A Treatise on Current Advances in Egg Activation Protocols

Kaniska Mukherjee, Rania Al-Hawas, Debasish Ghosh

Abstract: Ca²⁺-oscillations pave the way for activation of eggs in many animals that are discussed here. This so called “repetitive” rises is instrumental in embryonic development; the comparative analysis in various animals below illustrates the importance of Ca²⁺ and Sr²⁺ (to some extent), both being divalent cations, in the process of egg development. Only recently it has been found that a sperm-derived factor “phospholipase Cᶉ”, is the most likely sperm candidate,that is pivotal in the Ca²⁺-oscillations. Phospholipase Cᶉ is capable of converting phosphatidyl inositol 4,5 bis-phosphate into diacylglycerol (DAG) and Inositol 1,4,5-trisphosphate (IP₃). The latter binds to receptors present on the membrane of ER (endoplasmic Reticulum), thereby opening up and aiding in the transport of Ca²⁺ oscillatory rise in the cytosol. This event is instrumental in activating various dormant processes in eggs (described below). Inhibition of the IP₃R1 receptors by function-blocking specific antibodies resulted in lack of embryonic development. At the same time aberrant phopholipase Cᶉ expression or the lack of it in males confers sterility for obvious reasons. Artificial activation of eggs is a prerequisite for parthenogenetic activation of eggs in various animals as described below. Egg activation protocols is designed to mimic the [Ca²⁺]_i responses induced by sperm or to replicate the [Ca²⁺]_i-oscillations which results in inactivation of M-phase kinases leading to release of eggs from MII arrest. . Exposing eggs to ethanol and Ca²⁺-ionophores viz, A23187 and ionomycin causes a single and sustained [Ca²⁺]_I increase. It can be concluded fairly certainly that “a single [Ca²⁺]_i –rise” is insufficient to induce complete cyclin B degradation. Inhibitors of protein synthesis and kinases, like cycloheximide (CHX) and 6-dimethylaminopurine (6-DAMP) are added to the medium for several hours after treatment with the ionophores. In direct comparison to Ca²⁺ionophores, a brief exposure to SrCl₂ or Acetylcholine or thimerosal generates repetitive Ca²⁺ that could last for several hours, resulting in Ca²⁺-ion signals much closer to the mode of activation by sperm. Moreover administration of agonists of IP₃Rs, such as IP₃ or adenophostin A also induces [Ca²⁺]_i- oscillations. Instead of chemical reagents, multiple [Ca²⁺]_i –rises is possible if evoked by applying electrical DC pulses in the presence of extracellular Ca²⁺. Injection of recombinant human PLCζ was tried out in the year 2012 and it induced [Ca²⁺]_i-oscillations in both mouse and human eggs. The production of pure recombinant form of PLCζ has been a goal in recent years and several methods have been employed to ensure the production of the same. Recombinant human PLCζ protein prepared in this way was able to generate Ca²⁺in a physiological range in mouse and human eggs. Moreover such studies also demonstrated the deleterious effect of mutant PLCζ and the possibility of the same to be overcome by recombinant PLCζleading eggs to develop to blastocyst stage. However this work seems to be extremely encouraging, careful extrapolation may be necessary to arrive at after applying the same in the laboratory and progressing to clinical settings.