RRBS of Bovine ICM. Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during bovine oocyte in vitro maturation

Background: In vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation. Objectives: To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM. Materials & methods: The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass. Results: Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts. Conclusions: These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal.

背景：体外成熟（in vitro maturation, IVM）是指体外受精（in vitro fertilization, IVF）前对生发泡完整卵母细胞进行的成熟培养技术，该方法在动物实验中已得到广泛应用。在人类辅助生殖领域，IVM通常仅用于生育力保存或存在卵巢刺激禁忌证的场景。当前标准IVM培养体系会添加血清和/或白蛋白形式的复合蛋白（complex proteins, CP），以模拟卵巢卵泡的生长微环境。但复合蛋白成分未明确，且存在批次差异及来源相关的伦理顾虑，因此亟需开发成分完全明确的IVM培养配方。褪黑素（melatonin）是卵泡液的已知组分之一，兼具代谢调节与抗氧化活性，在特定条件下可促进卵母细胞成熟。处于该发育阶段的生发泡完整卵母细胞易出现非整倍体及表观遗传失调。
研究目标：明确牛体外成熟过程中移除复合蛋白并添加褪黑素对卵母细胞发育、细胞遗传学及表观遗传学的影响。
材料与方法：本研究采用2×2析因设计，分别设置（i）是否添加复合蛋白，（ii）是否添加100 nM褪黑素两个变量，开展IVM培养。研究所用卵丘-卵母细胞复合物（cumulus-oocyte complexes, COCs）取自促排卵周期。经IVM与IVF后，将疑似合子置于标准培养体系中培养至第8天。分别对分离得到的卵丘细胞进行RNA测序（RNA sequencing, RNAseq），对分离的滋养外胚层细胞开展基于单核苷酸多态性（single nucleotide polymorphism, SNP）的细胞遗传学分析，并对内细胞团细胞采用简化代表性亚硫酸氢盐测序（reduced representation bisulfite sequencing, RRBS）进行DNA甲基化分析。
结果：IVM过程中移除复合蛋白会导致囊胚发育率小幅下降；添加褪黑素在保留复合蛋白的体系中可改善囊胚发育，但在无复合蛋白的体系中则产生不利影响。IVM培养体系的组成不会改变染色体异常的类型与发生率，但卵丘细胞的转录本表达谱显示COCs的代谢（主要为脂质代谢）发生改变。上述变化先于后续扩张期及孵出囊胚中独特代谢与表观遗传特征的建立。
结论：本研究结果凸显了IVM过程中COCs的脂质（尤其是固醇）代谢的重要性，为后续开发成分明确的IVM培养体系奠定了基础，该体系可用于获取细胞遗传学及表观遗传学均正常的可移植胚胎。