Molecular characterization and functional analysis of buffalo prolactin (PRL) in mammary gland development and lactation

Abstract. This study investigated the molecular characteristics and functions of buffalo prolactin (PRL) in the lactation. The buffalo PRL gene's complete coding sequence (CDS) is 690 bp, encoding 229 amino acids, with structure and function highly consistent with other Bovidae species. PRL expression was significantly higher in the buffalo mammary gland during lactation compared to the non-lactating period, highlighting its crucial role in lactation. PRL overexpression in buffalo mammary epithelial cells (BuMECs) promotes cell proliferation and increases the casein secretion and triglyceride (TAG) accumulation. This occurs through the activation of the JAK2-STAT5 (cell differentiation, casein (CSN) gene transcription, and lipid factor transcription), PI3K-AKT-mTOR (cell growth, milk protein gene translation, and lipid factor activation), and mitogen-activated protein kinase (MAPK; cell proliferation and stress mitigation) signaling pathways. Specifically, PRL overexpression upregulated the mRNA expression of genes in these pathways, such as JAK2, STAT5A, STAT5B, PI3K, AKT1, mTOR, and ERK2, while decreasing JNK1 and P38. Overexpression also increased the expression of cell cycle genes (CDK2, CDK4, Cyclin D1/E1), and enhanced cell viability. Furthermore, PRL overexpression led to increased expression of casein genes (CSN2, CSN3) and milk-fat-synthesis-related genes (FASN, SCD, ACACA, PPARG, SREBF1). Population genetic analysis identified five single-nucleotide polymorphisms (SNPs) in the buffalo PRL CDS, with c.34C > T and c.430T > C being non-synonymous substitutions that were predicted to affect protein function. This research provides a theoretical foundation for genetic interventions aimed at improving buffalo lactation traits.