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

大多数人都同意生命始于卵子和精子的融合。射精后，数百万的精子被重新释放到输卵管中。很少有人考虑过，精子是如何从数百万释放到女性生殖道的精子中挑选出来的。在它们沿着输卵管沿着的过程中--相当于一个人在24小时内从法兰克福走到柏林--许多选择性过程决定哪些精子可能是使卵子受精的候选者。不超过10-20个精子会接近到足以有机会使卵母细胞受精。我们计划应用数字全息显微镜（DHM）的尖端技术对精子运动进行四维分析。在过去的几年里，我们已经发展了这项技术，我们不能只应用精子的头部跟踪，而是在所有维度上观察整个鞭毛在空间和时间上的运动。这具有仅受所使用的相机的速度限制的时空分辨率。我们可以证明，小鼠精子在头部和鞭毛中具有明确的手性运动，它们彼此独立地工作。有了这个赠款提案，我们计划在施肥过程中将DHM应用于某些明确定义的点。这些时间点将是早期激活、线性运动、获能、精卵结合/融合和运动过度。我们选择这些时间点，因为它们非常明确，并且可以很容易地在体外模拟。我们将在携带CatSper通道突变的转基因小鼠的帮助下进行这些实验。CatSper是许多物种中成功受精的最重要的离子通道之一。我们也将使用其他物种的精子，除了像老鼠一样的牛，人类和海胆，使我们的研究结果的翻译重要性的结论。海胆精子将使我们有机会研究外致密纤维和纤维鞘对脊椎动物精子手性的重要性，因为海胆精子缺乏这样的结构。此外，卵母细胞将帮助我们模拟卵-精体外结合前的时间点。在这方面，我们将使用人工合成的透明质酸蛋白2（ZP 2251 -149）的肽，来模拟精子-卵子结合，以研究精子在四维空间的波形。在这个项目中，我们计划根据精子的不同关键功能来研究上述精子头部和鞭毛手性的重要性。如果头部和鞭毛的手性改变对精子功能的调节很重要，我们将专门检验这一假设。我们希望能为受精的基本生物学知识以及有关可能导致精子运动障碍的重要因素的信息产生重要的新发现，从而在受精过程中难以进行精子选择。

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.