Sperm chemotaxis toward eggs before fertilization has been demonstrated in many animals and plants, and several peptides and small organic compounds acting as chemoattractants have been identified. We previously showed that sperm of the ascidians Ciona intestinalis and Ciona savignyi are activated and then attracted toward the egg by a common factor released from the egg. In this study, we purified sperm-activating and -attracting factor (SAAF) from the egg-conditioning medium of C. intestinalis by using several steps of column chromatography. Determination of the molecular structure by NMR and MS͞MS analysis revealed that SAAF is a previously uncharacterized sulfated steroid: 3,4,7,26-tetrahydroxycholestane-3,26-disulfate. Furthermore, it was shown that the SAAF of C. savignyi was indistinguishable from that of C. intestinalis in terms of the chromatographic behavior and molecular weight, indicating that the same compound might be responsible for sperm activation and chemotaxis in both the species. Furthermore, we established a method for quantitative analysis of sperm chemotaxis and showed that the chemotactic behavior of Ciona sperm is controlled by the ''chemotactic turn'' associated with decrease in the concentration of SAAF. C hemotactic behavior is an important communication system among cells. Chemotaxis of spermatozoa toward eggs during fertilization is known in most animals and lower plants (1, 2). The chemical nature of the sperm attractants has been determined in the bracken fern to be a bimalate ion (3) and in brown algae to be unsaturated hydrocarbons (4-6). In animal species also, some candidates of sperm attractants have been reported (7,8), and the chemical structures of the sperm chemoattractants in three species, the sea urchin Arbacia punctulata (9), the coral Montipora digitata (10), and Xenopus laevis, have been identified (11). Despite much effort having been devoted to clarification of the mechanism underlying the chemotaxis, the absence of reliable bioassay methods has hampered the quantitative evaluation of sperm chemotaxis.Spermatozoa of the ascidian Ciona intestinalis are either immotile or only slightly motile when suspended in seawater. However, when an unfertilized egg is set in the sperm suspension, the spermatozoa near the egg are intensely activated and begin to show chemotactic behavior toward the egg (12-14). We showed in a previous study that the eggs probably release a sperm-activating and -attracting factor (SAAF) from their vegetal pole (14,15). SAAF induces entry of extracellular Ca 2ϩ and an increase in intracellular cAMP in the sperm (15, 16), which induces protein kinase A-dependent phosphorylation of 21-and 26-kDa axonemal proteins and activation of sperm motility (17). On the other hand, the chemotactic behavior of the ascidian sperm also requires extracellular Ca 2ϩ , but theophyllineactivated sperm, in which the drug induces increase in the [cAMP] i by virtue of being a phosphodiesterase inhibitor, show similar chemotactic behavior to that of normal sperm (14, 15)....

Ca 2؉ is known to have important roles in sperm chemotaxis, although the relationship between intracellular Ca 2؉ concentration ([Ca 2؉ ]i) and modulation of the swimming and chemotactic behavior of spermatozoa has not been elucidated. Using a highspeed Ca 2؉ imaging system, we examined the chemotactic behavior and [Ca 2؉ ]i in individual ascidian sperm cells exhibiting chemotactic responses toward sperm activating and attracting factor (SAAF), a chemoattractant released by eggs. In this study, we found that transient [Ca 2؉ ]i increased in the flagellum (Ca 2؉ bursts) concomitantly with a change in the swimming direction in an SAAF gradient field. During the initial phase of the Ca 2؉ bursts, the flagellum of the spermatozoon exhibited highly asymmetric waveforms enabling the quick turning of the swimming path. However, the flagellum subsequently changed to symmetric beating, causing the spermatozoon to swim straight. Interestingly, during such responses, [Ca 2؉ ]i remained higher than the basal level, indicating that the series of responses was not simply determined by Ca 2؉ concentrations. Also, we found that Ca 2؉ bursts were consistently evoked at points at which the spermatozoon attained around a temporally minimal value for a given SAAF concentration. We concluded that Ca 2؉ bursts induced around a local minimal SAAF concentration trigger a sequence of flagellar responses comprising quick turning followed by straight swimming to direct spermatozoa efficiently toward eggs.ascidian ͉ calcium ͉ fertilization ͉ flagella ͉ chemotaxis S perm chemotaxis during fertilization is a widely observed phenomenon across most species (1-3). Although sperm chemoattractants released by ova or their accessory organs are species-specific and differ among species, they induce similar behaviors, sudden quick changes in the direction of swimming paths from circular or helical trajectories (1, 2). Spermatozoa of the ascidian Ciona intestinalis are generally known to exhibit clear chemotaxis toward eggs (4, 5), and the chemoattractant released by the eggs has been identified as (25S)-3␣,4␤,7␣,26-tetrahydroxy-5␣-cholestane-3,26-disulfate, designated sperm activating and attracting factor (SAAF) (6, 7). Without SAAF, ascidian spermatozoa swim in circles with stable swimming-path curvatures, whereas the spermatozoa under SAAF stimulus frequently exhibit abrupt ''turns'' in their swimming directions; these behaviors are associated with highly asymmetric flagellar movements (5,8,9). The regulation of this turning seems to be an important feature of sperm (6,8,9).Ca 2ϩ is known to be an important factor involved in the regulation of flagellar beating. In the case of sea urchin spermatozoa, intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), appears to be correlated with the asymmetric beating of sperm flagella (10, 11), and it has been shown that sperm-activating peptides trigger the increase in [Ca 2ϩ ] i (12, 13). Also, [Ca 2ϩ ] i fluctuations in swimming sea urchin spermatozoa were observed to be closely related to the changes i...

SME1 was cloned due to its high copy number effect: it enabled MATa/MAT alpha diploid cells to undergo meiosis and sporulation in a vegetative medium. Disruption of SME1 resulted in a recessive Spo- phenotype. These results suggest that SME1 is a positive regulator for meiosis. DNA sequencing analysis revealed an open reading frame of 645 amino acids. An amino terminal peptide of ca 400 amino acids in the deduced protein was similar to known protein kinases. Transcription of SME1 was regulated negatively by nitrogen and glucose and positively by MATa/MAT alpha and IME1, another positive regulator gene of meiosis. By complementation analysis, SME1 was found to be identical to IME2, which had been shown to be important in meiosis. These results suggest that IME1 product stimulates meiosis by activating transcription of SME1 (IME2) and that protein phosphorylation is required for initiation of meiosis.