Evidence from genome-wide association studies (GWAS) links genetic variants near the LYPLA1 locus to variations in sheep muscle development. To further investigate whether LYPLA1 expression plays a role in fetal sheep myoblast development, we performed gain-of-function/loss-of-function analyses targeting LYPLA1 using siRNA and recombinant vectors. CCK-8 assays, EdU staining, and flow cytometry were used to investigate the effects of both LYPLA1 knockdown and overexpression on the proliferation of fetal sheep myoblasts. Furthermore, this study investigated the effect of LYPLA1 on myogenic differentiation by means of indirect immunofluorescence staining with MyHC and quantitative analysis of myogenic regulatory factor (MRF) expression. The results showed that downregulation of LYPLA1 significantly reduced cell viability and proliferation rate, arrested myoblasts in the G2 phase, and inhibited proliferation. These effects were reversed when LYPLA1 was overexpressed. Furthermore, LYPLA1 knockdown significantly impaired myotube formation during terminal differentiation (days 5–7). This defect was correlated with a marked reduction in the expression of myogenic regulatory factors (MRFs). This effect was reversed when LYPLA1 was highly expressed. In summary, this study demonstrates that LYPLA1 promotes the proliferation and differentiation of fetal sheep myoblasts, establishing its potential as a target for the molecular breeding of sheep.

Previous studies have indicated that changes in LYPLA1 expression affect the proliferation and differentiation of fetal sheep myoblasts; however, the underlying molecular mechanisms remain unclear. This study examined transcriptional responses to LYPLA1 overexpression and knockdown in primary myoblasts derived from one Hu sheep fetus and explored the involvement of PI3K-Akt signaling in LYPLA1-associated proliferation. RNA sequencing showed that no differentially expressed genes remained significant after false discovery rate correction following LYPLA1 knockdown, whereas LYPLA1 overexpression yielded 362 significant genes, including 219 upregulated and 143 downregulated genes. Exploratory enrichment analysis of nominally significant candidate genes implicated extracellular and immune-related functions, steroid hormone biosynthesis, PPAR signaling, cytokine–cytokine receptor interaction, and PI3K-Akt signaling. Pharmacological inhibition with 0.5 μM PI3K/AKT-IN-1 partially attenuated the increases in EdU-positive cells and Cyclin D1 mRNA levels associated with LYPLA1 overexpression. These findings indicate that LYPLA1 overexpression is associated with broad transcriptional changes in ovine myoblasts and suggest that PI3K-Akt signaling contributes to the associated proliferative phenotype. However, the underlying mechanism requires further validation.