To examine the release of the sperm attractant from the eggs, the egg-conditioned seawater was used [25 (link)]. One volume of eggs was suspended in 15 volumes of ASW with different pH and incubated for 16–20 h at 4 °C. The egg suspensions were centrifuged at 20,000× g for 15 min at 4 °C and the obtained supernatant was designated as the egg-conditioned seawater.
Sperm motility and chemotaxis were examined, as previously described [40 (link)]. Pre-activation of sperm motility was performed, as described above. The pre-activated sperm suspension was mounted on the observation chamber pretreated with bovine serum albumin, and sperm movements around a micropipette tip containing the egg-conditioned seawater or 1 µM SAAF were recorded with a digital camera (Nikon1, Nikon, Tokyo, Japan) attached to a BX51 microscope (Olympus, Tokyo, Japan) under darkfield illumination. To analyze sperm chemotaxis around the egg, the dechorionated egg was placed directly in the observation chamber. For the chemotaxis assay, forced punctuation of the eggs was performed using a glass needle. The velocities and trajectories of spermatozoa were analyzed using the Bohboh software (BohbohSoft, Tokyo, Japan) [41 ]. The linear equation-based chemotaxis index (LECI) was calculated, as described previously [42 (link)].
The motility parameters, including curvilinear velocity (VCL) of the Ciona sperm, were observed by a microscope (BX 51, Olympus) equipped with the CCD camera (acA1300-200uc, Basler, Ahrensburg, Germany), and analyzed using the CASA system (SCA, Microptic, Barcelona, Spain) with sperm counting chamber slides (SC20-01-04-B, Leja, Nieuw-Vennep, The Netherlands). Spermatozoa with STR > 80 (%) and VCL > 10 (µm/s) were classed as motile. For precise information on the individual movement parameters, please see [43 (link)].
Sperm motility and chemotaxis were examined, as previously described [40 (link)]. Pre-activation of sperm motility was performed, as described above. The pre-activated sperm suspension was mounted on the observation chamber pretreated with bovine serum albumin, and sperm movements around a micropipette tip containing the egg-conditioned seawater or 1 µM SAAF were recorded with a digital camera (Nikon1, Nikon, Tokyo, Japan) attached to a BX51 microscope (Olympus, Tokyo, Japan) under darkfield illumination. To analyze sperm chemotaxis around the egg, the dechorionated egg was placed directly in the observation chamber. For the chemotaxis assay, forced punctuation of the eggs was performed using a glass needle. The velocities and trajectories of spermatozoa were analyzed using the Bohboh software (BohbohSoft, Tokyo, Japan) [41 ]. The linear equation-based chemotaxis index (LECI) was calculated, as described previously [42 (link)].
The motility parameters, including curvilinear velocity (VCL) of the Ciona sperm, were observed by a microscope (BX 51, Olympus) equipped with the CCD camera (acA1300-200uc, Basler, Ahrensburg, Germany), and analyzed using the CASA system (SCA, Microptic, Barcelona, Spain) with sperm counting chamber slides (SC20-01-04-B, Leja, Nieuw-Vennep, The Netherlands). Spermatozoa with STR > 80 (%) and VCL > 10 (µm/s) were classed as motile. For precise information on the individual movement parameters, please see [43 (link)].
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