Breaking the egg coat twice: sperm penetration and embryo hatching

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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.