title: Sperm proteases that may be involved in the initiation of sperm motility in the newt, Cynops pyrrhogaster.
abstract: A protease of sperm in the newt Cynops pyrrhogaster that is released after the acrosome reaction (AR) is proposed to lyse the sheet structure on the outer surface of egg jelly and release sperm motility-initiating substance (SMIS). Here, we found that protease activity in the sperm head was potent to widely digest substrates beneath the sperm. The protease activity measured by fluorescein thiocarbamoyl-casein digestion was detected in the supernatant of the sperm after the AR and the activity was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), an inhibitor for serine or cysteine protease, suggesting the release of serine and/or cysteine proteases by AR. In an in silico analysis of the testes, acrosins and 20S proteasome were identified as possible candidates of the acrosomal proteases. We also detected another AEBSF-sensitive protease activity on the sperm surface. Fluorescence staining with AlexaFluor 488-labeled AEBSF revealed a cysteine protease in the principal piece; it is localized in the joint region between the axial rod and undulating membrane, which includes an axoneme and produces powerful undulation of the membrane for forward sperm motility. These results indicate that AEBSF-sensitive proteases in the acrosome and principal piece may participate in the initiation of sperm motility on the surface of egg jelly.
authors.affiliation: Department of Biology, Faculty of Science, Yamagata University, 1-4-12 Kojirakawa, Yamagata 990-8560, Japan. yokoe@sbiol.kj.yamagata-u.ac.jp.
authors.forename: Misato
authors.initials: M
authors.lastname: Yokoe
authors.affiliation: Department of Biology, Faculty of Science, Yamagata University, 1-4-12 Kojirakawa, Yamagata 990-8560, Japan. sano@sbiol.kj.yamagata-u.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Sano
authors.affiliation: Department of Biology, Faculty of Science, Yamagata University, 1-4-12 Kojirakawa, Yamagata 990-8560, Japan. shibata@sbiol.sci.kj.yamagata-u.ac.jp.
authors.forename: Honami
authors.initials: H
authors.lastname: Shibata
authors.affiliation: Shimoda Marine Research Center, University of Tsukuba, 5-10-1 Shimoda, Shizuoka 415-0025, Japan. shibata@kurofune.shimoda.tsukuba.ac.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Shibata
authors.affiliation: Institute of Arts and Sciences, Yamagata University, 1-4-12 Kojirakawa, Yamagata 990-8560, Japan. ewatanabe@kdw.kj.yamagata-u.ac.jp.
authors.forename: Eriko
authors.initials: E
authors.lastname: Takayama-Watanabe
authors.affiliation: Shimoda Marine Research Center, University of Tsukuba, 5-10-1 Shimoda, Shizuoka 415-0025, Japan. kinaba@kurofune.shimoda.tsukuba.ac.jp.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Inaba
authors.affiliation: Department of Biology, Faculty of Science, Yamagata University, 1-4-12 Kojirakawa, Yamagata 990-8560, Japan. watan@sci.kj.yamagata-u.ac.jp.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Watanabe
mesh_terms: D000176:Acrosin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000177:Acrosome / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D057056:Cysteine Proteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D012455:Salamandridae / Q000378:metabolism* / Q000502:physiology; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013081:Sperm Motility; D013379:Substrate Specificity; D013450:Sulfones / Q000494:pharmacology
chemical_list: D015853:Cysteine Proteinase Inhibitors; D015842:Serine Proteinase Inhibitors; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D057056:Cysteine Proteases; D057057:Serine Proteases; D000176:Acrosin; D046988:Proteasome Endopeptidase Complex