Oviductal regulation of gamete interaction and reproductive function

配子相互作用和生殖功能的输卵管调节

• 批准号: RGPIN-2020-05467
• 负责人: Kan, Frederick
• 金额: $ 2.04万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=717213
• 关键词: Oviductal; regulation; gamete; interaction; reproductive

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.

哺乳动物的输卵管在女性生殖道中占有独特的位置，为许多关键生殖事件的发生提供了最佳的微环境。这项建议研究了仓鼠输卵管特异糖蛋白(HamOVGP1)在生殖过程的早期事件中的功能，并建立在我上一个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。我们之前已经证明了HamOVGP1通过增强精子蛋白质子集的酪氨酸磷酸化来增强精子获能的能力，这是顶体反应的先决条件。在目前的方案中，我们的第一个目标是进行高通量的磷酸蛋白质组分析，以进一步表征rHamOVGP1增强的酪氨酸磷酸化精子蛋白及其功能。 在我们上一次的NSERC-DG中，我们假设仓鼠子宫上皮细胞表面存在假定的HamOVGP1结合位点。为了验证这一假设，我们使用了原位和体外方法来证明在一组子宫上皮细胞的细胞表面存在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来验证这样的假设，即子宫上皮细胞表面的HamOVGP1结合位点具有抗黏附特性，作为种植的屏障，而缺乏表面HamOVGP1结合位点的细胞则作为种植的位点。我们的第二个目标是研究HamOVGP1在子宫容受性中的作用。我们已经成功地建立了仓鼠子宫上皮细胞的原代培养。我们将分离和培养这两个细胞群的子宫细胞，然后确定仓鼠胚胎将附着在哪个细胞群上。 最近，我在日本的合作者Y.Araki博士建立了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的表型，并被发现表现出与人类异位妊娠相似的表型，这强化了我们的假设，即HamOVGP1在子宫容受性中发挥作用。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。 利用我们在我最后一次NSERC DG任期内成功生产的重组HamOVGP1和ovgp1缺失仓鼠的可用性，现在可以揭示HamOVGP1是如何调控一些导致成功受精的关键事件及其在早期植入中的作用。这项研究的结果可用于开发加拿大农业中改进的家畜人工授精程序，以及在研究实验室中使用啮齿动物和在试管受精诊所中使用人类的试管受精程序。