Exosome-Enriched Maternal Serum Proteins Contain Signals of Conceptus Growth and Estrous Cycle in Buffalo (Bubalus bubalis)

Author(s): Jithil VR, Sujoy K Dhara, and Jyotirmoy Ghosh

The membrane-bound extracellular vesicles (EVs) the exosomes, that carry proteins, nucleic acids, and lipids in their cargo from the cells of origin, play a significant role during pregnancy establishment in different species. This study was thus designed to understand whether the precipitated fraction of maternal serum proteins by commercial exosome isolation reagents helps to understand the cycle and early-pregnancy signals by two-dimensional electrophoresis and to identify the differentially expressed protein spots by LC-MS/MS mass spectrometry analysis for their involvement in the different functional pathways and networks. Serum exosome-enriched fraction 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. The protein in the precipitate were sonicated, and passed through Sephadex G25 spin columns, quantified, and analyzed by one- (1D) and two-dimensional (2D) gel electrophoresis. Differentially expressed proteins were analyzed to understand the cycle and pregnancy stage-specific changes. A total of 9 unique spots were analysed by mass spectrometry and identified by NCBI non-redundant protein database search. Results indicated exosome protein contents at cycle day 10 were significantly lower than the other days of the cycle and early pregnancy samples. The 1D electrophoresis did not reveal any difference but the 2D analysis revealed 8 pregnancy-specific and one cycle-specific protein spots that are significantly upregulated. Mass spectrometry analysis of these 9 spots identified 19 different proteins by database search. The proteins SLAM9 and MARK1 were identified in multiple spots. The proteins identified are indicated in the mitosis, cell cycle regulation, morphogenesis, and regulation of several cellular and molecular pathways relating to conceptus development. In conclusion, the exosome-enriched maternal serum proteins contained signals of the cycle, early pregnancy, and conceptus growth. The proteins identified by this approach might be good targets for defining the critical period of pregnancy/cycle in buffaloes and future research on exosomes.