We have previously identified and cloned a human gene, D40, that is preferentially expressed in testis among normal organs, while it is widely expressed in various human tumor cell lines and primary tumors derived from different organs. In this report, we have examined the expression and localization of this protein in human testis with an antibody specific to D40 protein. In Western analyses, the anti-D40 antibody recognized a major band with a molecular mass of 300 kDa and a minor band of 250 kDa. These bands were not observed in the testis lysates from patients with Sertoli-cell-only syndrome and with Kleinfelter syndrome, who lack germ cells of the testis, indicating that D40 protein is expressed in the germ cells of normal testis. Immunohistochemical studies have revealed that D40 protein is highly expressed in spermatocytes and in the pre-acrosome of round spermatids. In the acrosome, D40 protein expression is observed not inside but outside the acrosome membrane. This is consistent with the finding that the amino-acid sequence at the amino terminal of the D40 protein lacks a hydrophobic signal peptide that is required for proteins to translocate to the membrane. Expression of D40 protein is observed in the acrosome of ejaculated spermatozoa as well, although the level is low compared with that in the pre-acrosome of spermatids. These results suggest that D40 protein plays important roles in spermatogenesis, especially in the formation and maintenance of the acrosome.
Takumi Sasao, Naoki Itoh, Hiroko Takano, Satoshi Watanabe, Gang Wei, Taiji Tsukamoto, Noboru Kuzumaki and Masato Takimoto
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+.