Functional dissection of Tmem95: a sperm cell surface protein essential for mammalian fertilization

在人类中，新的生命开始于受精，当精子和卵子相互识别，它们周围的膜融合形成一个新的胚胎。精子和卵子通过细胞表面识别分子相互作用，这些分子特异性地结合在一起，使它们融合在一起。尽管受精是一个基本的生物学过程，但我们对其中涉及的分子的了解非常少，尤其是在哺乳动物中；例如，我们不知道是什么分子使精子和卵子融合在一起。这些知识差距在人类中可以部分解释，因为围绕受精的伦理问题，以及研究细胞表面分子的困难。例如，卵子是一种非常罕见的细胞类型，人类通常在每个生育周期中只释放一个卵子，这限制了可用生物材料的数量。此外，已知细胞表面分子之间的物理相互作用非常弱（半衰期仅为几秒钟），需要使用专门的方法来检测它们。桑格研究所的细胞表面信号实验室专门研究识别这些短暂的相互作用，我们已经开发了一套技术来规避这些困难。我们以前使用这些技术来鉴定第一对必要的精子-卵子受体（见Bianchi等人）。Nature 2014 v508 p483)，为理解受精的分子机制做出了重要贡献。我们对一种新的精子表面蛋白感到兴奋，它的名称为Tmem95（跨膜蛋白95号），它对精子与卵子融合的能力至关重要。Tmem95是从养牛业的一项研究中确定的，该研究调查了一例特发性男性不育症（尽管精子看起来和运动正常，但却不能生育）。研究人员使用了在奶牛身上进行的数千次人工授精的结果，并通过仔细的记录保存，确定了TMEM95基因突变的来源。进一步的研究表明，在Tmem95基因上有基因工程突变的雄性老鼠也会不育，因为在体外受精试验中，看起来正常的精子无法与卵子融合。人类也有一个看起来非常相似的TMEM95基因，我们相信它也将在人类中发挥重要作用；然而，我们对这种蛋白质知之甚少。这项拨款申请旨在更多地了解TMEM95在施肥中的重要作用。具体来说，我们将使用我们的专业技术来确定它在卵子表面与受体相互作用的分子特性，描述蛋白质在受精过程中如何在精子中重新分配，确定Tmem95在融合膜中是否具有特定作用，并研究它如何与另一种重要的精子细胞表面蛋白质Izumo1相互作用。这些研究将进一步加深我们对基本生物学过程的分子理解，并为开发更好的人类和农业避孕和生育治疗提供基础。快速增长的人口（目前超过70亿，预计到2050年将达到100亿）引起了人们的担忧，即地球上有限的资源将无法维持持续的扩张。矛盾的是，不孕不育是一个日益严重的问题，特别是在西方国家，近年来，夫妇生第一个孩子的平均年龄有所增加。通过发现新的不育基因，这项研究可能为不育夫妇提供简单而廉价的基因筛查测试，从而指导他们的生育治疗，并节省试管受精失败的费用和痛苦。这些研究还可以改进畜牧业的繁殖技术，以及开发避孕疫苗，以控制野生动物种群，并以道德上更可接受的方式，而不是扑杀或绝育，使伴侣动物绝育。

In humans, new life starts at fertilization when a sperm and an egg recognise each other and their surrounding membranes fuse to form a new embryo. Sperm and egg interact using cell surface recognition molecules which bind specifically and enable them to fuse together. Although fertilization is a fundamental biological process, our understanding of the molecules involved is remarkably poor, especially in mammals; for example, we do not know the molecule/s that are responsible for fusing the sperm and egg together. These knowledge gaps can be partly explained in humans due to the ethical issues surrounding fertilization, and the difficulties in studying cell surface molecules. For example, eggs are a very rare cell type, with humans usually releasing just a single egg in each fertility cycle limiting the amount of biological material available. In addition, the physical interactions between cell surface molecules are known to be very weak (having half-lives of just seconds), requiring the use of specialised methods to detect them. The Cell Surface Signalling Laboratory at the Sanger Institute specializes in identifying these fleeting interactions and we have developed a set of techniques to circumvent these difficulties. We have previously used these techniques to identify the first essential sperm-egg receptor pair (see Bianchi et al. Nature 2014 v508 p483), which made an important contribution to the molecular understanding of fertilization.We are excited about a new sperm surface protein prosaically named Tmem95 (for TransMEMbrane protein number 95) that is essential for the ability of sperm to fuse with eggs. Tmem95 was identified from research in the cattle industry where a case of idiopathic male infertility (sperm that is infertile despite looking and moving normally) was investigated. Researchers used the results of thousands of artificial inseminations performed in cows, and by careful record-keeping, identified the source to a mutation in the TMEM95 gene. Further research has shown that male mice which have an engineered mutation in their Tmem95 gene are also infertile because normal-looking sperm are unable to fuse with eggs in IVF assays. Humans also have a TMEM95 gene which looks very similar and we believe that it will have an important role in humans too; however, we know very little about this protein. This grant application is aimed at understanding more about the important role TMEM95 has in fertilization. Specifically, we will use our specialized technologies to determine the molecular identity the receptor it interacts with on the egg surface, describe how the protein is redistributed in the sperm during fertilization, establish if Tmem95 has a specific role in fusing membranes together, and investigate how it interacts with another essential sperm cell surface proteins called Izumo1.These studies will both further our molecular understanding of a fundamental biological process, and provide a foundation to develop better contraceptives and fertility treatments both in humans and in the farming industry. The rapidly expanding human population (currently over 7 billion and predicted to reach 10 billion by 2050) has raised concerns that the limited resources on the planet will not sustain a continued expansion. Paradoxically, infertility is a growing problem, particularly in Western countries where the average age of couples having their first child has increased in recent years. By discovering new infertility genes, this research could open up the possibility of offering simple and inexpensive genetic screening tests to infertile couples that may guide their fertility treatment and save the expense and pain of failed rounds of IVF. These studies could also improve reproduction technologies in the livestock industry and also in the development of contraceptive vaccines to control wild animal populations and sterilize companion animals in an ethically more acceptable way other than culling or neutering.