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Gene editing and in vitro approaches to understand conceptus elongation in ungulates

Periodic Reporting for period 2 - ELONGAN (Gene editing and in vitro approaches to understand conceptus elongation in ungulates)

Reporting period: 2019-04-01 to 2020-09-30

The project aims to understand conceptus elongation, a largely unexplored developmental period exclusive from ungulates, which include the four most relevant mammalian farming species in Europe: cows, pigs, sheep and goat. Key developmental processes occurring during this period include the formation of a flat embryonic disc resembling that of humans, and gastrulation, which entails the differentiation of epiblast cells into the three germ layers. The importance of studying these developmental processes is dual. On one side, failures during this period account for most reproductive losses in livestock species, exerting a negative impact on European Bioeconomy. On the other, given the similarities in embryonic disc formation between humans and ungulates, ungulate embryos serve as a model to understand human diseases and conditions that origin during early development, such as spina bifida. The overall objectives of this project are 1) to uncover the role of specific genes on developmental processes by means of CRISPR-mediated ablation, 2) to determine the metabolic and proteomic composition of the uterine fluid to understand the requirements for conceptus elongation, and 3) to develop an in vitro system to achieve conceptus elongation without the need of experimental animals.
Relevant advances have been achieved in the development of CRISPR technology to edit the genome of livestock species, particularly in bovine. We have boosted the efficiency of precise gene ablation (KO) from <5 % to ~30 % by diminishing the incidence of a troublesome phenomenon called mosaicism. We have also improved targeted insertion rates from ~25 to ~50 % by chemical activation of a DNA repair pathway (HDR).
Using CRISPR technology, we have uncovered the essential roles of two proteins, one required to provide embryonic protection before implantation (ZP4) and other needed for fertilization (TMEM95). These novel findings are relevant for human or animal reproduction, as alteration of these proteins can be a root cause for infertility. Conversely, these proteins can be used as target to develop contraceptive methods. We have also explored the developmental failure caused by a deleterious haplotype (HH1) naturally occurring on dairy cows, observing that double carriers (embryos lacking the protein APAF1) develop normally through conceptus elongation, leading to developmental failures beyond maternal recognition of pregnancy, a relevant aspect for the reproductive management of this animals.
We have also analysed the compounds present in the bovine uterine fluid (UF) nourishing embryos before implantation. On this analysis we have observed great differences between the UF nourishing Day 7 blastocysts and that nourishing Day 14 elongated conceptuses, suggesting that embryo metabolic requirements are stage specific and providing relevant clues for the development of in vitro systems to develop embryos beyond the blastocyst stage.
Finally, we have developed an in vitro system that support bovine embryo development to the embryonic disc stage. Such advanced developmental stage had not been reached before in vitro in any farm animal and allow to study early developmental processes without the need of experimental animals. The system can be also used to test the success of diverse in vitro embryo production methods routinely used in the cattle industry without the need of transferring embryos to recipient animals.
The knowledge generated has been disseminated through 6 publications in indexed journals, 27 communications presented in 13 international scientific conferences, 2 conferences, 1 workshop and a training course oriented towards cattle industry, 2 workshops oriented towards policy makers, and 6 press releases.
The project has achieved relevant improvements for genome editing in livestock species. The efficiency of direct KO generation in bovine embryos has been boosted from <5 % to ~30 %, and the rate of targeted insertion of small sequences has been improved from ~25 to 50 %. These major improvements avoid the requirement of a colony of genetically modified animals from which to obtain the embryos lacking a specific gene (KO) required for developmental studies, as these embryos can now be directly obtained from in vitro produced embryos (i.e. without the need of experimental animals). One-step genome modification in livestock species is also extremely useful to generate large animal models required to advance in the knowledge, prevention and treatment of congenital or infectious diseases in animals and humans. The work plan aims to take advantage of the improved genome editing efficiencies to elucidate the function of candidate genes putatively involved in the developmental processes occurring during conceptus elongation. In this line, we have uncovered the third sperm protein being proved to be essential for mammalian fertilization (TMEM95), and the essential role on embryonic protection played by the protein ZP4, which is present in both ungulates and humans, but absent in mice, the only mammalian species where KO models were readily available.

The pioneer development of a fully in vitro system attaining bovine embryonic disc formation constitutes a significant milestone in developmental biology. This system will be extremely useful to decipher early gastrulation, which remains being a black box in developmental biology due to the prior absolute requirement for in vivo studies. The results we have obtained from the analysis of uterine fluids supporting the development of embryos at different stages have provided relevant clues to tune the in vitro system to achieve further stages of development on future experiments.