Connexin Channel Block with Gap26 Mimetic Peptide Improves Blastocyst Outcome and Development after Vitrification, Storage and Warming
Authors: Szymanska Katarzyna Joanna , Ortiz‐Escribano Nerea , Bol Melissa , Vandenberghe Lynn , Decrock Elke , Van den Abbeel Etienne , Van Soom Ann , Leybaert Lcu 
Published: November 2023

Connexin proteins assemble into hemichannels and gap junctions in the plasma membrane, and are abundantly expressed in mammalian embryos. Gap junctions facilitate cell‐cell communication and are crucial for embryo development. However, under cellular stress conditions, gap junctions can promote cell death by bystander effects. Additionally, hemichannels, which are normally closed, open under stress conditions and create a leakage pore through which crucial ions and metabolites, including Ca2+ and ATP, diffuse thereby leading to cell injury/death. We previously demonstrated that blocking gap junctions/hemichannels with connexin mimetic peptides protects against biobanking induced cell injury/death in human blood vessels (1).Vitrification is a relatively new method of cell and tissue cryopreservation without ice crystal formation that relies on solidification of a liquid by extreme elevation in viscosity and rapid freezing/thawing. This technique is widely used for oocytes and embryos but the process is not optimal and reduces their developmental capabilities. We hypothesize that vitrification/warming and the use of cryoprotectants are a strong stimulus for connexin hemichannel opening and subsequent cell injury in blastomeres, leading to impairment of embryonic development. While a couple of major improvements have been made in vitrification of human and cattle oocytes and blastocysts, the outcome is still not satisfactory.Preliminary experiments on bovine in vitro matured oocytes show improved survival and embryo development. We investigated if Gap26 peptide, which is identical to a short sequence on the first extracellular loop of connexin43 and interferes with channel function, can improve the outcome and development of vitrified bovine embryos. Blastocysts were cultured and hatching from the zona pellucida was evaluated. We performed TUNEL assays to assess the effect of vitrification on cell death in blastocysts. We found that supplementation of the vitrification and warming solutions with Gap26 had significantly improved survival and development rates. Interestingly, we also observed a higher hatching rate compared to the non‐supplemented control group. The number of TUNEL positive cells in all groups were similar. Additional experiments confirmed that Gap26 can only block hemichannels within minutes of exposure with no effect on gap junctions.These results indicate that blocking of connexin hemichannels plays an important role in reducing the negative effects of vitrification on cells. TUNEL analysis suggests that the effects of the cell injury caused by vitrification mainly present later during development, rather than immediate cell death. In conclusion, vitrification with Gap26 improves the functional state of the cells and entire embryo, and enhances their developmental potential. Gap26 supplementation may be a promising strategy for embryo and oocyte vitrification, also in other species.Support or Funding InformationResearch supported by the Fund for Scientific Research Flanders, Belgium (FWO, grant number G.0A54.13 and G.0320.15) and the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT, grant number 121446)