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

我们对诱导钙离子的精子分子和机制的了解一直存在空白。 哺乳动物卵子的摆动。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。一种精子分子，精子因子，SF，后来被确认为 磷脂酶Zeta，PLCz，被认为是唯一负责诱导钙释放的SF，并且 PLCz失活突变会导致人类男性不育。然而，PLCz基因敲除小鼠是不能生育的 并引起残留的钙振荡，暗示了备用机制的功能。组件(S) 而后备系统的生理作用是我们知识中的空白。这些差距代表着严重的 障碍，因为在我们克服它们之前，我们不了解卵子激活的钙调节。 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不育。这个 长期目标是了解钙离子振荡的分子基础，这种振荡揭示了发育的 计划在受精。目的是确定触发这些钙振荡的精子分子。 在鸡蛋里。老鼠和它的配子是完美的模型，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PLC外，PLC还参与钙信号的传递，并可作为后备机制 在PLCz故障的情况下。这一假设是基于广泛的初步数据而提出的。这个 这项拟议研究的理论基础是，一旦所有引发振荡的精子分子都已知， 它们的调节评估、诊断价值和在生殖管理中的应用 充分探索的物种。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) 确定PLC1Z-KO精子中诱导受精时钙振荡的分子(S)；2)测定 后备系统对典型受精的钙反应和卵子激活事件的贡献。 在目标1下，我们将使用TRIM-21mRNA、特异性抗体、CRISPR产生的KO小鼠系、 免疫荧光、钙监测和胚胎培养以确定PLCD4是否是 备份系统。在目标2下，我们将使用类似的方法，但针对不同的分子和 确定钙备份系统对典型动物钙信号贡献的机制 受精。这一应用的研究具有创新性，因为它代表了对 其他精子PLC，包括其缺失导致男性不育的一种，在精子的振荡中的作用 受精。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，发现男性生育指标，以及 确定新的避孕目标。最后，我们期待在理解分子方面取得进展。 以及诱导哺乳动物胚胎发育的调节机制。

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.