36 Comparative proteomic analysis of the extracellular vesicles secreted by the oviduct of turkey hens in vivo and in in vitro cultured oviductal cells

In the oviduct of turkey hens, viable spermatozoa are preserved for more than 2 months at cryptic structures called sperm storage tubules (SST). Previously we determined the size, concentration and specific markers of oviducal extracellular vesicles (EV) isolated from separate flushing of the uterovaginal junction containing SST and of magnum, not containing SST, as well as from in vitro cell cultures derived from these flushing. We propose that EV might be involved in sperm preservation at the SST. With the aim to stablish a suitable source of EV for potential identification of mechanisms involved in sperm preservation in SST, here we evaluated the proteomic content and the differential expression of proteins in the EV of oviducal lavages and in the supernatant of cell cultures isolated from said lavages. We used hens in the ovulation window during which, active preservation of sperm occurs at the SST. Collection of samples and isolation of EV was described in Rubilar et al. (2022 Reprod. Fert. Dev. 35, 225). Briefly, oviduct tracts of sacrificed hens were flushed with 5 mL of 1× phosphate-buffered saline and 2% of antibiotic-antimycotic solution, at 38.5°C. From these washings, oviducal EV were isolated (O-EV group). The oviducts of 10 hens were flushed and the oviducal tissue from the same animals was digested (1 mg/mL of collagenase type II at 38.5°C for 40/60 min) to generate cell cultures (S-EV group). Ten lavages and 10 primary cell cultures were generated and all produced EV which were concentrated by ultracentrifugation from each source. The EV were further analysed by nanoparticle tracking analysis. After EV collection, proteins were extracted, quantified and analysed by mass spectrometry (liquid chromatography with tandem mass spectrometry) using data dependent acquisition as data capture method. Results were analysed using the FragPipe software with the MSFragger search engine, using a Meleagris gallopavo proteome database available from Uniprot. To identify differentially expressed proteins (DEPs), label-free quantification (LFQ) of proteins based on intensity values, was performed only for proteins present in at least 3 of the 5 samples analysed by condition, using the Perseus software v2.0.7. Intensities were transformed to log2 filtered and normalized by medians using T-Test with a Benjamini–Horschberg multiple correction test (adjusted P < 0.05). A total of 764 proteins were identified by LFQ, of these, a set of 272 proteins were differentially expressed when EV were isolated from oviduct lavage (O-EV), compared to those secreted by cultured cells from these lavages. A total of 123 proteins were down-regulated and 149 were up-regulated in the lavages. The gene ontology and biological functions of some of these proteins is discussed. The biological functions represented the most in the overexpressed proteins were cell adhesion, GTPase activity and cell differentiation, while immune response and zymogen activation were the most un-represented. These findings support our previous study in which amount and homogeneity of EV was better in O-EV compared to S-EV. In conclusion, lavage of oviducal tissue yields EV with a set of DEP that can be of value to study preservation of avian sperm in SST.

This research was supported by FCYT regular 1210349.