AQUAPORIN-1aa and -8b are differentially localized in gilthead sea bream (Sparus aurata) spermatozoa and play distinct roles during the activation of sperm motility

10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 pageIntroduction In marine teleosts, such as the gilthead seabream, aquaporin water channels are implicated in sperm activation through mediation of water efflux from ejaculated spermatozoa exposed to hyperosmotic seawater. We have recently shown that seabream spermatozoa express up to five different aquaporin paralogs (Aqp1aa, -1ab, -7, -8b and -10b) with a segregated spatial distribution. However, the specific contribution of each paralog to the initiation and maintenance of sperm motility remains unknown. Here, we used paralog-specific antibodies as functional inhibitors to investigate the different roles of Aqp1aa and -8b during the hyperosmotic activation of sperm motility in the gilthead seabream. Methods Sperm was collected during the natural spawning season and used for immunofluorescence microscopy or activation assays. Aqp1aa and -8b mediated transport of water, urea or ammonia was determined from ectopic expression in Xenopus laevis oocytes, in the presence or absence of Aqp1aa and -8b affinity-purified antibodies. Motility parameters on seawater-diluted sperm were recorded using the Integrated Semen Analysis System software (ISAS version 1) in the presence of IgG (controls) or Aqp1aa or -8b antisera. Results and Discussion Expression of Aqp1aa and -8b in X. laevis oocytes revealed that Aqp1aa is water-specific, whereas Aqp8b is permeable to water, urea and ammonia. Double immunolocalization of Aqp1aa and -8b in ejaculated immotile spermatozoa confirmed that Aqp1aa is exclusively localized along the flagellum, whereas Aqp8b is dispersed in the head and the anterior flagellum. Upon activation, Aqp1aa localization is maintained along the tail while Aqp8b almost completely accumulates in the mitochondrion of the spermatozoa. Exposure of oocytes expressing Aqp1aa and -8b to their corresponding antibodies inhibited the osmotic water permeability of oocytes in a dose-dependent manner, and no cross-reaction of the antibodies was detected. Using these antibodies on sperm activation assays in seawater, we found that inhibition of Aqp1aa decreased most of the motion parameters (% of motile sperm, velocity, progressive motility), whereas Aqp8b inhibition only reduced the % of motile and progressive sperm. Conclusion These data suggest for the first time that Aqp1aa and -8b play distinct roles in the activation of teleost sperm. Aqp1aa possibly facilitates flagellar beating while Aqp8b may be required for the maintenance of spermatozoa progressivity. The function of Aqp8b-mediated mitochondrial transport of solutes (i.e. ammonia, urea) in this mechanism is under investigationPeer Reviewe