82 First characterisation of extracellular vesicle population from caprine oviduct fluid

Extracellular vesicles (EVs) are phospholipidic nanoparticles from plasmatic membrane origin that are present in many biological fluids. These nanoparticles are responsible for transporting amino acids, lipids, and nucleic acids through intercellular communication processes. EVs are often classified as microvesicles (100–1000 nm) or exosomes (30–150 nm), which are currently reported by several studies as an important component present in murine, feline, bovine, and swine oviduct fluid (OF). EVs from the OF are used to improve in vitro fertilisation and embryo development. However, there is no evidence about characterisation of caprine oviduct EVs (oEVs). Thus, the aim of this study was to isolate and characterise, for the first time, caprine oEVs. Goat oviducts (n = 22) were obtained from 11 females without defined racial pattern at a local slaughterhouse, and dry transported to the laboratory in a thermal container at 4°C. Each oviduct was flushed with phosphate-buffered saline (PBS) to maximise the recovery of OF. Then, the OF samples were centrifuged twice: at 300 g and 12,000 g for 15 min at 4°C, and EVs were later isolated from caprine OF by the serial ultracentrifugation method. Goat oEVs were isolated from a single animal (n = 5) or in pool (n = 2; three animals/pool). Each OF sample was ultracentrifuged twice at 100,000 g for 90 min at 4°C. The final pellet was resuspended in 150 µL PBS, aliquoted and stored at −80°C until EVs characterisation procedures. The EVs characterisation was performed by nanoparticle tracking analysis to measure population size, distribution, and concentration. The transmission electron microscopy (TEM) was conducted to confirm the presence and characterise the morphology of EVs in caprine OF. Evaluation by TEM of caprine OF showed the identification of two different populations of EVs: vesicles with diameter ranging 150–350 nm, similar to microvesicles, and vesicles with diameter ranging 50–150 nm and cup shaped, like exosomes. The nanoparticles concentration showed a considerable variation among evaluated groups. In pool samples, the EVs concentration ranged between 0.86 ± 0.03 × 10⁹ and 2.07 ± 0.13 × 10⁹ particles/mL, while the concentration of individually isolated samples ranged between 0.42 ± 0.02 × 10⁹ and 3.17 ± 0.02 × 10⁹ particles/mL. Nevertheless, EVs size distribution from both groups showed a homogeneous population with mean ranging from 187 ± 2 to 273 ± 2 nm and mode ranging from 136 ± 5 to 196 ± 2 nm, respectively. Additionally, the cumulative frequency distribution in the goat OF showed a similar proportion in both sample types. It was observed that 10% of the vesicles had a diameter up to 120 ± 3 to 159 ± 3 nm (D10), while 50% ranged between 165 ± 2 to 242 ± 2 nm (D50; median), and 90% had a diameter up to 290 ± 17 to 430 ± 8 nm (D90). In conclusion, our first report about the presence of EVs in caprine OF is the first step of studying the involvement of these microvesicles and exosomes on goat reproductive events and opens the possibility of their use as a tool to improve different in vitro reproductive biotechnologies.

This work was supported by CNPq and FUNCAP.