P-977: Removal of sperm plasma membranes immediately before ICSI prompts rapid oocyte activation and better post-implantation embryonic development

Abstract

We have previously demonstrated that the contents of the acrosome is potentially harmful to oocytes. Even though we do not have to remove the plasma membrane and acrosome from mouse and human spermatozoa prior to ICSI, removal of both plasma membrane and acrosome is preferable to mimic natural fertilization and to reduce potentially harmful effects of acrosomal enzymes on zygotes and embryos. In this study we exmined the effect of removal of both the plasma membrane and acrosome from spermatozoa prior to ICSI on oocyte activation and embryonic development. An experimental research. Oocytes were obtained from superovulated 7-15 week old B6D2F1 female mice. Mouse, human, bull and bore sperm were individually injected into mouse oocytes. Four types of spermatozoa were injected: (a) motile spermatozoa with intact plasma membranes, (b) spermatozoa immobilized by Piezo pulse, perhaps with locally disrupting plasma membranes, (c) spermatozoa treated for 1 min with 0.02-1.0 % Triton X-100 and (d) spermatozoon treated for 1 min with 0.02-0.2 % lysolecithin (LL). The speed of oocyte activation was assessed in two different ways. In the first, the extrusion of the second polar body was considered as a visible indication of oocyte activation. ICSI oocytes were examined at 30 min intervals to determine the proportion of the oocytes with completely extruded the second polar bodies. In the second, the onset and pattern of intracellular Ca2+ oscillations were determined after ICSI. To see the term developmental ability of ICSI oocytes was determined by transferring two-cell embryos into oviducts of pseudopregnant surrogate females. When the extrusion of the second polar body was considered as an indication of oocyte activation, it was clear that oocytes activate considerably faster when sperm plasma membranes were removed by Triton or LL prior to ICSI than when sperm plasma membranes were intact during ICSI. When the mouse or human spermatozoa were injected into mouse oocytes, the onset of intracellular Ca2+ oscillations was faster when sperm plasma membranes had been disrupted than when the membranes were intact at injection. The post implantation embryo development was the best when spermatozoa were free from plasma membrane and acrosome using LL immediately before injection. Removal of the plasma membrane and acrosome from spermatozoon prior to ICSI prompted rapid oocyte activation and better embryo development at least in the mouse. Rapid and consistent oscillations of intracellular Ca2+ oscillations took place when plasma membrane- and acrosome-free human spermatozoa were injected into mouse oocytes. Since sperm plasma membrane and acrosome never enter the oocyte during normal fertilization, removal of both plasma membrane and acrosome from spermatozoa immediately before ICSI is recommended.