Formation and characterization of vesicles from day-10 horse conceptuses

The objectives of this research were 1) to determine if cellular vesicles could be formed from Day-10 horse conceptuses similar to trophoblastic vesicles reported for other domestic species and 2) to characterize various aspects of their development in vitro and their ability to withstand the freeze-thaw process. Twenty-five conceptuses were recovered from lighthorse mares on Day 10 after ovulation. After two washes in 0.05% trypsin, each conceptus was placed in 0.25% trypsin until the capsule thinned. Mechanical dispersion with a glass pipette resulted in a combination of individual cells and cell clumps. When cultured in vitro, all preparations exhibited both partially and completely formed vesicles within 24 h. Cellular monolayers also developed within the first 24 h of culture and were predominant by 96 h. Within 13 to 20 d, all monolayers developed dense areas of cells that eventually released from the cell matrix and aggregated to form vesicles. Progesterone and total estrogen concentrations in media samples were lower (P < 0.05) for conceptuses that had required long trypsinization periods for dispersion. Pregnant mare serum gonadotropin was not detectable in any samples. Vesicles cultured in nontreated tissue culture flasks.doubled in size within 24 h but did not increase further by 48 h. Of 234 vesicles frozen after 48 h of culture, the postthaw viability, as measured by the ability to return to prefreeze characteristics, was 34, 21 and 11% after 24, 96 and 168 h in culture, respectively. We conclude that vesicles can be formed by enzymatic dispersion of Day-10 horse conceptuses. However, monolayers were the predominant result of both the initial dispersions and the long-term culture of vesicles. Vesicles showed a limited ability to grow in vitro.