Oviductal regulation of gamete interaction and reproductive function

哺乳动物输卵管在雌性生殖道中占据独特的位置，它为许多关键生殖事件的发生提供了最佳的微环境。该提案研究了仓鼠输卵管特异性糖蛋白（HamOVGP 1）在生殖过程的早期事件中的功能，并建立在我的最后一个NSERC-DG任期内完成的工作以及最近获得的初步结果的基础上。我们以前已经表明，HamOVGP 1的能力，以提高精子获能，顶体反应的先决条件，通过增强精子蛋白的一个子集的酪氨酸磷酸化。在本提案中，我们的第一个目标是进行高通量磷酸化蛋白质组学分析，以进一步表征rHamOVGP 1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，我们假设假定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，我们使用原位和体外方法来证明子宫上皮细胞群体的细胞表面上存在HamOVGP1结合位点。为了进一步开展这项工作，我们设计了一个实验方案，以测试子宫上皮细胞表面上的HamOVGP 1结合位点具有抗粘附特性作为植入屏障的假设，而缺乏表面HamOVGP 1结合位点的细胞作为植入位点。我们的第二个目的是研究HamOVGP 1在子宫容受性中的作用。我们已经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粘附于哪一个细胞群。 最近，我在日本的合作者，Y。荒木，建立ovgp1-null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，加强了我们的假设，即HamOVGP1在子宫容受性中起作用。基于这些前提，我们的第三个目标是进一步表征的ovgp1-/-和ovgp1 +/-仓鼠的表型，使用分子，细胞和免疫细胞化学的方法，侧重于HamOVGP 1在子宫容受性的作用。 使用我们在我的最后一个NSERC DG任期内成功生产的重组HamOVGP1和ovgp1缺失仓鼠的可用性，现在可以解开HamOVGP1如何调节一些导致成功受精的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为发展改进的授精程序的农场动物在加拿大农业和体外受精程序涉及啮齿动物在研究实验室和涉及人类在体外受精生育诊所。

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.