Final Report Summary - ZETA-STIM (The molecular basis for the ability of PLC zeta to stimulate mammalian embryo development.)
The earliest fundamental event during the creation of a new life occurs when the fertilizing sperm initiates embryo development by activating the female oocyte. In mammals, oocyte activation is triggered by a characteristic series of cytoplasmic calcium transients (calcium oscillations), which are widely believed to be initiated by a sperm-specific protein factor, termed PLC zeta. Calcium oscillations may directly influence the efficacy of subsequent embryo development and may underlie the drastically low success rates of current fertility treatments. Considering that infertility affects 1 in 7 (~70 million) couples, while ‘sub-fertility’ affects 1 in 15 men worldwide, it was important to delineate the role of PLC zeta during mammalian fertilization. Thus, the main purpose of this proposal was to establish the molecular mechanism of fertilization in mammals by demonstrating whether PLC zeta is the sole ‘sperm factor’ responsible for the generation of calcium oscillations during mammalian fertilization. This could facilitate the potential application of PLC zeta as a therapeutic for PLC zeta-mediated male infertility and enable a novel treatment to be provided by IVF clinics. Our findings have allowed reproduction/fertilization field to move forward. We demonstrated that sperm PLC zeta is the sole ‘sperm factor’ responsible for the generation of Ca2+ oscillations during mammalian fertilization, as we showed that a recent candidate sperm protein called PAWP, which was proposed as an alternative ‘sperm factor’, was unable to elicit Ca2+ oscillations in mouse and human oocytes. Our findings were consistent with a very recent study published by a Japanese group, suggesting that PAWP is not required for mouse egg activation. These findings suggest that sperm PLC zeta is the sole ‘sperm factor’ responsible for the oocyte activation at mammalian fertilization.
Another goal of the proposal in understanding the complex regulatory mechanism of PLC zeta was achieved by a study led by Dr. Nomikos, which suggested that the N- terminal lobe of the EF-hand domain of PLC zeta has an essential role in the interaction of this enzyme with its target membrane, which together with the XY-linker, may combine to provide a tether that facilitates proper PIP2 substrate access and binding in the PLC zeta active site.
In addition, during the two years of the Marie Curie Fellowship we have developed various protocols to produce recombinant human PLC zeta that can be used from IVF clinics as a therapeutic agent to overcome certain forms of male infertility and Dr. Nomikos has co-authored a significant publication, which demonstrated that recombinant human PLC zeta could rescue failed oocyte activation after ICSI in a mouse model of male factor infertility.
Moreover, a recent genetic study reported a male infertility case (oocyte activation failure) that was directly associated with a point mutation in PLC zeta C2 domain. This was the first clinical support for the vital role of this domain on PLC zeta function and we used a number of biochemical and biophysical techniques to demonstrate that this is purely due to the novel binding properties of the C2 domain to PI(3)P and PI(5)P or potentially to other unidentified egg protein(s), (unpublished data; under review for publication). We have also discovered a novel binding partner of sperm PLC zeta and this interaction might play a vital role during the first steps of mammalian fertilization (unpublished data; under review for publication).
Although the discovery of PLC has represented an important breakthrough in the field, we currently still do not understand how PLC works. This fellowship has generated new data that can provide the basis for such an understanding and can therefore create new advances in clinical medicine.
In parallel with his work on PLC zeta and mammalian fertilization, a major contribution made by Dr. Nomikos was to biochemically characterize clinical mutations in Calmodulin (CaM), shedding light on the previously poorly characterized biochemical mechanisms that underlie some forms of heart disease, giving therapeutic and diagnostic hope to patients suffering from Europe’s biggest killer. Finally, Dr. Nomikos recently characterized the molecular properties of phospholipase C delta-1 mutants associated with hereditary leukonychia, a rare genetic nail disorder characterized by distinctive whitening of the nail plate of all twenty nails. Hereditary leukonychia may exist as an isolated feature, or in simultaneous occurrence with other cutaneous or systemic pathologies. In this study, we demonstrated for the first time the importance of PLC-mediated calcium signalling within the manifestation of hereditary leukonychia.
Another goal of the proposal in understanding the complex regulatory mechanism of PLC zeta was achieved by a study led by Dr. Nomikos, which suggested that the N- terminal lobe of the EF-hand domain of PLC zeta has an essential role in the interaction of this enzyme with its target membrane, which together with the XY-linker, may combine to provide a tether that facilitates proper PIP2 substrate access and binding in the PLC zeta active site.
In addition, during the two years of the Marie Curie Fellowship we have developed various protocols to produce recombinant human PLC zeta that can be used from IVF clinics as a therapeutic agent to overcome certain forms of male infertility and Dr. Nomikos has co-authored a significant publication, which demonstrated that recombinant human PLC zeta could rescue failed oocyte activation after ICSI in a mouse model of male factor infertility.
Moreover, a recent genetic study reported a male infertility case (oocyte activation failure) that was directly associated with a point mutation in PLC zeta C2 domain. This was the first clinical support for the vital role of this domain on PLC zeta function and we used a number of biochemical and biophysical techniques to demonstrate that this is purely due to the novel binding properties of the C2 domain to PI(3)P and PI(5)P or potentially to other unidentified egg protein(s), (unpublished data; under review for publication). We have also discovered a novel binding partner of sperm PLC zeta and this interaction might play a vital role during the first steps of mammalian fertilization (unpublished data; under review for publication).
Although the discovery of PLC has represented an important breakthrough in the field, we currently still do not understand how PLC works. This fellowship has generated new data that can provide the basis for such an understanding and can therefore create new advances in clinical medicine.
In parallel with his work on PLC zeta and mammalian fertilization, a major contribution made by Dr. Nomikos was to biochemically characterize clinical mutations in Calmodulin (CaM), shedding light on the previously poorly characterized biochemical mechanisms that underlie some forms of heart disease, giving therapeutic and diagnostic hope to patients suffering from Europe’s biggest killer. Finally, Dr. Nomikos recently characterized the molecular properties of phospholipase C delta-1 mutants associated with hereditary leukonychia, a rare genetic nail disorder characterized by distinctive whitening of the nail plate of all twenty nails. Hereditary leukonychia may exist as an isolated feature, or in simultaneous occurrence with other cutaneous or systemic pathologies. In this study, we demonstrated for the first time the importance of PLC-mediated calcium signalling within the manifestation of hereditary leukonychia.