Breaking the egg coat twice: sperm penetration and embryo hatching

为了使卵母细胞受精，精子必须穿透卵的外壳，即透明带（ZP）。在植入子宫之后，哺乳动物胚胎必须从它的ZP中孵化出来，ZP在它通过雌性生殖道的过程中起到保护作用。因此，在胚胎形成和发育过程中有两个时间点，ZP裂解和相应的酶是产生新生命所必需的。人类辅助生殖技术（ART）每天都在进行。目前，为了克服受精时的ZP，胞浆内单精子注射（ICSI）比标准的体外受精（IVF）更常用。此外，经常进行辅助孵化以增加胚胎着床。这两种方法都依赖于破坏性的机械、化学或激光介导技术。因此，抗逆转录病毒治疗经常失败，而且由于伦理原因，对人类生殖缺陷进行研究几乎是不可能的。为了克服与人类有关的伦理问题，所谓的“人源化老鼠”被发明出来。这些小鼠的卵母细胞表达人ZP而不是小鼠ZP。这些卵母细胞现在被用于研究人类ZP蛋白与人类精子之间的相互作用，以获得有关人类生育障碍的进一步信息。我将研究哺乳动物带溶酶的分子基础，使用两种小鼠胚胎重组表达系统。我计划在哺乳动物细胞中表达acrosin（一种精子衍生的带溶酶），纯化该酶并测试其生物学功能。此外，我将对小鼠卵母细胞和囊胚进行转录组和蛋白质组分析，最终目的是确定哺乳动物孵化酶。这些潜在的孵化酶将进行表达、纯化和功能测试。为了更详细地了解这些分子，我还将通过x射线晶体学研究带裂解酶及其复合物与其各自底物的结构。最后，我将使用带溶酶辅助孵化，并将结果与已有的辅助孵化方法进行比较。我相信，在抗逆转录病毒治疗期间使用纯酶可能是一种更温和和更具体的方法，以帮助孵化和提高着床率。此外，我认为在已经存在的人源化小鼠中替换额外的蛋白质是进一步优化当前模型系统和更可靠地复制人类生殖的基础。

In order to fertilise an oocyte, sperm has to penetrate the egg coat, the zona pellucida (ZP). Afterwards to implant into the uterus, the mammalian embryo has to hatch from its ZP, which protects it during its passage through the female reproductive tract. Thus there are two time points during embryo formation and development, where ZP lysis and the corresponding enzymes are essential to give rise to new life.Assisted reproductive techniques (ART) in humans are carried out on a daily basis. Currently to overcome the ZP at fertilisation, intracytoplasmic sperm injection (ICSI) is performed more often than standard in vitro fertilisation (IVF). Moreover, assisted hatching is often carried out to increase embryo implantation. Both methods rely on disruptive mechanical, chemical or laser-mediated technologies. Consequently ART quite often fails and doing research on human reproductive defects is almost impossible due to ethics. To overcome the ethical aspect regarding to humans, so-called humanised mice were invented. Oocytes of these mice express human ZP instead of mouse ZP. These oocytes are now used to study interaction between human ZP proteins and human sperm, to get further information on human fertility disorders. I will study the molecular basis of mammalian zona lytic enzymes, using both mouse embryos andrecombinant expression systems. I am planning to express acrosin (a sperm derived zona lytic enzyme) in mammalian cells, purify the enzyme and test its biological function. In addition I will do transcriptome as well as proteome analysis of mouse oocytes and blastocysts with the ultimate aim of conclusively identifying the mammalian hatching enzyme(s). These potential hatching enzyme(s) will be expressed, purified and functional tested as well. To gain a more detailed understanding of these molecules, I will also investigate the structure of the zona lytic enzymes and complexes with their respective substrates by means of X-ray crystallography. Finally I will use the zona lytic enzymes to assist hatching and will compare the outcome with already existing assisted hatching methods. I am convinced that the use of a pure enzyme during ART could be a more gentle and specific method to assist hatching and to increase the implantation rate. Furthermore I think that the replacement of additional proteins in the already existing humanised mice is fundamental to further optimise the current model system and more reliably phenocopy human reproduction.