P-021 Development of recombinant Phospholipase C Zeta protein as a therapeutic intervention to overcome certain types of male infertility

Abstract

Abstract Study question Developing recombinant Phospholipase C Zeta (PLCζ) protein using a bacterial expression system to be utilized for potential future replacement of Ca2+ ionophores during IVF treatments. Summary answer Our findings support the use of MBP-PLCζ recombinant protein as a therapeutic tool for treating certain types of male factor infertility associated with fertilization failure What is known already The long-term safety of calcium ionophores, which are exclusively used for treating cases of fertilization failure, is still questionable. PLCζ is an endogenous agent that may represent a better replacement for treating male infertility cases related to fertilization failure Study design, size, duration Maltose binding protein (MBP)-tagged PLCζ was produced by cloning wild-type PLCζ into a pET41MM vector and purified using affinity chromatography techniques. Circular dichroism (CD) was employed to evaluate the secondary structure and the thermal denaturation of PLCζ protein. Moreover, the PIP2 hydrolysis assay was used to test the enzymatic activity of PLCζ protein over time and under different storage conditions. Furthermore, the embryonic toxicity of the protein was tested using a chick embryo model. Participants/materials, setting, methods Several methods were used in this study including Molecular cloning, circular dichroism, and chick embryo model to investigate the embryonic viability. Main results and the role of chance Our findings demonstrated that PLCζ recombinant protein displayed a properly folded secondary structure mainly composed of alpha-helices. Moreover, the protein was effective in maintaining the enzymatic activity and Ca2+ sensitivity for 90 days when stored at -80oC. Moreover, exposing chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control group. Kaplan-Meier survival curve showed that the rate of survival did not differ between the treatment, control and sham groups until the endpoint of the study. Limitations, reasons for caution The toxicology assays used are not sufficient to confirm the safety of the protein. Further in vivo studies are required to investigate the effect of MBP-PLCζ protein on early embryonic development before being used as a replacement for calcium ionophores in cases of oocyte activation failure. Wider implications of the findings These findings can help develop a new therapeutic tool for treating cases of male infertility associated with oocyte activation failure. Trial registration number not applicable