Endometrial and oviduct extra-cellular vescicles for in vitro equine sperm hyperactivation and oocyte fertilization.

This study explores the potential use of extracellular vesicles (EVs) from the female genital tract to promote in vitro fertilization (IVF) in horses, a process that is conventionally unsuccessful in this species. The research showed that these EVs could be involved in triggering hyperactivation in equine sperm and improve in vitro fertilization rates.

Methodology

• In an effort to replicate the natural equine spermatozoon-oviduct interaction, the researchers sourced extracellular vesicles (little “packages” of molecules) from female horse genital tracts.
• The genital tracts were collected during late estrus from three mares. After digestion with collagenase and trypsin, the cells were cultured in a specialized system to mimic their natural configuration.
• Extracellular vesicles were produced by culturing endometrial cells and oviductal spheroids in a serum-free medium for three days.
• The interaction between these EVs and spermatozoa was examined using fluorescence microscopy, with different labels used on the EVs.
• Sperm samples, taken from three stallions, were incubated in capacitating medium for 6 hours. Afterwards, they were induced to hyperactivate by further incubation in the same medium, this time supplemented with various combinations of EVs. A control group was treated the same way but without the addition of EVs.
• The researchers assessed sperm motility, EV incorporation, and acrosomal reactions (a vital process in fertilization) by using a system called CASA and staining with FITC-PNA/PI (a fluorescence technique).

Findings

• Extracellular vesicles from the apical horn were incorporated into the sperm head after 4 hours, while EVs from oviductal spheroids were incorporated into the middle tract after 1 hour. In contrast, EVs from the proximal horn were not incorporated.
• The rate of sperm cells undergoing acrosomal reaction was 50.25% with apical horn EVs, 57.14% with oviductal spheroid EVs, and 71.42% when these two types of EVs were combined.
• In comparison, the rate in the control group (without EVs) was only 15%.
• After the process of in vitro fertilization, 44% of oocytes displayed both male and female pronuclei, indicating successful fertilization. The control group, on the other hand, showed no instances of fertilization.

Conclusion

• The researchers concluded that extracellular vesicles derived from the apical horn and oviduct could promote sperm hyperactivation, a critical precondition for successful in vitro fertilization.
• The findings could significantly impact equine reproductive biotechnology by providing a possible avenue for successful in vitro fertilization.