Mouse phospholipase C, zeta 1 (PLCZ1), a strong candidate of egg-activating sperm factor, induces Ca(2+) oscillations and accumulates into formed pronucleus (PN) when expressed by cRNA injection. These activities were compared among mouse and human PLCZ1, newly cloned rat Plcz1, and medaka fish plcz1. The PLCZ1 proteins of the four species have an approximately homologous sequence of nuclear localization signal. However, the nuclear translocation ability was defective in rat, human, and medaka PLCZ1 expressed in mouse eggs. Rat PLCZ1 could not enter rat PN, whereas mouse PLCZ1 could. Mouse and human PLCZ1 translocated into the nucleus of COS-7 cells transfected with cDNA. There was little medaka PLCZ1 accumulated in the nucleus, and rat PLCZ1 was never located in the nucleus. All PLCZ1 proteins including fish could induce Ca(2+) oscillations in mouse eggs, but the activity was variable in the order of human >> mouse > medaka >> rat, estimated from minimal RNA concentration to induce Ca(2+) spikes. Ca(2+) oscillations by human PLCZ1 continued far beyond the time of PN formation (T(PN)), whereas those by mouse PLCZ1 ceased slightly before T(PN). High-frequency Ca(2+) spikes by overexpressed rat PLCZ1 stopped far before T(PN), possibly by feedback inhibition. Ca(2+) oscillations by fertilization of rat eggs stopped at T(PN), despite defective nuclear translocation of rat PLCZ1. Thus, PLCZ1 sequestration into PN participates in termination of Ca(2+) oscillations at the interphase of mouse embryos but does not always operate in other mammals, notably in rat embryos.

It is well known that the motility of spermatozoa in rainbow trout is suppressed by K+. We showed here that although trout sperm are completely immotile in medium containing 5 mM K+, motility was initiated by the subsequent addition of several mM Ca2+, suggesting that both K+ and Ca2+ are related to the process of the initiation of sperm motility. It was further found that Kt channel blockers tetraethylammonium, nonyltriethylammonium, Ba2+ and Cs+ , as well as the Ca2+ channel blocker verapamil, inhibited the initiation of sperm motility at doses at which these reagents inhibit chnnel-related functions in other cells. However, Na+ channel blocker, tetrodotoxin and anion channel blocker 4, 4-diisothiocyatatostilbene-2, 2'-disulfonic acid inhibited the motility only at extremely high doses. These results suggest that transport of K+ and Ca2+ through ion channels at the plasma membrane of spermatozoa is the first event that triggers the initiation of sperm motility in rainbow trout.The mechanism underlying the initiation of sperm motility is attracting much attention and one of the most successful investigations concerning this phenomenon were performed in studies using fish sperm, especially the sperm of salmonid fishes (15). The sperm motility of rainbow trout (22,28) as well as other salmonid fishes (19,22) is suppressed completely in the medium containing K f , which explains why the motility of trout sperm is suppressed in the sperm duct by seminal K+ surrounding the spermatozoa. Motility is initiated under experimental conditions when the semen is diluted into K+-free solution (19, 22, 28), suggesting that spermatozoa become motile when they are released into fresh water at natural spawning. In addition, it was also established that the decrease of K+ immediately caused the significant and transient increase of intracellular cAMP (17) which triggers the initiation of sperm motility (18) , through the CAMP-dependent phosphorylation of axonemal protein (16) in rainbow trout. However, how the decrease of K+ outside of the cell induces the synthesis of intracellular cAMP and causes the initiation of axoneme movement is poorly understood.Baynes et al. has suggested that inhibition of sperm motility by K+ is antagonized by the presence of divalent cations such as Ca2+ and Mg2+ ( 3 ) . We confirmed the above result and furthermore demonstrated that intracellular cAMP increased and spermatozoa became motile when several mM of Ca2+ was added to the sperm suspension in which spermatozoa were immotile in the presence of K+ (15). These imply that transport of Kf and Ca2+ may occur through plasma membrane, inducing the synthesis of cAMP in the cell, resulting in the initation of trout sperm motility.In order to clarify the contribution of ion channels to the process of initiation of sperm 117

Flux of K+ and changes in intracellular Ca2+ in the sperm of salmonid fishes were measured with spectrophotometry, ion electrode, microscopic fluorometry, and radioisotope accumulation. Release of K+ occurred at the initiation of sperm motility which is induced by decrease in external K+ and the K+ efflux and sperm motility were inhibited by K+ channel blockers. Intracellular Ca2+ increased within a short period in K(+)-free condition, and the accumulation of 45Ca in sperm cells was higher in motile sperm than that in immotile sperm. The efflux of K+ and the increase in intracellular Ca2+ were suppressed when external K+ concentration increased, i.e., sperm remained immotile. These results suggest that efflux of K+ through K+ channel and subsequent increase in intracellular Ca2+ are prerequisite for the initiation of sperm motility.

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Spermatozoa of the herring, Clupea palasii, were immotile in a solution of isotonic to seminal plasma but swimming was initiated in a hypertonic solution such as 60% seawater. This suggests that hyperosmolality is a factor for the initiation of sperm motility in herring. The motility of herring spermatozoa in hypertonic solution remained very low but became active in "egg water," suggesting that a sperm-activating substance is released from unfertilized eggs and diffuses into the surrounding environment to promote fertilization. The activity of the egg water was heat stable and nondialyzable, and was lost upon digestion with the proteolytic enzyme, pronase E; thus the substance is a protein. During procedures for partial purification using gel-filtration chromatography on Sephadex G-75, the substance was found to be very adherent to the Sephadex beads when buffer solution or the lower salt solution was used as the eluate instead of 0.5 M NaC1. This suggests that the sperm-activating substance might be attached to the surface of eggs in the relatively lower ionic environment of the female reproductive organ and is released from the eggs when they are spawned into the high salt environment of seawater. o 1992 Wiley-Liss, Inc.