Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer

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.The research was granted by São Paulo Research Foundation (FAPESP; grant numbers 2009/00938-3, 2010/13384-3, 2010/09561-7, 2011/14207-0 and 2012/07510-1), National Counsel of Technological and Scientific Development (CNPq; grant number 476229/2011-1) and Coordination for the Improvement of Higher Level Personnel (CAPES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Study of the effect of mitochondrial DNA amount on embryonic development : implications for fertility and mitochondrial inheritance

Resumo: O DNA mitocondrial (mtDNA) dos mamíferos é composto por cerca de 16.500 pares de bases, tem herança exclusivamente materna, e codifica 13 polipeptídios essenciais para a função mitocondrial. Centenas a milhares de cópias de mtDNA estão presentes nas células somáticas dependendo da necessidade energética do tecido. No entanto, oócitos contêm mais de 150.000 cópias, no mínimo uma ordem de magnitude maior que a quantidade presente na maioria das células somáticas. Além disso, uma vez que o mtDNA não é replicado durante o desenvolvimento inicial, a quantidade de mtDNA por célula diminui a cada ciclo celular. Portanto, o número de cópias presentes no oócito deve ser suficiente para atender às necessidade energéticas das células embrionárias até que a replicação do mtDNA seja restabelecida. Considerando que há uma grande variabilidade da quantidade de mtDNA entre oócitos e que alguns trabalhos têm relacionado infertilidade e cópias de mtDNA no oócito, a quantidade de mtDNA poderia ser utilizada para selecionar embriões mais competentes a se desenvolverem. Para testar esta hipótese utilizou-se como modelo experimental o bovino uma vez que o desenvolvimento embrionário desta espécie é muito mais similar ao do humano que o de camundongo. Para tanto, em um primeiro experimento foram utilizados oócitos bovinos provenientes de folículos de diferentes tamanhos. Oócitos oriundos de folículos pequenos, os quais são sabidamente menos competentes a se desenvolverem a blastocisto, continham menos mtDNA comparado com oócitos oriundos de folículos maiores. No entanto, devido a grande variabilidade do número de cópias, num segundo experimento embriões partenogenéticos no estádio de uma célula sofreram biópsia para se determinar o conteúdo de mtDNA antes de serem cultivados para acessar a capacidade de desenvolvimento. Em contraste com achados prévios, o número de cópias de mtDNA nas biópsias não diferiu entre embriões competentes e incompetentes, indicando que o conteúdo de mtDNA não está relacionado com a competência de desenvolvimento a blastocisto. Para confirmar este achado, embriões no estádio de uma célula foram depletados em mais de 60% do seu conteúdo de mtDNA por centrifugação seguido da remoção de parte da fração citoplasmática rica em mitocôndrias. Surpreendentemente, os embriões depletados desenvolveram-se normalmente a blastocisto, os quais continham número de cópias de mtDNA similar a controles não manipulados. O desenvolvimento dos embriões depletados foi acompanhado por um aumento na expressão de genes (TFAM e NRF1) que controlam a replicação e transcrição do mtDNA, indicando uma habilidade intrínseca do embrião bovino em restaurar o conteúdo de mtDNA no estádio de blastocisto. Em conclusão, embriões bovinos competentes são capazes de regular o conteúdo de mtDNA no estádio de blastocisto independentemente do número de cópias presente no oócito. Estes achados contrariam o que foi descrito em camundongos, ressaltando a necessidade de estudos com espécies mais semelhantes ao homem antes do uso clínico do mtDNA como ferramenta para o diagnóstico de fertilidade em mulheres. Além disso, este trabalho tem implicação na manipulação da herança mitocondrial e, portanto, na prevenção da transmissão de sérias patologias causadas por mutações no mtDNA ;;Abstract:The mammalian mitochondrial DNA (mtDNA) is composed by only about 16,500 base pairs, is exclusively inherited from the mother, and encodes 13 polypeptides essential for mitochondrial function. Hundreds to thousands mtDNA copies are found in somatic cells depending on the energetic requirement of the tissue. However, oocytes contain more than 150,000 copies, at least an order of magnitude greater than most somatic cells. Moreover, since replication of mtDNA is downregulated during early development, the mtDNA content per cell decreases after each cell cycle. Therefore, mtDNA copy number in oocytes should be enough to support the energetic requirement of embryonic cells until mtDNA replication to be restablished. Considering there is a wide variability of mtDNA copy number among oocytes and there are reports showing a link between infertility and oocyte mtDNA copy number, the content of mtDNA could be used to select embryos more competent to develop. To test this hypothesis we used the bovine as an experimental model since its embryonic development is more similar to human than the murine is. Therefore, in a first experiment bovine oocytes derived from follicles of different sizes were used. Oocytes obtained from small follicles, known to be less competent to develop into blastocysts, contained less mtDNA than those originated from larger follicles. However, due to the high variability in copy number, in a second experiment a more accurate approach was examined in which parthenogenetic one-cell embryos were biopsied to measure their mtDNA content and then cultured to assess development capacity. Contrasting with previous findings, mtDNA copy number in biopsies was not different between competent and incompetent embryos, indicating that mtDNA content is not related to early developmental competence. To further examine the importance of mtDNA on development, one-cell embryos were partially depleted of over than 60% of their mtDNA by centrifugation followed by the removal of the mitochondrial-enriched cytoplasmic fraction. Surprisingly, depleted embryos developed normally into blastocysts, which contained mtDNA copy numbers similar to non-manipulated controls. Development in depleted embryos was accompanied by an increase in the expression of genes (TFAM and NRF1) controlling mtDNA replication and transcription, indicating an intrinsic ability to restore the content of mtDNA at the blastocyst stage. In conclusion, competent bovine embryos are able to regulate their mtDNA content at the blastocyst stage regardless of the copy numbers present in oocytes. These findings are in disagreement with that reported for mice, highlighting the need for studies using species more similar to human before the clinical use of mtDNA as a diagnostic tool in woman fertility. Moreover, these findings are important to manipulate mitochondrial inheritance and, therefore, to prevent transmission of important disorders caused by mtDNA mutation

Mitochondrial Dna Copy Number, A Marker Of Viability For Oocytes.

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Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains

<div><p>Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals.</p></div

Pronounced Segregation Of Donor Mitochondria Introduced By Bovine Ooplasmic Transfer To The Female Germ-line.

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.82563-7

Pronounced Segregation of Donor Mitochondria Introduced by Bovine Ooplasmic Transfer to the Female Germ-Line1

Ooplasmic transfer (01) has been used in basic mouse research for studying lhe 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 lhe moderate amount of mitochondria introduced by OT is transmitted to lhe offspring's oocytes; e.g., modifies lhe germ fine. The danar 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 lhe female germ line. Centrifugation-mediated enrichment of mitochondria il1 lhe pole zone of lhe recipient zygote's ooplasm and its substitution by danar ooplasm led to elevated proportions of danar 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 lhe proliferation of mitochondria from lhe OT parents-the recipient zygote (Bos primigenius taurus type) and lhe danar ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from lhe seven OT fetuses at different points during fetal development found a decreasing proportion of donor mt DNA (r2=0.78). This indicates a preferred proliferation of recipient of the OT recipeint expressinf a B. primigenius indicus phenotype

Comparison of mtDNA copy number among tissues of heteroplasmic males.

<p>Bars depict mtDNA copy number per cell from mice aged four months. Tissues analyzed included brain, heart, liver and tail. Values are reported as mean ± SEM. Bars with different letters denote a significant difference among tissues (P < 0.05).</p

Level of NZB mtDNA in heteroplasmic mouse embryos produced by cytoplasmic transfer.

<p>Zygotes were centrifuged prior to cytoplasmic transfer to enable production of embryos with higher levels of NZB mtDNA (filled circles). For comparison, cytoplasmic transfer was also performed without centrifugation (empty circles). Embryos from both groups were evaluated with regards to the level of NZB mtDNA at the zygote and blastocyst stages by ARMS-qPCR. Bars represent the means. P values for the effect of Group, Stage and Group x Stage are denoted in the inset.</p

Specificity of the ARMS-qPCR approach for amplification of target mtDNA.

<p>Using samples from homoplasmic mice (either NZB or B6), non-specific amplification of non-target mtDNA accounted for only 0.01% of target amplification. Analysis of samples from heteroplasmic mice showed that levels as low as 0.1% of mtDNA of NZB or B6 origin could be detected. Individual Ct values (cycle at which plots crossed the threshold) are denoted in the inset.</p