29 The in vitro culture of domestic cat embryos without zona pellucida increases the expression of YAP1 and EOMES at the blastocyst stage

Domestic cat blastocysts cultured without zona pellucida have a reduced implantation capacity after embryo transfer. The objective of this study was to evaluate the expression pattern of trophectoderm markers in domestic cat blastocysts cultured without zona pellucida. For this, two experimental groups were evaluated: (1) Domestic cat embryos generated by IVF and cultured in vitro (zona-intact group [ZI]), and (2) domestic cat embryos generated by IVF and cultured in vitro without zona pellucida (zona-free group [ZF]). Ovaries were collected by ovariohysterectomy. Immature cumulus-oocyte complexes (COCs) were matured in vitro in supplemented M-199 in a 5% CO₂ atmosphere at 38.5°C for 24 h. For IVF, 20–30 COCs were co-cultured with 1.5–2.5 × 10⁶ spermatozoa/mL in supplemented TALP medium in 5% CO₂ at 38.5°C for 24 h. For in vitro cultures in the ZF group, the zona pellucida was removed by incubation in 2 mg/mL pronase for 2–4 min. Embryos from the ZF group were cultured into microwells using the well of the well system. In the ZI and ZF groups, embryos were cultured in 500 µL of supplemented SOF medium, in a gas atmosphere of 5% O₂, 5% CO₂ and 90% N₂, at 38.5°C, for seven days. The cleavage, morula, and blastocyst rates were estimated. The relative expression of trophectoderm (YAP1, TEAD4, CDX2, and EOMES), cell adhesion (E-cadherin), and apoptosis (CASP3) markers was evaluated by RT-qPCR in the blastocysts. The gene SDHA was used as internal control. The Wilcoxon nonparametric test was used to evaluate statistical differences (P < 0.05). Regarding the in vitro development (% mean ± s.d.), no differences were observed in the cleavage rate: ZI = 271/530 (51.4 ± 14.1) and ZF = 216/375 (57.6 ± 10.6); morula rate: ZI = 140/271 (51.6 ± 12.6) and ZF = 124/216 (57.4 ± 13.6); and blastocyst rate: ZI = 60/271 (22.1 ± 12.0) and ZF = 55/216 (25.5 ± 14.0). No statistical differences were observed in the relative expression of TEAD4, CDX2, E-cadherin, and CASP3 between blastocysts from the ZI and ZF groups. The relative expression of the trophectoderm markers YAP1 and EOMES was significantly higher in ZF-blastocysts compared to ZI-blastocysts. In conclusion, the culture of domestic cat embryos without zona pellucida does not affect in vitro development. However, it generates an overexpression of the trophectoderm markers YAP1 and EOMES at the blastocyst stage. More studies are needed to elucidate whether this overexpression might affect the in vivo development.

This research was supported by ANID Fondecyt Postdoctorado 3200352.