The mRNA "epitranscriptome": delineating its role in regulating gene expression in mammalian oocytes and the impact of assisted reproductive technolog

不孕症影响着全球约六分之一的夫妇，到2018年，有300万婴儿使用辅助生殖技术（ART）出生。然而，每个周期的成功率仅为20-35%，NHS资源限制意味着每对夫妇的周期数量有限。42岁以后，女性的成功率骤降至4%，这是一个问题，因为女性越来越多地推迟生育。此外，癌症幸存者的治疗需求也有所增加，因为化疗药物会对生育能力产生负面影响。因此，为了能够预测个体的生育能力并改善对生育能力受损者的治疗，需要了解支持生殖细胞生存和发育的基本机制。在大约50%的情况下，生育能力受损的伴侣是女性。女性出生时具有有限数量的卵母细胞，当这些卵母细胞耗尽时发生更年期。卵母细胞维持在与称为颗粒细胞的支持细胞密切相关的“卵泡”中。该池中的卵泡在排卵前被激活并生长。卵泡的生存、生长和发育依赖于基因表达的复杂控制。新的测序技术的出现现在提供了了解基因表达的复杂性的可能性，基因表达是早期胚胎生长、成熟、受精和发育的基础，这在以前是无法想象的。这些研究揭示了体内和体外成熟卵母细胞之间转录组（或mRNA的互补）的差异。然而，转录后调控机制，如调控mRNA翻译/稳定性，对维持生育力同样重要，但了解得少得多。重要的是，在其他领域的工作，特别是那些研究癌症，细胞分化和大脑的工作，已经发现了一个令人兴奋的新机制，调节mRNA翻译/稳定性，启动了一个新的领域称为“epitranscriptomics”，类似于表观遗传学领域。这一进展来自下一代测序（NGS），它揭示了mRNA核苷酸的修饰（甲基化，最常见的是m6 A）经常发生。虽然DNA甲基化及其在表观遗传学中的作用已被很好地理解，但RNA中这些修饰的功能才刚刚开始出现。然而，它们似乎通过影响RNA结合蛋白的结合来控制mRNA的翻译/稳定性，重要的是，这种类型的调节对哺乳动物雌性生育力的影响仍有待探索。最近的研究表明，它对斑马鱼卵母细胞向受精卵的转变（1）和小鼠精子发生过程中的减数分裂过程（2）至关重要，支持了它对哺乳动物雌性生育力也很重要的假设。因此，该项目将使用尖端的NGS应用程序来绘制发育中卵母细胞的表观转录组，并将其与体外发育的卵母细胞进行比较，了解ART技术对基因表达的影响。这将在小鼠中完成，但在适当的情况下，将使用爱丁堡的独特资源将研究结果转化为人类。

Infertility affects roughly 1 in 6 couples worldwide, with 3 million babies having being born using assisted reproduction technologies (ART) by 2018. However, success rates lie at only 20-35% per cycle, with NHS resource restrictions meaning that the number of cycles per couple is limited. After age 42, success rates for women plummet to 4%, which is problematic as women are increasingly delaying childbirth. Moreover there is also an increased treatment need from cancer survivors as chemotherapeutics can negatively affect fertility. Thus to be able to predict individual fertility and to improve treatments for those with compromised fertility, there is a need to understand the basic mechanisms that underpin the survival and development of germ cells.In roughly 50% of cases the partner with compromised fertility is female. Women are born with a finite number of oocytes, with menopause occurring when these become depleted. Oocytes are maintained in "follicles" in close association with support cells known as granulosa cells. Follicles from this pool become activated and grow prior to ovulation. The survival, growth and development of follicles relies on intricate control of gene expression.The advent of new sequencing technologies now offers the possibility of understanding the complexity of gene expression that underlies the growth, maturation, fertilisation and development of early embryos, in detail previously unimaginable. Such studies have revealed differences in the transcriptome (or complement of mRNAs) between in vivo and in vitro matured oocytes. However, post-transcriptional control mechanisms, such as regulated mRNA translation/stability are equally key to maintaining fertility but much less well understood.Importantly, work in other fields particularly from those studying cancer, cell differentiation and the brain has uncovered an exciting new mechanism of regulating mRNA translation/stability, launching a new field known as "epitranscriptomics" in analogy to the field of epigenetics. This advance came from Next Generation Sequencing (NGS) which revealed that modifications (methylations, most frequently m6A) of the nucleotides of mRNAs occur frequently. Whilst DNA methylation and its role in epigenetics is well understood, the function of these modifications in RNA is only just starting to emerge. However they appear to play critical roles on controlling the translation/stability of mRNAs by affecting the binding of RNA-binding proteins.Importantly, the impact of this type of regulation on female fertility in mammals remains to be explored. Recent work has shown it is critical for the oocyte to zygote transition in zebrafish (1) and meiotic progression during mouse spermatogenesis (2), supporting the hypothesis that it will also be important for female fertility in mammals.Thus this project will use cutting-edge NGS applications to map the epitranscriptome of developing oocytes and compare them to those developed in vitro, to understand the impact of ART technologies on gene expression. This will be done in mouse but where appropriate, findings will be translated in humans using unique resources available within Edinburgh.