Abstract
ObjectiveTo determine the effect of infertility-linked sperm phospholipase Cζ (PLCζ) mutations on their ability to trigger oocyte Ca2+ oscillations and development, and also to evaluate the potential therapeutic utility of wild-type, recombinant PLCζ protein for rescuing failed oocyte activation and embryo development.DesignTest of a novel therapeutic approach to male factor infertility.SettingUniversity medical school research laboratory.Patient(s)Donated unfertilized human oocytes from follicle reduction.Intervention(s)Microinjection of oocytes with recombinant human PLCζ protein or PLCζ cRNA and a Ca2+-sensitive fluorescent dye.Main Outcome Measure(s)Measurement of the efficacy of mutant and wild-type PLCζ-mediated enzyme activity, oocyte Ca2+ oscillations, activation, and early embryo development.Result(s)In contrast to the wild-type protein, mutant forms of human sperm PLCζ display aberrant enzyme activity and a total failure to activate unfertilized oocytes. Subsequent microinjection of recombinant human PLCζ protein reliably triggers the characteristic pattern of cytoplasmic Ca2+ oscillations at fertilization, which are required for normal oocyte activation and successful embryo development to the blastocyst stage.Conclusion(s)Dysfunctional sperm PLCζ cannot trigger oocyte activation and results in male factor infertility, so a potential therapeutic approach is oocyte microinjection of active, wild-type PLCζ protein. We have demonstrated that recombinant human PLCζ can phenotypically rescue failed activation in oocytes that express dysfunctional PLCζ, and that this intervention culminates in efficient blastocyst formation.
Highlights
Our findings demonstrate the potential utility of phospholipase Cz (PLCz) protein in in vitro fertilization (IVF) treatment, providing a novel therapeutic agent that may help to overcome those cases of egg activation failure caused by deficient or defective forms of PLCz in human sperm
The intracellular Ca2þ release triggered by InsP3 produces the characteristic cytoplasmic Ca2þ oscillations that result in egg activation, and this initiates the embryo development process
PLCz has been identified in many different mammalian species, suggesting that it could play a pivotal role at fertilization in all mammals
Summary
To determine the effect of infertility-linked sperm phospholipase Cz (PLCz) mutations on their ability to trigger oocyte Ca2þ oscillations and development, and to evaluate the potential therapeutic utility of wild-type, recombinant PLCz protein for rescuing failed oocyte activation and embryo development. Subsequent microinjection of recombinant human PLCz protein reliably triggers the characteristic pattern of cytoplasmic Ca2þ oscillations at fertilization, which are required for normal oocyte activation and successful embryo development to the blastocyst stage. Mouse eggs expressing the mutant human PLCz were unable to activate normally and failed to commence embryo development This infertile phenotype could be effectively rescued by microinjection of the wild-type human PLCz protein, leading to Ca2þ oscillations and successful early embryogenesis up to the blastocyst stage. Our findings demonstrate the potential utility of PLCz protein in in vitro fertilization (IVF) treatment, providing a novel therapeutic agent that may help to overcome those cases of egg activation failure caused by deficient or defective forms of PLCz in human sperm
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