25 Comparative expression of specific microRNAs between domestic cat blastocysts cultured with and without the zona pellucida on Days 8 and 10 of in vitro development

Domestic cat blastocysts cultured without the zona pellucida have a reduced implantation capacity and an altered expression and release of specific miRNAs at Day 7 of in vitro culture (IVC). However, the expression pattern and release of these miRNAs at later developmental stages have not been previously analyzed. The objective of this study was to evaluate the expression of specific miRNAs released into the culture medium by domestic cat blastocysts with and without zona pellucida at Days 8 and 10 of IVC. The study had two experimental groups: (1) domestic cat blastocysts generated by IVF and cultured in vitro (zona intact, ZI group) and (2) domestic cat embryos generated by IVF and cultured in vitro without the zona pellucida (zona free, ZF group). Ovaries and testicles from domestic cats were obtained by ovariohysterectomy and orchiectomy, respectively. The cumulus–oocyte complexes (COCs) were subjected to IVM, in 5% CO₂, at 38.5°C, for 26 h. For IVF, 20–30 matured COCs were co-cultured 1.5 to 2.5 × 10⁶ sperm mL⁻¹, in 5% CO₂, at 38.5°C, for 24 h. In the ZF group, the zona pellucida was removed after IVF by incubation in 2 mg mL⁻¹ pronase for 4 min. Then, ZF-embryos were cultured in microwells to prevent blastomere disaggregation until the blastocyst stage was reached at Day 7. In both experimental groups, IVC was done in supplement synthetic oviductal fluid (SOF) medium with 10 µL mL⁻¹ insulin-transferrin-selenium and 0.3% BSA (without FBS) in a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 38.5°C for 5 days. At Day 5 SOF was replaced by medium-199. One proportion of blastocysts were cultured until Day 8 and the other proportion until Day 10; the culture medium was collected at both of these days. The relative expression of miRNAs related to trophoblast differentiation (miR21, miR29, miR96), IVF failure (miR25), blastocyst formation (miR24, miR199), and implantation and immune response (miR98) was analyzed by RT-qPCR, using the standard curve method. miR191 was used as internal control. The Kruskal-Wallis nonparametric test was used to estimate significant differences (P < 0.05). No statistical differences were observed in the relative expression of miR21, miR29, miR96, and miR199 at Day 8 between groups. At Day 8 of IVC, the relative expression of miR24 and miR25 was higher in the conditioned medium of ZF-blastocysts than in the medium of ZI-blastocysts. Meanwhile, the relative expression of miR98 was significantly higher in the conditioned medium of ZI-blastocysts. Furthermore, the relative expression of miR98 significantly increased from Day 8 to Day 10 in the conditioned medium of ZI-blastocysts. However, no differences in the relative expression of all other miRNAs were observed at Day 10 between groups. In conclusion, ZF-blastocysts maintain an altered release of miR24, miR25, and miR98 at Day 8. Interestingly, the expression of miR98 increased from Day 8 to Day 10 in the ZI group, but this remained unchanged in the ZF group. The altered release of these miRNAs might be related to the reduced implantation capacity of feline blastocysts cultured without the zona pellucida. Identify the functions of these miRNAs will apport new information about the influence of the zona pellucida during embryo development and implantation in this species.