Vitrification of immature germinal vesicle-stage oocytes is a promising method in assisted reproduction but is associated with reduced developmental potential and low birth rates. Cumulus-oocyte complexes (COCs) express several connexins that form hexameric hemichannels, which interact head to head to create a gap junction or exist as unopposed free hemichannels. The latter are normally closed but open under stress conditions and may exert detrimental effects. We determined whether minimizing hemichannel opening and cell death during vitrification could improve COC quality. Bovine immature COCs underwent vitrification, storage and warming, followed by dye uptake to assess hemichannel opening and TUNEL staining to detect cell death. Based on these scores, we optimized the procedure by tuning the equilibration time, temperature, cryoprotectant concentration and extracellular Ca2+ concentration and assessed its impact on maturation, cleavage and blastocyst formation after parthenogenetic activation. We found that the major stressor resides in the cooling/warming phase of the vitrification procedure and observed that hemichannel opening and cell death in cumulus cells measure different aspects of cell stress. Optimization of the hemichannel and cell death readouts demonstrated that combined minimal hemichannel opening/cell death gave the highest cleavage rates but had no effect on maturation and blastocyst formation. Neither hemichannel nor cell death optimization performed better than the non-optimized protocol, leading to the conclusion that cell stress factors other than those detected by hemichannel dye uptake or TUNEL positivity are involved.
Katarzyna Joanna Szymańska, Nerea Ortiz-Escribano, Etienne Van den Abbeel, Ann Van Soom and Luc Leybaert
Minerva Ferrer-Buitrago, Davina Bonte, Petra De Sutter, Luc Leybaert and Björn Heindryckx
Oocyte activation is a calcium (Ca2+)-dependent process that has been investigated in depth, in particular, regarding its impact on assisted reproduction technology (ART). Following a standard model of signal transduction, Ca2+ drives the meiotic progression upon fertilization in all species studied to date. However, Ca2+ changes during oocyte activation are species specific, and they can be classified in two modalities based on the pattern defined by the Ca2+ signature: a single Ca2+ transient (e.g. amphibians) or repetitive Ca2+ transients called Ca2+ oscillations (e.g. mammals). Interestingly, assisted oocyte activation (AOA) methods have highlighted the ability of mammalian oocytes to respond to single Ca2+ transients with normal embryonic development. In this regard, there is evidence supporting that cellular events during the process of oocyte activation are initiated by different number of Ca2+ oscillations. Moreover, it was proposed that oocyte activation and subsequent embryonic development are dependent on the total summation of the Ca2+ peaks, rather than to a specific frequency pattern of Ca2+ oscillations. The present review aims to demonstrate the complexity of mammalian oocyte activation by describing the series of Ca2+-linked physiological events involved in mediating the egg-to-embryo transition. Furthermore, mechanisms of AOA and the limitations and benefits associated with the application of different activation agents are discussed.