Table1_Tetraploid embryo aggregation produces high-quality blastocysts with an increased trophectoderm in pigs.docx

Tetraploid complementation is an ideal method for demonstrating the differentiation potential of pluripotent stem cells. In this study, we selected the most efficient tetraploid production method for porcine embryos and investigated whether tetraploid blastomere aggregation could enhance the quality of tetraploid embryos. Three methods were investigated to produce tetraploid embryos: First, tetraploid embryos were produced using electro-fusion of two-cell stage parthenogenetic blastomere (FUTP). Second, somatic cell was injected into the mature oocyte and fused to produce tetraploid embryos. Third, oocytes were matured with Cytochalasin B (CB) for the late 22 h of in vitro maturation to inhibit the first polar body (PB1). Following that, non-PB1 oocytes were treated with CB for 4 h after parthenogenetic activation. There was no significant difference in the blastocyst development rate and tetraploid production rate of the embryos produced through the three methods. However, FUTP-derived blastocysts had a significantly lower percentage of apoptotic cells compared to other methods. The developmental competence of embryos, expression of trophectoderm cell marker genes, and distribution of YAP1 protein were investigated in tetraploid embryos produced using the FUTP method. The FUTP method most effectively prevented apoptosis during porcine tetraploid embryo formation. Tetraploid aggregation-derived blastocysts have a high proportion of trophectoderm with increased expression of the CDX2 mRNA and high YAP1 intensity. High-quality blastocysts derived from a tetraploid embryo aggregation can serve as suitable source material for testing the differentiation potential of pluripotent stem cells for blastocyst complementation in pigs.

四倍体补偿（tetraploid complementation）是验证多能干细胞（pluripotent stem cells）分化潜能的理想方法。本研究筛选出适用于猪胚胎的高效四倍体制备方法，并探究四倍体卵裂球聚集是否可提升四倍体胚胎的质量。本研究共探究了三种四倍体胚胎制备方法：其一，采用二细胞期孤雌卵裂球电融合法制备四倍体胚胎（FUTP）；其二，将体细胞注射至成熟卵母细胞后经融合制备四倍体胚胎；其三，在卵母细胞体外成熟的最后22小时采用细胞松弛素B（Cytochalasin B, CB）处理，以抑制第一极体（PB1）的排出。随后，对未排出PB1的卵母细胞在孤雌激活后继续用CB处理4小时。三种方法制备的胚胎在囊胚发育率与四倍体形成率上无显著差异，但相较于其余两种方法，FUTP法制备的囊胚凋亡细胞占比显著更低。本研究进一步针对FUTP法制备的四倍体胚胎，探究其胚胎发育能力、滋养层细胞标记基因表达特征及YAP1蛋白分布模式，结果表明FUTP法可最为有效地抑制猪四倍体胚胎形成过程中的细胞凋亡。经四倍体聚集制备的囊胚拥有更高比例的滋养层细胞，其CDX2 mRNA表达量与YAP1蛋白信号强度均显著升高。通过四倍体胚胎聚集获得的高质量囊胚，可作为验证猪胚胎囊胚补偿中多能干细胞分化潜能的理想实验材料。