5 Characterization of plasmatic extracellular vesicles during pregnancy in beef cattle

Extracellular vesicles (EVs) are bilipid membrane structures that mediate intercellular communication by carrying proteins, miRNA, and DNA. Previous evidence has suggested the potential role of EVs in the crosstalk between the embryo and the mother. It has been reported that EVs derived from embryonic tissue are present in the mother’s bloodstream in humans, mice, sheep, and cattle. Our study aimed to characterize the plasmatic EVs in pregnant cows carrying male or female calves. Angus cows (n = 60) were subjected to timed AI (TAI) using the 7-Day CoSynch + CIDR protocol. Thirty days after TAI, pregnancy diagnoses were conducted using gray mode ultrasonography. Thirty pregnant cows were randomly selected for the experiment. Blood samples were taken from the jugular vein on Days 45, 60, 90, 120, 150, 180, 210, 240, and 270 of gestation. Blood samples from each individual were centrifugated to separate the plasma, and 1-mL aliquots were stored at −80°C. After parturition, the sex of the calf was recorded, and samples from five male-carrying gestations (MCGs) and five female-carrying gestations (FCGs) were selected for EV analysis. Plasmatic EVs were isolated by ultracentrifugation with an SW55Ti swinging-bucket rotor for 90 min at 4°C. To measure the mean size and concentration of EVs, samples were diluted at a 1:40 ratio with PBS and run through nano flow cytometry using the NanoFCM. This novel technology combines traditional flow cytometry and flow nanoparticle tracking analysis. Additionally, electron microscopy and western blotting were used to further characterize the particles isolated from plasma. Data were analyzed with Proc MIXED (SAS), including fetal sex, day of gestation, and the interaction of these variables as fixed effects. The visualized size of the EVs ranged between 50 and 200 nm, with an average size of 87–105 nm, classifying most EVs as small EVs. The size of EVs was not affected by fetal sex or gestation day (P < 0.05). The concentration of the EVs did not follow a clear pattern in either MCGs or FCGs throughout pregnancy. Additionally, fetal sex or day of gestation had no significant effect on the EVs’ concentration (P < 0.05). Negative staining and electron microscopy revealed disc-like lipid bi-layered vesicles with size variations from 50 to 300 nm, confirming the previous classification done by flow nanoparticle analysis. To further classify the EVs by pathway of release, as recommended by the International Society for Extracellular Vesicles, we performed a western blot to test the presence of CD63, CD9, CD81, and TSG101 proteins, leading us to characterize them as exosomes. Overall, these results indicate that most EVs found in plasma during the pregnancy of beef cattle, regardless of the fetus’s sex, are primarily small EVs and exosomes. No difference was found between MCGs and FCGs in size, concentration, or type of EV during the whole gestation. The following characterization step would involve sequencing the EV cargo to determine whether the exosomes mainly originate from the fetus and fetal membranes or maternal tissues.