Efficient pig ICSI using Percoll-selected spermatozoa; evidence for the essential role of phospholipase C-ζ in ICSI success

Abstract

In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used. A proportion of the oocytes injected with control sperm were subjected to electrical stimulation at 1 h after ICSI (Cont + ES group). It was found that the Percoll group showed a large amount of PLCζ in comparison with the Control group. Furthermore, application of Percoll-selected sperm for ICSI increased the efficiency of fertilization and embryo development. Thus, these results indicate the Percoll-selected sperm have sufficient PLCζ and high oocyte-activating ability after ICSI in pigs.

Lack of calcium oscillation causes failure of oocyte activation after intracytoplasmic sperm injection in pigs

“…Indeed, immediately after thawing, more than half of the pig sperm were found to have lost PLCζ immunoreactivity, leading to failure of oocyte activation after ICSI [33]. Use of sperm with appropriate levels of PLCζ for ICSI actually increases the efficiency of oocyte activation and normal fertilization [33]. Our findings suggest that the primary reason for failure of oocyte activation after ICSI may be a deficit, rather than a difference, in the pattern of Ca 2+ oscillation.…”

“…The membranes of pig sperm can be damaged during the freezing and thawing processes associated with cryopreservation [32], and such damage can lead to leakage of intracellular PLCζ [29]. Indeed, immediately after thawing, more than half of the pig sperm were found to have lost PLCζ immunoreactivity, leading to failure of oocyte activation after ICSI [33]. Use of sperm with appropriate levels of PLCζ for ICSI actually increases the efficiency of oocyte activation and normal fertilization [33].…”

Zeta method: A noninvasive method based on membrane charge for selecting spermatozoa expressing high level of phospholipaseCζ

“…The result of a recent study showed that DGC procedure is effective in the separation of spermatozoa with PLCζ, and these spermatozoa result in high oocyte activation after ICSI in pig (Nakai et al, ). Furthermore, Kashir et al () demonstrated that percentage of spermatozoa containing PLCζ was significantly higher following DGC procedure compared to neat semen while cryopreservation of semen has significant detrimental effects upon spermatozoa containing PLCζ.…”

Challenges and Considerations during In Vitro Production of Porcine Embryos

“…These decreases in development may be due to selection of damaged sperm and/or incomplete oocyte activation. Importantly, boar sperm selected by a Percoll gradient were shown to have higher abundance of phospholipase C-ζ (PLCζ), a well-studied sperm-borne oocyte activation factor, and the use of Percoll-selected sperm for ICSI increased monospermic fertilization and oocyte activation as well as development to the blastocyst stage [47]. Several protocols use polyvinylpyrrolidone (PVP) for sperm immobilization before ICSI; however, this compound is toxic when injected into oocytes.…”