207 Cat oviductal exosomes as a tool to improve gamete rescue of endangered felids: effects on sperm capacitation and in vitro fertilization

Abstract

Exosomes play important roles in reproduction, for example, facilitating fetal-maternal interaction and establishment of pregnancy. However, little is known about how oviductal exosomes affect sperm function. We investigated the effects of oviductal exosomes on sperm capacitation and fertilizing ability in domestic cats as a model for endangered felids. Oviducts were collected from cats (1-5 years) after elective spaying and flushed with 1mL of PBS. Exosomes (EX) were isolated using the Total Exosome Isolation kit (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) and labelled with BODIPY dye (boron dipyrromethene). Unattached dye was removed by using Exosome Spin Columns (MW 3000, Invitrogen). Presence and purity of EX was confirmed by transmission electron microscopy (TEM). To investigate the effects of EX on sperm capacitation, semen was recovered from epididymis of 5 cats (3 replicates) after elective neutering. One million spermatozoa mL−1 were incubated with or without EX (1V sperm to 2V total exosomes from 2 oviducts) for 1h in PBS. The samples were then incubated at 38.5°C in 1 of the 2 conditions: (1) capacitation (SOF medium+1000IU of penicillin, 10μg mL−1 streptomycin, 10μg mL−1 heparin, 20 µM penicillamine, 10 µM hypotaurine, 1 µM epinephrine); and (2) non-capacitation (capacitation medium without heparin, penicillamine, or hypotaurine) for up to 24h. Total motility, hyperactive and progressive motility, and percentage of intact acrosomes (FITC-PNA) were assessed at 0, 1, 2, 18, and 24h. Data were analysed by using SPSS software (IBM Corp., Armonk, NY) using a paired samples t-test with 95% CI. Vesicles of 30-100nm were observed by TEM, indicating successful isolation of EX, which were found to bind to both acrosome and mid-piece (BODIPY labelling). Sperm incubated with EX exhibited higher motility than those without EX (P<0.05 for all comparisons) at 1h (capacitation: 80±3.7v. 70±3.5; non-capacitation: 78±1.6v. 66±2.5%), 2h (capacitation: 77±1.3v. 61±1.1; non-capacitation: 74±0.9v. 55±3.5%), 18h (capacitation: 53±1.5v. 40±4.7; non-capacitation: 30±1.2v. 21±3.7%), and 24h (capacitation: 30±2.5v. 12±1.1; non-capacitation: 21±2.1v. 8±0.9%). Regarding progressive and hyperactive motility and acrosome integrity, only hyperactive motility at 1h using capacitation medium was significantly higher in the presence of EX than without it (18±1.5v. 9±2.7%; P<0.05). Next, we performed IVF using sperm with or without 1h incubation with EX. After 18h IVF, presumptive zygotes were stained with Hoechst 33342 and observed under a fluorescence microscope to assess fertilization and polyspermy. Preliminary data (30 oocytes/group) revealed that sperm incubation with EX reduced polyspermy (6±4% v. 20±8%) and improved normal fertilization (28±14vs 8±4%), although the differences were not significantly different (P>0.05 for both groups). In conclusion, the findings indicate that oviductal EX play roles in regulating sperm function by enhancing sperm motility. Further studies are needed to confirm the impact of EX on fertilization and how this strategy can be applied to endangered felid conservation.