Effects of Cycloastragenol on Cellular Senescence of Pig Donor Fibroblast, Cytoskeletal Dynamic, and Early Developmental Stage of Nuclear Transfer Embryo

Germplasm resources are the foundation of the revitalization of the swine breeding industry, and their protection and utilization are critical. However, the outbreak of African swine fever and increasing market competition have caused a significant decline in the number of local Chinese pig breeds, therefore threatening the genetic diversity of pig germplasm resources, with many breeds now facing endangerment. Somatic cell nuclear transfer (SCNT) is a promising strategy for restoring endangered pig breeds, but the aging of donor cells severely limits the efficiency of pig SCNT. Cycloastragenol (CAG) has demonstrated anti-aging effects in earlier studies.

This study aimed to evaluate the impact of CAG on aging-related phenotypes of pig ear marginal fibroblasts (PEMFs) and to explore the molecular mechanisms by which CAG regulates cellular senescence using RNA sequencing. Additionally, the potential of CAG as a pre-treatment for SCNT donor cells was further investigated.

PEMFs exhibiting aging phenotypes were obtained through continuous passaging. Cell proliferation assays and flow cytometry were performed to detect Senescence-associated β-galactosidase (SA-β-Gal) activity, in order to analyze the optimal concentrations and treatment times of CAG for PEMFs at different stages of aging. The anti-aging effects of CAG were further assessed by measuring p53 protein levels and the expression of Senescence-associated secretory phenotype (SASP) markers, coupled with immunofluorescence staining of the cytoskeleton. By integrating phenotypic analysis with transcriptomic data, the potential mechanisms by which CAG slowed down PEMF senescence were elucidated. Additionally, SCNT embryo cleavage and blastocyst rates were statistically analyzed, followed by staining and quantification of the total cell number and trophectoderm cell number in SCNT blastocysts to investigate the effects of CAG-pretreated PEMFs on SCNT embryo developmental potential.

SA-β-Gal assay and CCK-8 cell proliferation assay demonstrated that the anti-aging and proliferation-inhibitory effects of CAG were concentration- and time-dependent. Based on dose-effect balance analysis, the recommended treatment conditions were 100 μmol·L^-1 CAG for 24 hours in P3/P6 PEMFs and 12 hours in P9 PEMFs. Under these conditions, CAG significantly reduced SA-β-Gal activity, p53 protein expression, and the secretion of certain SASP factors in PEMFs. Additionally, CAG treatment led to a significant reduction in cell surface area, focal adhesion number, and stress fiber thickness and quantity, while also reorganizing the cytoskeleton. Transcriptomic analysis and subsequent validation results revealed that CAG modulates pathways such as focal adhesion and extracellular matrix (ECM) signaling, thereby affecting the stability of the cytoskeleton. In embryo culture experiments, PEMFs treated with CAG exhibited higher cleavage and blastocyst formation rates, as well as greater blastocyst total cell numbers and trophoblast cell counts, indicating a significant improvement in the quality and developmental potential of early embryos.

CAG effectively delayed donor cell senescence, reorganized the cytoskeleton, and significantly enhanced SCNT efficiency and embryo developmental potential. This discovery offered a novel approach to addressing the challenges of pig germplasm conservation and low cloning efficiency.