In vivo analysis of mammalian fertilization

项目概要/摘要 发情周期、排卵、受精和植入前妊娠是基本的生殖过程 具有临床意义。虽然研究揭示了介导的细胞和分子机制 这些事件，其中大部分数据来自静态组织学分析、低分辨率可视化， 以及无脊椎动物模型（例如海胆）的研究。因此，任何关于哺乳动物的结论 受精发生在身体深处，是推断出来的，并不一定代表 原生状态。如果克服了这一技术限制，我们可能会更全面地了解 哺乳动物的生殖导致更好的生育治疗和辅助生殖的发展 技术（ART）。 通过整合活体功能性光学相干断层扫描 (OCT) 和生殖生物学方面的专业知识，我们 最近建立了一套独特的雌性小鼠生殖道体内成像方法。 我们的方法允许（i）使用显微技术对小鼠输卵管（输卵管）进行实时动态体积成像 尺度空间分辨率，(ii) 输卵管纤毛位置和纤毛拍频 (CBF) 的深度分辨映射；和 (iii) 追踪单个精子及其在输卵管内的运动能力。目前这些测量值均未 可以用其他方法实现，且女性生殖道的动态环境过于复杂，无法实现 模型。因此，我们处于独特的地位，可以直接可视化特定哺乳动物的生殖过程 从一个全新的角度来看。 我们提出了第一个哺乳动物受精体内体积成像研究。这项研究正利用 OCT 成像新技术的发展，将允许对荷尔蒙进行定量评估 小鼠输卵管纤毛跳动和肌肉收缩的调节以及生育失败的功能分析 人类缺陷模型。这项研究可能会为哺乳动物受精过程提供新的见解 其天然状态，并有助于更好地了解导致不孕症的病理学。它还将建立新的 功能性实时成像工具，这将是生殖研究向前迈出的重要一步。 科学前提、科学严谨性和相关生物变量：该提案旨在填补 通过高度创新的实时成像方法，我们在生殖生物学领域缩小了重大差距 开发的。所有提出的实验均得到强有力的初步数据的支持，这些数据已发表在 四份同行评审出版物；目前正在审查另一份出版物。我们仔细地阐述了 使用的实验动物数量以及选择模型的理由。 “性作为一种生物 变量”不适用于我们的研究设计。我们已发布的协议的详细信息和参考是 提供以确保初步和拟议的实验可以在其他实验室复制。

PROJECT SUMMARY/ABSTRACT Estrous cycle, ovulation, fertilization, and pre-implantation pregnancy are fundamental reproductive processes of clinical importance. While research has shed light on the cellular and molecular mechanisms mediating these events, much of these data are derived from static histological analysis, low-resolution visualizations, and studies of invertebrate models (e.g. sea urchin). Therefore, any conclusions regarding mammalian fertilization, which takes place deep inside the body, are extrapolated and do not necessarily represent the native state. If this technical limitation was overcome, we may gain a more complete understanding of mammalian reproduction leading to the development of better fertility treatments and Assisted Reproductive Technologies (ART). By integrating expertise in live, functional optical coherence tomography (OCT) and reproductive biology, we recently established a set of unique methods for in vivo imaging of the female mouse reproductive tract. Our approach allows for (i) live, dynamic volumetric imaging of the mouse Fallopian tube (oviduct) with micro- scale spatial resolution, (ii) depth-resolved mapping of oviduct cilia location and cilia beat frequency (CBF); and (iii) tracking of individual sperm and their motility within the oviduct. None of these measurements are currently possible with other methods, and the dynamic environment of the female reproductive tract is too complex to model. Therefore, we are in a unique position to directly visualize specific mammalian reproductive processes from an entirely new vantage point. We propose the first in vivo volumetric imaging study of mammalian fertilization. This study is taking advantage of new technological developments in OCT imaging and will allow for quantitative assessment of hormonal regulation of oviduct cilia beating and muscle contractions, and functional analysis of fertility failures in mouse models of human defects. This study will likely provide new insight on the process of mammalian fertilization in its native state and lead to a better understanding of pathologies resulting in infertility. It will also establish new functional live imaging tools, which will be a major step forward in reproductive research. Scientific Premise, Scientific Rigor, and Relevant Biological Variables: This proposal is aimed to fill a significant gap in the field of reproductive biology through highly innovative live imaging methods, which we developed. All proposed experiments are supported by strong preliminary data, which have been published in four peer-reviewed publications; one more publication is currently under review. We carefully articulated the number of experimental animals to be used, and the rationale for the choice of the models. “Sex as a biological variable” does not apply to our study design. Extensive details and references to our published protocols are provided to ensure that preliminary and proposed experiments can be replicated in other laboratories.