Hyperactivated motility, a swimming pattern displayed by mammalian sperm in the oviduct around the time of ovulation, is essential to fertilization. Ca(2+) has been shown to be crucial for the initiation and maintenance of hyperactivated motility. Nevertheless, how Ca(2+) reaches the axoneme in the core of the flagellum to switch on hyperactivation is unknown. Ca(2+)-releasing agents were used to determine whether an intracellular store provides Ca(2+) to the axoneme. Hyperactivation was induced immediately in bull sperm by thapsigargin, caffeine, and thimerosal. The responses were dose-dependent and were induced in both capacitated and uncapacitated sperm. When external Ca(2+) was buffered below 50 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the response to caffeine was significantly reduced; however, the responses to thapsigargin and thimerosal were not affected. This indicates caffeine-induced hyperactivation depends on external Ca(2+) influx, whereas hyperactivation by thapsigargin and thimerosal do not. Acrosome reactions were not induced by these treatments; therefore, an acrosomal store was probably not involved. Indirect immunofluorescence labeling showed type I inositol 1,4,5-trisphosphate receptors (IP(3)R) in the acrosome and neck region, but no ryanodine receptors (RyR) were found using anti-RyR antibodies or BODIPY FL-X ryanodine. These data indicate that there is an IP(3)R-gated Ca(2+) store in the neck region of sperm that regulates hyperactivated motility.

Hyperactivated motility, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization in vivo. It is characterized by high-amplitude flagellar waves and, usually, highly asymmetrical flagellar beating. It had been suggested, but not tested, that Ca2+ and cAMP switch on hyperactivation by directly affecting the flagellar axoneme. In this study, the direct affects of these agents on the axoneme were tested by using detergent-demembranated bull sperm. As confirmed by TEM, treatment of sperm with 0.2% Triton X-100 disrupted the plasma, acrosomal, and inner mitochondrial membranes, leaving axonemes intact. In the presence of 2 mM ATP, the percentage of reactivated sperm that were hyperactivated increased to 80% when free Ca2+ was increased from 50 to 400 nM. The effect of the Ca2+ in this range was to increase beat asymmetry by increasing the curvature of the principal bend. No additional increases were observed above 400 nM free Ca2+, but motility was suppressed at 1 mM. The ability of Ca2+ to produce hyperactivation depended on ATP availability, such that more ATP was required to produce the high amplitude flagellar bends characteristic of hyperactivated motility than to produce activated motility. Cyclic AMP was not required for reactivation, nor for hyperactivation. Production of hyperactivated motility also required an alkaline environment (pH 7.9-8.5). These results suggest that, provided sufficient ATP is present and pH is sufficiently alkaline, Ca2+ switches on hyperactivation by enabling curvature of the principal bends to increase.

Hyperactivated movement of spermatozoa was first reported by Yanagimachi (1969), who observed that, as spermatozoa gained the ability to fertilize oocytes in vitro, their flagella began beating more vigorously than before. He proposed that the vigorous beating plays a vital role in the penetration of the zona pellucida.Yanagimachi's proposal that hyperactivated motility enables spermatozoa to penetrate the zona pellucida was later confirmed by experiments. Stauss et al. (1995) used various methods that prevented hyperactivation but did not inhibit the acrosome reaction, and demonstrated that hyperactivated hamster spermatozoa were more successful at penetrating the zonae pellucidae of oocytes in vitro than spermatozoa that were not hyperactivated.Additional functions have been ascribed to hyperactivation. These were based on consideration of the obstacles encountered by spermatozoa in the oviduct. Mucus is present in the isthmus of the oviduct of rabbits (Jansen, 1978), humans (Jansen, 1980), pigs (Suarez et al., 1992) and cattle (Suarez et al., 1997). In many species, spermatozoa also encounter another type of mucus, consisting primarily of hyaluronic acid, while passing through the cumulus oophorus. Mucus increases the viscosity and elasticity of the aqueous milieu in which spermatozoa swim. Both hamster (Suarez et al., 1991) and mouse (Suarez and Dai, 1992) spermatozoa penetrated viscoelastic substances more effectively when they were hyperactivated; therefore, hyperactivated spermatozoa are likely to be more effective at penetrating oviductal mucus and the cumulus matrix in vivo.The behaviour of hamster and mouse spermatozoa within transilluminated oviducts indicated that hyperactivation enables spermatozoa to move about effectively in the lumen until they encounter the oocyte. Hyperactivated spermatozoa placed on a microscope slide in simple aqueous medium often spend a lot of time swimming in circles, whereas within the oviduct, the walls of which are not hard and flat like those of a slide, hyperactivated spermatozoa appear to cover space effectively in a search pattern. Mouse spermatozoa used the deep flagellar bends characteristic of hyperactivation to turn around within pockets of mucosa and escape out into the central lumen (Suarez and Osman, 1987). Hyperactivated hamster spermatozoa glided rapidly over the mucosal surface of the ampulla (Katz and Yanagimachi, 1980).In several species of mammal, a reservoir of spermatozoa forms within the isthmus of the oviduct. The reservoir is thought to preserve spermatozoa and ensure that they are in the proper state for fertilization when oocytes enter the oviduct, and is formed when spermatozoa are trapped by Hyperactivation is a movement pattern observed in spermatozoa at the site and time of fertilization in mammals. It may be critical to the success of fertilization, because it enhances the ability of spermatozoa to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to...

Hyperactivated sperm motility is usually characterized by high-amplitude flagellar bends and asymmetrical flagellar beating. There is evidence that an inositol 1,4,5-trisphosphate (IP3) receptor-gated Ca2+ store in the base of the flagellum provides Ca2+ to initiate hyperactivation; however, the identity of the store was not known. Ca2+ stores are membrane-bounded organelles, and the only two membrane-bounded organelles found in this region of sperm are the redundant nuclear envelope (RNE) and mitochondria. Transmission electron micrographs revealed two different compartments of RNE, one enriched with nuclear pores and the other containing few pores but extensive membranous structures with enlarged cisternae. Immunolabeling showed that IP3 receptors and calreticulin are located in the region containing enlarged cisternae. In other cell types, mitochondria adjacent to Ca2+ stores are actively involved in modulating Ca2+ signals by taking up Ca2+ released from stores and also may respond by increasing production of NADH and ATP to support increased energy demand. Nevertheless, bull sperm did not show an increase in NADH when Ca2+ was released from intracellular stores by thapsigargin to induce hyperactivation. Consistently, no net increase in ATP production was detected when sperm were hyperactivated, although ATP was hydrolyzed at a greater rate. Furthermore, blocking Ca2+ efflux from mitochondria by CGP-37157, a specific inhibitor of the mitochondrial Na+/Ca2+ exchanger, did not inhibit the development of hyperactivated motility. We concluded that the intracellular Ca2+ store is the part of RNE that contains enlarged cisternae and that Ca2+ is released directly to the axoneme to trigger hyperactivated motility without the active participation of mitochondria.

Dihydroceramide desaturases are evolutionarily conserved enzymes that convert dihydroceramide (dhCer) to ceramide (Cer). While elevated Cer levels cause neurodegenerative diseases, the neuronal activity of its direct precursor, dhCer, remains unclear. We show that knockout of the fly dhCer desaturase gene, (), results in larval lethality with increased dhCer and decreased Cer levels. Light stimulation leads to ROS increase and apoptotic cell death in -KO photoreceptors, resulting in activity-dependent neurodegeneration. Lipid-containing Atg8/LC3-positive puncta accumulate in-KO photoreceptors, suggesting lipophagy activation. Further enhancing lipophagy reduces lipid droplet accumulation and rescues -KO defects, indicating that lipophagy plays a protective role. Reducing dhCer synthesis prevents photoreceptor degeneration and rescues-KO lethality, while supplementing downstream sphingolipids does not. These results pinpoint that dhCer accumulation is responsible for -KO defects. Human dhCer desaturase rescues-KO larval lethality, and rapamycin reverses defects caused by dhCer accumulation in human neuroblastoma cells, suggesting evolutionarily conserved functions. This study demonstrates a novel requirement for dhCer desaturase in neuronal maintenance and shows that lipophagy activation prevents activity-dependent degeneration caused by dhCer accumulation.