提高猪体外生产胚胎效率和移植效率，对畜牧生产和生物医学研究具有重要的意义。体外胚胎生产已被广泛研究和应用在各个领域，但胚胎质量和妊娠率仍处于相对较低的水平。因此，如何挑选优质胚胎成为提高体外胚胎生产效率的关键环节。首次卵裂形态是挑选优质胚胎的有效指标，然而猪胚胎的形态评估参数尚不完善。本研究以孤雌激活胚胎（Parthenogenetic Activation，PA）和体外受精胚胎（In Vitro Fertilization，IVF）作为研究对象，探究猪体外生产胚胎的首次卵裂模式及其对胚胎发育潜能和基因表达的影响，并通过定量蛋白质组学揭示了首次卵裂不同卵裂球的蛋白差异，为首次卵裂模式评价猪胚胎的发育潜能和质量评估提供参考依据。

1. 猪胚胎首次卵裂模式及其对发育潜能的影响

基于首次卵裂模式对PA胚胎和IVF胚胎进行比较分析，发现两种胚胎具有相似比例的形态分布。随后将横截面积比为1~1.1的2-cell胚胎列为均匀分裂（Even Division，ED）胚胎，将横截面积比为1.2以上的2-cell胚胎列为不均匀分裂（Uneven Dicision，UD）胚胎，两种类型胚胎（PA和IVF）中ED和UD组胚胎所占比例无显著差异。卵裂期胚胎统计结果显示，UD组胚胎发育停滞在9~16-cell时期的比例显著高于ED组，与ED组相比，UD组γ-H2A.X的蛋白表达水平，DNA损伤修复基因（ATM和ATR）和细胞周期检查点相关基因CHEK1的mRNA水平显著更高。进一步对囊胚率和囊胚质量进行评估后发现，UD组的囊胚率和细胞数量显著低于ED组胚胎，滋养层标记基因CDX2和凋亡相关基因CASP-3的mRNA表达水平显著高于ED组，此外UD组中γ-H2A.X和Cleaved caspase-3的蛋白表达水平显著更高。最后，利用二元logistic回归分析证实首次卵裂模式是形成优质囊胚质的影响因素（OR = 0.378， P < 0.05）。

2.  首次卵裂不同卵裂球的蛋白质组学分析

为了探究首次卵裂不同卵裂球的分子差异，本研究对PA和IVF中ED胚胎中的中等卵裂球（Medium Blastomeres，MBs）、UD胚胎中大卵裂球（Large Blastomeres，LBs）和小卵裂球（Small Blastomeres，SBs）进行了蛋白质组学定量分析，并利用差异表达蛋白（Differentially Expressed Proteins, DEPs）进行了生物信息学分析。组学分析结果显示，不同大小的卵裂球（LBs、MBs和SBs）中共检测到109个DEPs。DEPs的细胞定位主要在细胞质基质、细胞核和线粒体中，其中HADHA、UGGT2、NDUFS1和PLIN2与线粒体和脂滴功能相关。GO分析显示，DEPs参与蛋白质代谢和加工，LBs与SBs中的DEPs集中在“RNA结合”和“肌动蛋白细胞骨架组织”。KEGG分析显示，在LBs vs. MBs和MBs vs. SBs中均检测到“核糖体”信号通路，而在LBs vs. SBs中检测到代谢相关通路。随后，通过对LBs和SBs中DEPs的蛋白互作网络（Protein Interaction Network，PPI）分析，揭示了两个与RNA结合和细胞骨架相关的蛋白质密集网络。最后，分别通过蛋白免疫印迹和免疫荧光对与RNA结合相关的DDX1和与细胞骨架相关的ACTB进行了验证。因此，本研究揭示了猪胚胎中LBs、MBs和SBs之间的DEPs，进一步表明细胞骨架调控首次卵裂模式；能量代谢、RNA结合和核糖体决定子细胞的发育能力，这可能会影响整个胚胎的发育潜力。

综上所述，本研究表明猪胚胎首次卵裂过程中的不均匀分裂是普遍现象。不均匀分裂的发生与细胞骨架相关蛋白的不对称分布有关。不均匀分裂行为不仅导致了子细胞中某些特定蛋白的差异分布以及卵裂球内区域分子机制的特异性变化，而且影响胚胎的发育能力。

It is of great significance for animal husbandry and biomedical research to improve the efficiency of embryo production and transfer in vitro. In vitro embryo production has been widely studied and applied in various fields, but the embryo quality and pregnancy rate are still at a relatively low level. Therefore, the embryos selection with the high quality has been a key factor affecting the efficiency of in vitro embryo production. The morphology of first cleavage is an effective index for selecting high-quality embryos, but the morphological parameters of porcine embryos are not available so far. In this study, the first cleavage pattern of porcine embryos and its effects on embryonic development potential were explored with parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos. Subsequently, the quantitative proteomics were performed on the blastomeres with different sizes collected from the embryos after the first cleavage. And the protein differences of different blastomes of the first cleavage were revealed by, Results of which might provid a reference for embryos evulation in pig.

1.  Effects of first cleavage pattern on the developmental potential of porcine embryos

A comparative analysis of PA embryos and IVF embryos based on the first cleavage pattern of pig embryos were firstly observed on PA and IVF embryos, and results showed that the similar proportions of embryos with different size blastomeres in both PA and IVF embryos. Then a cross-sectional area ratio of 1 ~ 1.1 at 2-cell embryos were classified as even division (ED) embryos, and a cross-sectional area ratio of 1.2 or more at 2-cell embryos were classified as uneven division (UD) embryos. Between PA and IVF embryos, no significant difference was observed on the proportion of ED and UD embryos. The statistical results of embryo development at cleavage stage showed that the proportion of embryo development retardation at 9-16-cell stage in UD group was significantly higher than in ED group. Compared with ED group, the protein level of γ-H2A.X, mRNA level of DNA damage repair genes (ATM and ATR) and cell cycle checkpoint related gene CHEK1 were significantly higher in UD group. Further assessment of blastocyst rate and cell number in UD group were significantly lower than those in ED group, and mRNA level of trophoblast marker CDX2 and apoptosis-related gene CASP-3 were significantly higher than those in ED group. In addition, at blastocyst stage, protein level of γ-H2A.X and Cleaved caspase-3 were significantly higher in UD group. Finally, binary logistic regression analysis proved that the first cleavage pattern might affect the formation of high-quality blastocysts (OR = 0.378，P < 0.05).

2.  Proteomic analysis of blastomes of different sizes in 2-cell embryo

Medium blastomeres (MBs) were collected from ED embryos, as well as large blastomeres (LBs) and small blastomeres (SBs) were collected from UD embryos. Then proteomic quantitative analysis was performed on LBs, MBs, and SBs to get the differentially expressed proteins (DEPs), and subsequent bioinformatics analysis was conducted on these DEPs. By the proteomic analysis, a total of 109 DEPs were detected in blastomeres with different size of LBs, MBs vs. SBs. Cellular location of DEPs was mainly in the cytosol, nucleus, and mitochondria, from which HADHA, UGGT2, NDUFS1 and PLIN2 with mitochondria and lipid droplet function were noted. GO analysis showed that DEPs involved in protein metabolism and processing, and DEPs in LBs vs. SBs were focused in “RNA binding” and “actin cytoskeletal tissue”. KEGG pathway of “ribosome” was detected in both LBs vs. MBs and MBs vs. SBs, while metabolism pathways were found in LBs vs. SBs. Subsequently, two protein-dense networks associated with RNA binding and cytoskeleton were revealed by (Protein interaction network, PPI) analysis of DEPs in LBs vs. SBs. Finally, DDX1 protein associated with RNA binding and ACTB protein associated with cytoskeleton were verified by western blotting and immunofluorescence, respectively. Therefore, the present study firstly detected DEPs in LBs, MBs and SBs of 2-cell stage pig embryos, and further indicated that the formation of uneven division might be determined by the cytoskeleton, while the developmental capacity of daughter cells after the first division of pig embryos might be affected by the energy metabolism, RNA binding and ribosome determine.

In summary, the present study confirmed that the uneven division during the first cleavage might be a common phenomenon in pig. The occurrence of the uneven division is related to the asymmetric distribution of cytoskeleton-associated proteins. The uneven division behavior might not only lead to the differential distribution of some specific proteins in daughter cells, but also might affect the subsequent developmental capacity of pig embryos.

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