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 1 mL 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 1 h in PBS. The samples were then incubated at 38.5°C in 1 of the 2 conditions: (1) capacitation (SOF medium + 1000 IU of penicillin, 10 μg mL⁻¹ streptomycin, 10 μg 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 24 h. Total motility, hyperactive and progressive motility, and percentage of intact acrosomes (FITC-PNA) were assessed at 0, 1, 2, 18, and 24 h. Data were analysed by using SPSS software (IBM Corp., Armonk, NY) using a paired samples t-test with 95% CI. Vesicles of 30-100 nm 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 1 h (capacitation: 80 ± 3.7 v. 70 ± 3.5; non-capacitation: 78 ± 1.6 v. 66 ± 2.5%), 2 h (capacitation: 77 ± 1.3 v. 61 ± 1.1; non-capacitation: 74 ± 0.9 v. 55 ± 3.5%), 18 h (capacitation: 53 ± 1.5 v. 40 ± 4.7; non-capacitation: 30 ± 1.2 v. 21 ± 3.7%), and 24 h (capacitation: 30 ± 2.5 v. 12 ± 1.1; non-capacitation: 21 ± 2.1 v. 8 ± 0.9%). Regarding progressive and hyperactive motility and acrosome integrity, only hyperactive motility at 1 h using capacitation medium was significantly higher in the presence of EX than without it (18 ± 1.5 v. 9 ± 2.7%; P < 0.05). Next, we performed IVF using sperm with or without 1 h incubation with EX. After 18 h 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.