EXTRACELLULAR VESICLES FROM RAM SEMINAL PLASMA TRANSPORT DECAPACITATION PROTEINS

ARMANI TOMAS; NICOLLI A. ; ZALAZAR L. ; PEREZ MARTINEZ S. ; CESARI A.

Mar del Plata

Congreso; REUNIÓN CONJUNTA SAIC, SAB, AAFE y AACYTAL 2023; 2023

SAIC, SAB, AAFE y AACYTAL

Extracellular vesicles (EVs) are micro and nanovesicles present in body fluids. Seminal plasma (SP) contains EVs derived from the epididymis, seminal vesicle, prostate and bulbourethral glands. They contain lipids, proteins and small regulatory RNAs from the original tissues and play a key role in the exchange of information with the sperm. Although EVs of various species have been characterized, there are not many studies in ram SP. This species of productive interest, presents a low performance in artificial insemination and in vitro fertilization using cryopreserved sperm. We have studied the role of small SP proteins termed as “decapacitation factors (DFs)” as suitable treatments to reduce and revert sperm cryodamage. The aim of this work was to isolate and characterize EVs from ram SP and determine if they participate in the transport of DFs. Three methods were used: ultracentrifugation (UC), precipitation with different ratios of polyethylene glycol (PEG) PEG:PS, and molecular size exclusion column (SEC). Total protein content, protein profile and the presence of CD9 tetraspanin as a vesicle marker were analyzed.Anti fibronectin (Fn) was used to study the presence of DFs from the Binder of Sperm Proteins family. Morphology was characterized by transmission electron microscopy. The total protein content was higher in EVs obtained by UC and SEC. The protein profile analyzed by SDS-PAGE showed that the EVs obtained by UC have a wide range of molecular weight (Mw) proteins, while those obtained by the other methods showed predominance of high or low Mw, according to the method. The EVs obtained by UC, SEC and PEG:PS 1:3 and 1:5 revealed positive for anti CD9 and Fn antibodies. This allows us to select three methods for the isolation of SP EVs and demonstrate that EVs transport decapacitation proteins, being a potential tool for supplementation of seminal extenders and to deep study the interaction of EV and ram sperm.