Unraveling the Signal Transduction Pathways of Novel Mitochondrial Peroxiporins in Activated Piscine Spermatozoa

Abstract

Recent studies of how ejaculated marine teleost spermatozoa survive the hyperosmotic shock of seawater (SW) have uncovered a key role of an aquaporin-8 type water channel, termed Aqp8bb, in an activated sperm. Aqp8bb alleviates oxidative stress by acting as a mitochondrial peroxiporin to facilitate the efflux of reactive oxygen species (ROS), thereby allowing ATP production and the maintenance of flagellar motility. It is not known, however, whether the ejaculated spermatozoa of freshwater (FW) teleosts, which experience an opposing hyposmotic shock during activation, display similar detoxification mechanisms, and whether conserved or divergent signaling pathways controlling Aqp8bb mitochondrial targeting, exist in the different lineages. To uncover the intracellular signaling cascade in marine teleosts, we initially used a repertoire of activators and inhibitors of different transduction pathways on SW-activated seabream sperm. These experiments suggested that c-Jun N-terminal kinase (JNK), protein kinase C (PKC), and glycogen synthase kinase-3 (GSK3) are involved in Aqp8bb phosphorylation and transport to the mitochondrion. Accordingly, inhibition of JNK and GSK3 caused ROS accumulation in the mitochondrion and a decline of sperm motility, and thus, established the importance of these kinases in the regulatory transduction cascade. To determine the molecular initiators of this signaling cascade, we incubated nonactivated sperm with a Ca2+ ionophore in the presence of external Ca2+. This induced the phosphorylation of GSK3 and of Aqp8bb, and the accumulation of the channel in the mitochondrion, all of which could be prevented with an inhibitor of PKC. In turn, exposure of immotile spermatozoa to ROS, or SW-activation of sperm in the presence of a Ca2+ chelator, promoted the induction of the JNK-GSK3 signaling pathway, with the resultant triggering of Aqp8bb phosphorylation and mitochondrial membrane trafficking. To identify the specific target residues that are phosphorylated in Aqp8bb, we heterologously expressed the seabream channel in human liver hepatocellular (HepG2) cells. These experiments coupled with site-directed mutagenesis of the identified residues confirmed that both intracellular Ca2+ and oxidative stress regulate Aqp8bb mitochondrial trafficking through the respective GSK3- and JNK-mediated phosphorylation of Ser16 and Thr24 in the Aqp8bb N-terminus. Intriguingly, investigations of FW-activated salmon spermatozoa revealed that Aqp8bb is also trafficked to the mitochondria, but is only regulated by the PKC-GSK3 pathway. In conclusion, these results reveal for the first time that Aqp8bb channels function as mitochondrial peroxiporins in the activated spermatozoa of both FW and SW teleosts, but that Ca2+-activated PKC is the primary signaling pathway in FW teleost spermatozoa, while both Ca2+-activated PKC and ROS-activated JNK are the primary signaling pathways that regulate the Aqp8bb-mediated detoxification mechanism in the spermatozoa of SW teleosts.