Fertilization and early embryo development are regulated by a unique maternal-gamete/embryo cross-talk within the oviduct. Recent studies have shown that extracellular vesicles (EVs) within the oviduct play important roles in mediating this developmental process. Here, we examined the influence of oviductal EVs on sperm function in the domestic cat. We demonstrated that (1) EVs are enriched in proteins related to energy metabolism, membrane modification, and reproductive function; (2) EVs bound and fused with the membranes of the acrosome and mid piece; and (3) incubating sperm with EVs improved motility, fertilizing capacity of cat spermatozoa and prevented acrosomal exocytosis in vitro . These findings indicated that oviductal EVs mediate sperm function and fertilization in the cat and provides new insights to improve sperm cryopreservation and in vitro fertilization in the domestic and wild felids and human.

Zinc is a critical component in a number of conserved processes that regulate female germ cell growth, fertility, and pregnancy. During follicle development a sufficient intracellular concentration of zinc in the oocyte maintains meiotic arrest at prophase I until the germ cell is ready to undergo maturation. An adequate supply of zinc is necessary for the oocyte to form a fertilization-competent egg as dietary zinc deficiency (ZD) or chelation of zinc disrupts maturation and reduces oocyte quality. Following sperm fusion to the egg to initiate the acrosomal reaction, a quick release of zinc, known as the zinc spark, induces egg activation in addition to facilitating zona pellucida hardening and reducing sperm motility to prevent polyspermy. Symmetric division, proliferation, and differentiation of the pre-implantation embryo rely on zinc availability both during oocyte development and post-fertilization. Further, the fetal contribution to the placenta, fetal limb growth, and neural tube development are hindered in females challenged with ZD during pregnancy. In this review, we discuss the role of zinc in germ cell development, fertilization, and pregnancy with a focus on recent studies in mammalian females. We further detail the fundamental zinc-mediated reproductive processes that have only been explored in non-mammalian species and speculate on the role of zinc in similar mechanisms of female mammals. The evidence collected over the last decade highlights the necessity of zinc for normal fertility and healthy pregnancy outcomes which suggests zinc supplementation should be considered for reproductive age women at risk of ZD.

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.