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Vertebrate reproductive science and technology

6 CRISPR-Generated ZP4 Knockout Rabbits Exhibit Impaired In Vivo Pre-Implantation Development

I. Lamas-Toranzo A , N. F. Balvís A , P. L. Lorenzo B , P. G. Rebollar C , M. Avilés D and P. Bermejo-Álvarez A
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A Dpto Reproducción Animal, INIA, Madrid, Madrid, Spain;

B Dpto Fisiología Animal, Universidad Complutense de Madrid, Madrid, Madrid, Spain;

C Dpto Producción Animal, Universidad Politécnica de Madrid, Murcia, Murcia, Spain;

D Dpto Biología Celular e Histología, Universidad de Murcia, Murcia, Murcia, Spain

Reproduction, Fertility and Development 30(1) 142-142
Published: 4 December 2017


Mammalian oocytes are surrounded by a glycoprotein layer termed the zona pellucida (ZP), which acts as a protective barrier during pre-implantation embryo development and plays essential roles during follicular development and fertilization. This structure is composed of 4 different proteins in several species such as rabbits and humans. However, the oocytes of the only species for which knockout (KO) embryos are readily available, the laboratory mouse, lacks one of these proteins (ZP4), so its function remains elusive. To determine the role of ZP4, we have generated ZP4 KO rabbits by CRISPR and compared their reproductive function to that of heterozygous (Hz) and wild type (wt) rabbits. Following mating with wt males, female KO rabbits showed a clear impairment in reproductive performance compared with Hz or wt females, with only 1 out of 5 KO females producing a small litter of 4 pups, whereas all mated Hz and wt females exhibited normal litter sizes (wt 9.2 ± 0.6; Hz 10.6 ± 0.5; mean ± SEM). In order to elucidate the cause for the impaired delivery rate, ovulation rates and cleavage rates were initially assessed, with no differences observed between groups (oocytes ovulated: wt 11.7 ± 1; Hz 15 ± 2.9; KO 13.3 ± 2.9; cleavage rates (%): wt 81.7 ± 0.1; Hz 95.5 ± 0.1; KO 87.3 ± 0.1), which suggests that ZP4 does not play a critical role in folliculogenesis or fertilization. However, when expanded blastocysts were recovered at Day 6 after mating, embryo development was found to be clearly impaired in embryos lacking ZP4, as none of the embryos obtained from 2 KO females were able to undergo blastocyst expansion. Morphological differences were observed in the zona pellucida from KO rabbits, being irregular, noticeably less elastic and easier to deform, and significantly thinner (thickness: wt 15.2 ± 1.5 µm; Hz 15.3 ± 1.4 µm; KO 10.9 ± 0.7 µm; ANOVA P < 0.05). These morphological differences suggest that rabbit ZP without ZP4 may fail to protect the embryo during development or may physically impede blastocyst expansion.

This study is supported by the projects AGL2014-58739-R and RYC-2012-10193 (to PBA), AGL2015-70159-P (to MA) and AGL2015-65572-C2-1-R (to PGR and PL). ILT and NFB are supported by FPI grants.

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