticity. Given that STMN1 affects fear behavior, we hypothesized that genetic variations in the STMN1 transcriptional regulatory region affect gene transcription activity and control fear behavior. In this study, two single nucleotide polymorphisms (SNPs), g. -327 A>G and g. -125 C>T, were identified in 317 English Springer Spaniels. A bioinformatics analysis revealed that both were loci located in the canine STMN1 putative promoter region and affected transcription factor binding. A statistical analysis revealed that the TT genotype at g.-125 C>T produced a significantly greater fear level than that of the CC genotype (P < 0.05). Furthermore, the H4H4 (GTGT) haplotype combination was significantly associated with canine fear behavior (P < 0.01). Using serially truncated constructs of the STMN1 promoters and the luciferase reporter, we found that a 395 bp (-312 nt to +83 nt) fragment constituted the core promoter region. The luciferase assay also revealed that the H4 (GT) haplotype promoter had higher activity than that of other haplotypes. Overall, our results suggest that the two SNPs in the canine STMN1 promoter region could affect canine fear behavior by altering STMN1 transcriptional activity.
Assuntos
Medo , Regiões Promotoras Genéticas/genética , Elementos Reguladores de Transcrição/genética , Estatmina/genética , Animais , Cães , Feminino , Variação Genética/genética , Genótipo , Haplótipos/genética , Masculino , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. Although the toxicity of ZEA have been widely studied in a number of cell types, the mechanistic role of ZEA on apoptosis of endometrial stromal cells (ESCs) remains poorly understood. The objective of this study was to determine the effects of ZEA on apoptosis of mouse ESCs and explore the signaling pathway underlying the cytotoxicity of ZEA. The results showed that ZEA treatment caused obvious apoptosis in ESCs as determined by the flow cytometry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR) revealed that ZEA treatment increased the ratio of Bax/Bcl-2. The enzymatic activity assays revealed that caspases-3 and caspase-9 were activated by ZEA treatment in a dose-dependent manner. In addition, flow cytometry show that the apoptotic percentages of cells pretreated with Z-VAD-FMK and Z-LEHD-FMK were markedly reduced compared to the ZEA-treated cells. Overall, the results suggested that ZEA induced obvious apoptosis in ESCs via a Bcl-2 family and caspases-dependent signaling pathway.
Assuntos
Caspases/metabolismo , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Estrogênios não Esteroides/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Zearalenona/toxicidade , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Inibidores de Caspase/farmacologia , Caspases/genética , Células Cultivadas , Endométrio/patologia , Feminino , Camundongos , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
To investigate the effect of inhibin gene immunization on antibody production and reproductive performance in broiler breeder females, Partridge Shank hens aged 380 days were immunized with inhibin recombinant plasmid pcISI. One hundred and twenty hens were randomly assigned to four groups and treated intramuscularly with 25, 75, or 125 µg/300-µL inhibin recombinant plasmid pcISI (T1â¼T3) or 300-µL saline as control (C), respectively. Booster immunization was given with the same dosage 20 days later. Blood and egg samples were collected to detect the antibody against inhibin by enzyme-linked immunosorbent assay and to evaluate egg performance. The ovaries were collected to classify the follicles and detect the FSH receptor (FSHR) messenger RNA (mRNA) expression by reverse transcription-PCR. The results showed that immunization against pcISI could elicit antibody against inhibin in both plasma and egg yolk compared with the control (P < 0.05), whereas booster immunization did not increase the antibody level in plasma. Vaccination promoted egg lay during the first 30 days after primary vaccination (P < 0.05) with no effect on egg quality and hatching rate. Immunization increased the amounts of dominant, small yellow and large white follicles in the ovary (P < 0.05). Reverse transcription-PCR results showed that immunization increased the FSHR mRNA in the large white follicles, whereas decreased the FSHR mRNA in the small yellow follicles (P < 0.05). In conclusion, inhibin vaccine pcISI can stimulate the production of antibody against inhibin as well as the follicle development and egg laying performance in Partridge Shank hens, which provides a good foundation for the application of inhibin DNA vaccine in avian production.
Assuntos
Formação de Anticorpos , Galinhas/crescimento & desenvolvimento , Inibinas/imunologia , Óvulo/crescimento & desenvolvimento , Animais , Galinhas/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Imunização/veterinária , Folículo Ovariano/crescimento & desenvolvimento , Óvulo/fisiologia , Reprodução/imunologiaRESUMO
Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin produced mainly by Fusarium. ZEA causes reproductive disorders and is both cytotoxic and genotoxic in animals; however, little is known regarding the molecular mechanism(s) leading to ZEA toxicity. Sertoli cells are somatic cells that support the development of spermatogenic cells. The objective of this study was to explore the effects of ZEA on the proliferation, apoptosis, and necrosis of rat Sertoli cells to uncover signaling pathways underlying ZEA cytotoxicity. ZEA reduced the proliferation of rat Sertoli cells in a dose-dependent manner, as indicated by a CCK8 assay, while flow cytometry revealed that ZEA caused both apoptosis and necrosis. Immunoblotting revealed that ZEA treatment increased the ratio of Bax/Bcl-2, as well as the expression of FasL and caspases-3, -8, and -9, in a dose-dependent manner. Collectively, these data suggest that ZEA induced apoptosis and necrosis in rat Sertoli cells via extrinsic and intrinsic apoptotic pathways. This study provides new insights into the molecular mechanisms by which ZEA exhibits cytotoxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1731-1739, 2016.
Assuntos
Apoptose/efeitos dos fármacos , Estrogênios não Esteroides/toxicidade , Células de Sertoli/efeitos dos fármacos , Zearalenona/toxicidade , Animais , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Células Cultivadas , Proteína Ligante Fas/metabolismo , Masculino , Necrose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Sprague-Dawley , Células de Sertoli/citologia , Transdução de SinaisRESUMO
The object of this study was to evaluate associations between the adenosine monophosphate deaminase 1 (AMPD1) gene polymorphisms and inosine monophosphate acid (IMP) contents of chicken to provide a molecular marker for breeding. Three single nucleotide polymorphisms (SNPs), g.4064G/A, g.5573A/G and g.6805G/A were detected in exons IV, VI, and VIII of the AMPD1 gene in Fast Partridge and Lingshan chickens, respectively. All were purine conversion and caused no alteration in amino acid sequence. Statistical analysis revealed that Lingshan chicken with the homozygous genotype AA at position 4064 and 6805 had a significantly greater IMP content than those with the GG genotype (P<0.05). Fast Partridge chicken with the genotype GG at position 6805 had a significantly greater IMP content compared with those with the AA genotype (P<0.05). In conclusion, the polymorphism at g.6805A/G was correlated with IMP content (P<0.05) in both Fast Partridge and Lingshan chickens. The results in our study suggest that SNP 6805A/G can be used as a possible candidate marker of IMP content of chicken.
Assuntos
AMP Desaminase/genética , Galinhas/genética , Inosina Monofosfato/metabolismo , AMP Desaminase/metabolismo , Animais , Sequência de Bases , Cruzamento , Galinhas/metabolismo , Estudos de Associação Genética , Marcadores Genéticos , Carne , Fases de Leitura Aberta/genética , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Distribuição TecidualRESUMO
Sirtuins have been implicated in diverse biological processes, including oxidative stress, energy metabolism, cell migration, and aging. Here, we employed Sirtuin inhibitors, nicotinamide (NAM) and Sirtinol, to investigate their effects on porcine oocyte maturation respectively. The rate of polar body extrusion in porcine oocytes decreased after treatment with NAM and Sirtinol, accompanied with the failure of cumulus cell expansion. We further found that NAM and Sirtinol significantly disrupted oocyte polarity, and inhibited the formation of actin cap and cortical granule-free domain (CGFD). Moreover, the abnormal spindles and misaligned chromosomes were readily detected during porcine oocyte maturation after treatment with NAM and Sirtinol. Together, these results suggest that Sirtuins are involved in cortical polarity and spindle organization in porcine oocytes.
Assuntos
Benzamidas/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Meiose/efeitos dos fármacos , Naftóis/farmacologia , Niacinamida/farmacologia , Oócitos/efeitos dos fármacos , Sirtuínas/antagonistas & inibidores , Actinas/metabolismo , Animais , Polaridade Celular , Cromossomos de Mamíferos/efeitos dos fármacos , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Feminino , Oócitos/citologia , Oócitos/enzimologia , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/enzimologia , Corpos Polares/efeitos dos fármacos , Cultura Primária de Células , Sirtuínas/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/ultraestrutura , SuínosRESUMO
Neutrophil cytosolic factor 1 (NCF1) plays a crucial role in host defense against microbial pathogens. In this study, we investigated the potential alternative splicing patterns, expression and splice-relevant single nucleotide polymorphisms (SNPs) of the bovine NCF1 gene to increase insights into its potential role against bovine mastitis caused by Escherichia coli infection. Using RT-PCR and clone sequencing methods, we found two novel splice variants designed as NCF1-TV1 (retained intron 6) and NCF1-TV2 (retained part of intron 8), respectively, encoding two putative truncated proteins (239AA and 283AA). Two splice variants were drastically up-regulated in the mastitis-infected cows' mammary tissues, blood and neutrophils compared with these of healthy cows using real-time RT-PCR. Genomic sequencing analysis identified four novel SNPs g.10112 G>A, g.10766 T>C, SNPs g.12085 G>A and g.12430 T>C at the ends of intron 6 and intron 8 of NCF1. ESE motif predicted that the SNP (g.10766 T>C) might affect the binding with splicing-related factors and subsequently caused the production of aberrant splice variant NCF1-TV1, which is one of the potential reasons that the functional SNP was associated with increased milk somatic cell score in cow. Our results would help in better understanding the NCF1 gene function in the process against pathogen infection, and the effect of splicing-related SNP on the production of aberrant splice variant and careful functional characterization in dairy cattle.
Assuntos
Infecções por Escherichia coli/veterinária , Mastite Bovina/imunologia , NADPH Oxidases/biossíntese , NADPH Oxidases/genética , Polimorfismo de Nucleotídeo Único , Splicing de RNA , Regulação para Cima , Animais , Sangue/imunologia , Bovinos , Clonagem Molecular , Infecções por Escherichia coli/imunologia , Perfilação da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/imunologia , Mastite Bovina/microbiologia , Neutrófilos/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Maternal obese environment has been reported to induce oxidative stress and meiotic defects in oocytes, however the underlying molecular mechanism remains unclear. Here, using mice fed a high fat diet (HFD) as an obesity model, we first detected enhanced reactive oxygen species (ROS) content and reduced Sirt3 expression in HFD oocytes. We further observed that specific depletion of Sirt3 in control oocytes elevates ROS levels while Sirt3 overexpression attenuates ROS production in HFD oocytes, with significant suppression of spindle disorganization and chromosome misalignment phenotypes that have been reported in the obesity model. Candidate screening revealed that the acetylation status of lysine 68 on superoxide dismutase (SOD2K68) is dependent on Sirt3 deacetylase activity in oocytes, and acetylation-mimetic mutant SOD2K68Q results in almost threefold increase in intracellular ROS. Moreover, we found that acetylation levels of SOD2K68 are increased by ~80% in HFD oocytes and importantly, that the non-acetylatable-mimetic mutant SOD2K68R is capable of partially rescuing their deficient phenotypes. Together, our data identify Sirt3 as an important player in modulating ROS homeostasis during oocyte development, and indicate that Sirt3-dependent deacetylation of SOD2 plays a protective role against oxidative stress and meiotic defects in oocytes under maternal obese conditions.
Assuntos
Meiose/fisiologia , Obesidade/complicações , Oócitos/patologia , Estresse Oxidativo , Sirtuína 3/fisiologia , Acetilação , Animais , Dieta Hiperlipídica , Feminino , Homeostase , Camundongos , Oócitos/metabolismo , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Superóxido Dismutase/metabolismoRESUMO
Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN-treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule-free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose-dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse.