194 Regions of the oviduct of Creole turkey hens—with and without sperm-storage tubules—secrete extracellular vesicles, according to tissue, reproductive status, and origin of collection (in vivo-in vitro)

Turkey hens have the ability to preserve viable sperm in the so-called sperm storage tubules (SST) for up to 70 days, though the molecular mechanisms involved are unknown. We hypothesised that extracellular vesicles (EV) secreted by SSTs might be involved in this process. The objective was to characterise the EV of two oviducal regions of Creole turkey hens: (1) uterovaginal junction (UVJ) containing SST and (2) magnum (M) not containing SST. The samplings were carried out in three reproductive stages: negative photoperiod (NP), positive photoperiod (PP), and positive photoperiod in ovulation (PPinO). EV were collected from oviducal fluid (in vivo) from UVJ and M in NP, PP, and PPinO, and from supernatants of cultured oviducal cells in vitro from UVJ and M in PP and PPinO. After slaughter of the hens, the oviducal fluid was collected by flushing (5 mL of PBS 1% and AAM 2% at 38.5°C) and tissue was isolated and digested to carry out primary cell cultures (1 mg/mL of collagenase type II at 38.5°C for 40/60 min). Cells were cultured with DMEM/F12 without phenol red, 10% FBS, 2% turkey hen serum, 1% AAM, 1% MEM NEAA, 1% pyruvate, 0.55 mM β-mercaptoethanol, and 10 ng/mL epidermal growth factor (EGF) at 39°C and 5% CO₂. Cultures with >70% confluence and >60% epithelial cells (morphology) were selected. The cells were cultured for 24 h in the same medium, but with EV-depleted FBS. EVs were concentrated by ultracentrifugation and processed by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blot (WB). The results (log10) of NTA were evaluated by ANOVA and l.s.d. Fisher (P < 0.05). In in vivo samples of NP, PP, and PPinO, the size variable showed that the reproductive stage factor (RS) presents differences: higher in PP and lower in PPinO. The concentration variable presents differences in RS, greater in PPinO and PP, and in tissue (T), greater in UVJ. The combined effect of RS + T shows differences with a lower value in NP-M. In in vivo and in vitro samples of PP and PPinO, the size variable presented differences in RS, greater in PP, and the origin (O), greater in vitro. The combined effects showed differences in; RS+T, higher in PP-UVJ and PP-M and lower in PPinO-UVJ and PPinO-M; RS+O, higher in PP-in vitro and lower in PPinO-in vivo; T+O, higher in M-in vitro and UVJ-in vitro and lower in M-in vivo and UVJ-in vivo. The concentration variable presents differences in O, greater in vivo. The combined effects showed differences in; RS+O, higher for PP-in vivo and PPinO-in vivo and lower for PP-in vitro; T+O, higher in UVJ-in vivo and M-in vivo and lower in M-in vitro and UVJ-in vitro. TEM showed characteristic EV morphology (cup shape) and WB confirms the presence of EV. Currently, transcriptomic and proteomic analysis of EV content is ongoing. In conclusion, there are differences in the characteristics and secretion of EV according to RS, O, and T. This can be of importance to understand the molecular mechanisms involving EV in prolonged semen storage in turkeys.

This research was supported by FCYT REGULAR 1210349.