33 Domestic cat embryos cultured without the zona pellucida have an altered protein pattern at the blastocyst stage

Domestic cat blastocysts cultured without the zona pellucida have a reduced implantation capacity and an altered expression of pluripotency and differentiation genes. Despite that, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were made: (1) domestic cat embryos 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 complex (COC) were subjected to in vitro maturation (IVM), in 5% CO₂, at 38.5°C, for 26 h. The IVF was done co-cultured 1.5 to 2.5 × 10⁶ spermatozoa/mL with 20–30 COCs. In the ZF group, after IVF the zona pellucida was removed by incubation in 2 mg/mL pronase for 4 min. Then, ZF embryos were cultured in microwells to prevent blastomere disaggregation. In both experimental groups, in vitro culture was done in supplement M199, without fetal bovine serum and with 10 µL/mL insulin-transferrin-selenium, in a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂, at 38.5°C for 7 days. Cleavage, morula, and blastocyst rates were estimated at Days 2, 5, and 7, respectively. Day 7 blastocysts were subjected to liquid chromatography tandem mass spectrometry. For this, protein extraction was done, and then protein digestion was made using trypsin; 200 ng of the peptides was injected in a nano-ultra-high-performance liquid chromatography (nanoElute, Bruker Daltonics) coupled to a mass spectrometer timsTOF Pro (Bruker Daltonics). The data were analysed using the software MSFragger 3.5 and the plataform Fragpipe v18.0, the Felis catus database from Uniprot was used to identify the standard proteome. The Wilcoxon nonparametric test was used to evaluated differences in in vitro embryo development (P < 0.05). For the proteomic analysis, t-test along with the multiple correction Benjamini-Horschberg test was used to identify differentially expressed proteins (P < 0.05). No statistical differences (%Mean ± standard deviation) were found in the cleavage: ZI = 140/351 (39.9 ± 11.6), ZF = 201/470 (42.8 ± 15.7); morula: ZI = 71/140 (50.7 ± 14.3), ZF = 95/201 (47.3 ± 27.9); and blastocyst rates: ZI = 44/140 (31.4 ± 8.5), ZF = 63/201 (31.3 ± 17.8). Regarding to proteomic analysis, 443 proteins were commonly shared by all the ZF blastocyst, and 142 proteins were identified in all the ZI blastocyst. From these proteins, 42 were differentially expressed between ZF and ZI blastocysts. In the ZF blastocysts, 22 proteins were up-regulated, among which we identified different types of heat shock proteins (HSPA8, HSP90B1, HSPA5), proteins related to metabolic process (IGF2BP1, COX5B, RPL8) and biological regulation (TLE6, CCT8, RNPS1), among others. Furthermore, 20 proteins were down-regulated, and some of them have an important role in embryo development (HMGA1, MOEP19, PLK1). In conclusion, embryo culture without the zona pellucida did not affect in vitro development but alters the protein profile of domestic cat blastocysts.