Granier, Shelby K., authorMcCue, Patrick, advisorGraham, James K., advisorHatzel, Jennifer, committee memberTesfaye, Dawit, committee member2023-06-012023-06-012023https://hdl.handle.net/10217/236570Fertilization in mammalian species relies on the activation of spermatozoa in the female reproductive tract by a consecutive series of events termed 'capacitation'. In vivo, ejaculated equine spermatozoa are deposited directly into the uterus and eventually arrive in the ampulla of the oviduct, which is the site of fertilization. However, the roles of the uterus, oviduct, and their secretions have on equine sperm capacitation is largely unknown. Extracellular Vesicles (EVs), including microvesicles and exosomes, are membrane enclosed nanoparticles released from most cell types that carry cargos of biologically active molecules that can affect nearby or distant recipient cells. EVs have recently been identified as playing a role in reproductive functions including sperm capacitation. The aims of the present study were: first characterize EVs collected from the uterine lumen of mares in both the estrus and diestrus phases of their reproductive cycles; and second investigate the effect these uterine EVs have on stallion sperm function. Uterine fluid from 6 mares was collected during both estrus and diestrus using a low volume uterine lavage then EVs were isolated from the fluid by ultracentrifugation, and EV concentration determined by nano-tracker analysis. The concentration of EVs obtained from estrus fluids (EEV) was 235 ± 164.029 billion EVs/mL and tended to be higher (p=0.07) than those obtained in diestrus fluids (DEV) (83.67 ± 89.328 billion EVs/mL). The average size of EVs were similar (p > 0.05) with values of 148.633 ± 11.35 nm for EEV and 146.183 ± 11.89 nm for DEV. Transmission electron microscopy delivered images of vesicles with characteristic cup-shape morphology and size consistent with NTA results. Immunoblotting confirmed the particles contained exosome markers TSG-101 and CD-63, and were negative for cytochrome C, a mitochondrial organelle marker, indicating these vesicles were indeed EVs. To determine the effect EVs have on sperm, semen from 3 Quarter Horse stallions were cryopreserved, and EVs added to samples after thawing. In the first experiment, EVs or PBS void of EVs were fluorescently labeled and incubated with frozen-thawed stallion spermatozoa for one hour and uptake was evaluated by fluorescent microscopy. Fluorescence was observed only in sperm incubated with EVs, and a greater fluorescent intensity detected in EEV treated sperm. In a second experiment, spermatozoa from each stallion were co-cultured with EEV, DEV, and PBS void of EVs (control) for 90 minutes and sperm functions associated with capacitation, including hyperactivated motility, and acrosome reactions, were evaluated using a computer assisted semen analysis unit (CASA) and flow cytometry. The percentages of hyperactively motile sperm were higher (p < 0.05) for EEV treated sperm compared to control and DEV. In addition, the percentage of acrosome reacted sperm was higher (p < 0.05) for sperm treated with EEV and DEV when compared to control. In summary, these results confirm that: 1) EVs can be isolated from uterine fluid of mares, 2) uterine derived EVs can be taken up by stallion spermatozoa, and 3) uterine derived EVs have a biological effect on stallion spermatozoa function in vitro. Consequently, it is hypothesized that EVs from the mare reproductive tract will have similar biological effects on stallion sperm function in vivo.born digitalmasters thesesengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.equine reproductionfertilitytheriogenologyextracellular vesiclescapacitationspermatozoaText