Release and molecular composition of mammalian SFs

University Of Massachusetts Amherst, Amherst MA

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Abstract

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.