Release and molecular composition of mammalian SFs

描述（由申请人提供）：哺乳动物卵的受精特征在于存在细胞内钙（[Ca 2 +]i）振荡，其负责诱导卵活化。精子如何启动和维持这些振荡尚不清楚，尽管最近的证据，包括来自胞浆内精子注射实验（ICSI）的证据，表明精子融合后可能会释放到卵质中，[Ca 2 +]i振荡诱导因子（SF）。本实验室的长期目标是分离和纯化精子中的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性以及精子中不同Ca 2+活性组分的分子组成。 具体目的1：研究SF的时间释放。该假设将被检查，SF成为迅速解离的精子头部，以诱导持续振荡，然后，在原核（PN）形成的时间，它与原核（PN）/周围PN结构，其目标是在释放时收到。为了验证这一假设，将Hoechst标记的精子头部从受精卵中取出，并在秋水仙胺存在下监测这些卵中振荡的持续性。将通过PN-包膜破裂时[Ca 2 v]i升高的存在来评价去核卵中SF与PN的相关性，这将由暴露于冈田酸诱导。 目的2：研究不同精子钙活性制剂的分子组成.本研究假设精子中不同的Ca 2+活性组分，即可溶性SF和低可溶性（TritonX-100和pH可溶性）SF，具有相同的生化特性，实际上可能具有共同的Ca 2+活性分子。将使用柱色谱法和使用生物素化肽A7的亲和沉淀进行生物化学分级分离，生物素化肽A7消耗SF中的Ca 2 v活性。在SDS-PAGE后，通过基质辅助激光解吸-质谱法对重要多肽条带进行测序。磷脂酶C（PLC）zeta，一种新的睾丸PLC的存在和功能，将通过蛋白质印迹法和从馏分中的分子消耗进行评估。重要性：1）这些目的的结果将首次描述受精过程中SF的暂时释放，并将建立精子中所有Ca 2+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，一种通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。

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.