Brad Stith, Ph.D is Professor in the Department of Biology at University of Colorado Denver, the Downtown Campus. He received his BS in Biology from Ohio State University in 1974 and his Ph.D from Washington State University in 1982. He was a Post-doctoral Fellow in Pharmacology at UC School of Medicine and in 1987 was appointed Assistant Professor in Biology at University of Colorado Denver. Brad Stith, PhD.
Although there are numerous membrane events associated with fertilization (membrane fusion during the acrosome reaction of sperm, sperm-egg merger, and cortical granule exocytosis), there have been few studies on lipids except for the activation of phospholipase C. Brad's laboratory studies the ctivation of kinases such as Src tyrosine kinase by lipid second messengers. In the 1990's, Brad's laboratory was the first to report the elevation of IP3 and DAG mass (products of phospholipase C catalysis) during fertilization and this and other work led to the suggestion that phospholipase D1b (PLD), phospholipase A2, autotoxin, and sphingomyelinase are activated at fertilization. This work was aided by his new lipid analysis protocol for HPLC and evaporative light scattering mass detection (developed in collaboration with Avanti Polar Lipids, Inc). Brad measured the major classes of phospholipids, plasmalogens, sphingolipids, and unsaturated lipids through fertilization. Fertilization is associated with an increase in phosphatidic acid mass (PLD catalyzes the production of this signaling lipid), and he suggests that this lipid may activate Src tyrosine kinase which is know to activate phospholipase C. He has measured the affinity of PA and Src (with fat blots where the binding of Src to 15 different lipids is determined), and theKd (using artificial vesicles, he used a vesicle sedimentation procedure to quantify the affinity of Src and PA). Compared to other lipids, Src bound specifically and with a much higher affinity to PA (phospholipase C did not bind PA). Addition of artificial PA to eggs releases intracellular calcium (measured by our intracellular calcium imaging system), opens calcium dependent chloride channels(measured by a membrane potential recording and voltage clamping system), increases Src and phospholpase C activity (phosphospecific antibody Western blotting), and increases IP3 mass. Important controls involve the use of other ineffective negative phospholipids (similar in charge to PA), and that tyrosine kinase, phospholipase C or IP3 receptor inhibitors block the action of PA. Thus, Brad has found a new step in the pathway from sperm binding to Src activation during fertilization in Xenopus laevis (an NIH recognized model system). Furthermore, this is the first suggestion that production of PA leads to Src activation, which in turn leads to phospholipase C activation and the major fertilization event of intracellular calcium release. Not only does this work impact other work on fertilization or the release of intracellular calcium, but it could also impact cancer studies where Src plays a major role. Brad also finds an increase in ceramide production through sphingomyelinase activation, and ceramidemight also play a role in fertilization.
Below is a microscope video that shows fertilization in the Xenopus frog. It shows both the surface contraction wave, the wrinkle that travels from the sperm binding site across the egg) and gravitational rotation (where the whole cell rotates to heavy white side down). Note the wave crosses the cell from left to right (from where the sperm entered) and then at around 9-10 min (note the times in the top left; hundredths of a second, seconds, and minutes reading from the right), the cell rotates (gravitational rotation). Insemination was at time zero. A second wave is starting as the video ends at about 15 min after insemination. I captured one image every 20 sec and the replay is at 10 frames per sec. The zygote is about 1.25mm across.