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）：哺乳动物卵中的受精为特征，其特征是存在细胞内钙（[Ca2+] i）振荡，这些振荡负责诱导卵子激活。精子如何启动和维持这些振荡尚不清楚，尽管最近的证据，包括胞质内精子注射实验（ICSI）的证据表明，融合后，精子可能释放到卵子中，A [CA2+] I振荡诱导因子（SF）。实验室的长期目标是隔离和净化精子的Ca 2+活性分子，目前的特定目的将确定SF的时间释放和细胞靶向以及精子中不同Ca 2+活性分数的分子组成。 特定目的1：研究SF的时间释放。该假设将检查SF从精子头迅速解离以诱导持续的振荡，然后在前核（PN）形成时，它与核（PN）/Peri-PN结构相关联，该结构是在释放时接收到的靶向。为了检验这一假设，将从受精卵中去除hoechst标记的精子头，并在粘膜剂存在下监测的这些卵中的振荡持续存在。 SF与PN中的PN缔合卵中的缔合将通过存在[Ca2v] I在PN-Envelope崩溃时上升来评估，这将通过暴露于Okadaic Acid引起。 具体目标2：研究不同精子Ca 2+活性制剂的分子组成。将研究该假设，即可溶性的SF和较少可溶性（Triton X-100和pH溶解性）SF，具有生化特性，实际上可能具有常见的Ca 2V活性分子（S）。生化分级将使用柱色谱法进行，并使用生物素化肽A7亲和沉淀，从而耗尽SF的Ca 2V活性。 SDS-PAGE在SDS-PAGE后通过基质辅助激光脱附质量光谱法进行测序。新型睾丸PLC磷脂酶C（PLC）的存在和功能，将通过分数的蛋白质印迹和耗竭来评估。意义：1）这些目标的结果将提供受精过程中SF时间释放的首次描述，并将建立精子中所有Ca 2交流制剂的多肽组成，这将导致SF的鉴定； 2）可以评估精子操纵对ICSI发起的振荡模式的生理相关性和影响，这是一种通常用于治疗人类不孕症的技术，该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所致。