RESUMO
OBJECTIVE: This study was designed in two-legs. In the in vivo, we explored the potential of a rinse solution containing a combination (Comb) of 0.1 mg/mL CaneCPI-5 (sugarcane-derive cystatin), 1.88 × 10- 5M StN15 (statherin-derived peptide) and 1.0 mg/mL hemoglobin (Hb) to change the protein profile of the acquired enamel pellicle(AEP) and the microbiome of the enamel biofilm. The in vitro, was designed to reveal the effects of Comb on the viability and bacterial composition of the microcosm biofilm, as well as on enamel demineralization. MATERIALS AND METHODS: In vivo study, 10 participants rinsed (10mL,1 min) with either deionized water (H2O-control) or Comb. AEP and biofilm were collected after 2 and 3 h, respectively, after rinsing. AEP samples underwent proteomics analysis, while biofilm microbiome was assessed via 16 S-rRNA Next Generation Sequencing(NGS). In vitro study, a microcosm biofilm protocol was employed. Ninety-six enamel specimens were treated with: 1)Phosphate-Buffered Solution-PBS(negative-control), 2)0.12%Chlorhexidine, 3)500ppmNaF and 4)Comb. Resazurin, colony-forming-units(CFU) and Transversal Microradiography(TMR) were performed. RESULTS: The proteomic results revealed higher quantity of proteins in the Comb compared to control associated with immune system response and oral microbial adhesion. Microbiome showed a significant increase in bacteria linked to a healthy microbiota, in the Comb group. In the in vitro study, Comb group was only efficient in reducing mineral-loss and lesion-depth compared to the PBS. CONCLUSIONS: The AEP modification altered the subsequent layers, affecting the initial process of bacterial adhesion of pathogenic and commensal bacteria, as well as enamel demineralization. CLINICAL RELEVANCE: Comb group shows promise in shaping oral health by potentially introducing innovative approaches to prevent enamel demineralization and deter tooth decay.
Assuntos
Cárie Dentária , Desmineralização do Dente , Humanos , Película Dentária/química , Película Dentária/microbiologia , Cárie Dentária/prevenção & controle , Proteômica , Biofilmes , Hemoglobinas/análise , Desmineralização do Dente/prevenção & controleRESUMO
In brief: Pyruvate metabolism is one of the main metabolic pathways during oocyte maturation. This study demonstrates that pyruvate metabolism also regulates the epigenetic and molecular maturation in bovine oocytes. Abstract: Pyruvate, the final product of glycolysis, undergoes conversion into acetyl-CoA within the mitochondria of oocytes, serving as a primary fuel source for the tricarboxylic acid (TCA) cycle. The citrate generated in the TCA cycle can be transported to the cytoplasm and converted back into acetyl-CoA. This acetyl-CoA can either fuel lipid synthesis or act as a substrate for histone acetylation. This study aimed to investigate how pyruvate metabolism influences lysine 9 histone 3 acetylation (H3K9ac) dynamics and RNA transcription in bovine oocytes during in vitro maturation (IVM). Bovine cumulus-oocyte complexes were cultured in vitro for 24 h, considering three experimental groups: Control (IVM medium only), DCA (IVM supplemented with sodium dichloroacetate, a stimulant of pyruvate oxidation into acetyl-CoA), or IA (IVM supplemented with sodium iodoacetate, a glycolysis inhibitor). The results revealed significant alterations in oocyte metabolism in both treatments, promoting the utilization of lipids as an energy source. These changes during IVM affected the dynamics of H3K9ac, subsequently influencing the oocyte's transcriptional activity. In the DCA and IA groups, a total of 148 and 356 differentially expressed genes were identified, respectively, compared to the control group. These findings suggest that modifications in pyruvate metabolism trigger the activation of metabolic pathways, particularly lipid metabolism, changing acetyl-CoA availability and H3K9ac levels, ultimately impacting the mRNA content of in vitro matured bovine oocytes.
Assuntos
Histonas , Técnicas de Maturação in Vitro de Oócitos , Animais , Bovinos , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Acetilcoenzima A/metabolismo , Histonas/metabolismo , Oócitos/metabolismo , Ácido Pirúvico/farmacologia , Ácido Pirúvico/metabolismo , Epigênese Genética , Células do CúmuloRESUMO
This study investigated the skeletal muscle proteome of crossbred bulls and steers with the aim of explaining the differences in carcass and meat quality traits. Therefore, 640 post-weaning Angus-Nellore calves were fed a high-energy diet for a period of 180 days. In the feedlot trial, comparisons of steers (n = 320) and bulls (n = 320) showed lower (P < 0.01) average daily gain (1.38 vs. 1.60 ± 0.05 kg/d), final body weight (547.4 vs. 585.1 ± 9.3 kg), which resulted in lower hot carcass weight (298.4 vs. 333.7 ± 7.7 kg) and ribeye area (68.6 vs. 81.0 ± 2.56 cm2). Steers had higher (P < 0.01) carcass fatness, meat color parameters (L*, a*, b*, chroma (C*), hue (h°)) and lower ultimate pH. Moreover, lower (P < 0.01) Warner-Bratzler shear force (WBSF) were observed in steers compared to bulls (WBSF = 3.68 vs. 4.97 ± 0.08 kg; and 3.19 vs. 4.08 ± 0.08 kg). The proteomic approach using two-dimensional electrophoresis, mass spectrometry and bioinformatics procedures revealed several differentially expressed proteins between steers and bulls (P < 0.05). Interconnected pathways and substantial changes were revealed in biological processes, molecular functions, and cellular components between the post-mortem muscle proteomes of the compared animals. Steers had increased (P < 0.05) abundance of proteins related to energy metabolism (CKM, ALDOA, and GAPDH), and bulls had greater abundance of proteins associated with catabolic (glycolysis) processes (PGM1); oxidative stress (HSP60, HSPA8 and GSTP1); and muscle structure and contraction (TNNI2 and TNNT3). The better carcass (fatness and marbling degree) and meat quality traits (tenderness and color parameters) of steers were associated with higher abundance of key proteins of energy metabolism and lower abundance of enzymes related to catabolic processes, oxidative stress, and proteins of muscle contraction SIGNIFICANCE: Sexual condition of cattle is known to be an important factor affecting animal performances and growth as well as the carcass and meat quality traits. The investigation of skeletal muscle proteome help a better understanding of the origin of the differences in quality traits between bulls and steers. The inferior meat quality of bulls was found to be due to the greater expression of proteins associated with primary and catabolic processes, oxidative stress, and muscle contraction. Steers had greater expression of proteins, from which several are known biomarkers of beef quality (mainly tenderness).
Assuntos
Proteoma , Proteômica , Bovinos , Animais , Masculino , Proteoma/análise , Carne/análise , Músculo Esquelético/química , Tecido AdiposoRESUMO
The aim of this study was to identify differentially expressed genes, biological processes, and metabolic pathways related to adipogenesis and lipogenesis in calves receiving different diets during the cow-calf phase. Forty-eight uncastrated F1 Angus × Nellore males were randomly assigned to two treatments from thirty days of age to weaning: no creep feeding (G1) or creep feeding (G2). The creep feed offered contained ground corn (44.8%), soybean meal (40.4%), and mineral core (14.8%), with 22% crude protein and 65% total digestible nutrients in dry matter. After weaning, the animals were feedlot finished for 180 days and fed a single diet containing 12.6% forage and 87.4% corn-based concentrate. Longissimus thoracis muscle samples were collected by biopsy at weaning for transcriptome analysis and at slaughter for the measurement of intramuscular fat content (IMF) and marbling score (MS). Animals of G2 had 17.2% and 14.0% higher IMF and MS, respectively (p < 0.05). We identified 947 differentially expressed genes (log2 fold change 0.5, FDR 5%); of these, 504 were upregulated and 443 were downregulated in G2. Part of the genes upregulated in G2 were related to PPAR signaling (PPARA, SLC27A1, FABP3, and DBI), unsaturated fatty acid synthesis (FADS1, FADS2, SCD, and SCD5), and fatty acid metabolism (FASN, FADS1, FADS2, SCD, and SCD5). Regarding biological processes, the genes upregulated in G2 were related to cholesterol biosynthesis (EBP, CYP51A1, DHCR24, and LSS), unsaturated fatty acid biosynthesis (FADS2, SCD, SCD5, and FADS1), and insulin sensitivity (INSIG1 and LPIN2). Cow-calf supplementation G2 positively affected energy metabolism and lipid biosynthesis, and thus favored the deposition of marbling fat during the postweaning period, which was shown here in an unprecedented way, by analyzing the transcriptome, genes, pathways, and enriched processes due to the use of creep feeding.
RESUMO
MicroRNAs (miRNAs) are key regulators of gene expression, potentially affecting several biological processes, whose function can be altered by sequence variation. Hence, the integration of single nucleotide polymorphisms (SNP) and miRNAs can explain individual differences in economic traits. To provide new insights into the effects of SNPs on miRNAs and their related target genes, we carried out a multi-omic analysis to identify SNPs in miRNA mature sequences (miR-SNPs) associated with fatty acid (FA) composition in the Nelore cattle. As a result, we identified 3 miR-SNPs in different miRNAs (bta-miR-2419-3p, bta-miR-193a-2, and bta-miR-1291) significantly associated with FA traits (p-value < 0.02, Bonferroni corrected). Among these, the rs110817643C>T, located in the seed sequence of the bta-miR-1291, was associated with different ω6 FAs, polyunsaturated FA, and polyunsaturated:saturated FA ratios. Concerning the other two miR-SNPs, the rs43400521T>C (located in the bta-miR-2419-3p) was associated with C12:0 and C18:1 cis-11 FA, whereas the rs516857374A>G (located in the bta-miR-193a-2) was associated with C18:3 ω6 and ratio of ω6/ω3 traits. Additionally, to identify potential biomarkers for FA composition, we described target genes affected by these miR-SNPs at the mRNA or protein level. Our multi-omics analysis outlines the effects of genetic polymorphism on miRNA, and it highlights miR-SNPs and target candidate genes that control beef fatty acid composition.
Assuntos
Ácidos Graxos/análise , MicroRNAs/genética , Músculo Esquelético/metabolismo , Carne Vermelha/análise , Criação de Animais Domésticos , Animais , Brasil , Cruzamento , Bovinos , Ácidos Graxos/metabolismo , Feminino , Regulação da Expressão Gênica , Metabolismo dos Lipídeos/genética , Masculino , MicroRNAs/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The mitochondrial transcription factor A (TFAM) is considered a key factor in mitochondrial DNA (mtDNA) copy number. Given that the regulation of active copies of mtDNA is still not fully understood, we investigated the effects of CRISPR-Cas9 gene editing of TFAM in human embryonic kidney (HEK) 293T cells on mtDNA copy number. The aim of this study was to generate a new in vitro model by CRISPR-Cas9 system by editing the TFAM locus in HEK293T cells. Among the resulting single-cell clones, seven had high mutation rates (67-96%) and showed a decrease in mtDNA copy number compared to control. Cell staining with Mitotracker Red showed a reduction in fluorescence in the edited cells compared to the non-edited cells. Our findings suggest that the mtDNA copy number is directly related to TFAM control and its disruption results in interference with mitochondrial stability and maintenance.
RESUMO
Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. Recently, several strategies have been applied in order to improve reprogramming efficiency, mainly focused on removing repressive epigenetic marks such as histone methylation from the somatic nucleus. Herein we used the specific and non-toxic chemical probe UNC0638 to inhibit the catalytic activity of the histone methyltransferases EHMT1 and EHMT2. Either the donor cell (before reconstruction) or the early embryo was exposed to the probe to assess its effect on developmental rates and epigenetic marks. First, we showed that the treatment of bovine fibroblasts with UNC0638 did mitigate the levels of H3K9me2. Moreover, H3K9me2 levels were decreased in cloned embryos regardless of treating either donor cells or early embryos with UNC0638. Additional epigenetic marks such as H3K9me3, 5mC, and 5hmC were also affected by the UNC0638 treatment. Therefore, the use of UNC0638 did diminish the levels of H3K9me2 and H3K9me3 in SCNT-derived blastocysts, but this was unable to improve their preimplantation development. These results indicate that the specific reduction of H3K9me2 by inhibiting EHMT1/2 during nuclear reprogramming impacts the levels of H3K9me3, 5mC, and 5hmC in preimplantation bovine embryos.
Assuntos
Reprogramação Celular/genética , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Histona Metiltransferases/genética , Animais , Blastocisto , Bovinos , Diferenciação Celular , Clonagem de Organismos/métodos , Transferência Embrionária/métodos , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Técnicas de Transferência Nuclear , Oócitos/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional/genética , Quinazolinas/farmacologiaRESUMO
Gene editing in large animal models for future applications in translational medicine and food production must be deeply investigated for an increase of knowledge. The mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters. This gene maintains mtDNA, and it is essential for the initiation of mtDNA transcription. Lately, we generated a new cell line through the disruption of the TFAM gene in bovine fibroblast cells by CRISPR/Cas 9 technology. We showed that the CRISPR/Cas9 design was efficient through the generation of heterozygous mutant clones. In this context, once this gene regulates the mtDNA replication specificity, the study aimed to determine if the post-edited cells are capable of in vitro maintenance and assess if they present changes in mtDNA copies and mitochondrial membrane potential after successive passages in culture. The post-edited cells were expanded in culture, and we performed a growth curve, doubling time, cell viability, mitochondrial DNA copy number, and mitochondrial membrane potential assays. The editing process did not make cell culture unfeasible, even though cell growth rate and viability were decreased compared to control since we observed the cells grow well when cultured in a medium supplemented with uridine and pyruvate. They also exhibited a classical fibroblastoid appearance. The RT-qPCR to determine the mtDNA copy number showed a decrease in the edited clones compared to the non-edited ones (control) in different cell passages. Cell staining with Mitotracker Green and red suggests a reduction in red fluorescence in the edited cells compared to the non-edited cells. Thus, through characterization, we demonstrated that the TFAM gene is critical to mitochondrial maintenance due to its interference in the stability of the mitochondrial DNA copy number in different cell passages and membrane potential confirming the decrease in mitochondrial activity in cells edited in heterozygosis.
Assuntos
Sistemas CRISPR-Cas , Bovinos/genética , Proteínas de Ligação a DNA/genética , Edição de Genes , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética , Animais , Células Cultivadas , Replicação do DNA , DNA Mitocondrial/genética , Fibroblastos/metabolismo , Dosagem de Genes , Mitocôndrias/genéticaRESUMO
Mitochondria play a fundamental role during development of the female germline. They are fragmented, round, and small. Despite these characteristics suggesting that they are inactive, there is accumulating evidence that mitochondrial dysfunctions are a major cause of infertility and generation of aneuploidies in humans. In addition, mitochondria and their own genomes (mitochondrial DNA-mtDNA) may become damaged with time, which might be one reason why aging leads to infertility. As a result, mitochondria have been proposed as an important target for evaluating oocyte and embryo quality, and developing treatments for female infertility. On the other hand, mutations in mtDNA may cause mitochondrial dysfunctions, leading to severe diseases that affect 1 in 4,300 people. Moreover, very low levels of mutated mtDNA seem to be present in every person worldwide. These may increase with time and associate with late-onset degenerative diseases such as Parkinson disease, Alzheimer disease, and common cancers. Mutations in mtDNA are transmitted down the maternal lineage, following a poorly understood pattern of inheritance. Recent findings have indicated existence in the female germline of a purifying filter against deleterious mtDNA variants. Although the underlying mechanism of this filter is largely unknown, it has been suggested to rely on autophagic degradation of dysfunctional mitochondria or selective replication/transmission of non-deleterious variants. Thus, understanding the mechanisms regulating mitochondrial inheritance is important both to improve diagnosis and develop therapeutic tools for preventing transmission of mtDNA-encoded diseases.
Assuntos
Fertilidade , Células Germinativas/metabolismo , Mitocôndrias/genética , Doenças Mitocondriais/patologia , DNA Mitocondrial/metabolismo , Desenvolvimento Embrionário , Feminino , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismoRESUMO
Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.
Assuntos
Expressão Gênica , Genes Essenciais , Oócitos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Animais , Búfalos , Bovinos , Indústria de Laticínios , Feminino , Perfilação da Expressão Gênica , Oócitos/citologia , Padrões de Referência , Estações do AnoRESUMO
In vitro-produced embryos store high lipid content in cytoplasmic lipid droplets (LD), and reduction or removal of LD has been demonstrated to improve freeze-thaw viability. The Perilipin Adipophilin Tail-interacting Protein of 47 kD (PAT) family of proteins is involved in the formation and regulation of LD in many cell types, but their presence has not been addressed either in cattle oocytes or preimplantation embryos. Therefore, this study aimed to detect the expression of PAT family transcripts (Perilipin-2 [PLIN2] and Perilipin-3 [PLIN3]) in immature and in vitro-matured (IVM) oocytes, and in in vitro-produced embryos at the stages of two to four cells, eight to 16 cells, morulae (MO), and blastocyst (BL). The expression of PLIN3 was downregulated in response to IVM, and PLIN2 was comparatively more expressed than PLIN3 in IVM oocytes (P < 0.001). During the early stages of embryo development, PLIN2 expression reached its peak at the MO stage (P < 0.001) and decreased again at the BL stage. In contrast, PLIN3 was expressed in low levels during the earliest stages of development, slightly upregulated at the MO stage (P < 0.05), and greatly increased its expression at the BL stage (15-fold; P < 0.001). PLIN3 was comparatively more expressed than PLIN2 during embryo culture in most stages analyzed (P < 0.05), except in eight- to 16-cell embryos. These results indicate that PLIN2 might be involved in the maintenance of lipid stocks necessary to support embryo development after fertilization of IVM oocytes. Also, we hypothesize that PLIN3 is the main PAT protein responsible for stabilization of LD formed in consequence of the acute lipid load seen during embryo development. We confirmed the presence of both PLIN2 and PLIN3 proteins in BL at Day 7 using immunocytochemistry: these PAT proteins colocalized with LD stained with BODIPY. PLIN3 seemed to be more ubiquitously spread out in the cytoplasm than PLIN2, consistent with the pattern seen in adipocytes. These findings suggest that both elderly (bigger) and newly formed (smaller) LD, positive for PLIN2 and PLIN3 respectively, coexist in blastocysts. To our knowledge this is the first report showing that transcripts of the PAT family are present in cattle oocytes and embryos.
Assuntos
Bovinos/embriologia , Desenvolvimento Embrionário , Proteínas de Membrana/metabolismo , Oócitos/crescimento & desenvolvimento , Proteínas de Transporte Vesicular/metabolismo , Animais , Fertilização in vitro/veterinária , Perilipina-2RESUMO
Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.
Assuntos
Bovinos , Núcleo Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Clonagem de Organismos/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Zigoto/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Bovinos/embriologia , Bovinos/genética , Bovinos/metabolismo , Núcleo Celular/fisiologia , Células Cultivadas , Decitabina , Feminino , Histonas/metabolismo , Hidralazina/farmacologia , Ácidos Hidroxâmicos/farmacologia , Técnicas de Transferência Nuclear/veterinária , Gravidez , Nascimento a Termo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Ácido Valproico/farmacologia , Zigoto/fisiologiaRESUMO
Ooplasm transfer has been used successfully to treat infertility in women with ooplasmic insufficiency and has culminated in the birth of healthy babies. To investigate whether mitochondrial dysfunction is a factor in ooplasmic insufficiency, bovine oocytes were exposed to ethidium bromide, an inhibitor of mitochondrial DNA replication and transcription, during in-vitro maturation (IVM). Exposure of immature oocytes to ethidium bromide for 24h during IVM hampered meiotic resumption and the migration of cortical granules. However, a briefer treatment with ethidium bromide during the last 4h of IVM led to partial arrest of preimplantation development without affecting oocyte maturation. Ooplasm transfer was then performed to rescue the oocytes with impaired development. In spite of this developmental hindrance, transfer of normal ooplasm into ethidium bromide-treated oocytes resulted in a complete rescue of embryonic development and the birth of heteroplasmic calves. Although this study unable to determine whether developmental rescue occurred exclusively through introduction of unaffected mitochondria into ethidium bromide-damaged oocytes, e.g. ethidium bromide may also affect other ooplasm components, these results clearly demonstrate that ooplasm transfer can completely rescue developmentally compromised oocytes, supporting the potential use of ooplasm transfer in therapeutic applications.
Assuntos
Citoplasma/transplante , Etídio/farmacologia , Oócitos/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Citoplasma/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Oócitos/citologia , Oócitos/metabolismoRESUMO
Nuclear-mitochondrial incompatibilities may be responsible for the development failure reported in embryos and fetuses produced by interspecies somatic cell nuclear transfer (iSCNT). Herein we performed xenooplasmic transfer (XOT) by introducing 10 to 15% of buffalo ooplasm into bovine zygotes to assess its effect on the persistence of buffalo mitochondrial DNA (mtDNA). Blastocyst rates were not compromised by XOT in comparison to both in vitro fertilized embryos and embryos produced by transfer of bovine ooplasm into bovine zygotes. Moreover, offspring were born after transfer of XOT embryos to recipient cows. Buffalo mtDNA introduced in zygotes was still present at the blastocyst stage (8.3 vs. 9.3%, p = 0.11), indicating unaltered heteroplasmy during early development. Nonetheless, no vestige of buffalo mtDNA was found in offspring, indicating a drift to homoplasmy during later stages of development. In conclusion, we show that the buffalo mtDNA introduced by XOT into a bovine zygote do not compromise embryo development. On the other hand, buffalo mtDNA was not inherited by offspring indicating a possible failure in the process of interspecies mtDNA replication.
Assuntos
Búfalos , Técnicas de Transferência Nuclear , Animais , Sequência de Bases , Bovinos , Primers do DNA , DNA Mitocondrial/genética , ZigotoAssuntos
DNA Mitocondrial , Dosagem de Genes , Oócitos/fisiologia , Animais , Sobrevivência Celular , FemininoRESUMO
Ooplasmic transfer (OT) has been used in basic mouse research for studying the segregation of mtDNA, as well as in human assisted reproduction for improving embryo development in cases of persistent developmental failure. Using cattle as a large-animal model, we demonstrate that the moderate amount of mitochondria introduced by OT is transmitted to the offspring's oocytes; e.g., modifies the germ line. The donor mtDNA was detectable in 25% and 65% of oocytes collected from two females. Its high variation in heteroplasmic oocytes, ranging from 1.1% to 33.5% and from 0.4% to 15.5%, can be explained by random genetic drift in the female germ line. Centrifugation-mediated enrichment of mitochondria in the pole zone of the recipient zygote's ooplasm and its substitution by donor ooplasm led to elevated proportions of donor mtDNA in reconstructed zygotes compared with zygotes produced by standard OT (23.6% +/- 9.6% versus 12.1% +/- 4.5%; P < 0.0001). We also characterized the proliferation of mitochondria from the OT parents-the recipient zygote (Bos primigenius taurus type) and the donor ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from the seven OT fetuses at different points during fetal development found a decreasing proportion of donor mtDNA (r(2) = 0.78). This indicates a preferred proliferation of recipient taurine mitochondria in the context of the nuclear genotype of the OT recipient expressing a B. primigenius indicus phenotype.
Assuntos
Citoplasma/transplante , Mitocôndrias/fisiologia , Técnicas de Transferência Nuclear , Oócitos/citologia , Animais , Bovinos , Células Cultivadas , Corrente Citoplasmática/fisiologia , DNA Mitocondrial/genética , Técnicas de Cultura Embrionária , Transferência Embrionária/veterinária , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Feminino , Desenvolvimento Fetal/fisiologia , Células Germinativas/citologia , Células Germinativas/ultraestrutura , Técnicas de Transferência Nuclear/veterinária , Oócitos/ultraestrutura , Gravidez , Doadores de TecidosRESUMO
Background: Heat stress (HS) can compromise the female reproductive system, resulting in a massive reduction of reproductive performance. During the period of follicular growth, HS may compromise the oocyte, either because of a direct effect of elevated temperature on the gamete or because of changes in follicular function that would damage oocyte quality. Oocytes harvested from Holstein cows during summer have reduced ability to develop to the blastocyst stage than those harvested during winter. Nevertheless, although there are reports showing a clear effect of HS on oocyte competence to development after IVF, the exact mechanisms by which the oocyte is compromised remain unknown. Review: Resistance to HS is dependent on cow's genotype. Bos indicus breeds (i.e. Gir) are more resistant to elevated temperature and humidity than breeds (i.e. Holstein) that evolved in a temperate climate. This resistant is, among other factors, due to the superior ability of certain breeds to regulate their body temperature. Thus, breed selection is present as a possible tool to minimize the effects of temperature and humidity on milk production and reproduction. On the other hand, Holstein cows are the most common breed used, even in tropical countries (i.e. Brazil), because of their yield in milk production. As a result, there is a considerable effect upon milk production and reproductive performance through the year in tropical and subtropical countries. A possible way to minimize this effect is the employment of reproductive biotechnologies such as fixed-time artificial insemination (FTAI) and embryo transfer (ET). FTAI has been shown to eliminate problems of estrus detection caused by HS. Nevertheless, it is not enough to complete restore herd pregnancy rates because oocytes and early embryos have already been damaged by HS. In contrast, embryos three days after conception orolder are less sensitive to HS. Thus, ET has been used to improve cow's reproductive performance during the hot months of the year (i.e. spring and summer). This is carried out by producing embryos during the fresh months but using them for ET during the HS period. Regarding fertility problems, it is recognized that repeat-breeder (RB) cows cause huge economic problems to the farmer. These cows are characterized by poor fertilization rates and/or early embryonic loss. It is still not certainly known why these cows become RB, when it is the critical period of the year for them, and what should be done to avoid this problem. We have observed that the conception rates in RB Holstein cows were greater after ET (41.7%) than after AI (17.9%). This indicates that ET may be an effective alternative to achieve satisfactory conception rates throughout the year in RB cows, especially during periods of HS. Moreover, when three categories of Holstein cows (heifers, high-producing cows in peak lactation and RB) were subjected to ovum pick up during the summer and the winter, blastocyst rates and quality were significantly affected by both, category and period, being RB cows mostly affected during the summer. Moreover, lower amounts of mitochondrial DNA were found in RB oocytes, suggesting that the low reproductive performance observed in these cows is related to some injure in oocyte quality. Conclusion: HS negatively affects the physiology and fertility of dairy cows and heifers. Concerning about the reproductive field, the effects of HS were detected in vitro and in vivo on oocytes and embryos and also on conception rates. Moreover, RB Holstein cows seem to be even more sensitive to HS and this can probably be related to their oocyte potential into develop in blastocysts. Therefore, efforts should be taken in order to improve the environmental conditions and heat resistance of dairy cows in tropical and subtropical areas. Studies are been conducted to understand the molecular level by which HS disrupt reproduction aiming to develop methods to attenuate or reverse HS consequences upon fertility of dairy cows.
Assuntos
Animais , Feminino , Bovinos , Oócitos , Transtornos de Estresse por Calor/complicações , Transtornos de Estresse por Calor/veterinária , Genitália Feminina/fisiopatologia , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere(NT-B) and fibroblast(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p<0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p<0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.
Assuntos
Blastômeros/citologia , Clonagem de Organismos/métodos , DNA Mitocondrial/metabolismo , Fibroblastos/citologia , Técnicas de Transferência Nuclear , Animais , Blastocisto/citologia , Bovinos , Transferência Embrionária , Embrião de Mamíferos/citologia , Cinética , Mitocôndrias/metabolismo , Modelos Biológicos , Oócitos/citologiaRESUMO
Bovine fetal fibroblast cells were treated with ethidium bromide at a low concentration for 15 passages in culture to determine its effect on mitochondrial DNA copy number and on cell metabolism. Mitochondrial membrane potential and lactate production were estimated in order to characterize cell metabolism. In addition, mitochondrial DNA ND5 in proportion to a nuclear gene (luteinizing hormone receptor) was determined at the 1st, 2nd, 3rd, 10th, and 15th passages using semi-quantitative PCR amplification. Treated cells showed a lower mitochondrial membrane potential and higher levels of lactate production compared with control cells. However, the mitochondrial DNA/nuclear DNA ratio was higher in treated cells compared with control cells at the 10th and 15th passages. This ratio changed between the 3rd and 10th passages. Despite a clear impairment in mitochondrial function, ethidium bromide treatment did not lead to mitochondrial DNA depletion. It is possible that in response to a lower synthesis of ATP, due to an impairment in oxidative phosphorylation, treated cells develop a mechanism to resist the ethidium bromide effect on mtDNA replication, resulting in an increase in mitochondrial DNA copy number.