127 Extracellular vesicles from regions of turkeys’ oviduct inhibit sperm motility without significant effect on sperm viability
M. Rubilar A , D. Caamaño A , L. Méndez A , Y. S. Wong A , I. Martínez A , E. Inostrosa A , L. L. Rodríguez-Alvarez A and F. O. Castro AA
Like most avian females, turkey hens have sustained fertilization over time, which relies on the female’s ability to store and release sperm during ovulatory cycles. Specialized structures in the uterovaginal junction (UVJ) called sperm storage tubules (SST) confer this capacity by inhibiting sperm motility while sustaining viability. In turkey hens, SSTs ensure sperm viability for up to 70 days. The mechanisms that allow the conservation and survival of avian spermatozoa still need to be discovered. We hypothesize that extracellular vesicles (EVs) in the oviductal fluid are crucial for long-lasting sperm conservation in the turkey hen’s oviduct. Earlier, we showed that EVs were more abundant in the UVJ than in the magnum (M, the most extensive section of the oviduct, which has no SSTs) of the same hen. In vivo flushed EVs have different characteristics, size, abundance, and proteomic content than EVs obtained from cultured oviductal cells in vitro. Here, we aimed to test the effect of EVs extracted from in vivo flushing of UVJ or M of turkey hens on the viability and motility of rooster spermatozoa in vitro. EVs were obtained by lavage of the oviducts from the M and the UVJ of actively reproductive hens and concentrated by ultracentrifugation (100 000g for 18 h). Six biological replicates were obtained. Size and concentration of EVs were measured using a nano-tracking system (NTA NanoSight300, Malvern Panalytical). Sperm were collected by dorso-abdominal massaging of five donor roosters, counted, and pooled. Three replicates were performed. The collected semen was centrifuged (500g for 10 min) and resuspended at a final concentration of 1 × 107 sperm mL−1 in sperm diluent (136 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 1.26 mM CaCl2, 4.2 mM NaHCO3, 5.6 mM glucose, and 10 mM HEPES, pH 7.4) that contained the following concentration of EVs: 0, 4 × 107, 4 × 108, or 4 × 1010 EVs mL−1 from M or UVJ, at 38.5°C, 5% CO2 for 2, 4, 6, or 10 h. After the incubation period, sperm viability and motility were evaluated. For the former, eosin staining was used; for the latter, visual observation of total motility was scored using an inverted microscope. All experiments were done in triplicate. Separate ANOVA tests were performed to analyze the effect of concentration of EVs and incubation times on sperm motility and viability. Normality was assessed via Shapiro-Wilk with log10 transformation for ANOVA. The viability of spermatozoa was affected according to the concentration of EVs and their origin. The higher the concentration of EVs from M (4 × 1010), the better the viability; for EVs from UVJ, 4 × 108 EVs mL−1 sufficed for the highest viability (P < 0.05). There was a decrease in viability of spermatozoa at 6 h in all groups, more marked in the control group (with no EVs; P < 0.05). Motility decreased as a function of time as a rule in both control and EV-treated groups; however, at 10 h, EV from UVJ was associated with lowered sperm motility compared with M and control without decreasing viability. We concluded that incubation of spermatozoa with EVs from the UVJ of turkey hens significantly diminished sperm motility without affecting viability. EVs might be a tool for preserving turkey semen without refrigeration.
Funding was provided by Fondecyt Regular 1210349, ANID, Gobierno de Chile.
