Germ Cell Differentiations During Spermatogenesis and Taxonomic Values of Mature Sperm Morphology of Atrina (Servatrina) Pectinata (Bivalvia, Pteriomorphia, Pinnidae)

Dev. Reprod. Vol. 16, No. 1, 19～29 (2012) 19

Germ Cell Differentiations during Spermatogenesis and Taxonomic Values of Mature Sperm Morphology of Atrina (Servatrina) pectinata (Bivalvia, Pteriomorphia, Pinnidae)

Hee-Woong Kang1, Ee-Yung Chung2,†, Jin-Hee Kim3, Jae Seung Chung4 and Ki-Young Lee2

1West Sea Fisheries Research Institute, National Fisheries Research & Development Institute, Incheon 400-420, Korea 2Dept. of Marine Biotechnology, Kunsan National University, Gunsan 573-701, Korea 3Marine Eco-Technology Institute, Busan 608-830, Korea 4Dept. of Urology, College of Medicine, Inje University, Busan 614-735, Korea

ABSTRACT : The ultrastructural characteristics of germ cell differentiations during spermatogenesis and mature sperm morphology in male Atrina (Servatrina) pectinata were evaluated via transmission electron microscopic observation. The accessory cells, which contained a large quantity of glycogen particles and lipid droplets in the cytoplasm, are assumed to be involved in nutrient supply for germ cell development. Morphologically, the sperm nucleus and acrosome of this species are ovoid and conical in shape, respectively. The acrosomal vesicle, which is formed by two kinds of electron-dense or lucent materials, appears from the base to the tip: a thick and slender elliptical line, which is composed of electron-dense opaque material, appears along the outer part (region) of the acrosomal vesicle from the base to the tip, whereas the inner part (region) of the acrosomal vesicle is composed of electron-lucent material in the acrosomal vesicle. Two special characteristics, which are found in the acrosomal vesicle of A. (S) pectinata in Pinnidae (subclass Pteriomorphia), can be employed for phylogenetic and taxonomic analyses as a taxonomic key or a significant tool. The spermatozoa were approximately 45-50 ㎛ in length, including a sperm nucleus (about 1.43 ㎛ in length), an acrosome (about 0.51 ㎛ in length), and a tail flagellum (about 46-47 ㎛). The axoneme of the sperm tail evidences a 9+2 structure. Key words : Atrina (Servatrina) pectinata, Spermatogenesis, Germ cell, Mature sperm morphology

INTRODUCTION Bernard, 1986; Healy, 1989; Kim & Kim, 2011). Thus far, there has only been one report regarding the The ultrastructures of germ cells and accessory cells spermiogenesis and sperm ultrastructure of A. pinnata during spermatogenesis and sperm morphology have been japonica, a Pinnidae species in subclass Pteriomorphia evaluated in many species of bivalve molluscs using electron (Kim, 2001). However, gaps in our knowledge regarding microscopy (Healy, 1989; Eckelbarger et al., 1990; Eckelbarger spermiogenesis and sperm morphology remain to be re- & Davis, 1996; Gaulejac et al., 1995; Chung & Ryou, solved. Little information is currently available regarding 2000; Chung et al., 2005). In general, ultrastructural studies germ cell differentiation during spermatogeneis and mature have been used to assess and describe spermatogenesis and sperm morphology. Therefore, it is important to clarify the mature sperm morphology in a number of bivalve species ultrastructures of germ cells and accessory cells during (Popham, 1979; Bernard & Hodgson, 1985; Hodgson & spermatogenesis and the taxonomic values of the sperm morphology of A. (S.) pectinata. In the bivalves generally, there is minimal variation in the fine structures of the †Corresponding author: Ee-Yung Chung, Dept. of Marine Biotechnology, Kunsan National University, Gunsan 573-701, Korea. Tel: +82-63-469-1832, nucleus and tail flagellum, but profound variation in the E-mail: [email protected] 2 0 H-W Kang, E-Y Chung, J-H Kim, JS Chung, K-Y Lee Dev. Reprod. Vol. 16, No. 1 (2012) form of the acrosome and mitochondria of the sperm of this species should be determined and compared with midpiece (Popham, 1979). other families in subclass Pteriomorphia. In particular, the Recently, several researchers (Franzén, 1970, 1983; Popham, results regarding the ultrastructural qualities of sperm mor- 1979; Hodgson & Bernard, 1986) have reported that sperm phology of this species should provide the information morphology can be an effective aid in phylogenic exami- necessary for the elucidation of relationship patterns among nations, and knowledge of the ultrastructure of bivalve the subclasses (Popham, 1979). Therefore, the principal spermatozoa can prove useful for taxonomic purposes. For objective of this study is to evaluate the ultrastructures of that reason, mature sperm ultrastructure has been viewed germ cells and accessory cells during spermatogenesis and as a particularly valuable tool in assessing taxonomic and to confirm sperm type with the acrosomal characteristics of phylogenetic problems within the Bivalvia (Popham, 1979; sperm ultrastructure via phylogenetic analyses of this Healy, 1989, 1995; Hodgson & Bernard, 1986), and spermio- species. cladistic analysis has been studied as a potentially useful tool for assessing the phylogenetic relationship in metazoa MATERIALAND METHODS (Jamieson, 1987, 1991). In particular, the acrosome of the sperm evidences extensive morphological diversity in the 1. Sampling bivalve sperm, and hence may be the most useful structure Male specimens of Atrina (Servatrina) pectinata were in terms of phylogenetic relational evaluations (Franzén, collected monthly in the subtidal zones (3-10 m in water 1956). With regard to acrosomal morphology, Popham depth) of coastal waters of Boryong, Choongchungnam-do, (1979) demonstrated that the acrosomal morphology of the Korea, from January to December, 2006. The clams were sperm can be used to organize bivalves into subclasses. transported to the laboratory where they were maintained Additionally, Healy (1989) previously reported that different in seawater at 20℃. subclasses of bivalves each evidence unique acrosomal morphologies. 2. Transmission Electron Microscopy Despite the subtle differences in acrosomal morphology For transmission electron microscopical observations, that exist between closely related families, an examination excised pieces of the gonads were cut into small pieces of published micrographs of sperm structure from families and fixed immediately in 2.5% paraformaldehyde glutaraldehyde in the same subclass of bivalve shows that each subclass in 0.1 M phosphate buffer solution (pH 7.4) for 2 hours at can be characterized by a basic acrosomal morphology or 4℃. After prefixation, the specimens were washed several position (Healy, 1989). times in the buffer solution and then postfixed in a 1% Therefore, the acrosomal morphology of the sperm in A. osmium tetroxide solution in 0.2 M phosphate buffer (pH (S.) pectinata in Pinnidae should be compared with that of 7.4) for 1 hour at 4℃. Specimens then were dehydrated in species belonging to other families in subclass Pteriomorphia, increasing concentrations of ethanol, cleared in propylene because A. (S.) pectinata is a member of subclass Pterio- oxide and embedded in an Epon-Araldite mixture. Ultrathin morphia. In addition, the number of mitochondria in the sections of Epon-embedded specimens were cut with glass sperm midpiece tends to be stable within any given family knives on a Sorvall MT-2 microtome and LKB ultramicrotome or superfamily, varying from a maximum of 14 in Modiotus at a thickness of about 80-100 nm. Tissue sections were difficiis (Drozdov & Reunov, 1986) to a minimum of 4 mounted on collodion-coated copper grids, doubly stained (common to many bivalve families) (Healy, 1989, 1995). with uranyl acetate followed by lead citrate, and observed Therefore, the number of mitochondria in sperm midpiece with a JEM 100 CX-Ⅱ (80-KV) electron microscope. Dev. Reprod. Vol. 16, No. 1 (2012) Spermatogenesis and Mature Sperm Morphology of A. (S.) pectinata 21

RESULTS be observed in the walls of the acini. The primary spermatocytes (approximately 5.5-6.2 ㎛ diameter) are similar 1. Germ Cell Differentiations during Spermatogenesis in size and shape to that of the secondary spermatogonia. The gonad of A. (S.) pectinata (Bivalvia, Pteriomorphia, However, at this stage, the nucleolus in the nucleus (about Pinnidae) is composed of a number of the acini. Spermato- 3.8-4.0 ㎛ diameter) of the primary spermatocyte is no genesis follows a centripedal pattern. The process of longer prominent, and the chromatin is in the form of a spermatogenesis of A. (S.) pectinata showed to be similar patchwork. Primary spermatocytes in three stages of first to those of other bivalves, During spermatogenesis, germ meiotic prophase have been distinguished, based on nuclear cell differentiations by spermatogenic stages occur in the size and morphology. In particular, zygotene/pachtene sper- numerous acini of the testis, and the mature testis normally matocytes contain nuclei with more highly conensed chromatin contains most stages of spermatogenensis. For that reason, and synaptonemal complexes are formed as a result of the the transition from spermatogonia to spermatozoon can be pairing of homologous chromosomes during the zygotene observed in one section. For convenient, spermatogenesis stage of prophase I. Diplotene spermatocyte nuclei also can be divided into four stages as follows: (1) spermatogonia, contain synaptonemal complexes but their nuclear chromatin (2) spermatocytes, (3) spermatids, and (4) spermatozoa. is less condensed. Both zygotene/patchtene and diplotene spermatocytes contain a few small mitochondria, and numerous 2. Spermatogonia membrane-bound (Fig. 1B). Spermatogonia are located along the internal wall of the The primary spermatocyte spermatocyte develops into acini. Two types of spermatogonia, designated primary and secondary spermatocytes by the meiotic division. The secondary secondary, occur (Fig. 1A). Primary spermatogonia are relatively spermatocyte (approximately 5.0-5.7 ㎛ diameter) is similar large (approximately 6.5-7.5 ㎛), irregularly shaped cells in size and organelle complement to the primary spermatocyte having mitochondria, rough endoplasmic reticulum and but is characterized by less osmiophilic chromatin. vacuoles in the vacant cytoplasm, and a prominent nucleus At this time, in paricular, secondary spermatocytes appear (4.0-4.5 ㎛) contains isolated patches of condensed chro- near the accessory cells containing a large nucleus and a matins. The primary spermatogonia differntiate into the number of mitochondria and a large quantity of glycogen secondary spermatogonia by the mitotic division. particles in the cytoplasm (Fig. 1C). In the stage of the The nuclear size (4.0-4.3 ㎛ diameter) of secondary secondary spermatocyte, several granules appear near the spermatogonia (about 6.2-7.0 ㎛) are slightly smaller than Golgi complex. These granules formed by the Golgi those of primary spermatogonia. At this stage, a prominent complex become the proacrosomal granule (Fig. 1D). electron-dense nucleolus appear in the nucleus, and the nucleus contains smalll clumps of electron-dense chromatin. 4. Spermatids The cytoplasm is largely devoid of cell organelles except The secondary spermatocyte develops into spermatids for several small mitochondria (0.3 ㎛ diameter), rough by the meiotic division. The spermatids contain electron endoplasmic reticulum, and vacuoles (Fig. 1A). dense hetero chromatins in the nucleus. At this stage, spermiogenesis occur in the stages of spermatids. As seen 3. Spermatocytes in bivalve species, spermiogenesis involves a number of The secondary spermatogonia differentiate into spermatocytes simultaneous cellular events including nuclear elongation by the mitotic division. At this stage, two stages of spermatocyte and condensation, fusion of proacrosomal vesicles and development, presumed to be primary and secondary, can their migration to the apical region of the cell, movement 2 2 H-W Kang, E-Y Chung, J-H Kim, JS Chung, K-Y Lee Dev. Reprod. Vol. 16, No. 1 (2012) of mitochondria to the caudal region of the cell, and the ellipsoidal line being composed of electron-dense opaque loss of superfluous cytoplasm occur. For comvenience, the material from the base to the tip, while the inner part spermatid development can be divided into two stages (region) of the acrosomal vesicle is composed of the during spermiogenesis: the early and late stages of spermatids. electron-dense lucent material from the base to the tip. In the early stage of spermiogenesis, early spermatids At this time, the acrosomal vesicle (about 0.51 ㎛ long) contain a spherical nucleus (approximately 3.0 ㎛ diameter) is occupied by subacrosomal material which comprises with electron-dense and lucent patches of the fine granular embedded in a coasely granular matrix, and forms membrane heterochromatin. The cytoplasm contains numerous mitochon- bound. However, the axial rod is not present in the dria, rough endoplasmic reticulum, Golgi complex, and the subacrosomal material in the anteriorly invaginated subacro- proacrosomal granules. In particular, several proacrosomal somal space of the acrosomal vesicle. granules appear in the same zone, in the vicinity of the During the late stage of the spermatid, the granular Golgi complex, and fuse to form a larger single oval- nucleus becomes smaller (approximately 1.43 ㎛), and the shaped vesicle (Fig. 1D, 1E). The proacrosomal vesicles nuclear contents continue to condense, there is continued migrate to the presumptive anterior end of the spermatid, loss of cytoplasm by sloughing. At the same time, in the where they coalesce to form a single electron-dense acrosomal midpiece of spermatozoa, mitochondria become reduced in vesicle (Fig. 1F). In the late stage of spermiogenesis, the number but increase in size. The larger mitochondria form early spermatids differentiate into the late spermatids, and a close association with the nucleus. As further development the cytoplasm is lost by sloughing. A single acrosomal occurs, the mitochondria come to occupy the end of the vesicle locates at the presumptive anterior pole of the cell opposite to the acrosome, thus forming the sperm spermatids. the acrosomal vesicle is initially oval-shape, midpiece (Fig. 1H). The spermatid middlepiece contains and a subacrosomal fossa appears as a shallow depression the distal centriole and an adjacent proximal centriole along the apical surface of the nucleus (Fig. 1G). As which lies next to the posterior nuclear envelope at a 90° differentiations continue, the a nuclear surface invaginates angle to the long axis of the cell. At the same time, the tail to form a digitate, subacrosomal space. At the same time, appears during the late spermatid stage, developing from several small mitochondria fuse to form large spherical the distal centriole; the proximal centriole occupies a mitochondria under the posterior nuclear fossa. position between the distal centriole and the nucleus. As shown in Fig. 1H, two components (the ultrastructure However, two lateral satellite fibers are not found near the of the acrosomal vesicle and deposit of subacrosomal distal centriole (Fig. 1I). material) of the acrosomal vesicle are easily recognized during the acrosomal formation. The acrosomal vesicle 5. Spermatozoa then begins to invaginate on its a nuclear surface, forming An acrosomal vesicle contains high electron dense opaque the characteristic hollow, conical-shaped acrosome. Finally, material from the base to the tip. In particular, the apex the morphology of the acrosomal vesicle becomes the part and right and left lateral basal rings of an acrosomal cone-shape by way of various morphological changes and vesicle show electron opaque part (regions), as seen in the course of invagination from initial oval shape. subclass Pteriomorphia species. Mature spermatozoa of A. At this stage during spermiogenesis, the acrosomal (S.) pectinata is approximately 50 ㎛ long, and consist of vesicle of the late spermatid is composed of two kinds of conical acrosome positioned at the top of an oval nucleus, obviously different regions (parts): the outer part (region) a pair of centrioles surrounded by five spherical mitochondria, of the acrosomal vesicle is formed by a thick and slender and a long flagellum. An acrosome on the nucleus is Dev. Reprod. Vol. 16, No. 1 (2012) Spermatogenesis and Mature Sperm Morphology of A. (S.) pectinata 23

Fig. 1. Spermatogenesis in male Atrina (Servatrina) pectinata (A-M). (A) Primary spermatogonia and secondary spermatogonia. Note a nucleolus and chromatins in the nucleus, and mitochondria in the cytoplasm; (B) Primary spermatocyte. Note several synaptonemal complexes in the nucleus during the prophase of the primary meiotic division, and mitochondria; (C) Secondary spermatocytes and the accessory cell. Note glycogen particles. Note the nuclei of primary spermatocytes, secondary spermatocytes, and the accessory cell containing a large quantity of glycogen particles and mitochondria in the cytoplasm; (D) A spermatid and proacrosomal granules. Note high electron dense heterochromatin in the nucleus, and granules and several proacrosomal granules in the cytoplasm; (E) A spermatid and in the early stage of spermiogenesis. Note high electron dense proacrosomal vesicle near the Golgi complex on the apical region of the nucleus of a spermatid; (F) A spermatid (ST) in the late stage of spermiogenesis. Note proacrosomal granule and a proacrosomal vesicle on the nucleus of a spermatid; (G) A spermatid and acrosomal vesicle. Note appearance of an acrosomal vesicle, subacrosomal material on the nucleus, centrioles, spherical mitochondria under the posterior nuclear fossa; (H) Spermiogenesis and acrosome formation of a spermatid. Note appearance of an acrosomal vesicle which consist of electron opaque region and electron lucent region subacrosomal material on the nucleus, centrioles, spherical mitochondria under the posterior nuclear fossa; (I) A spermatid and the midpiece of the sperm. Note spherical mitochondria, the proximal centriole and distal centriole and a flagellum; (J) A completed spermatozoon. Note appearance of an acrosomal vesicle which consist of electron opaque region and electron lucent region subacrosomal material on the nucleus, centrioles, spherical mitochondria and lacunae under the posterior nuclear fossa; (K) A spermatozoon and the midpiece of the sperm. Note the nucleus mitochondria, the proximal centriole and distal centriole and a flagellum; (L) Cross sectioned midpiece of the sperm. Note five mitochondria surrounding a pair of centrioles; (M) Cross sectioned tail flagellum of mature sperm. Note the axoneme showing a 9 + 2 structure (a pair of central doublets and nine pair of peripheral microtubules). 2 4 H-W Kang, E-Y Chung, J-H Kim, JS Chung, K-Y Lee Dev. Reprod. Vol. 16, No. 1 (2012) composed of an acrosomal vesicle (about 0.51 ㎛ long) 1995; Kim et al., 2010a,b,c). Numerous studies have shown and a granular subacrosomal material exist in a coasely that all bivalves have primitive spermatozoa (Franzén, 1970, granular matrix. At this stage, a thick and slender elliptical 1983) which is typical of animals that release their gametes line (region), which is composed of the electron-dense into the surrounding water (Gaulejac et al., 1995). It is opaque material, appears along the outer part (region) of generally well known that spermatogenesis occurs via the acrosomal vesicle from the base to the tip, while the inner interaction between germ cells and accessory cells within part (region) of the acrosomal vesicle is composed of the the acini. During spermatogenesis, the accessory cells, electron lucent material in the acrosomal vesicle from the which are attached to germ cells in the acinus, supply base to the tip (Fig. 1J). Two special characteristics men- nutrients necessary for germ cell development (Eckelbarger tioned above are only found in the acrosomal vesicle of A. et al., 1990; Eckelbarger & Davis, 1996; Chung et al., (S.) pectinata in Pinnidae. The central hollow of the acrosome 2007). The spermatogenesis of A. (S.) pectinata evidences contains a granular subacrosomal material. However, the some phenomena reminiscent of those of other bivalves axial rod do not exist in the granular subacrosomal material. (Eckelbarger et al., 1990; Chung et al., 2007; Kim et al., The acrosomal vesicle is somewhat deeply invaginated. 2010a,b,c). In this study, in the early stage of germ cell The invagination is narrow anteriorly. The shape of the development, the spermatogonia and primary and secondary nucleus (1.43 ㎛ diameter) is a short and oval-shaped. In spermatocytes are positioned nearby the accessory cells particular, irregular electron-lucent lacunae are present in within the acinus wall. The accessory cells, which are in the nucleus. And also a short, shallow basal invagination close contact with developing germ cells, were found to appears in the nucleus (Fig. 1K). The miedpiece includes contain a large quantity of glycogen particles and lipid five spherical mitochondria with well-develped cristae and droplets in their cytoplasm, as has previously been noted the proxiamal and distal centrioles (Fig. 1L). The proximal in Scobicularia plana (Sousa et al., 1989), Crassostrea centriole lies at 90° to the distal centriole and sperm virginica (Eckelbarger & Davis, 1996), and C. gigas (Kim longitudinal axis, and is connected to a shallow invagination et al., 2010a). Therefore, it is supposed that they are of the nucleus by a thin layer of dense material. The distal involved in the supply of nutrients for germ cell develop- centriole is attached to the plasma membrane, and its ment during spermatogenesis (Eckelbarger et al., 1990; continuous with the flagellum. However, sattelite fibers Eckelbarger & Davis, 1996). Recently, Sousa et al., (1989) disappear near the distal centriole. The flagellum is about suggested that the Golgi complex may form only a single 46-47 ㎛ long, and exhibits a classic 9+2 microtubular acrosomal vesicle, similar to what has been observed in substructure axoneme (that is, nine peripheral microtubules other molluscs. This has been demonstrated previously in surrounding a central doublets) (Fig. 1M). The flagellum, the spermatid stage of Perna perna (Bernard & Hodgson, which is originated from the distal centriole, is surrounded 1985) and Pecten maximus (Dorange & Le Pennec, 1989). at its base by plasma membrane (Figs. 1I, 1K, 1M). In this study, in the case of A. (S.) pectinata, the proacrosomal granule appeared near the Golgi complex in the DISCUSSION synthetic activity occurring during the early developmental stage of spermatids. At this time, several small electron- 1. Spermatogenesis dense granules were also observed in the vicinity of The general process of spermatogenesis in A. (S.) pectinata proacrosomal granule (Fig. 1D, E, F). It is probable that is similar to that of the other bivalves and, more specifically, the small granules, which are generated by the Golgi to that of other Pteriormorphia species (Gaulejac et al., complex, fused to form a single proacrosomal vesicle, as Dev. Reprod. Vol. 16, No. 1 (2012) Spermatogenesis and Mature Sperm Morphology of A. (S.) pectinata 25 has been previously noted in externally-fertilizing bivalves variation on the first mode of acrosomal development. In and other invertebrates (Bernard & Hodgson, 1985; Hodgson the case of the freshwater clam Neotrigonia (Unionoidea), & Bernard, 1986). The acrosome of A. (S.) pectinata acrosome formation occurs via the production of multiple appears in the anterior side of the spermatid. The acrosomal proacrosomal vesicles that do not fuse into a single acro- vesicle comprises the outer electron opaque region and an somal vesicle. Therefore, this pattern could be regarded as inner, more electron-lucent region. Such similar differentiation a variation on the first mode of acrosomal development. In has been recorded in the acrosome of other families as this study, in the early stage of the spermatid during well (Popham, 1979; Bernard & Hodgson, 1985; Hodgson spermiogenesis, a few electron-dense granules, which are & Bernard, 1986). These regions of differing electron initially formed by the Golgi complex, later became the opacity probably reflect the differing functions of the definitive acrosomal vesicle via the fusion of small proa- acrosome during the fertilization process (Hodgen & Bernard, crosomal granules. Therefore, among the three modes of 1986). The conical acrosomal vesicle in the spermatozoa acrosomal development and formation, the processes of of this species is quite similar to that described for the acrosomal development and formation of A. (S.) pectinata Isognomidae (Pterioidea) (Healy, 1989). To date, the modes can be viewed as characteristic of the first mode (Fig. 2A, of acrosomal developments and formations in bivalves and B, C, D). In this study, the morphologies and sizes of the gastropods associated with external or internal fertilizations sperm acrosomes in Pinnidae species evidenced similar have been studied by many researchers (Longo & Dornfeld, morphological and ultrastructural characteristics, as has 1967; Longo & Anderson, 1969; Baccetti & Afzelius, been noted in other families. Generally, the acrosome 1976; Bacetti, 1979; Bernard & Hodgson, 1985; Healy, could be classified into five shapes, namely: cone, long 1989). Healy (1989) reported that acrosomal development cone, modified cone, cap, and modified cap shapes. In this in the Mollusca can be classified into three modes: The study, the acrosomal morphology of the spermatozoon of first mode of the acrosomal development is observed in a this species was cone-shaped. However, among the Veneridae number of other externally-fertilizing bivalves and other species of subclass Heterodonta, C. sinensis and Phacosoma invertebrates. Numerous electron-dense proacrosomal vesicles, japonicus were cone-shaped, unlike Saxidomus japonicus, which are formed initially by the Golgi complex, sub- Meretrix lusoria, Notochione jedoensis, all of which had sequently become the definitive acrosomal vesicle via the cap-shaped acrosomes (Kim, 2001). By way of contrast, in fusion of several Golgi-derived vesicles. This pattern is a several species of the Mytilidae (Bernard & Hodgson, characteristic of the first mode of the acrosomal development. 1985; Hodgson & Bernard, 1986) and Ostreidae (Daniels The second mode of acrosomal development is observed et al., 1971), this absence of an axial rod in the acrosome in internally fertilizing molluscs (higher prosobranch gastropods, may be correlated with the thickness of the oocyte vitelline opisthobranch and pulmonate gastropods) and in many envelope (Verdonk et al., 1983). Popham (1979) noted that other internally fertilizing animal groups. In this mode, the the absence of an axial rod in the acrosome is reflective initial definitive acrosomal vesicle is formed by the Golgi of the primitive bivalve condition. complex of a large receptacle vesicle, the growth of which is then achieved via the fusion of small vesicles budded 2. Taxonomic Values of Sperm Morphology and from the Golgi cisternae, or from materials channeled Ultrastructure directly from the cisternae. This pattern is characteristic of Generally, the sizes of the sperm nuclei of bivalves prove the second mode of acrosomal development. The third unuseful in taxonomic analysis, because the morphological mode of acrosomal development could be regarded as a characteristics of sperm nuclei are irregular and vary 2 6 H-W Kang, E-Y Chung, J-H Kim, JS Chung, K-Y Lee Dev. Reprod. Vol. 16, No. 1 (2012) among the species in a family (Healy, 1995). However, is comprised. In this study, in particular, in A. (S.) pectinata with regard to the sperm ultrastructures of bivalves, in in Pinnidae, a slender elliptical line in the acrosomal particular, the acrosome morphology and the number of vesicle, which is composed of high electron-dense opaque mitochondria at the midpiece of the sperm are broadly material, appeared along the outer part (region) of the useful for the purposes of taxonomic analyses (Healy, acrosomal vesicle from the base to the tip of the acrosomal 1995; Popham, 1979). To date, the ultrastructures and mor- vesicle, and the inner part (region) of the acrosomal vesicle phologies of the acrosomes in many families in two sub- was composed of an electron-lucent part (region) from the classes (Pteriormorphia & Heterodonta) have been investigated base to the tip of the acrosomal vesicle. In particular, a by a number of researchers. In particular, Hodgson & slender elliptical line was not detected in the acrosomal Bernard (1986) reported that mature sperm morphologies vesicle in species of the Pteriidae, Ostreidae, Mytilidae, and ultrastructures can be used for the classification and and Arcidae (Fig. 2B, C, D); this particular substructure differentiation of genera and families, as is the case with was found only in the acrosomal vesicles of the sperm in the morphologies and positions of the acrosomal vesicles A. (S.) pectinata and Pinna nobilis (Gaulejac et al., 1995) of the species of families in subclasses Pteriomorphia and of the Pinnidae in subclass Pteriomorphia. Therefore, these Heterodonta. In order to classify the species by sperm special characteristics can be employed for phylogenetic and morphology, we have generally employed some structural and morphological differences in the acrosomal vesicle of the species as a key or a valuable tool for the classification of the species. Comparisons of mature sperm morphology of A. (S.) pectinata in Pinnidae with those in the species of four families in subclass Pteriomorphia. Hodgson and Bernard (1986) reported that in general, the bivalve subclass Pteriomorphia members evidence common acrosomal vesicular structural characteristics: they are cone-like in shape, and are composed of electron-dense opaque material from the base to the tip that is, "the apex part and the right and left lateral parts of basal rings". However, all species in subclass Heterodonta in class Bivalvia also have common structural characteristics: the acrosomal vesicles are modified cones, and are composed of high electron-dense opaque materials (the base and lateral parts of the basal rings) and electron-lucent materials (the apex part of the acrosomal vesicle). Regarding the existence of special substructures in the acrosomal vesicle in an acrosome, if the ultrastructures Fig. 2. A schematic diagram of the acrosomes and nuclei of of the acrosomal vesicle of this species are compared with spermatozoa in four families of the subclass Pteriomorphia (A-D). (A) Pinnidae: Atrina (Servatrina) pectinata; (B) those of species belonging to other families in subclass Pteriidae: Pinctata martensii; (C) Ostreidae: Crassostrea Pteriormorphia, as shown in Figure 2, unique structural gigas; (D) Mytilidae: Mytilus coruscus. A, acrosome; AR, characteristics appeared in the acrosomal vesicles of each axial rod; AV, acrosomal vesicle. Arrow head mark, species in the four families of which subclass Pteriomorphia special substructure. Dev. Reprod. Vol. 16, No. 1 (2012) Spermatogenesis and Mature Sperm Morphology of A. (S.) pectinata 27 taxonomic analyses as a taxonomic key or a significant Regarding an axial rod in the acrosome vesicle or sperm tool (Fig. 2A). In Pinctata martensii in Pteriidae, a special nucleus, in general, the axial rod, a special structure structure evidencing a thick and wide triangular shape associated with fertilization, is detected within the acrosome which is composed of electron-dense opaque material or sperm nucleus. Of four families in subclass Pteriomorphia, (occupying approximately 50% of the upper part of the and in A. (S.) pectinata in the Pinnidae and P. (F.) acrosomal vesicle), appeared in the upper portion (region) martensii in the Pteriidae, no axial rod was detected within of the acrosomal vesicle, whereas the lower portion (region) the acrosomal vesicle of the sperm, whereas certain Ostreidae of the acrosomal vesicle was shown to be composed of (C. gigas and C. nipponica) and Mytilidae (Mytilus coruscus electron-lucent material. Thus, this special structure, which and M. galloprovincialis) species in subclass Pteriomorphia exists in the upper part of the acrosomal vesicle in P. (F.) contained the axial rod in the subacrosomal material martensii, differs somewhat from that of A. (S.) pectinata, within the acrosomal vesicle in the Mytilidae species, the which was observed stretching from the base to the tip of axial rod was found in the nucleus (Kim, 2001; Kim et al., the acrosomal vesicle. Therefore, we assume that the 2010a,b). However, Kim (2001) reported that, interestingly, existence of a special structure evidencing a thick and the axial rod in the sperm was not found in only one broad triangular shape in the acrosomal vesicle of the species, namely the Mytilidae species, Septifer (Mytilisepta) spermatozoon can be employed as a key characteristic for virgatus. In this study, the satellite fibers, which are the identification of P. (F.) martensii (Kim, 2001) in the generally located nearby the distal centriole of the sperma- Pteriidae in subclass Pteriomorphia (Fig. 2B). Beside Pteriidae, tozoon, were not detected in A. (S.) pectinata in the this special structural characteristic was not detected in the Pinnidae. Additionally, Kim (2001) previously noted reported acrosomal vesicle in Pinnidae, Ostreidae, and Mytilidae that no satellite fibers were found in P. (F.) martensii in species (Fig. 2A, C, D). A few transverse bands (whorls) Pteriidae species, while these structures were detected in at the anterior part of the acrosomal vesicle were found species of the Ostreidae and Mytilidae of subclass only in Ostreidae species such as Crassostrea gigas (Kim Pteriomorphia (Kim et al., 2010a,b,c). Regarding number et al., 2010a) and C. nipponica (Kim et al., 2010b). However, of mitochondria at the sperm midpiece, Healy (1995) these structures were not detected in the Pinnidae (A. (S.) reported that with regard to the sperm ultrastructures of pectinata), Pteriidae, Mytilidae, or Arcidae (Fig. 2A, B bivalves, the numbers of mitochondria in the sperm and D). However, two Ostreidae species, C. gigas and C. midpiece now constitute a widely employed metric in nipponica, harbored 2-3 electron-dense and electron-lucent taxonomic analyses. This is why the number of mitochondria transverse bands (special substructure associated with fertili- in the sperm midpiece tends to be stable within any given zation), while, Saccostrea commercialis have 3-4 transverse family or superfamily (Healy, 1989, 1995). Recently, some bands (special substructure associated with cross incom- authors (Chung & Ryou, 2000; Kim, 2001; Chung et al., patibility between C. gigas and Saccostrea commercialis) 2007; 2010) have demonstrated that the number of mito- at the anterior region (part) of the acrosomal vesicle chondria at the midpiece of the spermatozoon were five in (Healy & Lester, 1991; Kim et al., 2010a). Therefore, the Pinnidae Arcidae, Mytilidae, and Pteriidae in subclass these patterns can be regarded as relevant differences in Pteriomorphia; four mitochondria were found in the midpiece acrosomal vesicular structures that are observed only in of the spermatozoon in only the Ostreidae family of Ostreidae species, thus differentiating them definitively subclass Pteriomorphia, and five were found in the Pinnidae, from species members of the Pinnidae, Pteriidae, and Pteriidae, Mytilidae, and Arcidae (Chung et al., 2011) in Mytilidae of subclass Pteriomorphia. subclass Pteriomorphia, as can be seen in most Veneridae, 2 8 H-W Kang, E-Y Chung, J-H Kim, JS Chung, K-Y Lee Dev. Reprod. Vol. 16, No. 1 (2012)

Solenidae, and Corbiculidae species of subclass Heterodonta Korea. Kor J Malacol 21:95-105. (Kim, 2001; Chung et al., 2007, 2010). Even among species Chung EY, Kim EJ, Park GM (2007) Spermatogenesis and within the same family, though, the number of mitochondria sexual maturation in male Mactra chinensis (Bivalvia: at the sperm midpiece may vary to some degree. Sometimes, Mactridae) of Korea. Integr Bios 11:227-234. even within a single species, this factor evidenced slight Chung EY, Chung CH, Kim JH, Park SW, Park KH (2010) variations. In this study, we determined there to be five Ultrastructures of germ cells and the accessory cells mitochondria in the midpiece of the sperm in A. (S.) during spermatogenesis in male Gomphina veneriformis pectinata, a Pinnidae species of subclass Pteriomorphia. (Bivalvia: Veneridae) on the east coast of Korea. Kor Therefore, the numbers of mitochondria in the sperm J Malacol 26:51-62. midpiece were not strictly linked to the subclass; however, Chung EY, Kim JH, Kim SH, Seo WJ (2011) Germ cell their numbers were clearly correlated with family and development during spermatogenesis and some characteris- superfamily (Healy, 1995). Thus, in the aggregate, our results tics of mature sperm morphology in male Scapharca regarding mitochondrial count in the sperm midpiece are subcrenata (Pteriomorphia: Arcidae) in Western Korea. consistent with the previous report of Healy (1995). Kor J Malacol 27:121-129. Daniels EW, Longwell AC, McNiff JM, Wolfgang RW ACKNOWLEDGEMENTS (1971) A reinvestigation of the ultrastructure of the spermatozoa from the American oyster Crassostrea virginica. The authors are grateful to Dr. Tae Hwan Lee of the Trans American Microscopy Society. 90:275-282. University of Michigan for helpful comments on the Dorange G, Le Pennec M (1989) Ultrastructural characteristics manuscript. This research was supported in part by funding of spermatogenesis in Pecten maximus (Mollusca, Bivalvia). from Research Projects on conservation and restoration of Inver Reprod & Develop 15:109-117. fisheries species, NFRDI, Korea (RP-2012-AQ-012). Drozdov TA, Reunov AA (1986) Spermatogenesis and the sperm ultrastructure in the mussel Modiolus difficillis. REFERENCES Tsitologiia 28:1069-1074. Eckelbarger KJ, Bieler R, Mikkelsen PM (1990) Ultra- Baccetti B, Afzelius BA (1976) Biology of sperm cell. S. structure of sperm development and mature sperm mor- Krager, New York. Monogr Devel Biol 10:1-254. phology in three species of commensal bivalves (Mollusca: Baccetti B (1979) The evolution of the acrosomal complex. Galeommatoidea). J Morph 205:63-75. In: Fawcett DW., Bedford JM (eds.) The Spermatozoon. Eckelbarger KJ, Davis CV (1996) Ultrastructure of the gonad Urban Schwarzenberg, Baltimore, pp 305-328. and gametogenesis in the eastern oyster, Crassostrea Bernard RTF, Hodgson AN (1985) The fine structure of virginica. II. Testis and spermatogenesis. Mar Biol 127: the sperm and spermatid differentiation in the brown 89-96. mussel Pernaperna. South Africa J Zool 20:5-9. Franzén Å (1956) On spermatogenesis, morphology of the Chung EY, Ryou DK (2000) Gametogenesis and sexual spermatozoon, and biology of fertilization among inverte- maturation of the surf clam, Mactra veneriformis on brates. Zoology of Bidr Uppsala 31:355-482. the west coast of Korea. Malacologia 42:149-163. Franzén Å (1970) Phylogenetic aspects of the mophology Chung EY, Park KY, Son PW (2005) Ultrastructural study spermatozoa and spermiogenesis in Baccetti B (ed): on spermatogenesis and sexual maturation of the mail Comparative Spermatology. Accademia Nationale Dei jicon scallop, Chlamys farreri on the west coast of Lincei, Rome pp 1-573. Dev. Reprod. Vol. 16, No. 1 (2012) Spermatogenesis and Mature Sperm Morphology of A. (S.) pectinata 29

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