Impact of genome variation on bovine embryo development

尽管广泛使用先进的生殖生物技术，但牛的总体生育力正以每十年约1.5%的速度下降，导致由于产量损失而导致的经济短缺。在奶牛中，生育率定义为单次授精的妊娠率，可能低至35%，大多数妊娠失败是由于受精后前2至3周内的胚胎丢失。 因此，迫切需要获得对牛胚胎发育和发育失败的新的和更好的理解，以制定新的战略来对抗这种经济损失的来源。 为了进一步探索牛胚胎发育，我们建议利用最近发现的基因组变异，这表明超过4%的牛基因组是由超过1000千碱基的DNA序列，重复不同的次数。这种变异被称为拷贝数变异或CNV。这些CNV区域包括功能基因，在人类中已被证明与各种复杂疾病相关，如帕金森病和癌症。 Y染色体特别富含多拷贝基因，其中一些显示CNV。 我们以前已经证明了一个基因，睾丸特异性蛋白Y编码（TSPY）的公牛生育力的CNV效应，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对胚胎发育的尚未探索的影响。我们推测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）确定在体外产生的和来源于不同公牛的单次射精的同胞胚胎中Y染色体连锁的多拷贝基因的CNV程度。2)确定mRNA表达水平和与囊胚期Y染色体连锁基因拷贝数的相关性。3)确定下调多拷贝Y染色体连锁转录物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果的影响。我们将采用最先进的工具来确定特定基因的拷贝数及其RNA和蛋白质水平与牛胚胎中DNA拷贝和发育速率的关系。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。这项新研究的结果将提供有关胚胎发育机制的关键信息，并有助于制定改善生殖技术的策略。这对于价值100亿美元的加拿大养牛业来说尤其重要，因为该行业依赖生殖生物技术来提高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