Regulation of Ca2+ influx in mouse oocytes and eggs during maturation and fertilization to improve assisted reproductive technologies and modulate fertility

调节小鼠卵母细胞和卵在成熟和受精过程中的 Ca2 流入，以改进辅助生殖技术并调节生育力

基本信息

批准号：
10401470
负责人：
Rafael Antonio Fissore
金额：
$ 25.33万
依托单位：
UNIVERSITY OF MASSACHUSETTS AMHERST
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-17 至 2024-05-31
项目状态：
已结题

项目摘要

There is a persistent gap in our knowledge of the molecules and mechanisms that mediate Ca2+ influx into oocytes and eggs. Ca2+ influx is required for egg activation and embryo development in all mammals, including human beings. This gap in our knowledge therefore represents a serious impediment, as until it is filled we cannot physiologically modulate Ca2+ influx or objectively diagnose and treat infertility associated with disturbances in Ca2+ homeostasis. The long-term goal is to understand how Ca2+ homeostasis is regulated in oocytes and eggs and identify its molecular effectors. The objective here is to identify the Ca2+ channel(s) that mediate Ca2+ influx during maturation and fertilization and characterize their regulatory mechanisms. Mouse oocytes/eggs are a great model because they display distinctive Ca2+ entry during maturation and fertilization, and Ca2+ release is required for egg activation. The central hypothesis is that expression and/or distinct regulation of underdetermined Ca2+ channels on the plasma membrane underlies Ca2+ influx in oocytes, its inactivation during maturation and its recovery after fertilization. This hypothesis was conceived based on extensive preliminary data. The rationale for this research is that once the channels are identified, a better understanding of the molecular determinants of oocyte maturation and fertilization will be gained. The findings here also have the potential to translate into therapeutic methods to assist infertile couples in this country. We plan to test our central hypothesis by pursuing the following specific aims: 1) Identify the Ca2+ channel(s) that mediate Ca2+ influx in oocytes prior to and during maturation; and 2) Identify the Ca2+ influx channel(s) that support oscillations after fertilization. Under Aim 1, Ca2+ imaging, pharmacology, conditional knockout mice, and electrophysiology will be used to identify the active channel(s) and to assess the impact of Ca2+ influx on maturation; the role of a TRPM7-like current recently discovered by the applicant and collaborators will be closely examined. Under Aim 2, the signaling mechanism(s) whereby fertilization stimulates Ca2+ influx and the contributing channel(s) will be determined. A novel approach that overcomes the inactivation of Ca2+ influx in eggs will facilitate these studies. Genetic models and electrophysiology will confirm the function of these channel(s) during fertilization. The research in this application is innovative because it combines electrophysiology, pharmacology and new KO lines, approach that has served to identify two new channels in oocytes, including TRPM7, whose global deletion is embryonic lethal at E7.5. The contribution of the proposed project is significant because it is expected to allow physiological modulation of Ca2+ entry in oocytes and eggs that will produce new conditions and activation protocols for use in the clinic. It will also expand our understanding of the impact of Ca2+ homeostasis on oocyte competence and infertility.

我们对介导 Ca2+ 内流的分子和机制的了解一直存在差距进入卵母细胞和卵子。所有哺乳动物的卵子激活和胚胎发育都需要 Ca2+ 流入，包括人类。因此，我们知识上的这种差距是一个严重的障碍，直到它已被填满，我们无法从生理上调节 Ca2+ 流入或客观地诊断和治疗不孕症与 Ca2+ 稳态紊乱有关。长期目标是了解 Ca2+ 如何卵母细胞和卵子中的稳态受到调节，并确定其分子效应器。这里的目标是识别在成熟和受精过程中介导 Ca2+ 流入的 Ca2+ 通道并表征他们的监管机制。小鼠卵母细胞/卵子是一个很好的模型，因为它们表现出独特的特征成熟和受精过程中 Ca2+ 进入，卵子激活需要 Ca2+ 释放。中央假设是血浆上未确定的 Ca2+ 通道的表达和/或独特调节膜是卵母细胞中 Ca2+ 流入、成熟过程中失活以及成熟后恢复的基础施肥。这一假设是基于大量的初步数据而提出的。这样做的理由研究表明，一旦确定了通道，就可以更好地了解分子决定因素将获得卵母细胞的成熟和受精。这里的发现也有可能转化为研究帮助该国不孕夫妇的治疗方法。我们计划检验我们的中心假设通过追求以下具体目标： 1) 识别介导卵母细胞 Ca2+ 流入的 Ca2+ 通道成熟之前和期间； 2) 识别支持振荡后的 Ca2+ 流入通道施肥。目标 1、Ca2+ 成像、药理学、条件基因敲除小鼠和电生理学将用于识别活性通道并评估 Ca2+ 流入对成熟的影响；这申请人和合作者最近发现的类TRPM7电流的作用将密切关注检查了。在目标 2 下，受精刺激 Ca2+ 流入的信号机制和将确定贡献渠道。克服 Ca2+ 失活的新方法鸡蛋的涌入将促进这些研究。遗传模型和电生理学将证实该功能受精期间这些通道的变化。该应用中的研究具有创新性，因为它结合了电生理学、药理学和新的 KO 系，这些方法已用于识别两种新的卵母细胞中的通道，包括 TRPM7，其整体缺失在 E7.5 时是胚胎致死的。这拟议项目的贡献是显着的，因为它有望实现生理调节 Ca2+ 进入卵母细胞和卵子中，这将产生新的条件和激活方案，用于诊所。它还将扩大我们对 Ca2+ 稳态对卵母细胞能力影响的理解和不孕症。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

SPERM FACTORS AND EGG ACTIVATION: ICSI and the discovery of the sperm factor and PLCZ1.

DOI：
10.1530/rep-21-0487
发表时间：
2022-05-23
期刊：
REPRODUCTION
影响因子：
3.8
作者：
Gupta, Neha;Akizawa, Hiroki;Lee, Hoi Chang;Fissore, Rafael A.
通讯作者：
Fissore, Rafael A.

Editorial: The Fertilization Success From the Oocyte's Perspective.

DOI：
10.3389/fcell.2021.810420
发表时间：
2021
期刊：
Frontiers in cell and developmental biology
影响因子：
5.5
作者：
Michaut MA;Souza-Fabjan JMG;Fissore RA
通讯作者：
Fissore RA

Multidrug resistance transporter-1 dysfunction perturbs meiosis and Ca2+ homeostasis in oocytes.

DOI：
10.1530/rep-22-0192
发表时间：
2023-01-01
期刊：
REPRODUCTION
影响因子：
3.8
作者：
Nabi, Dalileh;Bosi, Davide;Gupta, Neha;Thaker, Nidhi;Fissore, Rafael;Brayboy, Lynae M.
通讯作者：
Brayboy, Lynae M.

The eggstraordinary story of how life begins.