鯉魚Tsga2同源基因MSAP：分子特性、功能及演化之研究

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In order to explore the molecular mechanisms underlying spermatogenesis, genes from a subtractive screen of testicular cDNA library of common carp (Cyprinus carpio) were scrutinized. In this study, one of the positive cDNA clones, denoted as p196, which has shown to be expressed exclusively in adult testis of carp, is further characterized. The open reading frame of p196 encodes a 218-aa protein that has highly acidic and hydrophilic properties, as well as seven copies of the MORN (membrane occupation and recognition nexus) motif spanning more than half of N-terminal sequences of the protein. The intact sequences share high identity to the two-third segment of the murine Tsga2 (testis-specific gene A2) product (also called meichroacidin) N-terminally, a protein that may play roles in meiotic stages of spermatogenesis in mouse, indicating that the p196 cDNA could be a carp homologue of Tsga2 (or TSGA2 in human). However, the carp homologous protein is specifically expressed during late spermiogenesis rather than meiosis as murine Tsga2 reported previously and especially enriched in mature spermatozoa, in which the protein is localized to the basal bodies and flagella. Fractionation of sperm proteins and in vitro polymerization of flagellar tubulins revealed that the protein might be one of the axonemal components of carp sperm. Because of this distinctive distribution, the cyprinid gene is thus called MSAP (MORN motif-containing sperm-specific axonemal protein) to discriminate it from murine Tsga2. Database search and immunoblot analyses suggested that the MSAP/Tsga2 homologues were widespread in a variety of phyla but highly divergent in their C-terminal length and sequences. In contrast to the 2-DIGE (two-dimenaional gel electrophoresis) profile of Tsga2, existence of multiple pI variants of MSAP also displayed the difference in posttranslational regulation between the murine and cyprinid homologues. Divergence in the cellular distribution, C-terminal region and protein modification between the conserved molecules implicates an adaptive radiation of the MSAP/Tsga2 homologues during vertebrate phylogenesis and their distinct roles in male germline development among various vertebrates. In addition, possible MSAP-interacting proteins were examined by using blot overlay assay and tandem mass spectrometry. One of the interacting candidates was identified as the carp septin7, a member of a conserved GTPase family that may participate in cellular morphogenesis and cytokinesis. Immunoflourescence localization suggested that septin7 was associated with a subset of MSAP within the basal body of carp sperm. These findings lead to an MSAP/septin-mediated model for flagellar differentiation during spermiogenesis.