卵母细胞体外成熟过程中卵丘细胞生物标志及其预测和提高胚胎发育潜能的意义

Recovery of immature oocytes, as cumulus-oocyte complexes (COCs), followed by in vitro maturation (IVM) and IVF is a useful treatment for women with infertility, but IVM generated embryos have lower pregnancy rate and needed more improvement. Our previous study demonstrated that oocytes matured with and without cumulus cells have different protein synthesis patterns. More importantly, it has been noted that oocytes matured as COCs have significantly higher embryo developmental potential (EDP) and implantation potential (IP) than denuded oocytes. Recent studies indicated that analysis of gene activities in cumulus cells (CCs) may reveal the oocyte quality related to EDP and IP, indicating that there is a set of potential markers in CCs that could be employed for the purposes of oocyte selection. EDP may be used as a standard to select and improve the IVM medium composition. Therefore, once the biomarkers in CCs during IVM correlated with oocyte quality were identified, it is not only beneficial to predict the viability of IVM oocytes but also as accurate criteria is very useful to be used for improving the composition of IVM media. We hypothesize that the transcription profiles differ in CCs as a functional marker of IVM oocyte for EDP. The essential methodology behind the proposed study, using the mouse model, is to collect the CCs during IVM while keeping track of the competence of individual oocyte through the process of maturation, fertilization, and subsequent embryonic development. Genes differentially expressed in CCs and oocytes, which may be attributed to post-fertilization oocyte competence, will be identified by microarray analysis from the extracted RNA of CCs. These criteria (biomarkers) together with morphological assessment of EDP can also be used accurately to improve IVM medium. This study will be (1) To determine the gene expression in CCs responsible for EDP in IVM oocytes. (2) To improve the composition of IVM medium in order to maximize the EDP of IVM oocytes. If we can succeed in identifying CC-derived biomarkers during IVM culture that is able to reveal accurately high quality and viable oocytes, this will certainly bring a great improvement to infertility treatment by IVM. In addition, these biomarkers can be used as more accurate standard criteria in combination with morphological assessment of EDP to improve the composition of IVM medium. These potential markers could be employed for the purposes of IVM oocyte selection and for the improvement of IVM culture medium composition. The transcription profiles will enable us to improve the IVM medium formulation for the purpose of producing healthy embryos in culture.

获取未成熟卵母细胞，如卵丘复合体（COC），继之体外成熟（IVM）和IVF，是不孕症治疗的有益方法。但妊娠率低，急需改进。我们研究证明，有或无卵丘细胞(CCs)的成熟卵母细胞分泌不同的蛋白质谱；COCs的成熟卵母细胞具有更高的胚胎发育潜能（EDP）和种植潜能（IP）。分析CCs基因与蛋白表达，可无创性地预测卵母细胞质量、EDP和IP。本研究将（1）确定IVM中卵母细胞EDP相关的CCs基因表达特征谱，探讨卵母细胞成熟分子调节机制；（2）创新IVM培养基的构成，大大提高IVM的EDP。方法：收集小鼠模型IVM中的CCs，比较有/无CCs条件下的卵母细胞成熟潜能、受精潜能、EDP，采用芯片比较二种条件下CCs和卵母细胞中的差异表达基因，结合EDP、形态学，发现某些关键的生物学标志。这些生物学标志分子应用于预测卵母细胞质量，指导IVM中的卵母细胞选择；也作为创新研制IVM培养基的观察标志。

卵母细胞成熟过程中，卵丘细胞（CCs）基因表达谱和特异基因，可能作为卵母细胞潜能和IVF妊娠结局的生物标志，在人类可望作为无创性筛选卵母细胞的标志，或可成为改进体外卵母细胞成熟（IVM）培养液的因子。研究发现，IVM过程中随着卵母细胞成熟，MII期CCs上调的基因2615个，涉及基质金属蛋白酶调节及表皮生长因子受体结合等功能；下调的基因2810个，主要涉及细胞核内相关生物学事件。选择其中4个功能基因（EFEMP1, FDX1, GJA1, CCND2），研究发现IVM过程中，随卵母细胞成熟的进程，CCs 的EFEMP1和FDX1上调，GJA1和CCND2下调，可望作为卵母细胞质量相关的生物标记物。应用二代测序技术的RNA测序技术，以及深入生物信息学分析，进一步发现体外成熟与体内成熟过程中，卵丘细胞上9个基因（Arrb1, Atp2c1, Cdh5, Cntnap1, Mkln1, Lgr4, Rhobtb1, Smc2和 Six2）可能作为候选的靶基因，这些基因涉及调控G-蛋白偶联受体、Wnt和 MAPK信号、肌动蛋白维丝、细胞黏附等功能。选择Rhobtb1, Mkln1, Smc2, Arrb1, Atp2c1, Cdh5和Lgr4基因进行了扩大样本的研究。AIFM2和FDX1主要定位于卵丘细胞胞质中，在GV-COC中各层均有表达，且GV期卵母细胞中也有表达；IVM-COC和IVV-COC中表达阳性的细胞数目较GV期显著增多。因此，卵丘细胞上AIFM2和FDX1参与了卵母细胞成熟过程，有望作为预测卵母细胞成熟的CCs标记物。在人卵泡的研究发现，CCs的Arrb1, Cntnap1 和Lgr4与囊胚形成相关，因此可能成为卵母细胞受精后和胚胎早期发育潜能的标志物。在卵丘-卵母细胞复合体上，AMH主要表达于卵丘细胞，而其重要受体AMHR- II在卵母细胞及卵丘细胞上均有表达。在IVM培养液中添加AMH对卵母细胞成熟没有影响，但可以上调卵母细胞质量相关因子的表达与分泌。在IVM培养液中添加AMH可以提高胚胎的囊胚形成率及囊胚质量。基于以上研究，我们探讨了卵母细胞成熟过程中，CCs差异表达的基因参与卵丘细胞-卵母细胞直接的交流，调节卵母细胞体内和体外成熟，可以作为卵母细胞成熟的无创性的生物标志，并可能应用于创新IVM培养液，改善IVF临床结局。