Release and molecular composition of mammalian SFs

描述(申请人提供)：哺乳动物卵子受精的特征是存在细胞内钙([Ca~(2+)]i)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，包括来自卵胞浆内单精子注射实验(ICSI)的证据表明，精子融合后可能会释放一种[Ca2+]i振荡诱导因子(SF)到卵浆中。该实验室的长期目标是分离和纯化精子的钙活性分子，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钙活性组分的分子组成。具体目的1：考察顺铂的时间释放规律。这一假设将被检验，即SF迅速从精子头部解离以诱导持续的振荡，然后，在原核(PN)形成时，它与原核(PN)/周围-PN结构相关联，在释放时接收到靶向。为了验证这一假设，霍赫斯特标记的精子头将从受精卵中移除，并在秋水仙素存在的情况下监测这些卵子中振荡的持久性。去核卵子中SF与Pn的关系将通过在Pn-包膜破裂时[Ca2v]i的存在来评估，这将由冈田酸诱导。特定目的2：研究不同精子钙活性制剂的分子组成。这一假设将被研究，不同的精子钙活性部分，即可溶性SF和较难溶解的(Triton X-100和pH可溶的)SFS，具有共同的生化特性，并可能具有共同的Ca2+活性分子(S)。生化分离将使用柱层析进行，并使用生物素化多肽A7进行亲和沉淀，生物素化多肽A7将耗尽SF中的钙离子活性。在用基质辅助激光解吸-质谱仪进行SDS-PAGE后，将对重要的多肽条带进行测序。磷脂酶C(PLC)Zeta是一种新的睾丸PLC，它的存在和功能将通过Western blotting和去除其分子来评估。意义：1)这些目标的结果将首次描述受精过程中SF的时间释放，并将建立精子中所有钙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纵的生理相关性和对ICSI引发的振荡模式的影响，ICSI是一种常用的治疗人类男性不育的技术，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。

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.