22 The zona pellucida is required for normal development of in vitro-produced cat embryos

Abstract

Limited information have been reported regarding to the role of the zona pellucida during embryo development in the domestic cat. In the present research we compared in vitro and in vivo development of cat embryos generated with versus without the zona pellucida. Embryos were produced by IVF and cultured up to blastocyst stage accordingly to experimental group: 1) with intact zona pellucida (ZI) and 2) without zona pellucida (ZF). Ovaries of domestic cats were collected by ovariohysterectomy and cumulus-oocyte complexes (COCs) were recovered by slicing. For IVM, COCs were cultured in supplemented medium-199 for 26-28 h in a 5% CO₂, 5% O₂, and 90% N₂ atmosphere at 38.5°C. For IVF, 1.5-2.5 × 10⁶ epididymal spermatozoa/mL were incubated with 20-30 COCs in supplemented Tyrode's albumin lactate pyruvate medium for 18 h in a 5% CO₂ atmosphere at 38.5°C. The zona pellucida of the presumed zygotes was removed by 2-4 min of incubation in 2 mg mL⁻¹ pronase. In ZI and ZF groups, embryo culture was done in 4-well dishes; in the ZF group the well of the well system was used. The IVC was done in supplemented synthetic oviductal fluid medium, in a 5% CO₂, 5% O₂, and 90% N₂ atmosphere at 38.5°C for 8 days. The frequencies of cleavage and development to the morula and blastocyst stages were determined. The relative expression of the pluripotency markers OCT4, SOX2, and NANOG was evaluated by RT-qPCR in the blastocysts using the standard curve method. The SDHA gene was used as internal control. Additionally, the in vivo development was evaluated. For this, cat recipients were synchronized with 200 IU equine chorionic gonadotrophin followed by 100 IU human chorionic gonadotrophin 4 days later, and embryo transfers (ET) were made by mid-ventral laparotomy. The Wilcoxon nonparametric test was used to evaluate the developmental competence and the gene expression analysis. Nine and six replicates were performed in the ZI and ZF groups, respectively. No differences were observed between the ZI and ZF groups in the cleavage rate: 155/239 (64.9%) and 116/177 (65.5%), morulae rate: 115/155 (74.2%) and 68/116 (58.6%), and blastocysts rate: 51/155 (32.9%) and 36/116 (31.0), respectively (P > 0.05). No differences were observed in the expression of OCT4 (P > 0.05). However, the expression of SOX2 and NANOG was lower in blastocysts from the ZF group than in those from the ZI group (P < 0.05). Finally, three ET were done at the blastocyst stage in both groups. In the ZI group, 8-16 Day 7 blastocysts were transferred per cat, two pregnancies were obtained with one gestational sac in each cat (2/3, 66.6%), and one live kitten was born at Day 64 of pregnancy (1/3, 33.3%). In the ZF group, 7-14 were transferred per cat, and no pregnancies were obtained after ET. In conclusion, in the domestic cat, zona pellucida removal at the presumed zygote stage did not affect in vitro development to blastocyst but affect negatively the expression of SOX2 and NANOG. However, the low survival after ET of ZI embryos is a possible indicator that embryo quality might be affecting survival rates. Nonetheless, these results add credence to previous ET studies that provide indirect evidence of a crucial role of the zona pellucida for successful implantation of cat embryos.