Release and molecular composition of mammalian SFs

哺乳动物 SF 的释放和分子组成

• 批准号: 6772484
• 负责人: Rafael Antonio Fissore
• 金额: $ 7.6万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6772484
• 关键词: SDS polyacrylamide gel electrophoresis; artificial fertilization; calcium flux; chromatography; colchicine; egg /ovum; fertilization; hydroxyapatites; laboratory mouse; matrix assisted laser desorption ionization; nuclear membrane; okadaic acid; phospholipase C; protein sequence; sperm; sperm analysis; swine; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): Fertilization in mammalian eggs is characterized by the presence of intracellular calcium ([Ca2+]i) oscillations that are responsible for inducing egg activation. How the sperm initiates and sustains these oscillations is not known, although recent evidence, including that from intracytoplasmic sperm injection experiments (ICSI), suggests that the sperm may release into the ooplasm, after fusion, a [Ca2+]i oscillation-inducing factor (SF). The long-term goal of the laboratory is to isolate and purify the sperm's Ca 2+ active molecule, and the present specific aims will ascertain the temporal release and cellular targeting of SF and the molecular composition of the different Ca 2+active fractions in sperm. Specific Aim 1: To investigate the temporal release of SF. The hypothesis will be examined that SF becomes rapidly dissociated from the sperm head to induce persistent oscillations and then, at the time of pronuclear (PN) formation, it associates with pronuclear (PN)/peri-PN structures, the targeting for which is received at the time of release. To test this hypothesis, Hoechst-labeled sperm heads will be removed from fertilized eggs, and the persistence of oscillations in these eggs monitored in the presence of colcemid. Association of SF with the PN in enucleated eggs will be evaluated by the presence of a [Ca2v]i rise at PN-envelope breakdown, which will be induced by exposure to okadaic acid. Specific Aim 2: To investigate the molecular composition of the different sperm Ca 2+ active preparations. The hypothesis will be studied that distinct sperm Ca 2+ active fractions, i.e. soluble SF and less soluble (Triton X-100 and pH-soluble) SFs, share biochemical properties and may have, in fact, a common Ca 2v active molecule(s). Biochemical fractionation will be carried out using column chromatography, and affinity precipitation using biotinylated peptide A7, which depletes Ca 2v activity from SF. Sequencing of significant polypeptide bands will be performed after SDS-PAGE by Matrix Assisted Laser Desorption-Mass Spectrometry. The presence and function of phospholipase C (PLC) zeta, a novel testis PLC, will be assessed by Western blotting and depletion of the molecule from the fractions. Significance: 1) Results from these aims will provide the first description of temporal release of SF during fertilization, and will establish the polypeptide composition of all the Ca 2vactive preparations in sperm, which will lead to the identification of SF; 2) It will be possible to assess the physiological relevance and impact of sperm manipulations on the pattern of oscillations initiated by ICSI, a technique commonly used to treat human male infertility, which has been shown to have detrimental effects on development, and some of which may be due to activation defects.

描述（由申请人提供）：哺乳动物卵子受精的特征是细胞内钙（[Ca2+]i）振荡的存在，其负责诱导卵子活化。精子如何启动和维持这些振荡尚不清楚，尽管最近的证据，包括来自胞浆内单精子注射实验 (ICSI) 的证据，表明精子在融合后可能会释放到卵质中，一种 [Ca2+]i 振荡诱导因子。 SF）。实验室的长期目标是分离和纯化精子的Ca 2+ 活性分子，目前的具体目标是确定SF的时间释放和细胞靶向以及精子中不同Ca 2+ 活性组分的分子组成。 具体目标 1：研究 SF 的暂时释放。将检验以下假设：SF 快速从精子头部解离以诱导持续振荡，然后在原核 (PN) 形成时，它与原核 (PN)/peri-PN 结构相关联，其目标被接收发布时。为了检验这一假设，将从受精卵中取出赫斯特标记的精子头，并在秋水酰胺存在的情况下监测这些卵子振荡的持续性。去核蛋中 SF 与 PN 的关联将通过 PN 包膜破裂时 [Ca2v]i 升高的存在进行评估，这将由暴露于冈田酸诱导。 具体目标 2：研究不同精子 Ca 2+ 活性制剂的分子组成。将研究以下假设：不同的精子 Ca 2+ 活性组分，即可溶性 SF 和难溶性（Triton X-100 和 pH 溶性）SF，具有相同的生化特性，并且实际上可能具有共同的 Ca 2v 活性分子）。将使用柱色谱进行生化分级分离，并使用生物素化肽 A7 进行亲和沉淀，该肽会消耗 SF 中的 Ca 2v 活性。 SDS-PAGE 后，将通过基质辅助激光解吸质谱法对重要的多肽条带进行测序。磷脂酶 C (PLC) zeta（一种新型睾丸 PLC）的存在和功能将通过蛋白质印迹和从级分中去除该分子来评估。意义： 1) 这些目标的结果将首次描述受精过程中 SF 的暂时释放，并将确定精子中所有 Ca 2v 活性制剂的多肽组成，这将导致 SF 的鉴定； 2）将有可能评估精子操作对 ICSI 引发的振荡模式的生理相关性和影响，ICSI 是一种常用于治疗人类男性不育症的技术，已被证明对发育有不利影响，其中一些可能是由于激活缺陷造成的。