Spermatozoa of Viviparus ater (Mollusca, Gastropoda, Prosobranchia): An ultrastructural study

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Spermatozoa of Viviparus ater (Mollusca, Gastropoda, Prosobranchia): An ultrastructural study a

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Enzo Ottaviani , Barbara Rebecchi , Anna Maria & Bolognani Fantin

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Dipartimento di Biologia Animale , Università di Modena e Reggio Emilia , via Campi 213/D, I‐41100, Modena, Italy Published online: 28 Jan 2009.

To cite this article: Enzo Ottaviani , Barbara Rebecchi , Anna Maria & Bolognani Fantin (2001) Spermatozoa of Viviparus ater (Mollusca, Gastropoda, Prosobranchia): An ultrastructural study, Italian Journal of Zoology, 68:3, 187-194, DOI: 10.1080/11250000109356407 To link to this article: http://dx.doi.org/10.1080/11250000109356407

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Ital. J. Zool., 68: 187-194 (2001)

Spermatozoa of Viviparus ater (Mollusca, Gastropoda, Prosobranchia): an ultrastructural study ENZO OTTAVIANI BARBARA REBECCHI ANNA MARIA BOLOGNANI FANTIN

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Dipartimento di Biologia Animale, Università di Modena e Reggio Emilia, via Campi 213/D, I-41100 Modena (Italy)

ABSTRACT The fine structure of the two types of sperm, euspermatozoa and paraspermatozoa, present in Viviparus ater was studied. Under optical observation, the mature euspermatozoon showed a spiral head, with a highly condensed chromatin, and single flagellar structure. Ultrastructural examination revealed that the head is almost completely filled by the spiral, strongly electron-dense nucleus. The acrosome is a simplified horse shoe-like structure. The mid-piece begins in the infolded basal zone of the nuclear invagination, where a proximal centriole occurs and microtubular axonemes appear. In the mid-piece, the mitochondria are arranged in a helical fashion around the axonemes. Glycogen is not present. The passage to the flagellum is not marked by specialized structures: the mitochondrial sheath stops and a thick layer of glycogen envelops the axonemes. A typical 9+2 structure is evident. On the basis of the nuclear characteristics, the paraspermatozoon can be classified as oligopyrene. It is bigger than the euspermatozoon, shows a small elongated head which is poorly stained with the basic stains, an expanded mid-piece with a thick sheath, and 10-12 flagella. Under transmission electron microscopy, the head reveals a number of axonemes emerging at the top from clearly evident, microtubule-organizing centres. The poorly electrondense nucleus wraps around the axonemes. In the mid-piece, a line of mitochondria runs along the centre surrounded by the cytoplasm containing several axonemes (9+2). Various typical, electron-dense structures, defined by a membrane and regularly disposed between the plasmamembrane and axonemes, correspond to the thick elements of the sheath observed by scanning electron microscopy. At the end of the mid-piece, each axoneme enters a flagellum where it is surrounded by a thin layer of glycogen. KEY WORDS: Viviparus ater - Mollusca - Spermatozoa - Scanning electron microscopy - Transmission electron microscopy. ACKNOWLEDGEMENTS This work was supported by a 60% MURST (Italy) grant to A.M.B.F. (Received 7 February 2001 - Accepted 16 May 2001)

INTRODUCTION Maxwell (1983) and Healy (1988) reviewed the literature on molluscan spermatozoon form and Fretter (1984) and Koike (1985) that on prosobranchs, while the genesis of paraspermatozoa in gastropod molluscs was described by Buckland-Nicks et al. (1982) and Hodgson (1997). One of the major distinctions in molluscan sperm morphology regards the species with external fertilization that present so-called 'primitive' spermatozoa, and those with internal fertilization associated with the so-called modified form (Franzen, 1955; Maxwell, 1983). Except for the freshwater bivalve Curbicula, which has biflagellate sperm, fertile spermatozoa in molluscs are uniflagellate. However, they present the broadest diversity within the animal kingdom, in particular with regard to the mode of chromatin condensation and the basic proteins associated with the chromatin. Dimorphic spermatozoa in prosobranchs were first observed in the gastropod Paludina vivipara by von Siebold (1836). Since that initial observation, dimorphic sperms have been found to be common in the prosobranchs with internal fertilization, and numerous light and ultrastructural microscope studies have examined and discussed the dimorphic spermatozoa of a variety of meso and neogastropod prosobranchs (for references, see Healy & Jamieson, 1981). In order to differentiate the two types of spermatozoa, several terms have been employed, including typical and atypical sperm; eupyrene, oligopyrene or apyrene sperm (on the basis of chromatin content); normal and abnormal sperm. Healy & Jamieson (1981) reviewed the terminology and proposed the terms 'euspermatozoa' and 'paraspermatozoa' to replace the unsatisfactory or inappropriate earlier terminology. These terms will be used in the present study. In contrast to the general evolutionary conservation of histones from somatic cell nuclei, basic proteins from sperm nuclei display great variability in different animal species. Various Authors have studied basic sperm proteins in molluscs and reported very marked variability, particularly in the content, size and amino-acid composition of protamines and protamine-like components (Subirana et al, 1973; Ausio, 1986, 1992, 1995; Subirana & Colom, 1987; Chiva et al, 1989; Daban et al, 1990, 199D. The structure and development of spermatozoa in Viviparus viviparus (formerly called Paludina vivipara) were described in detail by Meves (1900 and 1903, quoted by Hanson, 1952) who based most of piece of observations on sections of the testis. Further papers have added little to Meves' data, with the exception of that of Pollister & Pollister (1943, quoted by Hanson, 1952) who accurately described the behaviour of chromosomes during the divisions of the spermatocytes. Gall (1961) described the replication of centrioles during spermatogenesis in Japanese V. malleatus. The first ultrastructural studies were carried out on sperm from V. viviparus

188 (Hanson et al, 1952), V. malleatus (Ishizaki & Kato, 1958) and Cipangopaludina malleata (Yasuzumi & Tanaka, 1958). More recently Griffond (1980, 1981) investigated both typical and atypical spermatogenesis in an ultrastructural study of V. viviparus, while histochemical studies (Dembski, 1968) were performed on sperm from V. contectus. In the present paper, we have examined the fine structure of the two types of sperms present in Viviparus ater(J)e Cristofori and Jan, 1832), a prosobranch common in Northern Italy, with a view to providing further information on their function.

MATERIALS AND METHODS

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Animals Adult males of V. ater collected in the spring and autumn in canals near Modena (Italy) and selected on the basis of the modified right tentacle (fused with the penis in the males) were maintained in the laboratory for no more than 1-2 days in dechlorinated freshwater at room temperature, and then sacrificed. Morphological reactions After removing the shell, the testis was dissected out. Several pieces of it were fixed in Bouin's fluid and processed for histological studies. Haematoxylin-eosin, Azan-Mallory, and Azan-Malloryfast green pH 1 stains were carried out on 7-um slices. The seminal liquid was collected directly from testis tubula using a Pasteur pipette and diluted (v/v) with an isotonic solution to Viviparus hemolymph. Smears prepared with the diluted seminal liquid were dried and stained by Giemsa stain. Further smears were fixed with the following solutions: 1) methanol/acetic acid (3:1), for 5 min; 2) 2% p-formaldehyde in PBS fixed smears pH 7.3, for 5 min; 3) 2% glutaraldehyde in 20 mmol/1 phosphate buffer pH 7.2, for 30 min. PAS reaction with and without a-amylase (Type XIII-A from human saliva; Sigma, St. Louis, Missouri, USA) digestion (1 mg/ml for 1 h at 37° C), aniline blue and fast green pH 8 stains were performed on fixed smears. Scanning electron microscopy (SEM) Diluted seminal liquid was spotted on cover slips covered by a 0.1% polylysine solution in borate buffer, 10 mmol/1, pH 8.2. After 20 min, the cover slips were fixed (2 h 30 min at 4° C) with a 1:1 mixture of 0.8 % glutaraldehyde and 1% osmium tetroxide in phosphate buffer 10 mmol/1, pH 7.3. After three washings with the same buffer, the samples were dehydrated with a graded series of ethanol solution reaching 99%. Dehydration was completed in a critical point drying apparatus (Balzers Union 010 CPD) with CO2. Dry specimens were coated with a thin layer of gold-palladium and examined by a Philips SEM XL40 at the Centra Interdipartimentale Grandi Strumenti, University of Modena and Reggio Emilia. Trasmission electron microscopy (JEM) Small fragments of testis from specimens collected in both spring and autumn were fixed in a mixture of glutaraldehyde (final concentration 0.8%) and osmium tetroxide (final concentration 1%) in 5 mmol/1 phosphate buffer, pH 7.3. After dehydration, the specimens were embedded in Durcupan ACM resin (Fluka Chemie AG, Buchs, Switzerland). Ultrathin sections were prepared with a Reichert ultracut using a diamond knife, mounted on coated copper grids, and then stained with uranyl acetate and lead citrate. Preparations were examined with a 400 T Philips electron microscope at the Centro Interdipartimentale Grandi Strumenti, University of Modena and Reggio Emilia.

E. OTTAVIANI, B. REBECCHI, A. M. BOLOGNANI FANTIN

RESULTS

Light and SEM observations Two types of sperm, euspermatozoon and paraspermatozoon, were present in the testis and in the seminal liquid smears of V. ater. The mature euspermatozoon was about 60 um in length and, at optical observation, showed a spiral head with a completely condensed chromatin and a unique flagellar structure (Fig. 1, arrow). The head stained darkly with basic stains (haematoxylin, aniline blue), while only flagellum appeared positive to PAS reaction. The SEM observations did not add any important structural information, with the exception of the presence at the top of the head of a small enlargement, probably corresponding to the acrosomal region (Fig. 2, arrow). The paraspermatozoon was classified as an oligopyrene type on the basis of nuclear characteristics. With a length of about 120 pm, it was larger than the euspermatozoon and showed a small, elongated head poorly stained with the basic stains (Fig. 1, arrowheads), an expanded midpiece and 10-12 flagella. In the paraspermatozoa, flagella also appeared positive to PAS reaction. The SEM observations showed some longitudinal structures in the head (Fig. 3), and a thick sheath in the mid-piece (Fig. 4). The flagellar fibrils did not show any particular feature. Observations by phase contrast microscopy of live sperms in isosmotic solution demonstrated that paraspermatozoa are more motile than euspermatozoa. TEM observations Euspermatozoa The euspermatozoon head was almost completely filled by the spiral, strongly electron-dense nucleus (Figs 5, 6). The acrosome positioned anteriorly to the nucleus was a simplified, horseshoe-like structure containing amorphous or granulated material (Fig. 7). In some not completely mature sperms, the Golgi apparatus could be seen shifting over the nucleus that was beginning to change its structure. At this moment, the Golgi apparatus appears to secrete the pro-acrosome vesicles. A short mid-zone not identifiable at optical or SEM investigation was also seen. The mid-piece initiated in the infolded basal zone of the nuclear invagination which contained an amorphous, electron-dense mass in close contact with the basal nuclear envelope and a ringshaped centriole. In the mid-piece where microtubular axonemes originated (Fig. 8), the mitochondria were arranged in a helical fashion around the axoneme without forming a ribbon-like, or so-called paracrystalline structure, as described for other prosobranchs and opistobranchs (Fig. 9, longitudinal section). Glycogen was not present in the mid-piece either between or within the mitochondria. The passage to the flagellum was not marked by specialized structures: the mitochondrial sheath disappeared and a thick layer of glycogen enveloped the axonemes (Fig. 9). The typical 9+2 axone-

VIVIPARUS ATER SPERMATOZOA

mal structure (Fig. 10) was evident, and the glycogen layer extended towards the end of the flagellum. Some not completely mature euspermatozoa were observed (Fig. 17). The main feature was an elongated, uncoiled head with poorly condensed chromatin. The midpiece and tail showed a well-defined structure (Fig. 17).

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Paraspermatozoa The head showed a particular structure (Fig. 11). Some axonemes were present at the top of the cytoplasm initiating from some very clearly defined microtubule-organizing centres (Fig. 12). The poorly electron-dense nucleus wrapped around the axonemes, and a very thin cytoplasm layer separated the nucleus from the plasma membrane (Fig. 13). In the mid-piece, there was a line of mitochondria in the centre surrounded by cytoplasm containing several axonemes (9+2) (Figs 14, 15, cross and longitudinal sections, respectively). Several electrondense structures defined by a membrane (vesicles) were regularly distributed between the plasma membrane and

189

axonemes. These corresponded to the thick elements of the sheath observed by SEM (Figs 14, 15, cross and longitudinal sections, respectively). At the end of the midpiece, each axoneme entered a flagellum where it was surrounded by a thin glycogen layer (Fig. 16). Some incompletely mature paraspermatozoa were also observed (Fig. 18). At this phase of spermiogenesis, axonemes were present and the mitochondria were moving towards the mid-piece where the sheath was absent. Numerous small vesicles containing electron-dense material were scattered in the cytoplasm around the nucleus without forming the thick sheath structures of mature sperms.

DISCUSSION Viviparus ater, in common with a wide variety of meso and neogastropods, produces two types of sperm: euspermatozoa and paraspermatozoa. The main characteristics of euspermatozoon are the spiral head with

Figs 1-4 - Morphological and SEM images of euspermatozoa and paraspermatozoa. 1 - Seminal liquid smear stained with Giemsa; the spiral heads of euspermatozoon (arrow) and paraspermatozoon (arrowheads) are shown (bar = 10 pm). 2 - Head of euspermatozoon; arrow marks the acrosome (SEM; bar = 10 pm). 3 - Head of paraspermatozoon (SEM; bar = 5 pm). 4 - Sheath of mid-piece of paraspermatozoon (SEM; bar = 5 pm).

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Figs 5-10 - TEM images of euspermatozoa. 5 - Cross section: head showing highly condensed chromatin (arrow) and mid-piece (arrowhead); the sperms are still surrounded by the cytoplasmic processes of the Sertoli cells (bar = 5 um). 6 - Longitudinal section: spiral electron-dense chromatin; GA, Golgi apparatus (bar = 5 um). 7 - The Golgi apparatus with a horseshoe-like form (bar = 1 pm). 8 - Zone between the head and mid-piece: a ring-shaped centriole marks the passage (arrow) (bar = 5 pm). 9 - Zone between the mid-piece and flagellum: at the mid-piece, the axoneme is surrounded by the mitochondrial sheath (arrows), while at the flagellum, there is a thick layer of glycogen (bar = 1 pm). 10 - Cross section of the flagellum showing the typical 9+2 microtubular structure (bar = 1 pm).

VMPARUS ATER SPERMATOZOA

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191

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Figs 11-16 - TEM images of paraspermatozoa. H - Head (H) and mid-piece (MP) (bar = 5 pm). 12 - Head: microtubule-organizing centres (arrows) and some axonemes (arrowheads) (bar = 2 pm). 13 - Head: presence of uncondensed chromatin between axonemes (bar = 2 pm). 14 - Cross section of mid-piece (bar = 2 pm). 15 - Longitudinal section of mid-piece (bar = 5 pm). 16 - Transition from the mid-piece to flagella (bar = 2 pm)..

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Figs 17-18 - TEM images. 17 - Immature form of euspermatozoon (bar = 5 prn). 18 - Immature form of paraspermatozoon (bar = 10 pm).

highly condensed chromatin and the presence of only one ring-shaped centriole at the nucleus base, unlike the two cylindrical perpendicular centrioles described, for example, in V. malleatus (Gall, 1961). The paraspermatozoa show some features observed in other prosobranchs, such as the presence of several axonemes initiating at the top of the head from microtubule-organizing centres, and the vesicles containing an electron-dense material in the mid-piece. However, the nuclear feature and the thick sheath observed by SEM are quite peculiar. A number of studies on the ultrastructural characteristics of both prosobranch euspermatozoa (Bulnhem, 1962; Selmi & Giusti, 1980; Giusti & Selmi, 1982a; Healy, 1982a, b, 1983a, b; Afzelius et al., 1989) and paraspermatozoa (Bulnhem, 1962; Koike & Nishiwaki, 1980; Melone et al, 1980; Selmi & Giusti, 1980; Giusti &

Selmi, 1982b, 1983; Healy, 1982c, 1986; Afzelius et al, 1989) have been published. The euspermatozoon of V. ater with its very extended head and the increased volume of mitochondria may be considered a better specialized example within invertebrates. This could be connected with the internal fertilization typical of Viviparidae, as proposed by Griffond (1980). The structure of the V. ater euspermatozoon differs from that of other prosobranchs, such as Nucella lapillus (Walker & Mac Gregor, 1968), TheodoxusJluviatilis (Giusti & Selmi, 1982a), and Cerithiidae (group 1 and 2 species), Potamididae and Turritellidae (Healy, 1983b). On the other hand, it shares some common characteristics with euspermatozoa from other species of Viviparidae, such as the presence of a homogeneous helical nucleus in the head (Cipangopaludina malleata,

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VIVIPARUS ATER SPERMATOZOA

Yasuzumi & Tanaka, 1958; V. viviparus, Griffond, 1980). However, the structure of the acrosome and the feature of the mitochondria differ from those in V. viviparus (Hanson et al, 1952; Griffond, 1980) and C. malleata (Yasuzumi & Tanaka, 1958). With regard to the paraspermatozoon, the structure seen in V. ater is quite different from that observed in T. fluviatilis (Selmi & Giusti, 1983) or in Diala cento (Healy, 1986), where the sperm appears as a thin filament without a nucleus. There are, however, several resemblances with paraspermatozoa from species belonging to the families of Cerithiidae, Potamididae, and Planaxidae (Healy, 1986; Giusti & Selmi, 1982b). In particular, the almost invariable absence of acrosomes, the presence of a great number of vesicles containing an electron-dense material in the mid-piece and the retention of multiple mobile tails. In the paraspermatozoon of V. ater, the nuclear material is dispersed around the axonemes. This contrasts with V. viviparus, where the nucleus appears as an irregular ring surrounding the axoneme bases (Giusti & Selmi, 1982b), Cochlostoma montanum and Cerithium vulgatum (Giusti & Selmi, 1982b) and Rhinoclavis vertagus and Clypeomorus moniliferus (Healy, 1986), where the nucleus appears as a small condensed cylinder surrounded by axonemes or by vesicles. Moreover, in the mid-piece of V. ater, the regularly-shaped vesicles surround a group of axonemes and a central mitochondrion line as in V. viviparus (Griffond, 1981) and C. vulgatum, but unlike in C. montanum (Giusti & Selmi, 1982b) or Zeacumantus subcarinatus (Healy, 1986). Given the large amount of chromatin, the euspermatozoon is undoubtedly the germ cell liable to fertilization. Several hypotheses have been made on the function of paraspermatozoon. It may be involved in euspermatozoon transport (Ankel, 1924; Woodward, 1940; Bulnhem, 1962); in activation resulting from substances produced by the breakdown of the paraspermatozoon vesicles (Hanson et al, 1952), or in nutrition (Dembski, 1968; Griffond, 1981). In histological sections of V. ater ovary, we observed the presence of both types of sperm near mature ovocytes. Moreover, the paraspermatozoa morphology remains unchanged. This suggests that the paraspermatozoa may carry out functions of transport and trophism for euspermatozoa. The paraspermatozoa do not present a large amount of glycogen, suggesting that these sperms do not contain nutrient material, but they are mobile specialized cells which carry the less mobile euspermatozoa. The packing of the cytoplasm gives to the paraspermatozoa rigidity without loss of flexibility, and may act as a mechanical support for euspermatozoa, as hypothesized for probranch molluscs by Giusti & Selmi (1982).

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