Impact of genome variation on bovine embryo development

尽管先进的生殖生物技术被广泛使用，但牛的总体生育力正在以每十年约1.5%的速度下降，导致由于产量损失而造成的经济缺口。在奶牛中，受精率定义为每一次受精的妊娠率可能低至35%，大多数失败怀孕是由于受精后头2至3周内的胚胎丢失造成的。因此，迫切需要对牛胚胎发育和发育失败有新的和更好的理解，以制定新的战略来对抗这一经济损失的来源。为了进一步探索牛胚胎的发育，我们建议利用关于基因组变异的最新发现，这些发现表明，超过4%的牛基因组由超过1000千克碱基的DNA序列组成，这些DNA序列重复不同的次数。这种变异称为拷贝数变异或CNV。这些CNV区域包括功能基因，在人类中已被证明与多种复杂疾病有关，如帕金森氏症和癌症。Y染色体特别富含多拷贝基因，其中几个显示CNV。我们之前已经证明了睾丸特异蛋白Y编码(TSPY)基因的CNV对公牛生育能力的影响，初步数据表明，它也可能影响胚胎发育。因此，我们现在建议详细研究CNV对胚胎发育的影响。我们假设多拷贝基因的拷贝数和表达水平影响胚胎发育。我们的具体目标包括：1)确定Y染色体连锁多拷贝基因在不同公牛单个射精产生和衍生的同胞胚胎中的CNV程度。2)检测囊胚期Y染色体连锁基因的mRNA表达水平及其与拷贝数的相关性。3)确定下调多拷贝Y染色体连锁转录本对早期胚胎发育的影响。4)确定多拷贝Y染色体连锁基因拷贝数减少对胚胎发育结局的影响。我们将使用最先进的工具来确定特定基因的拷贝数及其RNA和蛋白质水平与DNA拷贝数和牛胚胎发育速度的关系。随后，我们将改变Y染色体连锁基因的拷贝数和/或相应的mRNA转录本，以确定影响胚胎发育的作用模式。这项新颖的研究结果将提供有关胚胎发育机制的关键信息，并有助于制定改进生殖技术的战略。这与价值100亿美元的加拿大养牛业尤其相关，该行业依赖生殖生物技术来实现遗传进步和生产效率。

In spite of the widespread use of advanced reproductive biotechnologies, the overall fertility of cattle is declining at a rate of about 1.5% per decade leading to economic shortfall due to loss in production. In dairy cattle, fertility defined as pregnancy rate per single insemination, may be as low as 35%, with the majority of failed pregnancies resulting from embryo loss within the first 2 to 3 weeks after fertilization. It is therefore imperative that a new and better understanding of bovine embryo development and developmental failure be obtained to devise new strategies to combat this source of economic loss. To further explore bovine embryo development we propose to take advantage of recent discoveries concerning genomic variation, which suggest that over 4% of the bovine genome is composed of DNA sequence of over 1000kilo bases, that are repeated a varying number of times. This variation is known as copy number variation or CNV. These CNV regions include functional genes, which in humans have been shown to be associated with a wide variety of complex diseases, such as Parkinson's and cancer. The Y-chromosome is particularly rich in multi-copied genes, several of which display CNVs. We have previously shown a CNV effect on bull fertility for a gene, the Testis specific Protein Y-encoded (TSPY), with preliminary data suggests that it could also affect embryo development. Therefore, we are now proposing to examine in details the as yet unexplored effect of CNVs on embryo development. We hypothesize that the number of copies and the expression levels of multi-copied genes influence embryo development. Our specific objectives include: 1) Determine the extent of CNV for Y chromosome-linked multi-copied genes in sibling embryos in vitro produced and derived from single ejaculates of different bulls. 2) Determine mRNA expression levels and correlation to copy number of Y-chromosome linked genes at the blastocyst stage. 3) Determine the effect of down-regulating multi-copied Y-chromosome linked transcripts on early embryonic development. 4) Determine the effect of copy number reduction of multi-copied Y chromosome -linked genes on the embryo developmental outcome. We will employ state of the art tools to determine the number of copies of specific genes and their RNA and protein levels in relation to DNA copies and rate of development in bovine embryos. Subsequently we will alter the number of copies of Y chromosome linked genes and/or the corresponding mRNA transcripts to pin-point the mode of action that affect embryonic development. The results of this novel study will provide key information on the mechanisms of embryo development and assist in the development of strategies for improving reproductive technologies. This is particularly relevant for the 10 billion dollar Canadian cattle industry, which relies on reproductive biotechnologies for genetic advancement and production efficiency