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Sayyed Mohammad Hadi Alavi, Natsuki Matsumura, Kogiku Shiba, Naoki Itoh, Keisuke G Takahashi, Kazuo Inaba and Makoto Osada

Factors that inhibit and stimulate the initiation of sperm motility were determined for Manila clam (Ruditapes philippinarum), Pacific oyster (Crassostrea gigas), and Japanese scallop (Patinopecten yessoensis). Compared with artificial seawater (ASW), serotonin (5-hydroxytryptamine creatinine sulfate, 5-HT) could fully trigger sperm motility and increase sperm velocity and motility duration. Sperm motility was decreased in ASW at pH 6.5–7.0 and suppressed at pH 4.0. In Manila clam and Pacific oyster, 5-HT could overcome the inhibitory effects of acidic pH on sperm motility. In the presence of nigericin (a K+/H+ exchanger), sperm motility was only triggered at pH 8.3. Testicular fluid K+ concentrations were two- to fourfold higher than that in ASW. Sperm motility and velocity were decreased in ASW or 5-HT containing ≥40 mM K+ or ≥2.5 mM 4-aminopyridine, suggesting K+ efflux requirement to initiate motility. Sperm motility and velocity were reduced in ASW or 5-HT containing EGTA or W-7, suggesting that extracellular Ca2 + is required for Ca2 +/calmodulin-dependent flagellar beating. Ca2 + influx occurs via Ca2 + channels because sperm motility and velocity were decreased in both ASW and 5-HT containing T-type and L-type Ca2 + channel blockers. 5-HT-dependent initiation of sperm motility was associated with intracellular Ca2 + rise, which was comparable to that seen in ASW but was not observed in the presence of EGTA or a Ca2 + channel blocker. Extracellular Na+ is also essential for sperm motility initiation via regulation of Na+/Ca2 + exchange. Overall, 5-HT-dependent initiation of sperm motility in marine bivalve mollusks is an osmolality-independent mechanism and regulated by extracellular pH, K+, Ca2 +, and Na+.

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Gen Hiyama, Mei Matsuzaki, Shusei Mizushima, Hideo Dohra, Keisuke Ikegami, Takashi Yoshimura, Kogiku Shiba, Kazuo Inaba and Tomohiro Sasanami

Systems for maintaining the viability of ejaculated sperm in the female reproductive tract are widespread among vertebrates and invertebrates. In birds, this sperm storage function is performed by specialized simple tubular invaginations called sperm storage tubules (SSTs) in the uterovaginal junction (UVJ) of the oviduct. Although the incidence and physiological reasons for sperm storage in birds have been reported extensively, the mechanisms of sperm uptake by the SSTs, sperm maintenance within the SSTs, and control of sperm release from the SSTs are poorly understood. In this study, we demonstrated that the highly conserved heat shock protein 70 (HSP70) stimulates sperm motility in vitro and also that HSP70 expressed in the UVJ may facilitate the migration of sperm released from the SSTs. Quantitative RT-PCR analysis demonstrated that the expression of HSP70 mRNA in the UVJ increases before ovulation/oviposition. Gene-specific in situ hybridization and immunohistochemical analysis with a specific antibody to HSP70 demonstrated that HSP70 is localized in the surface epithelium of the UVJ. Furthermore, injection of anti-HSP70 antibody into the vagina significantly inhibited fertilization in vivo. In addition, we found that recombinant HSP70 activates flagellar movement in the sperm and that the binding of recombinant HSP70 to the sperm surface is mediated through an interaction with voltage-dependent anion channel protein 2 (VDAC2). Our results suggest that HSP70 binds to the sperm surface by interacting with VDAC2 and activating sperm motility. This binding appears to play an important role in sperm migration within the oviduct.