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 MS/MS 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.

Zona pellucida (ZP)–induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCδ4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca²⁺ responses in single sperm, and we report that the [Ca²⁺]_i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCδ4^−/− sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca²⁺]_i increases in PLCδ4^−/− sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca²⁺]_i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca²⁺]_i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca²⁺ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCδ4^−/− sperm. These results indicate that PLCδ4 is an important enzyme for intracellular [Ca²⁺]_i mobilization in the ZP-induced acrosome reaction and for sustained [Ca²⁺]_i increases through SOC induced by ZP and progesterone in sperm.

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 high-speed 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.

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+-induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.

The chemotaxis of sperm towards eggs is a widespread phenomenon that occurs in most forms of life from lower plants to mammals and plays important roles in ensuring fertilization. In spermatozoa, the attractants act as beacons, indicating the path leading to the eggs from the same species. The existence of species-specific sperm chemotaxis has been demonstrated in marine invertebrates; thus, sperm chemotaxis may be involved in preventing crossbreeding, especially in marine invertebrates with external fertilization. However, the mechanisms of sperm chemotaxis in mammalian species differ from those of marine invertebrates. In mammals, the attractant source is not the egg, but follicular fluids or cumulus cells and chemotactic behaviour is shown only in small populations of sperm. Nevertheless, the fundamental mechanisms underlying sperm chemotaxis are likely to be common among all species. Among these mechanisms, intracellular Ca²⁺ concentration ([Ca²⁺]_i) is an important factor for the regulation of chemotactic behaviour in spermatozoa. Sperm attractants induce the entry of extracellular Ca²⁺, resulting in [Ca²⁺]_i increase in the sperm cells. Furthermore, [Ca²⁺]_i modulates sperm flagellar movement. However, the relationship between [Ca²⁺]_i and the chemotactic response of a sperm flagellum is not well known. Investigation of the dynamic responses of sperm cells to their attractants is important for our understanding of the regulation of fertilization. Here, we reviewed sperm chemotaxis focusing on the mechanisms that regulate sperm flagellar beating during the chemotactic response.