4-dimensional analysis of chiral movement and waveform of sperm flagella during fertilization with digital-holographic microscopy

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Most would agree that life starts with the fusion of egg and sperm. Millions of sperm are rereleased into the oviduct after ejaculation. Few have considered how the fertilizing sperm is selected from the millions of sperm released to the female genital tract. During their way along the oviduct – a journey that is equivalent to a human walking from Frankfurt to Berlin within 24 hours – many selective processes decide which sperm are possible candidates to fertilize the egg. No more than 10-20 sperm will come even close enough to have a chance to fertilize the oocyte. We plan to apply the cutting-edge technique of digital-holographic Microscopy (DHM) on sperm movement to analyze their trajectories and flagellar wave in four dimensions. We have developed this technique in the last years that far, that we cannot apply only a head tracking of the sperm but observe the whole flagellar movement in space and time in all dimensions. This with a spatiotemporal resolution which is only limited by the speed of the camera used. We could show that murine sperm have a well-defined chiral movement in the head as well as in the flagellum, which work independently from each other. With this grant proposal we plan to apply DHM to certain and well-defined points during fertilization. Those time points will be early activation, linear movement, capacitation, sperm-egg binding/fusion, and hypermotility. We choose these time points, because they are very well defined and can easily be simulated in-vitro. We will perform these experiments in part with the help of transgenic mice which carry mutations for the CatSper channel. CatSper is across many species one of the most important ion channels for successful fertilization. We will also use sperm of other species beside mouse - like bovine, human and sea urchin to make conclusion about translational importance of our findings. Sea urchin sperm will give us in addition the chance to investigate the importance of outer dense fibers and the fibrous sheath for the chirality of vertebrate spermatozoa, because sea urchin sperm are lacking such structures. In addition, oocytes will help us to simulate the time point just before egg-sperm binding in vitro. In this context we will use a synthetic peptide of the zona pellucida protein 2 (ZP2251-149), to simulate the sperm-egg binding to investigate the wave form of sperm in four dimensions. With this project we plan to study the importance of the above mentioned chiralities of the sperm head and flagellum in the light of different key functions of spermatozoa. We will specifically test the hypothesis, if altering chiralities of the head and flagellum are important for the regulation of sperm functions. We expect to generate significant new findings for the basic biological knowledge of fertilization as well as information about important factors which can lead to disturbance of sperm movement and in turn to difficulties of sperm selection during fertilization.