Effect of donor cell nuclei with Xist shRNA treatment on the differential protein expression in 4-cell stage porcine embryos derived from somatic cell nuclear transfer

報告時間：2024-04-12
報告地點：Room 407
指導老師：San-Yuan Huang&Pin-Chi Tang
學生：Shu-Ping Chang
摘要

The embryos derived from the somatic cell nuclear transfer (SCNT) technique have encountered cloning deficiency and multiple developmental problems, which are highly related to incomplete epigenetic cell reprogramming. Pigs is not only an important farm animal, but also a suitable animal model for therapeutic application because of their sharing physiological similarities with humans. Therefore, enhancing the cloning efficiency of SCNT porcine embryos may benefit both animal and medical fields. However, the special zygotic genomic activation stage at the 4-cell stage of porcine embryos differs from most mammals, deepening the difficulties in early porcine embryogenesis researches. Previous studies revealed that abnormal Xist gene expression was a common defect of epigenetics observed in SCNT porcine embryos. Knockout of the Xist gene significantly improved developmental competence. Hence, this study aimed to investigate the effects of Xist shRNA treatment on differentially expressed proteins (DEPs) in 4-cell stage SCNT porcine embryos. Male somatic cells with or without treatment of Xist T4 pU6-shRNA plasmid electroporation were used as donor cells for SCNT. A total of 100 4-cell stage SCNT embryos from in vitro culture were collected in both treatments. Another 100 parthenogenetically activated (PA) embryos were used as the positive control. Embryonic proteins were purified by stacking gel-aided separation and subjected to label-free proteomic quantification with mass spectrometry. The DEPs were annotated by bioinformatic analysis with gene ontology, biological pathway, and protein-protein interaction database searches. Results showed that there were 25 proteins upregulated and 15 proteins downregulated between Xist shRNA treated and the non-treated SCNT embryos, respectively. Gene ontology analysis revealed that the majority of the DEPs were involved in protein metabolism, signal transduction, and developmental processes. The biological pathway enrichment analysis showed that the DEPs were mainly enriched in endoplasmic reticulum protein processing. The protein-protein interaction clustered the DEPs into functions of endoplasmic reticulum stress, intermediate filament cytoskeleton organization, DNA binding and methylation maintenance, and carbon metabolism. The metabolism-related DEPs (ENO1, ME3, SMS, and DLST) upregulated in Xist shRNA SCNT embryos suggested that Xist shRNA treatment might improve the energy metabolism of SCNT embryos. However, their expression levels were downregulated compared to those in the PA embryos. Both the 4-cell stage SCNT embryos showed downregulated expression of proteins related to protein folding and endoplasmic reticulum stress (CALR, PDIA3, HSPA5, HSP90B1, PDIA4, and BCAP31) which may indicate that early stage SCNT embryos still face endoplasmic reticulum stress, leading to misfolded or unfolded protein induction of apoptosis. In addition, the downregulated expression of HSPA1B/Hsp70.2, HSP90AA1, and HSPA8 in SCNT embryos may suggest that the dysfunction in chaperone-mediated autophagy (CMA) regulation of early stage embryonic development, leading to ineffective degradation of maternal factors to induce zygotic genome activation, which may associate with the phenomenon of 4-cell stage block encountered by SCNT porcine embryos.

Keywords: somatic cell nuclear transfer, 4-cell stage porcine embryos, Xist shRNA, Proteomics
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