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

利用数字全息显微镜对受精过程中精子鞭毛的手性运动和波形进行4维分析

• 批准号: 227377324
• 负责人: Professor Dr. Gunther Wennemuth
• 金额: --
• 依托单位: Institut für Anatomie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/227377324?language=en
• 关键词: dimensional; analysis; chiral; movement; waveform

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.

大多数人会同意，生命始于卵子和精子的融合。数百万的精子在射精后被重新释放到输卵管中。很少有人考虑过如何从释放到女性生殖道的数百万精子中挑选受精的精子。在他们沿着输卵管的过程中--相当于一个人在24小时内从法兰克福走到柏林--许多选择性的过程决定了哪些精子可能使卵子受精。不会有超过10-20个精子接近到有机会使卵母细胞受精。我们计划将数字全息显微镜(DHM)的尖端技术应用于精子运动，从四个维度分析他们的轨迹和鞭毛波。到目前为止，我们在过去的几年里已经开发出了这项技术，我们不能只应用头部跟踪精子，而是在所有维度上观察整个鞭毛在空间和时间上的运动。这是一种时空分辨率，只受所用相机速度的限制。我们可以证明，小鼠的精子在头部和鞭毛中都有明确的手性运动，它们相互独立地工作。有了这项赠款建议，我们计划在受精过程中将DHM应用于某些明确定义的点。这些时间点将是早期激活、直线运动、获能、精子-卵子结合/融合和运动过度。我们选择这些时间点，因为它们定义得很好，可以很容易地在体外模拟。我们将在一定程度上借助携带CatSper通道突变的转基因小鼠进行这些实验。CatSper在许多物种中都存在，是成功受精的最重要的离子通道之一。我们还将使用老鼠类牛、人和海胆以外的其他物种的精子来得出我们发现的翻译重要性的结论。此外，海胆精子将使我们有机会研究外部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在体外模拟卵子与精子结合之前的时间点。在此背景下，我们将使用合成的透明带蛋白2(ZP2251-149)的多肽来模拟精子与卵子的结合，从四个维度来研究精子的波形。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的手性的重要性。如果改变头部和鞭毛的手性对调节精子功能很重要，我们将专门测试这一假设。我们希望为受精的基本生物学知识以及可能导致精子运动障碍并进而导致受精过程中精子选择困难的重要因素的信息产生重要的新发现。