WWW.WAS.ORG • WORLD AQUACULTURE • SEPTEMBER 2013 49 fish spermatozoa. Regulation of sperm motility is linked to cAMP signaling pathways in several animal species, including mammals, salmonids, sparids, and sea urchins or ascidians, including the contribution of the cAMP-dependent protein kinase A (PKA) as a regulator of sperm motility in some animal species. The link between cAMP concentration increase and motility initiation at the axoneme level was investigated mainly in salmonids. It involves a complex series of phosphorylation and dephosphorylation events. This includes the cAMP-dependent phosphorylation of the 15 kDa movement-initiating phosphoprotein (Hayashi et al. 1987, Jin et al. 1994) of a PKA (Itoh et al. 2003) and of the 22 kDa dynein light chain (Inaba et al. 1998). Protein phosphorylation is also regulated by proteasomes (Inaba et al. 1993, Inaba et al. 1998). Investigation of the dependence between protein phosphorylation and microtubule sliding and movement initiation is ongoing. Energetic Aspects Regulating Fish Sperm Motility In fish with external fertilization, shed spermatozoa depend entirely on energy previously stored or produced (but at too slow a rate) by mitochondria. The cellular sites of energy production (the mid-piece, containing mitochondria) are not the same as the sites of energy consumption (dynein ATPases along axoneme). So, energy must be transferred from one site to the other. An ATP shuttle plays this role in spermatozoa of salmonids and cyprinids. These fish possess an energetic compound, phosphocreatine (PCr), and creatine kinase enzyme (CK) that catalyzes the formation of ATP from PCr and ADP (Saudrais et al. 1998). Therefore, spermatozoa regenerate ATP from PCr and motility-generated ADP via CK. In addition, they can regenerate ATP through adenylate kinase (AK), which catalyzes the formation of ATP from ADP. The elucidation of fish speciesspecific peculiarities of the PCr shuttle and AK system participation in the supply of energy to the axoneme is of great interest. Initiation of fish sperm motility is followed immediately by a decrease of ATP level. The pattern observed in spermatozoa of sturgeon (Billard et al. 1999), trout, turbot and seabass (Cosson et al. 2008b) is a sharp decrease of ATP level in the first seconds of motility initiation and some stable concentration until the end of the investigated time. Similarly, a reduction of ATP content within 1-2 min of motility initiation is followed by a plateau until 5 min in carp spermatozoa (Perchec et al. 1995). spermatozoa in a dose- and time-dependent manner (Cosson et al. 1999). After cessation of movement, sperm cells undergo an unusual curling process, mainly a twisting of the flagellum, easily observed by dark-field microscopy and taking several seconds to reach completion in turbot. In seabass, sperm motility is prevented by 0.1 ppm HgCl2, but effects are greatly reduced when it is applied to demembranated sperm, inasmuch as it is assumed that metal binds and inhibits water channels present in the membrane. Combining the above features with knowledge of osmotic signals, SACs and Hg-sensitive water channels should be included in the paradigm describing the signaling pathway for activation of fish sperm (Cosson et al. 2008a). Aquaporin genes have been identified in the genome of several fish species, with some gene products localized in the spermatozoon. In Sparus aurata, the first event is water efflux that leads to local distortions of the flagellar membrane that, in turn, activate water channels (Zilli et al. 2012). Zilli et al. (2011) confirms the important role of aquaporins in initiating sperm motility. When these proteins are inhibited by HgCl2, the phosphorylation1 level, playing a crucial signal role, affects specific aquaporin proteins of specific molecular size (one of 174 kDa located in the head while others of 147, 97, and 33 kDa size in the flagella). In gilthead sea bream spermatozoa, the rapid water efflux across aquaporins determines a reduction in cell volume resulting from the increase in intracellular ionic concentration (Zilli et al. 2012). Signal Transfer to the Axonemal Motor of Flagella There are several candidates for such a role among ions. The increase of intracellular ion concentration could lead to the activation of adenylyl cyclase, which, in turn, would determine motility initiation by a mechanism of cAMP-dependent protein phosphorylation and dephosphorylation (Fig. 4). Reactive oxygen and nitrogen species, such as the superoxide anion (O2 −) and nitric oxide (NO), are modulators of signal transduction cascades. Some, especially H2O2, may enhance tyrosine phosphorylation through selective suppression of tyrosine phosphatase activity (Aitken et al. 2006) or activation of adenylyl cyclase, thus producing a higher cAMP level, leading to subsequent activation of serine/threonine kinase A. The various steps by which a signal is transferred from membrane to axoneme are complex and are not fully deciphered in FIGURE 4. Schematic drawing of signaling in fish sperm describing successive steps from contact with the surrounding fluid (seawater) to full activation of flagellar motility. A phosphorylation process occurs in specific axonemal proteins between steps 7 and 10 that leads to full activation at step 10 (Zilli et al. 2011, 2012). (CONTINUED ON PAGE 48)
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