Trim-away depletion of Zona Pellucida 3 (ZP3) protein in mouse zygotes (analysis after 10h)

Mutation studies always defined the functions of the zona pellucida (ZP) as extracellular, namely: to encase the oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, mutations in the three ZP mouse genes ZP1, ZP2 or ZP3 cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryos and oviductal epithelium. However, the concepti of ZP2-null and ZP3-null oocytes were still unviable also when the defects were obviated by monospermic fertilization in vitro and blastocyst transfer to uterus (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed, but there may be also intracellular functions yet to be discovered. The present study tested if experimentally induced degradation of intracellular ZP3 impacted on the development and transcriptome of mouse embryos. To this end we degraded ZP3 using its antibody in conjunction with the ubiquitin-protein ligase TRIM21. This method is known as 'Trim-away' (PMID 29153837). Briefly, in this method a cell (e.g. oocyte) expressing TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest, in this case ZP3. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. TRIM21 is here always to be understood as translation product of microinjected mCherry-Trim21 mRNA. We compared two experimental groups, as follows. Pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with approx. 100 picoliters of mix comprised of mCherry-Trim21 mRNA 0.2 mg/mL + anti-ZP3 antibody (Proteintech 21279-1-AP) 1 mg/mL + dextran beads 0.02 mg/mL, forming a group named 'Trim-away ZP3' group, in triplicate. As a reference, pronuclear-stage oocytes were microinjected with the same mixture as above, except that the antibody buffer was used in lieu of the antibody itself, in triplicate, forming a group named ‚no Trim'. To identify differently expressed genes we compared group 'Trim-away ZP3' with group ‘no Trim’. Ten hours after microinjection, embryos were collected and lysed for transcriptome analysis. Transcriptome analysis revealed that embryos of group 'Trim-away ZP3' and group ‘no Trim' differed in gene expression and were resolved in principal component analysis. The data support a conclusion that ZP3 found inside the embryo was not merely a remnant from oogenesis, but served an intracellular, post-fertilization role during mouse preimplantation development. Biological replicates were analyzed for each of two experimental conditions: 'Trim-away of ZP3' (protein degradation) in triplicate (r1, r2, r3) and ‚no Trim’ (Trim-away cocktail containing the buffer of the antibody in place of the antibody) in triplicate (r1, r2, r3). Trim-away followed to the coinjection of antibody anti ZP3 (Proteintech 21279-1-AP; 1 mg/mL), mCherry-Trim21 mRNA (0.2 mg/mL) and oregon green dextran beads (0.02 mg/mL), resulting in protein degradation in the proteasome. No Trim-away reaction took place after coinjection of mCherry-Trim21 mRNA (0.2 mg/mL), ZP3 antibody buffer, and oregon green dextran beads (0.02 mg/mL). RNA was extracted and analyzed at 10 hours past injection, when the embryos were at the pronuclear stage.

以往的突变研究均将透明带（zona pellucida, ZP）的功能定义为胞外功能，具体包括：包裹卵巢滤泡中的卵母细胞、确保物种特异性的精子结合，以及缓解胚胎表面所受的剪切应力。因此，小鼠体内3种透明带基因ZP1、ZP2或ZP3发生突变后，会因滤泡空虚、多精受精或胚胎与输卵管上皮间的异常接触而引发原发性不孕。然而，即便通过体外单精受精规避上述缺陷并将囊胚移植至子宫内，ZP2基因敲除与ZP3基因敲除卵母细胞所形成的胚胎仍无法存活（PMID 11245577）。这表明透明带的功能并非如此前所认为的仅局限于胞外空间，其可能还存在尚未被发现的胞内功能。本研究旨在探究实验诱导的胞内ZP3降解是否会对小鼠胚胎的发育及转录组产生影响。为此，我们利用靶蛋白抗体结合泛素连接酶TRIM21的方式降解ZP3，该方法被称为"Trim-away技术"（PMID 29153837）。简言之，该技术的操作流程为：向表达TRIM21的细胞（如卵母细胞）中注入针对目标蛋白（此处为ZP3）的特异性抗体，由此形成的三元复合物（靶蛋白-抗体-TRIM21）会在蛋白酶体中被降解。本研究中所使用的TRIM21均为显微注射的mCherry-Trim21 mRNA的翻译产物。本研究设置两组对照进行比较，具体如下：将原核期卵母细胞（B6C3F1 x CD1）显微注射约100皮升的混合液，该混合液包含0.2 mg/mL的mCherry-Trim21 mRNA、1 mg/mL的抗ZP3抗体（Proteintech 21279-1-AP）以及0.02 mg/mL的葡聚糖微球，该实验组命名为"Trim-away ZP3"组，共设置3个生物学重复。作为对照组，将原核期卵母细胞显微注射与上述成分一致的混合液，仅以抗体缓冲液替代抗ZP3抗体，同样设置3个生物学重复，该实验组命名为"no Trim"组。为筛选差异表达基因，本研究对"Trim-away ZP3"组与"no Trim"组的样本进行了比较分析。显微注射10小时后，收集胚胎并裂解以进行转录组分析。转录组分析结果显示，"Trim-away ZP3"组与"no Trim"组的胚胎在基因表达上存在显著差异，且主成分分析可将两组样本清晰区分。本研究数据证实，胚胎内部的ZP3并非仅为卵发生过程遗留的产物，而是在小鼠胚胎植入前发育阶段发挥了胞内受精后功能。本研究对两种实验条件均设置了3个生物学重复："ZP3 Trim-away降解"组（即蛋白降解组，r1、r2、r3）与"no Trim"组（即仅含抗体缓冲液的Trim-away混合液对照组，r1、r2、r3）。ZP3 Trim-away降解组的处理方式为：共注射抗ZP3抗体（Proteintech 21279-1-AP；1 mg/mL）、mCherry-Trim21 mRNA（0.2 mg/mL）以及俄勒冈绿葡聚糖微球（0.02 mg/mL），使靶蛋白在蛋白酶体中被降解。"no Trim"组的处理方式为：共注射mCherry-Trim21 mRNA（0.2 mg/mL）、ZP3抗体缓冲液以及俄勒冈绿葡聚糖微球（0.02 mg/mL），不会发生蛋白降解反应。注射后10小时（此时胚胎处于原核期），提取RNA并进行转录组分析。