A Question-based Project that Uses a MAP Kinase Western Blot to Examine the Role of Calcium in Sea Urchin Fertilization

 

Tested Studies in Laboratory Teaching, 2012, Volume 33

Aaron Coleman

Abstract

Sea urchins are a well-established model system for studying fertilization and early development. Urchin gametes are also an excellent tool for teaching cell biology in the laboratory, allowing students to make direct connections between biochemical changes in the egg and the cellular events of fertilization that can be observed under the microscope. In this project, the students examine some of the signal transduction events that occur at fertilization, and answer two questions regarding the role of the cytoplasmic calcium influx in entry of the zygote into the cell cycle. The central experiment of the project is a Western blot that allows the students to determine the activity of mitogen-activated protein (MAP) kinase. MAP kinase becomes active during the maturation of oocytes in most animals, and this activity is thought to maintain cell cycle arrest and prevent parthenogenesis (Colledge et al, 1994). Sea urchin eggs, which complete meiosis during maturation, contain active MAP kinase that becomes inactivated upon fertilization, presumably removing the cell cycle brake and allowing the zygote to undergo cleavage (Carroll et al, 2000). At fertilization, sperm binding triggers the activation of phospholipase C, leading to rapid production of the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG), and the opening of Ca2+ channels on the endoplasmic reticulum. The resulting Ca2+ influx is a central node in fertilization signal transduction and it stimulates many of the events of egg activation, including exocytosis of the cortical granules and elevation of the fertilization envelope. The students are introduced to the major aspects of fertilization signal transduction in lecture, and are then directed to answer the following two questions with their laboratory experiments: 1) Is the calcium influx sufficient to inactivate MAP kinase? 2) Is the calcium influx sufficient to produce cell division (first cleavage)?

Keywords:  sea urchin, fertilization, signal transduction, MAP kinase

New Mexico State University (2011)