Mitochondrial Activation and Nitric Oxide (NO) Release at Fertilization in Echinoderm Eggs

Abstract

In the cell, mitochondria are important organelles to supply energy for essential cell activities. It is unclear when and how mitochondrial activity increases on fertilization. We refer to mitochondrial activation here as mitochondrial innermembrane hyperpolarization. We measured fl uorescence changes in mitochondrial inner-membrane potential (ΔΨ m ) during fertilization in eggs of the echinoderm Hemicentrotus pulcherrimus and the sand dollar Clypeaster japonicus using a mitochondrial dye, MitoTracker Red CMXR (MTR). Increase in fl uorescence was detected after insemination in most eggs, which indicates hyperpolarization in ΔΨ m . To obtain the relationship between changes in intracellular Ca 2+ ([Ca 2+ IntroductionFertilization is thought to be one of the most remarkable phenomena, in which a large amount of energy is required for a zygote to increase diverse cellular activities leading to structural and qualitative changes for development from a dormant unfertilized state. At fertilization, mitochondria could be signifi cant in increasing various cellular activities, including supplying the energy. Generally, mitochondria have been recognized as multifunctional organelles playing crucial roles in cellular control. Their roles are to provide the main cellular energy in the form of adenosine triphosphate (ATP), to generate and regulate reactive oxygen species, to buffer cytosolic Ca 2+ , and to regulate apoptosis through a mitochondrial permeability transition pore (Wallace et al. 2010 ). Therefore, it is assumed that mitochondrial activity should increase accompanying the sequential phenomena, called "egg activation," that comprise changes in intracellular concentration of Ca 2+ ([Ca 2+ ] i ), pH, nitric oxide (NO) release (ΔNO), other second-messenger signals, and cytoplasmic changes, leading to activation of many important enzymes and factors for development. In sea urchins, early studies on respiration demonstrated that a burst of respiration started immediately after fertilization and reached the peak level within 3 min (Foerder et al. 1978 ;Fujiwara and Yasumasu 1997 ;Warburg 1908 ). One third of the respiration at fertilization should be responsible for mitochondrial respiration. However, when and how the mitochondria really activate remains unclear. According to the chemiosmotic theory, measuring mitochondrial inner-membrane potential (ΔΨ m ) has been used as an index of mitochondrial activity because ΔΨ m is utilized to provide energy for ATP production (Solani et al. 2007 ). Mitochondrial activation is believed to be by changes in hyperpolarization of ΔΨ m (Fujihara et al. 2007 ). On the other hand, ΔNO at fertilization has been reported in sea urchin eggs, although its main function is still obscure (Leckie et al. 2003 ;Mohri et al. 2008 ). We found in our previous study that mitochondrial activity has some relationship to ΔNO because mitochondrial inhibitors such as CN − and NaN 3 inhibited ΔNO, and an egg stratifi ed by centrifugation showed colocalization by double-staining with the mito...