"Dr. Chandler's laboratory is concerned with sperm-egg interaction during fertilization in both vertebrates and invertebrates. Eggs of most animals are surrounded by extracellular matrix layers of fibrous glycoproteins that are instrumental in preparing the incoming sperm for fusion with the egg. In addition, these extracellular matrix layers are modified by secretory proteins at fertilization to produce a sperm blocking layer that aids in preventing polyspermy - the fertilization of an egg with multiple sperm. Inner ECM layers of many eggs (e.g., the zona pelucida in mammals and the vitelline envelope in amphibians) contain sperm binding proteins while the outer ECM layers (e.g., the cumulus oophorus in mammals and the jelly layers in amphibians) play a role in sperm chemotaxis, activation of sperm motility, and induction of the acrosome reaction, steps that are instrumental in the physiological preparation of sperm for interaction with the egg surface.
"Recently the laboratory cloned and sequenced Allurin, a 21 kDa protein released from frog egg jelly that acts as a sperm chemoattractant. Allurin was purified by anion exchange chromatography and its amino acid sequence confirmed by trypsin digestion and MALDI mass spectrometry. This protein is homologous to sperm-binding proteins in mammals that belong to the CRISP family and is expressed in a hormone-regulated manner and applied to the egg as it moves through the oviduct. Allurin binds to both amphibian and mammalian sperm in a dose-dependent manner. Currently, the laboratory is studying the signal transduction pathways by which allurin activates sperm, the distribution of allurin-like proteins within vertebrates and the tertiary structure of allurin. Chandler's group hopes to determine whether allurin-like proteins function in mammalian reproductive systems.
"To address these goals the laboratory uses a broad range of morphological, physiological, and molecular biological approaches. Quick-freeze/freeze fracture electron microscopy and laser scanning confocal microscopy are used to study the molecular structure of egg ECM layers. Video microscopy and immunocytochemistry are used to study sperm motility and expression of allurin in reproductive tissues. In vitro assays for sperm chemotaxis, sperm-egg binding and fertilization competence of sperm are employed to identify ECM proteins having essential biological activities. SDS PAGE, column chromatography, HPLC, and mass spectroscopy are used to isolate and characterize these glycoproteins. Ultimately, new ECM proteins will be sequenced, their genes identified, and sequence information used to search for related genes in mammals."