Mammalian Fertilization: Identifying the second sperm factor that induces residual calcium oscillations and its contributions to egg activation

哺乳动物受精：识别诱导残留钙振荡的第二个精子因子及其对卵子激活的贡献

• 批准号: 10574938
• 负责人: Rafael Antonio Fissore
• 金额: $ 23.48万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10574938
• 关键词: Affect; Animals; Antibodies; Calcium; Calcium Oscillations; Calcium Signaling; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Contraceptive methods; Data; Defect; Development; Diagnosis; Diagnostic; Embryonic Development; Embryonic Induction; Enzymes; Event; Failure; Fertility; Fertilization; Fertilization failure; Functional disorder; Genetic; Germ Cells; Goals; Health; Hour; Human; Immunofluorescence Immunologic; Infertility; Inositol; Investigation; Knockout Mice; Knowledge; Laboratories; Male Infertility; Mammals; Mediating; Messenger RNA; Methods; Mission; Modeling; Modification; Molecular; Monitor; Mus; Mutation; Outcome; Patients; Pattern; Periodicals; Phosphoinositide Pathway; Phospholipase; Phospholipase C; Physiological; Plants; Production; Protein Isoforms; Public Health; Regulation; Research; Residual state; Role; Signal Transduction; Site; System; Techniques; Testing; Therapeutic; United States National Institutes of Health; blastocyst; burden of illness; diagnostic value; egg; embryo culture; human male; improved; innovation; inorganic phosphate; male; male fertility; novel; prevent; programs; receptor; reproductive; response; spatiotemporal; sperm cell; zygote

There is a persistent gap in our knowledge of the sperm molecules and mechanisms that induce Ca2+ oscillations in mammalian eggs. Ca2+ release triggers egg activation and initiation of embryo development in all mammals, including human beings. A sperm molecule, the sperm factor, SF, later identified as Phospholipase zeta, PLCz, was thought to be the only SF responsible for inducing Ca2+ release, and PLCz inactivating mutations cause infertility in human males. However, Plcz knockout mice are subfertile and evoke residual Ca2+ oscillations, suggesting the function of a backup mechanism. The component(s) and the physiological role of the backup system are gaps in our knowledge. These gaps represent serious impediments, as until we overcome them, we do not understand the Ca2+ regulation of egg activation events or can objectively diagnose, prevent or treat infertility associated with egg activation failure. The long-term goal is to understand the molecular basis of the Ca2+ oscillations that unfold the developmental program at fertilization. The objective is to identify the sperm molecules that trigger these Ca2+ oscillations in the egg. The mouse and its gametes are perfect models because they are amenable to manipulation and genetic modifications. The central hypothesis underpinning this proposal is that one or more sperm PLCs besides PLCz contribute to delivering the Ca2+ signal and could serve as the backup mechanism in case of Plcz disfunction. This hypothesis was conceived based on extensive preliminary data. The rationale for the proposed research is that once all sperm molecules that induce oscillations are known, their regulation assessed, and their diagnostic value and application to the reproductive management of species fully explored. We plan to test our central hypothesis by pursuing the following specific aims: 1) Identify the molecule(s) in PLC1z-KO sperm that induce Ca2+ oscillations at fertilization; 2) Determine the contribution of the backup system to the Ca2+ responses and egg activation events of typical fertilization. Under Aim 1, we will use Trim-21 mRNA, specific antibodies, CRISPR-generated KO mouse lines, immunofluorescence, Ca2+ monitoring, and embryo culture to determine if PLCd4 is the active factor of the backup system. Under Aim 2, we will employ similar methods but target different molecules and mechanisms to ascertain the contribution of the Ca2+ backup system to the Ca2+ signal of typical fertilization. The research in this application is innovative because it represents a fresh examination of the role of other sperm PLCs, including one whose deletion causes male infertility, in the oscillations of fertilization. The contributions of the proposed project are significant because we aim to perfect the treatment of egg activation failure, improve embryo development, discover indicators of male fertility, and identify novel targets of contraception. Finally, we anticipate progress in understanding the molecules and regulatory mechanisms that induce embryo development in mammals.

我们对精子分子和诱导 Ca2+ 的机制的了解一直存在差距 哺乳动物卵的振荡。 Ca2+ 释放触发卵子激活和胚胎发育启动 在所有哺乳动物中，包括人类。精子分子，精子因子，SF，后来被确定为 磷脂酶 zeta (PLCz) 被认为是唯一负责诱导 Ca2+ 释放的 SF，并且 PLCz 失活突变会导致人类男性不育。然而，PLcz 基因敲除小鼠的生育力低下 并引起残余的 Ca2+ 振荡，表明有备用机制的功能。组件 而备份系统的生理作用是我们的知识空白。这些差距代表严重 障碍，因为在我们克服它们之前，我们不了解卵子激活的 Ca2+ 调节 事件或可以客观地诊断、预防或治疗与卵子激活失败相关的不孕症。这 长期目标是了解 Ca2+ 振荡的分子基础，该振荡展开发育 受精计划。目的是识别触发这些 Ca2+ 振荡的精子分子 在鸡蛋里。小鼠及其配子是完美的模型，因为它们易于操纵 和基因改造。支持这一提议的中心假设是一个或多个精子 除 PLCz 之外的 PLC 也有助于传递 Ca2+ 信号，并可作为备用机制 Plcz 故障时。这一假设是基于大量的初步数据而提出的。这 这项研究的基本原理是，一旦已知所有引起振荡的精子分子， 对它们的调节进行评估，以及它们的诊断价值和在生殖管理中的应用 物种得到充分探索。我们计划通过追求以下具体目标来检验我们的中心假设：1） 识别 PLC1z-KO 精子中在受精时诱导 Ca2+ 振荡的分子； 2）确定 备用系统对典型受精的 Ca2+ 反应和卵子激活事件的贡献。 在目标 1 下，我们将使用 Trim-21 mRNA、特异性抗体、CRISPR 生成的 KO 小鼠系、 免疫荧光、Ca2+ 监测和胚胎培养以确定 PLCd4 是否是 备份系统。在目标 2 下，我们将采用类似的方法，但针对不同的分子和 确定 Ca2+ 备用系统对典型 Ca2+ 信号的贡献的机制 施肥。该应用中的研究具有创新性，因为它代表了对 其他精子 PLC（包括删除导致男性不育的精子 PLC）在精子振荡中的作用 施肥。拟议项目的贡献是重大的，因为我们的目标是完善 治疗卵子激活失败，改善胚胎发育，发现男性生育力指标，以及 确定新的避孕目标。最后，我们预计在理解分子方面会取得进展 以及诱导哺乳动物胚胎发育的调节机制。