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Spermiogenesis and Taxonomic Value of Sperm Morphologies of Two Species in Veneridae (Bivalvia: Heterodonta)

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Spermiogenesis and Taxonomic Value of Sperm Morphologies of Two Species in Veneridae (Bivalvia: Heterodonta) Korean J. Malacol. 27(2): 149-157, 2011 Spermiogenesis and Taxonomic Value of Sperm Morphologies of Two Species in Veneridae (Bivalvia: Heterodonta) Jin Hee Kim1 and Sung Han Kim2 1Marine Eco-Technology Institute, Busan 608-830, Korea 2Department of Aquaculture and Aquatic Sciences, Kunsan National University, Gunsan 573-701, Korea ABSTRACT Some characteristics of the formations of acrosomal vesicles during the late stage of spermatids during spermiogenesis and taxonomical charateristics of sperm morphology in male two species (Saxidomus purpurata and Meretrix petechialis) in the family Veneridae were investigated by electron microscope observations. In two species, the morphologies of the spermatozoa have the primitive type and are similar to those of other bivalves in that it contains a short midpiece with five mitochondria surrounding the centrioles. The morphologies of the sperm nuclear types of S. purpurata and M. petechialis in Veneridae have the curved cylindrical and cylinderical type, respectively. And the acrosome shapes of two species are the same cap-shape type. In particular, the axial filament is not found in the lumen of the acrosome of two species, however, subacrosomal material are observed in the subacrosomal spaces between the anterior nuclear fossa and the acrosomal vesicle of two species. The spermatozoon of S. purpurata is approximately 46-52 µm in length, including a curved sperm nucleus (about 3.75 µm in length), a long acrosome (about 0.40 µm in length),and a tail flagellum (about 45-47 µm long). And the spermatozoon of M. petechialis is approximately 47-50 μm in length including a slightly curved sperm nucleus (about 1.50μm in length), an acrosome (about 0.56 μm in length) and tail flagellum (44-48 μm in length). In two species, the axoneme of the sperm tail flagellum of each species consists of nine pairs of microtubules at the periphery and a pair of cental doublets at the center. Therefore, the axoneme of the sperm tail flagellum shows a 9 + 2 structure. In particular, taxonomically important some charateristics of sperm morphologies of two species in the family Veneridae are acrosomal morphology of the sperm, The axial filament is not found in the acrosome as seen in a few species of the family Veneridae in the subclass Heterodonta. The acrosomal vesicle is composed of right, left basal rings and the apex part of the acrosomal vesicle. In particular, right and left basal rings show electron opaque part (region), while the apex part of the acrosomal vesicle shows electron lucent part (region). These charateristics belong to the subclass Heterodonta, unlikely a characteristic of the subclass Pteriomorphia showing all part of the acrosome being composed of electron opaque part (region). Therefore, it is easy to distinguish the families or the subclasses by the acrosomal structures. The number of mitochondria in the midpiece of the sperm of S. purpurata and M. petechialis in Veneridae are five. However, the number of mitochondria in the midpiece of the sperm in most species of Veneridae in the subclass Heterodonta are four. Therefore, the number of mitochondria of the sperm midpiece of two species are exceptionally 5, and it is only exceptional case in the species in Veneridae in the subclass Heterodonta. Except these cases, the number of mitochondria in the sperm midpiece in all families in the subclass Heterodontaare are 4, and now widely used in taxonomic analyses. Key words: Saxidomus purpurata, Meretrix petechialis, spermiogenesis, sperm morphology INTRODUTION Spermatogenesis and sperm morphology has been Received June 5, 2011 ; Accepted June 26, 2011 reported in many species of bivalve molluscs using Corresponding author: Kim, Jin Hee Tel: +82 (51) 621-6295 e-mail: [email protected] both light and electron microscopy (Eckelbarger et al., 1225-3480/24389 1990; Eckelbarger and Davis, 1996; Gaulejac et al., - 149 - Spermiogenesis and Sperm Morphologies of Two Species in Veneridae 1995; Chung and Ryou, 2000; Chung et al., 2007, spermatid and mature sperm morphologies of two 2010; Kim et al., 2010a,b). Recently, in the mollusca, species (Saxidomus purpurata and Meretrix petechialis sperm ultrastructure is considered a valuable tool in in Veneridae (Heterodonta) by electron microscope assessing taxonomic and phylogenetic problems within observation. the bivalvia (Franzén, 1970, 1983; Popham, 1979; Healy, 1989, 1995; Hodgson and Bernard, 1986; . MATERIALS AND METHODS Thus, sperm ultrastructures of bivalves are now 1. Sampling widely used in taxonomic analyses (Healy, 1995), and For collection of two species (Saxidomus purpurata is especially useful when comparing closely related and Meretrix petechialis), a total of 126 male species (Popham et al., 1974; Popham, 1979). individuals were collected at the intertidal and To date, there are several ultrastructural studies on subtidal zones of Simpo, Jollabuk-do, Korea, and used spermatogenesis of the family Veneridae in Korea: for transmission / scanning electron microscope Cyclina sinensis (Chung et al., 1991), Meretrix lusoria observations. (Kim, 2006), Gomphina melanaegis (Lee et al., 1999), Gomphina veneriformis (Park et al., 2003; Chung et 2. Transmission electron microscope observations al., 2010). Protothaca (Notochione) jedoensis (Kim et For transmission electron microscope observations, al., 2010c). excised pieces of the gonads were cut into small pieces and Although there have been several studies on sperm fixed immediately in 2.5% paraformaldehydeglutaraldehyde ultrastructure during spermatogenesis of two species in0.1 M phosphate buffer solution (pH 7.4) for 2 (S. purpurata and M. petechialis) have been hours at 4℃. After prefixation, the specimens were investigated by some authors, there are still gaps in washed several times in the buffer solution and then our knowledge on the use of taxonomic characteristics postfixed in a 1% osmium tetroxide solution in 0.2 M of mature sperm morphology as taxonomic tools to phosphate buffer (pH 7.4) for 1hour at 4℃. Specimens solve the taxonomic problems. Little information is then were dehydrated in increasing concentrations of available on the ultrastructural studies associated ethanol, cleared in propylene oxide and embedded in with spermatogenesis by electron microscopic an Epon-Araldite mixture. Ultrathin sections of observation. Epon-embedded specimens were cut with glass knives Recently, sperm ultrastructures of bivalves are now on a Sorvall MT-2 microtome and LKB widely used in taxonomic analyses (Healy, 1995). In ultramicrotome at a thickness of about 80 - 100 nm. particular, it is well-known that acrosomal Tissue sections were mounted on collodion-coated morphology of sperms has been used to organize copper grids, doubly stained with uranyl acetate bivalve subclasses (Popham, 1979), Therefore, it need followed by lead citrate, and observed with a JEM 100 to study acrosomal morphology of the sperm and the CX-Ⅱ (80-KV) electron microscope. number of mitochondria in the sperm midpiece for taxonomic analyses of this species. If some 3. Scanning electron microscope observations characteristics obtained from sperm ultrastructure A drop of sperm suspension was placed on a cover and the process of spermiogenesis are phylogenetically glass, prefixed with 2.5% glutaraldehyde and 2.5% analyzed, the results of the ultrastructural studies on paraformaldehyde in 0.1 M carcodylate buffer (pH bivalve spermatozoa will provide information needed 7.5) at 4°C for 15 min, and post fixed with 1% OsO4 for the elucidation of relationship patterns among for 10 min, before rinsing with carcodylate buffer. The several bivalve subclasses (Popham et al., 1974, specimens were dehydrated in a graded ethanol series, Popham, 1979; Healy, 1989, 1995). Therefore, the critical point dried, coated with gold, and observed main aim of the present study is to describe some under a scanning electron microscope (ISI-SS4D). In taxonomic characteristics for classification from the addition, after dehydration, some testes were - 150 - Korean J. Malacol. 27(2): 149-157, 2011 freeze-fractured in liquid nitrogen, and then part of the acrosomal vesicle, right and left basal submitted to the same procedure described above. rings, in paticular, right and left basal rings are electron dense opaque parts (region), while the apex RESULTS part of acrosomal vesicle is electron lucent part (region). In general, these characteristics are one of 1. Spermiogenesis and sperm morphology of Saxidomus purpurata and Meretrix petechialis the ultrastructures of the acrosomal vesicle in the In general, the process of spermiogenesis of acrosome. At this time, subacrosomal material are spermatids and mature process of spermatozoa appear present between the anterior part of nuclear fossa to be similar to those of other bivalve species. In this and the acrosomal vesicle. However, the axial study, to get some taxonomic characteristics for filaments, which are existed in Ruditapes classification from the spermatid and mature sperm philippinarum and Gomphina veneriformis in morphologies, morphological and ultrastuctural Veneridae, are not found in the subacrosomal characteristics of spermatids and mature spermatozoa material (Fig. 1B). Thereafter, a cap-shaped acrosomal in male Saxidomus purpurata and Meretrix petechialis vesicle becomes an acrosome. And then the acrosome in Veneridae (Bivalvia: Heterodonta) are investigated lying on the sperm nucleus become a cone in shape. by electron microscope observation. Studies on the Therefor, the completed acrosome
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