Change of osmolality surrounding spawned sperm from isotonic to hypotonic causes the initiation of sperm motility in the common carp. Here we show that membrane-permeable cAMP does not initiate motility of carp sperm that is quiescent in isotonic solution, and that motility of the demembranated sperm can be reactivated without cAMP. Furthermore, the cAMP level does not change during the initiation of sperm motility, and inhibitors of protein kinase do not affect sperm motility, suggesting that no cAMPdependent system is necessary for the regulation of sperm motility. Sperm motility could not be initiated in Ca 2؉ -free hypoosmotic solutions, and significant increase in the intracellular Ca 2؉ level was observed by a Ca-sensitive fluorescence dye during hypoosmolality-induced active motion period. The demembranated sperm cells were fully reactivated in the solutions containing 10 ؊7 to 10 ؊5 M Ca 2؉ . Ca 2؉ channel blockers such as verapamil and -conotoxin reversibly inhibited the initiation of sperm motility, suggesting that Ca 2؉ influx is the prerequisite for the initiation of carp sperm motility. Motility of intact sperm was completely blocked; however, that of the demembranated sperm was not inhibited by the calmodulin inhibitor W7, suggesting that the calmodulin bound close to the plasma membrane participated in the initiation of sperm motility. Flow cytometric membrane potential measurements and spectrophotometric measurements by using fluorescence dyes showed transient membrane hyperpolarization on hypoosmolality-induced motility. This article discusses the role of membrane hyperpolarization on removal of inactivation of Ca 2؉ channels, leading to Ca 2؉ influx at the initiation of carp sperm motility.

The sperm-activating and -attracting factor released from the eggs of the ascidians Ciona intestinalis and Ciona savignyi requires extracellular Ca 2؉ for activating sperm motility and eliciting chemotactic behavior of the activated sperm toward the egg. Here, we show that modulators of the store-operated Ca 2؉ channel, SK&F96365, Ni 2؉ , 2-aminoethoxydiphenylborane, and thapsigargin inhibit the chemotactic behavior of the ascidian sperm; on the other hand, blockers of voltage-dependent Ca 2؉ channels did not inhibit the chemotaxis, even though they inhibited the sperm activation operated by voltage-dependent Ca 2؉ channels. The blockers of store-operated Ca 2؉ channels also inhibited the asymmetrical flagellar beating and turning movements of the ascidian sperm, which are typical signs of sperm chemotaxis. Depletion of internal Ca 2؉ stores by thapsigargin induced capacitative Ca 2؉ entry into the sperm, which was blocked by SK&F96365. These results suggest that the intracellular Ca 2؉ concentration increase through the store-operated Ca 2؉ channels induces asymmetrical flagellar movements to establish the chemotactic behavior of the sperm. I t is now well known that many cells, e.g., leukocytes, starved amoebae of Dictyostelium discoideum, bacteria, and migrating axon cells during development, can detect an extracellular chemical gradient and migrate toward the source of the chemicals, that is, exhibit chemotactic behavior. There is a variety of factors inducing chemotaxis of cells, e.g., food, pathogens, partner cells, etc., and chemotactic behavior of cells is one of the important communication systems between separated cells. Chemotactic behavior of the sperm toward an egg during fertilization in animal species is a typical case, first reported in hydrozoans (1). This phenomenon is now widely known to occur, from sponges to humans (2-4), and much effort has been devoted to clarifying the mechanisms underlying sperm chemotaxis.Extracellular Ca 2ϩ is generally accepted as being an important factor in the induction of chemotactic behavior of sperm in many plant and animal species (5-8). In the case of the sea urchin, Arbacia punctulata, a peptide named resact in the jelly layer of the egg has been shown to have sperm-activating and -attracting activities (7). The peptide increases the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) via cAMP-gated Ca 2ϩ channels (9, 10), which, in turn, induces asymmetrical flagellum movements (5, 11, 12) that result in reorientation of the sperm swimming direction (13). The role of extracellular Ca 2ϩ -induced asymmetrical flagellar movement in inducing sperm chemotaxis has also been reported in hydrozoan species (14). Ascidian sperm chemotactic behavior is also characterized by abrupt turning movements of the sperm (15, 16). Furthermore, it has been shown that the sperm-attracting activity in the ascidians Ciona intestinalis and Ciona savignyi does not originate from the overall surface of the egg coats, such as follicle cells, but from the vegetal pole of the egg (17). We al...

Highlights･ Gsdf is expressed predominantly in XY gonads in the Nile tilapia.･ Gsdf expression pattern shows sexual dimorphism before morphological differentiation.･ Gsdf expression is the earliest gene expression detected in testis differentiation.･ Overexpression of gsdf is sufficient to induce testis differentiation in XX tilapia.Abbreviations: transforming growth factor-beta (TGF-beta), gonadal soma-derived factor (gsdf), doublesex/mab-3-related transcription factor-1 (Dmrt1), days post-hatching (dph), quantitative one-step RT-PCR (qRT-PCR), in situ hybridization (ISH) 2 ABSTRACTThe Nile tilapia, Oreochromis niloticus, is a gonochoristic teleost fish with an XX/XY genetic system and is an excellent model for gonadal sex differentiation. In the present study, we screened novel genes that were expressed predominantly in either XY or XX undifferentiated gonads during the critical period for differentiation of gonads into ovaries or testes using 5 microarray screening. We focused on one of the isolated 12 candidate genes, #9475, which was an ortholog of gsdf (gonadal soma-derived factor), a member of the transforming growth factor-beta superfamily. #9475/gsdf showed sexual dimorphism in expression in XY gonads before any other testis differentiation-related genes identified in this species thus far. We also overexpressed the #9475/gsdf gene in XX tilapia, and XX tilapia bearing the #9475/gsdf gene 10 showed normal testis development, which suggests that #9475/gsdf plays an important role in male determination and/or differentiation in tilapia.

In the ascidian Ciona intestinalis (and C. savignyi), sperm-activating and -attracting factor (SAAF) is released from the egg at fertilization and stimulates both Ca(2+) influx and a transient increase in cAMP level of the sperm, leading to the activation of sperm motility (M. Yoshida et al., 1994, Dev. Growth Differ. 36, 589-595). In this paper we show in C. intestinalis that valinomycin, a potassium-selective ionophore, as well as SAAF, activated sperm motility, and this activation was suppressed by extracellular high K(+). Membrane potential measurements showed that both SAAF and valinomycin increase K(+) permeability of sperm and induce membrane hyperpolarization, the amplitude of which depends on the external K(+) concentration. The membrane potential and intracellular K(+) concentration of Ciona sperm without SAAF were estimated to be about -50 mV and 560 +/- 40 mM, respectively. After treatment with SAAF or valinomycin the membrane potential became almost equal to the equilibrium potential of K(+) (-100 mV), and the cAMP level increased in artificial seawater. A potent voltage-dependent K(+) channel blocker, MCD peptide, at the concentration of 10 microM blocked SAAF-induced hyperpolarization of the cells, increase in cAMP, and sperm motility. These results suggest that membrane hyperpolarization produced by the opening of K(+) channels elevates cAMP synthesis and leads to the activation of sperm motility in Ciona.