Serum exosomes of pregnant and non-pregnant buffaloes revealed protein signatures of conceptus development and events of the non-fertile cycle

Abstract

AbstractExosomes are membrane-bound extracellular vesicles (EVs) that carry proteins, nucleic acids, and lipids in their cargo from the cells of origin. They are detected in circulatory body fluids and are indicated to play significant roles during the establishment of pregnancy in humans and animals. This study aimed to isolate, quantify, profile, compare, and identify the elected differentially expressed serum exosome proteins in cycling and early-pregnant buffaloes. Serum exosomes were isolated from 0.5 mL serum of non-pregnant day 0, day 10, and day 15 of the cycle (n = 6), and early pregnancy days 15, day 30, and day 60 (n = 5) buffaloes by precipitation method. The proteins of serum exosomes were harvested by sonication, passing through Sephadex G25 spin columns, quantified, and analyzed by one-(1D) and two-dimensional (2D) SDS-PAGE gel electrophoresis. Selected differentially expressed proteins were analyzed by mass spectrometry and generated peptide mass (m)/charge (z) ratio searched against NCBI cattle and buffalo database to identify the protein. The results indicated serum exosome protein on cycle day 10 was significantly lower than the tested other days of the cycle and early pregnancy samples. The 1D electrophoresis did not reveal any difference but the 2D analysis revealed subtle differences. Mass spectrometry analysis of 9 up-regulated spots (8 pregnancy-specific and one cycle-specific) identified 19 proteins under 11 categories by database search. They were functionally related to mitosis, cell cycle regulation, morphogenesis, and regulation of several cellular and molecular pathways which are essential for conceptus development. Out of 19 proteins 17 were found functionally interconnected due to their common properties indicating their importance during the period of critical period of cycle and early pregnancy. In summary, serum exosomes of pregnant and non-pregnant buffaloes revealed protein signatures of conceptus development and events of the non-fertile cycle.