Oviductal regulation of gamete interaction and reproductive function

哺乳动物的输卵管在雌性生殖道中占有独特的地位，为许多重要的生殖事件的发生提供了最佳的微环境。本研究旨在研究仓鼠输卵管特异性糖蛋白（HamOVGP1）在生殖过程早期的功能，并以我在NSERC-DG任职期间完成的工作以及最近获得的初步结果为基础。我们之前已经证明HamOVGP1能够通过增强一组精子蛋白的酪氨酸磷酸化来增强精子获能，这是顶体反应的先决条件。在本提案中，我们的第一个目标是进行高通量磷蛋白质组学分析，以进一步表征rhamovgp1增强的酪氨酸磷酸化精子蛋白及其功能。在我们最后的NSERC-DG中，我们假设hamovgp1结合位点存在于仓鼠子宫上皮细胞表面。为了验证这一假设，我们使用了原位和体外方法来证明hamovgp1结合位点存在于子宫上皮细胞群体的细胞表面。为了进一步开展这项工作，我们设计了一个实验方案来验证子宫上皮细胞表面hamovgp1结合位点具有抗粘附特性，作为着床屏障，而表面缺乏hamovgp1结合位点的细胞作为着床位点的假设。我们的第二个目标是研究HamOVGP1在子宫接受性中的作用。我们成功地建立了原代仓鼠子宫上皮细胞培养。我们将分离和培养子宫细胞的两个细胞群，然后确定仓鼠胚胎将依附于哪个细胞群。最近，我在日本的合作者荒木博士（Dr. Y. Araki）培育出了ovgp1缺失的仓鼠。我们的初步研究表明，ovgp1-/-雌性仓鼠是不育的。有趣的是，一只ovgp1-/-雌性仓鼠表现出不育表型，并被发现表现出与人类异位妊娠相似的表型，这加强了我们关于HamOVGP1在子宫容受性中发挥作用的假设。基于这些前提，我们的第三个目标是利用分子、细胞和免疫细胞化学方法进一步表征ovgp1-/-和ovgp1+/-仓鼠的表型，重点研究HamOVGP1在子宫容受性中的作用。利用我们在上一任NSERC DG任职期间成功生产的重组HamOVGP1和ovgp1缺失仓鼠的可用性，现在有可能揭示HamOVGP1如何调节导致成功受精的一些关键事件及其在早期植入中的作用。从这项研究中获得的结果可以外推到加拿大农业工业中农场动物的人工授精程序的改进，以及研究实验室中涉及啮齿动物的体外受精程序和体外受精生育诊所中涉及人类的体外受精程序。

The mammalian oviduct occupies a unique position in the female reproductive tract where it provides the optimal microenvironment for many critical reproductive events to occur. This proposal examines the function of hamster oviduct-specific glycoprotein (HamOVGP1) during the early events of the reproductive process and builds on work accomplished during the tenure of my last NSERC-DG as well as preliminary results recently obtained. We have previously shown the ability of HamOVGP1 to enhance sperm capacitation, a prerequisite for acrosome reaction, by potentiating tyrosine phosphorylation of a subset of sperm proteins. In the present proposal, our first objective is to perform high throughput phosphoproteomic analysis to further characterize the rHamOVGP1-enhanced tyrosine phosphorylated sperm proteins and their functions. In our last NSERC-DG, we hypothesized that putative HamOVGP1-binding sites are present on the surface of hamster uterine epithelial cells. To test this hypothesis, we used both in situ and in vitro approaches to demonstrate the presence of HamOVGP1-binding sites on the cell surface of a population of uterine epithelial cells. To carry this work further, we have designed an experimental scheme to test the hypothesis that HamOVGP1-binding sites on the surface of uterine epithelial cells have anti-adhesive property acting as a barrier for implantation whereas the cells that lack surface HamOVGP1-binding sites act as the sites for implantation. Our second objective is to examine the role of HamOVGP1 in uterine receptivity. We have successfully established primary hamster uterine epithelial cell cultures. We will isolate and culture the two cell populations of uterine cells and then determine which cell population hamster embryos will adhere to. Recently my collaborator in Japan, Dr. Y. Araki, has established ovgp1-null hamsters. Our preliminary studies showed that ovgp1-/- female hamsters are infertile. Interestingly, one ovgp1-/-female hamster exhibited infertile phenotype and was found to exhibit a phenotype similar to ectopic pregnancy in humans, reinforcing our hypothesis that HamOVGP1 plays a role in uterine receptivity. Based on these premises, our third objective is to further characterize the phenotypes of the ovgp1-/- and ovgp1+/- hamsters using molecular, cellular and immunocytochemical approaches focusing on the role of HamOVGP1 in uterine receptivity. Using the recombinant HamOVGP1 that we successfully produced during the tenure of my last NSERC DG and the availability of ovgp1-null hamsters, it is now possible to unravel how HamOVGP1 regulates some of the key events that lead to successful fertilization and its role in early implantation. Results obtained from this study can be extrapolated for the development of improved insemination procedures for farm animals in the Canadian agriculture industry and IVF procedures involving rodents in research laboratories and involving humans in IVF fertility clinics.