RESUMO
The difference between early pregnancy and delivery rate is quite large in assisted reproduction techniques (ARTs), including animal cloning. However, it is not clear why the implanted fetuses aborted after the early pregnancy stage. In the present study, we tried to evaluate the developmental and morphological characteristics of porcine parthenogenetically activated (PA) embryos or fetuses by electric stimulation during the early pregnancy period. The implanted PA and artificially inseminated (AI) embryos and fetuses were collected at day 26 and 35 after embryo transfer, respectively. The developmental and morphological parameters in the PA embryos at day 26 were similar to the AI embryos. The size, weight, formation of major organs, and apoptotic cells were not statistically different in both embryos at day 26. However, the PA fetuses at day 35 showed ceased fetal development and degenerated with abnormal morphologies in their organs. The day 35 PA fetuses showed significantly higher apoptotic cells and lower methylation status in three differentially methylated regions of the H19 gene compared to their comparators. Therefore, the normal development of PA embryos and fetuses during early gestation could lead to these pregnancies being misinterpreted as normal and become one of the main reasons for the gap between early pregnancy and delivery rate.
RESUMO
Most studies of xenografts have been carried out with complex immunosuppressive regimens to prevent immune rejection; however, such treatments may be fatal owing to unknown causes. Here, we performed immune molecular profiling following anti-CD154 monoclonal antibody (mAb) treatment in heterotopic abdominal cardiac xenografts from α-1,3-galactosyltransferase-knockout pigs into cynomolgus monkeys to elucidate the mechanisms mediating the undesirable fatal side effects of immunosuppressive agents. Blood samples were collected from healthy monkeys as control and then at 2 days after xenograft transplantation and just before humane euthanasia; 94 genes related to the immune system were analyzed. The basic immunosuppressive regimen included cobra venom factor, anti-thymocyte globulin, and rituximab, with and without anti-CD154 mAbs. The maintenance therapy was followed with tacrolimus, MMF, and methylprednisolone. The number of upregulated genes was initially decreased on Day 2 (-/+ anti-CD154 mAb, 22/13) and then increased before euthanasia in recipients treated with anti-CD154 mAbs (-/+ anti-CD154 mAb, 30/37). The number of downregulated genes was not affected by anti-CD154 mAb treatment. Additionally, the number of upregulated genes increased over time for both groups. Interestingly, treatment with anti-CD154 mAbs upregulated coagulation inducers (CCL2/IL6) before euthanasia. In conclusion, immunosuppressive regimens used for cardiac xenografting affected upregulation of 6 inflammation genes (CXCL10, MPO, MYD88, NLRP3, TNFα, and TLR1) and downregulation of 8 genes (CCR4, CCR6, CD40, CXCR3, FOXP3, GATA3, STAT4, and TBX21).
Assuntos
Anticorpos Monoclonais/farmacologia , Ligante de CD40/imunologia , Xenoenxertos/imunologia , Animais , Animais Geneticamente Modificados , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/imunologia , Humanos , Terapia de Imunossupressão/métodos , Imunossupressores/farmacologia , Transplante das Ilhotas Pancreáticas/imunologia , Macaca fascicularis , Suínos , Transplante Heterólogo/métodosRESUMO
Artificial selection has been demonstrated to have a rapid and significant effect on the phenotype and genome of an organism. However, most previous studies on artificial selection have focused solely on genomic sequences modified by artificial selection or genomic sequences associated with a specific trait. In this study, we generated whole genome sequencing data of 126 cattle under artificial selection, and 24,973,862 single nucleotide variants to investigate the relationship among artificial selection, genomic sequences and trait. Using runs of homozygosity detected by the variants, we showed increase of inbreeding for decades, and at the same time demonstrated a little influence of recent inbreeding on body weight. Also, we could identify ~0.2 Mb runs of homozygosity segment which may be created by recent artificial selection. This approach may aid in development of genetic markers directly influenced by artificial selection, and provide insight into the process of artificial selection.
Assuntos
Peso Corporal/genética , Homozigoto , Endogamia , Seleção Genética , Animais , Bovinos , Genoma , Genômica , Genótipo , Masculino , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
There are various hypotheses on dog domestication based on archeological and genetic studies. Although many studies have been conducted on the origin of dogs, the existing literature about the ancestry, diversity, and population structure of Korean dogs is sparse. Therefore, this study is focused on the origin, diversity and population structure of Korean dogs. The study sample comprised four major categories, including non-dogs (coyotes and wolves), ancient, modern and Korean dogs. Selected samples were genotyped using an Illumina CanineHD array containing 173,662 single nucleotide polymorphisms. The genome-wide data were filtered using quality control parameters in PLINK 1.9. Only autosomal chromosomes were used for further analysis. The negative off-diagonal variance of the genetic relationship matrix analysis depicted, the variability of samples in each population. FIS (inbreeding rate within a population) values indicated, a low level of inbreeding within populations, and the patterns were in concordance with the results of Nei's genetic distance analysis. The lowest FST (inbreeding rate between populations) values among Korean and Chinese breeds, using a phylogenetic tree, multi-dimensional scaling, and a TreeMix likelihood tree showed Korean breeds are highly related to Chinese breeds. The Korean breeds possessed a unique and large diversity of admixtures compared with other breeds. The highest and lowest effective population sizes were observed in Korean Jindo Black (485) and Korean Donggyeong White (109), respectively. The historical effective population size of all Korean dogs showed declining trend from the past to present. It is important to take immediate action to protect the Korean dog population while conserving their diversity. Furthermore, this study suggests that Korean dogs have unique diversity and are one of the basal lineages of East Asian dogs, originating from China.
Assuntos
Cães/genética , Genoma , Especificidade da Espécie , Animais , Cães/classificação , Desequilíbrio de LigaçãoRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. We generated an AD Tg pig by somatic cell nuclear transfer (SCNT) using a multi-cistronic vector that harbored three AD-related genes with a total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and hPS1 (M146V and L286P). Four AD Tg cell lines were established from Jeju black pig ear fibroblasts (JB-PEFs); the resultant JB-PEFAD cells harbored transgene integration, expressed transgene mRNAs, and had normal karyotypes. Tg line #2-1, which expressed high levels of the transgenes, was used for SCNT; cleavage and blastocyst rates of embryos derived from this line were lower than those of Non-Tg. These embryos yielded three piglets (Jeju National University AD-Tg pigs, JNUPIGs) revealed by microsatellite testing to be genetically identical to JB-PEFAD. Transgenes were expressed in multiple tissues, and at especially high levels in brain, and Aß-40/42, total Tau, and GFAP levels were high in brains of the Tg animals. Five or more copies of transgenes were inserted into chromosome X. This is the first report of an AD Tg pig derived from a multi-cistronic vector.
Assuntos
Doença de Alzheimer/genética , Animais Geneticamente Modificados/genética , Técnicas de Transferência Nuclear , Transgenes/genética , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Animais , Blastocisto/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Vetores Genéticos , Humanos , Mutação , SuínosRESUMO
Early chick development is a systematic process governed by the concerted action of multiple mechanisms that regulate transcription and post-transcriptional processes. Post-transcriptional microRNA-mediated regulation, with regard to lineage specification and differentiation in early chick development, requires further investigation. Here, we characterize the transcriptional and post-transcriptional regulation mechanisms in undifferentiated chick blastodermal cells. Expression of the miR-302 cluster, POUV, SOX2, and STAT5B decreased in a time-dependent manner in early chick development. We found that POUV, SOX2, and STAT5B regulate the transcription of the miR-302 cluster, as its 5'-flanking region contains binding elements for each transcription factor. Additionally, POUV, SOX2, and STAT5B maintain pluripotency by regulating genes containing the miR-302 cluster target sequence. For example, microRNAs from the miR-302 cluster can bind to PBX3 and E2F7 transcripts, thus acting as a post-transcriptional regulator that maintains the undifferentiated state of blastodermal cells by balancing the expression of genes related to pluripotency and differentiation. Based on these results, we suggest that both transcriptional and post-transcriptional regulation of the miR302 cluster is critical for intrinsically controlling the undifferentiated state of chick embryonic blastodermal cells. These findings may help our understanding of the cellular and molecular mechanisms that underlie developmental decisions during early chick development.
Assuntos
Embrião de Galinha/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , MicroRNAs/fisiologia , Modelos Biológicos , Fatores de Transcrição/fisiologia , Animais , Embrião de Galinha/metabolismo , Primers do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Luciferases , MicroRNAs/metabolismo , Interferência de RNA/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX/fisiologia , Fator de Transcrição STAT5/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Causal mutations affecting quantitative trait variation can be good targets for marker-assisted selection for carcass traits in beef cattle. In this study, linkage and linkage disequilibrium analysis (LDLA) for four carcass traits was undertaken using 19 markers on bovine chromosome 14. The LDLA analysis detected quantitative trait loci (QTL) for carcass weight (CWT) and eye muscle area (EMA) at the same position at around 50 cM and surrounded by the markers FABP4SNP2774C>G and FABP4_µsat3237. The QTL for marbling (MAR) was identified at the midpoint of markers BMS4513 and RM137 in a 3.5-cM marker interval. The most likely position for a second QTL for CWT was found at the midpoint of tenth marker bracket (FABP4SNP2774C>G and FABP4_µsat3237). For this marker bracket, the total number of haplotypes was 34 with a most common frequency of 0.118. Effects of haplotypes on CWT varied from a -5-kg deviation for haplotype 6 to +8 kg for haplotype 23. To determine which genes contribute to the QTL effect, gene expression analysis was performed in muscle for a wide range of phenotypes. The results demonstrate that two genes, LOC781182 (p = 0.002) and TRPS1 (p = 0.006) were upregulated with increasing CWT and EMA, whereas only LOC614744 (p = 0.04) has a significant effect on intramuscular fat (IMF) content. Two genetic markers detected in FABP4 were the most likely QTL position in this QTL study, but FABP4 did not show a significant effect on both traits (CWT and EMA) in gene expression analysis. We conclude that three genes could be potential causal genes affecting carcass traits CWT, EMA, and IMF in Hanwoo.
Assuntos
Bovinos/genética , Expressão Gênica , Locos de Características Quantitativas , Animais , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ligação Genética , Marcadores Genéticos , Haplótipos , Repetições de Microssatélites , Músculos/metabolismo , Fenótipo , Transcrição GênicaRESUMO
In previous proteomic studies, heat shock protein ß 1 (HSPB1) was detected as a candidate protein related to meat quality in cattle. This study sought to determine if its gene expression was associated with intramuscular fat content in the longissimus thoracis muscle of Korean cattle (Hanwoo). Tissue from two groups of 10 steers each, low-marbling (mean intramuscular fat content, 7.4 ± 1.5%) and high-marbling (23.5 ± 2.8%), were used for immunoblotting, real-time PCR, and statistical analyses. HSPB1 expression in both mRNA and protein was shown to be negatively related to intramuscular fat content (P < 0.05). Pathway analysis found two genes, TNF receptor superfamily member 6 (FAS) and angiotensinogen (AGT), that were regulators of the HSPB1 gene. The expression of the two genes showed a negative correlation with intramuscular fat content (P < 0.05). These results suggest that HSPB1, FAS, and AGT may be good candidate genes associated with intramuscular fat content in the longissimus muscle of Korean cattle.
Assuntos
Tecido Adiposo , Bovinos , Genes Reguladores/fisiologia , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/fisiologia , Músculo Esquelético/anatomia & histologia , Animais , Gorduras na Dieta/análise , Proteínas de Choque Térmico HSP27/análise , Masculino , Carne/análise , Músculo Esquelético/química , RNA Mensageiro/análise , República da CoreiaRESUMO
Obesity represents a major global public health problem that increases the risk for cardiovascular or metabolic disease. The pigs represent an exceptional biomedical model related to energy metabolism and obesity in humans. To pinpoint causal genetic factors for a common form of obesity, we conducted local genomic de novo sequencing, 18.2 Mb, of a porcine QTL region affecting fatness traits, and carried out SNP association studies for backfat thickness and intramuscular fat content in pigs. In order to relate the association studies in pigs to human obesity, we performed a targeted genome wide association study for subcutaneous fat thickness in a cohort population of 8,842 Korean individuals. These combined association studies in human and pig revealed a significant SNP located in a gene family with sequence similarity 73, member A (FAM73A) associated with subscapular skin-fold thickness in humans (rs4121165, GC-corrected p-value â=â0.0000175) and with backfat thickness in pigs (ASGA0029495, p-value â=â0.000031). Our combined association studies also suggest that eight neuronal genes are responsible for subcutaneous fat thickness: NEGR1, SLC44A5, PDE4B, LPHN2, ELTD1, ST6GALNAC3, ST6GALNAC5, and TTLL7. These results provide strong support for a major involvement of the CNS in the genetic predisposition to a common form of obesity.
Assuntos
Genes , Estudo de Associação Genômica Ampla , Neurônios/metabolismo , Análise de Sequência de DNA , Gordura Subcutânea/anatomia & histologia , Sus scrofa/genética , Adiposidade/genética , Adulto , Idoso , Animais , Estudos de Coortes , Feminino , Genes/fisiologia , Genoma , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Tamanho do Órgão , Polimorfismo de Nucleotídeo Único/fisiologia , Locos de Características Quantitativas/genética , Análise de Sequência de DNA/métodos , Dobras Cutâneas , Sus scrofa/anatomia & histologia , Sus scrofa/metabolismoRESUMO
BACKGROUND: Marbling (intramuscular fat) is a valuable trait that impacts on meat quality and an important factor determining price of beef in the Korean beef market. Animals that are destined for this high marbling market are fed a high concentrate ration for approximately 30 months in the Korean finishing farms. However, this feeding strategy leads to inefficiencies and excessive fat production. This study aimed to identify candidate genes and pathways associated with intramuscular fat deposition on highly divergent marbling phenotypes in adult Hanwoo cattle. RESULTS: Bovine genome array analysis was conducted to detect differentially expressed genes (DEGs) in m. longissimus with divergent marbling phenotype (marbling score 2 to 7). Three data-processing methods (MAS5.0, GCRMA and RMA) were used to test for differential expression (DE). Statistical analysis identified 21 significant transcripts from at least two data-processing methods (P < 0.01). All 21 differentially expressed genes were validated by real-time PCR. Results showed a high concordance in the gene expression fold change between the microarrays and the real time PCR data. Gene Ontology (GO) and pathway analysis demonstrated that some genes (ADAMTS4, CYP51A and SQLE) over expressed in high marbled animals are involved in a protein catabolic process and a cholesterol biosynthesis process. In addition, pathway analysis also revealed that ADAMTS4 is activated by three regulators (IL-17A, TNFα and TGFß1). QRT-PCR was used to investigate gene expression of these regulators in muscle with divergent intramuscular fat contents. The results demonstrate that ADAMTS4 and TGFß1 are associated with increasing marbling fat. An ADAMTS4/TGFß1 pathway seems to be associated with the phenotypic differences between high and low marbled groups. CONCLUSIONS: Marbling differences are possibly a function of complex signaling pathway interactions between muscle and fat. These results suggest that ADAMTS4, which is involved in connective tissue degradation, could play a role in an important biological pathway for building up marbling in cattle. Moreover, ADAMTS4 and TGFß1could potentially be used as an early biological marker for marbling fat content in the early stages of growth.
Assuntos
Adiposidade/genética , Bovinos/genética , Genoma/genética , Carne , Redes e Vias Metabólicas/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Músculos/metabolismo , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Reprodutibilidade dos Testes , República da Coreia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The pig genome contains the gamma 1 family of porcine endogenous retroviruses (PERVs), which are a major obstacle to the development of successful xenotransplantation from pig to human. Long terminal repeats (LTRs) found in PERVs are known to be essential elements for the control of the transcriptional activity of single virus by different transcription factors (TFs). To identify transcribed PERV LTR elements, RT-PCR and DNA sequencing analyses were performed. Twenty-nine actively transcribed LTR elements were identified in the kidney tissues of the NIH-Miniature pig. These elements were divided into two major groups (I and II), and four minor groups (I-1, I-2, I-3, and II-1), by the presence of insertion and deletion (INDEL) sequences. Group I elements showed strong transcriptional activity compared to group II elements. Four different LTR elements (PL1, PL2, PL3, and PL4) as representative of the groups were analyzed by using a transient transfection assay. The regulation of their promoter activity was investigated by treatment with M.SssI (CpG DNA methyltransferase) and garcinol (histone acetyltransferase inhibitor). The transcriptional activity of PERV LTR elements was significantly reduced by treatment with M.SssI. These data indicate that transcribed PERV LTR elements harbor sufficient promoter activity to regulate the transcription of a single virus, and the transcriptional activity of PERV LTRs may be controlled by DNA methylation events.
Assuntos
Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Provírus/genética , Provírus/metabolismo , Porco Miniatura , Sequências Repetidas Terminais/genética , Animais , Linhagem Celular , Metilação de DNA , Retrovirus Endógenos/química , Histona Acetiltransferases/antagonistas & inibidores , Humanos , Rim/química , Rim/virologia , Transplante de Rim , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Provírus/química , Fatores de Risco , Análise de Sequência de DNA , Suínos , Terpenos/farmacologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transplante HeterólogoRESUMO
In this paper, we report on the effects of a diet containing cloned-cattle meat on the reproductive parameters in pregnant rabbits. The artificially inseminated rabbits (gestation day 0) were fed a diet containing 5% or 10% of normal or cloned-cattle meat during the gestation period. Rabbits fed commercial pellet (no additional supplementations) were used as the control. Supplementation of cloned-cattle meat diets did not have any toxicologically significant effects on reproductive performance in dams (body weight, clinical signs, organ weight, and cesarean section analysis). And it also did not affect on fetal development (body and placental weight, and external, visceral and skeletal findings) compared to the controls. The only difference was a food consumption in the first week of gestation for all meat-based diet groups (p<0.05, 0.01, and 0.001, respectively). Our results collectively suggest that there are no obvious differences in reproductive parameters in pregnant rabbits fed cloned-cattle meat.
Assuntos
Clonagem de Organismos , Alimentos Geneticamente Modificados/toxicidade , Carne/efeitos adversos , Carne/análise , Reprodução/efeitos dos fármacos , Aminoácidos/análise , Animais , Apetite/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Bovinos , Dieta , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Inseminação Artificial , Técnicas de Transferência Nuclear , Tamanho do Órgão/efeitos dos fármacos , Placenta/efeitos dos fármacos , Gravidez , Coelhos , Segurança , Oligoelementos/análiseRESUMO
Using differential display reverse transcriptase polymerase chain reaction, we detected 11 differentially expressed genes between top round and loin muscle in Korean cattle (Hanwoo). In the loin muscle, the lightness (L*) value (P<0.01) and marbling fat content (P<0.01), which are important factors in determining meat quality, were higher than in top round muscle. Three of the 11 genes were validated as significant genes between two types of muscle by real-time polymerase chain reaction (P<0.05). To determine whether the three genes were associated with meat quality traits, a regression analysis was preformed. The result demonstrated that two genes (NADH dehydrogenase 2 and cytochrome oxidase III), which are involved in oxidative phosphorylation in mitochondria, were significantly correlated with marbling fat content in the loin muscle (P<0.01), while two genes were not significant with marbling fat content in top round muscle. No significant effects for two genes on other meat quality traits such as meat color (redness and yellowness value), Warner-Bratzler shear force, and water-holding capacity were detected in this study.
Assuntos
Perfilação da Expressão Gênica/veterinária , Carne/análise , Músculo Esquelético/química , Tecido Adiposo/química , Animais , Bovinos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Coreia (Geográfico) , Masculino , Mitocôndrias Musculares/enzimologia , NADH Desidrogenase/genética , Fosforilação Oxidativa , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The objective of this study was to identify proteins in the m. longissimus dorsi between early (12 months of age) and late (27 months of age) fattening stages of Hanwoo (Korean cattle) steers. Using two-dimensional electrophoresis and mass spectrometry, 8 proteins of 11 differentially expressed spots between the 12 and 27 month age groups were identified in the loin muscle. Among those that were differentially expressed, zinc finger 323 and myosin light chain were highly expressed in late-fattening stage, and two catabolic enzymes, triosephosphate isomerase (TPI) and succinate dehydrogenase (SDH) were expressed more in the early versus the late-fattening stage. In particular, the quantification of TPI and SDH by immunoblotting correlated well with fat content. Our data suggested that TPI and SDH are potential candidates as markers and their identification provides new insight into the molecular mechanisms and pathways associated with intramuscular fat contents of bovine skeletal muscle.
Assuntos
Tecido Adiposo/metabolismo , Bovinos/metabolismo , Músculo Esquelético/metabolismo , Proteoma/análise , Adiposidade/fisiologia , Fatores Etários , Animais , Eletroforese em Gel Bidimensional , Músculo Esquelético/química , ProteômicaRESUMO
Xenotransplantation from pigs offers an opportunity to resolve the shortage of human organs. The porcine endogenous retrovirus (PERV) cannot be eliminated because of its presence in the germline DNA. Three subgroups of the replication-competent PERV (PERV-A, PERV-B, and PERV-C) have been identified in pigs. We constructed a molecular clone of PERV-B from a Korean domestic pig BAC clone containing PERV genomes, and its replication competency was characterized in human cells. The pol region of PERV-B was detected in the genomic DNA of 293T cells transfected with PERV-B (465D1) and in the genomic DNA of 293T cells infected with PERV-B (465D1) viruses. The 293T cells transfected with PERV-B (465D1) were maintained for 140 days. PERV-B (465D1) showed the stationary replication competence with no toxicity in the cell growth.
Assuntos
Retrovirus Endógenos/genética , Retrovirus Endógenos/isolamento & purificação , Sus scrofa/virologia , Animais , Linhagem Celular , Sobrevivência Celular , Cromossomos Artificiais Bacterianos , Análise por Conglomerados , Retrovirus Endógenos/patogenicidade , Humanos , Coreia (Geográfico) , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Homologia de Sequência , Replicação ViralRESUMO
AIMS: The aim of the present study was to characterize genes regulated by protein kinase C PKCzeta inhibitor in the preovulatory granulosa cells following LH stimulation in the rat ovary. MAIN METHODS: Annealing control primer (ACP)-based polymerase chain reaction (PCR) method was used to identify differentially expressed genes in granulosa cells of preovulatory follicles cultured in the presence of luteinizing hormone (LH) and myristoylated PKCzeta pseudosubstrate peptide or a similarly sized control peptide. KEY FINDINGS: Among the 16 genes identified, five (testin, glypican-4, retrovirus SC1, aminolevulinic acid synthase 1 and serum-inducible kinase) experienced rapid and transient stimulation of gene expression upon exposure to human chorionic gonadotropin (hCG) in the ovary of immature rats primed with pregnant mare's serum gonadotropin (PMSG). In situ hybridization analysis revealed that hCG administration induced expression of these five genes in granulosa cells of preovulatory follicles. The Western analysis showed that the protein levels of testin and serum-inducible kinase were also increased by hCG. Expression of the eleven remaining genes in the ovary remained high at 24-72 h following hCG treatment. SIGNIFICANCE: The present data demonstrate the gonadotropin stimulation of genes differentially expressed by PKCzeta inhibitor, implicating that PKCzeta pathway possibly plays a role in controlling the ovulation process.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Hormônio Luteinizante/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Ovulação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Gonadotropina Coriônica/metabolismo , Gonadotropina Coriônica/farmacologia , Feminino , Perfilação da Expressão Gênica , Células da Granulosa/enzimologia , Humanos , Hormônio Luteinizante/farmacologia , Ovulação/genética , Ovulação/metabolismo , RatosRESUMO
Ectodermal neural cortex (ENC) 1, a member of the kelch family of genes, is an actin-binding protein and plays a pivotal role in neuronal and adipocyte differentiation. The present study was designed to examine the gonadotropin regulation and action of ENC1 during the ovulatory process in immature rats. The levels of ENC1 mRNA and protein were stimulated by LH/human chorionic gonadotropin (hCG) within 3 h both in vivo and in vitro. In situ hybridization analysis revealed that ENC1 mRNA was localized not only in theca/interstitial cells but also in granulosa cells of preovulatory follicles but not of growing follicles in pregnant mare's serum gonadotropin/hCG-treated ovaries. LH-induced ENC1 expression was suppressed by a high dose of protein kinase C inhibitor RO 31-8220 (10 microM) but not by low doses of RO 31-8220 (0.1-1.0 microM), suggesting the involvement of atypical protein kinase C. ENC1 was detected in both nucleus and cytoplasm that was increased by LH/hCG treatment. Both biochemical and morphological analysis revealed that LH/hCG treatment increased actin polymerization within 3 h in granulosa cells. Interestingly, ENC1 physically associated with actin and treatment with cytochalasin D, an actin-depolymerizing agent, abolished this association. Confocal microscopy further demonstrated the colocalization of ENC1 with filamentous actin (F-actin). The present study demonstrates that LH/hCG stimulates ENC1 expression and increases F-actin formation in granulosa cells. The present study further shows the physical association of ENC1 and F-actin, implicating the role of ENC1 in cytoskeletal reorganization during the differentiation of granulosa cells.
Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neuropeptídeos/metabolismo , Proteínas Nucleares/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismo , Animais , Northern Blotting , Western Blotting , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Imunofluorescência , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Imunoprecipitação , Hibridização In Situ , Técnicas In Vitro , Indóis/farmacologia , Hormônio Luteinizante/farmacologia , Proteínas dos Microfilamentos/genética , Neuropeptídeos/genética , Proteínas Nucleares/genética , Ligação Proteica , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Substâncias para o Controle da Reprodução/farmacologiaRESUMO
We established the Pig Genome Database (PiGenome) for pig genome research. The PiGenome integrates and analyzes all publicly available genome-wide data on pigs, including UniGenes, sequence tagged sites (STS) markers, quantitative trait loci (QTLs) data, and bacterial artificial chromosome (BAC) contigs. In addition, we produced 69,545 expressed sequence tags (ESTs) from the full-length enriched cDNA libraries of six tissues and 182 BAC contig sequences, which are also included in the database. QTLs, genetic markers, and BAC end-sequencing information were collected from public databases. The full-length enriched EST data were clustered and assembled into unique sequences, contigs, and singletons. The PiGenome provides functional annotation, identification of transcripts, mapping of coding sequences, and SNP information. It also provides an advanced search interface, a disease browser, alternative-splicing events, and a comparative gene map of the pig. A graphical map view and genome browser can map ESTs, contigs, BAC contigs (from the National Institute of Animal Science), Sino-Danish Pig Genome Project transcripts, and UniGene onto pig genome sequences which include our 182 BAC contigs and publically available BAC sequences of the Wellcome Trust Sanger Institute. The PiGenome is accessible at http://pigenome.nabc.go.kr/ .
Assuntos
Bases de Dados Genéticas , Genoma , Suínos/genética , Algoritmos , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Animais , Pesquisa Biomédica/métodos , Mapeamento Cromossômico/métodos , Biblioteca Gênica , Especificidade de Órgãos/genéticaRESUMO
Three isoforms of pig PDE4B were cloned and classified as two forms: PDE4B1 and PDE4B3, which contain UCR1 and UCR2; and PDE4B2, which contains only UCR2. The amino acid sequences of each isoform showed good conservation in human and rat. PDE4B2 is expressed in a wide range of tissues, but PDE4B1 and PDE4B3 are not. Using an informative SNP for the Iberian x Landrace intercross detected from intron 12, a linkage map was constructed. The location of PDE4B was estimated at 123.6 cM outside of the QTL-CI (124-128 cM) for IMF. However, the QTL-CI for IMF was reconfirmed with high significance, and its position was narrowed down to an interval of 4 cM (the region defined by markers PDE4B and SW1881). Using radiation hybrid mapping, LEPR, LEPROT, DNAJC6, AK3L1 and AK3L2 were selected as positional and/or functional candidates related to the QTL.
Assuntos
Tecido Adiposo/anatomia & histologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Músculos/anatomia & histologia , Locos de Características Quantitativas , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Primers do DNA , DNA Complementar , Ligação Genética , Humanos , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , SuínosRESUMO
To identify transcriptional markers for beef traits related to meat tenderness and moisture, we measured the transcriptome of the Longissimus dorsi skeletal muscle in 10 Korean native cattle (KNC). We analyzed the correlation between the beef transcriptome and measurements of four different beef traits, shear force (SF), water holding capacity (WHC), cooking loss (CL), and loin eye area (LEA). We obtained non-overlapping and unique panels of genes showing strong correlations (|r|>0.8) with SF, WHC, CL, and LEA, respectively. Functional studies of these genes indicated that SF was mainly related to energy metabolism, and LEA to rRNA processing. Interestingly, our data suggested that WHC is influenced by protein metabolism. Overall, the skeletal muscle transcriptome pointed to the importance of energy and protein metabolism in determining meat quality after the aging process. The panels of transcripts for beef traits may be useful for predicting meat tenderness and moisture.