Acta Zoologica (Stockholm) 80: 3545 (January 1999)

Spermiogenesis in barberi (Teleostei: : ), an oviparous, internally fertilizing Anna Pecio and Jan RafinÂski

Abstract Institute of Zoology Pecio, A. and Rafin ski, J. 1999. Spermiogenesis in Mimagoniates barberi Department of Comparative Anatomy (Teleostei: Ostariophysi: Characidae), an oviparous, internally fertilizing fish. Jagiellonian University Ð Acta Zoologica (Stockholm) 80: 35±45 Ingardena 6 PL 30060 KrakoÂw The ultrastructure of spermiogenesis of Mimagoniates barberi (Characidae), Poland an internally fertilizing oviparous is described. Spermiogenesis involves conspicuous changes of the topography of the cellular constituents Keywords: and modifications of their shape. The round nucleus elongates to become a spermiogenesis, ultrastructure, fish, very long flat plate. The long axis of the nucleus establishes the polarity of Mimagoniates, Glandulocaudinae, the spermatid and spermatozoon. The nucleus does not rotate and, as a consequence, the centrioles are located at the proximal tip of the spermato- Accepted for publication: zoon and the runs parallel to the long axis of the nucleus. Mito- 21 July 1998 chondria and most of the cytoplasm are shifted to the pole opposite the centrioles. The proximal portion of the flagellum is located in a long cytoplasmic canal which shortens during spermiogenesis and is free for most of its length in the mature spermatozoon flagellum. In the cytoplasm of elongated spermatids, close to the nuclear membrane, numerous micro- tubules appear which run from the centriolar pole to the mitochondrial pole forming a microtubular manchette. The microtubules persist in mature spermatozoa. It is concluded that the highly specialized structure of the spermatozoon of M. barberi is the result of only few developmental modifi- cations of spermiogenesis. Anna Pecio, Jan Rafin ski, Institute of Zoology, Department of Comparative Anatomy, Jagiellonian University, Ingardena 6, PL 30060 Krakow, Poland. E-mail: [email protected]

The evolution of special structural features of Introduction spermatozoa of internally fertilized must have Structure of fish spermatozoa is extremely diverse involved modifications of the process of spermiogenen- (Jamieson 1991). This diversity can be explained at least sis. Compared with the wealth of information on sper- partly by the diversity of modes of reproduction in fishes. matozoon structure, there are relatively few studies on Most externally fertilized teleosts have a simple type of the fish spermiogenesis. Most of these studies described spermatozoon, called aquasperm, characterized by a either spermiogenesis in fishes with simple spermatozoa roundish head and a short neck region with few mitochon- or in viviparous fishes belonging to advanced fishes of dria (Jamieson 1991; Mattei 1991). In contrast, the group Acanthopterygii (Billard 1970; Mattei 1970; spermatozoa of many internally fertilized teleosts have an Mattei and Mattei 1974; Grier 1975; Brusle 1981; elongated head and other derived features. These was Billard 1983; Billard 1986; Gwo and Gwo 1993). Here shown for example for groups which contain both we present results of an ultrastructural study of spermio- oviparous and viviparous species like Poeciliidae and Ze- genesis in Mimagoniates barberi Regan 1907, a small narchopteridae (Jamieson and Grier 1993). freshwater characid fish from streams of Paraguay. This

q 1999 The Royal Swedish Academy of Sciences 35 Spermiogenesis in the teleost Mimagoniates barberi . Pecio and RafinÂski Acta Zoologica (Stockholm) 80: 35±45 (January 1999) species is oviparous but females are inseminated matocytes are spheroid and their shape changes subse- internally (Pecio and Rafin ski 1994). This is a rare mode quently into ovoid, which in later stages of of reproduction in teleosts (Breder and Rosen 1966), spermiogenesis become even more elongated though internal fertilization must have preceded the (Fig. 1A,B). Mitochondria in secondary spermatocytes evolution of viviparity. Mimagoniates barberi belongs to are numerous and the Golgi apparatus is close to the Glandulocaudinae, a small group of oviparous South centriole (Fig. 2A). Clumped or slightly mottled American characids which all practice internal insemina- chromatin of the spermatocyte changes into more evenly tion (Weitzman and Fink 1985; Burns et al., 1995). The granular chromatin typical of early spermatids. The spermatozoon of Mimagoniates barberi has many derived spermatids are interconnected by cytoplasmic bridges, features when compared with spermatozoa of other spaces between them are partly filled with an electron characids with external fertilization (Pecio and Rafin ski dense material (Fig. 2B,C). The diameter of a chromatin 1994). The head is highly elongated, its long axis granuleisatthisstage 40 nm. The nucleus remains is not a continuation of the flagellar axis but runs more more or less spherical but the cell is already polarized. A or less paralell to the flagellum. Live spermatozoa are few, large mitochondria are shifted to one side of the flail-shaped and move with the centriolar part ahead. cell, the proximal centriole is in close contact with the There is no midpiece and the mitochondria are located nuclear membrane. The flagellum is situated within a on the posterior tip of the head. Such spermatozoon relatively short cytoplasmic canal (Fig. 2D). As the structure seems to be unique within Ostariophysi ± the nucleus of the spermatids elongates the cytoplasmic largest group of extant freshwater teleosts (Jamieson canal also elongates (Figs 2E, 3A). The flagellum is at 1991). Elongated spermatozoa have also been found in an angle of  458 relative the long axis of the nucleus, other glandulocaudine genera (Burns et al., 1995). The which shows a more or less flattened surface on the side present study gives the first description of the spermio- of the flagellum. Large flat cisternae develop parallel to genesis in this most unusual group of fishes. the flattened nucleus surface. The cisternae are often associated with the Golgi apparatus and other large vesicles (Fig. 3 B-D). At this stage the spermatid shows Materials and Methods a clear bilateral symmetry and polarity. Its long axis is defined not only by the shape of the nucleus but also by Sexually active males of M. barberi were taken from a the position of the proximal centriole which is situated breeding colony kept at our Department. Testes were on the apical side of the spermatid and by the position excised after the fishes were killed by an overdose of of the mitochondria on the opposite side. Most of the tricaine methanesulphonate (MS-222). Small pieces of cytoplasm is concentrated on the mitochondrial, either spermatogenic or aspermatogenic tissue of the testis posterior pole of the spermatid and on the flagellar side were fixed in 3% glutaraldehyde in cacodylate buffer (Fig. 3A). (pH = 7.4), post-fixed in 1% osmium tetroxide, The elongation of the nucleus and the spermatid dehydrated through ethanol series and embedded in Epon proceeds further. At this stage the nucleus is more or less 812. The ultrathin sections, stained with uranyl acetate tear-shaped (Fig. 4A). The nucleus has an elongated tip at and lead citrate, were examined in a JEOL JEM-100Sx the centriolar pole, while its mitochondrial tip is wide and electron microscope. obtuse. The chromatin is granular but the granules have For scanning electron microscopy, testes of four enlarged to 60±70 nm in diameter. The cytoplasm is specimens were fixed as for TEM, dehydrated in acetone, almost completely shifted to the mitochondrial pole and to critical-point dried, fractured, sputter-coated with gold the flagellar side of the spermatid (Fig. 4A). The flagellar and viewed in a JEOL JSM 35. canal elongates and reaches the mitochondrial pole (Fig. 4F). Results At later stages numerous microtubules appear in the cytoplasm. They originate in two groups situated laterally The testis of a mature M. barberi is composed of two to the distal centriole and continue along the centriolar- parts. In the anterior part of the testis spermatogenesis mitochondrial axis of the spermatid (Fig. 4 B-G). Closer takes place, whereas its caudal portion is devoid of any to the centriolar pole many rows of microtubules surround spermatogenic tissue and functions as a sperm storage the nucleus. They are also found in the cytoplasm adjacent organ(PecioandRafin ski, 1994). Germ cells are to the flagellar canal (Fig. 4 C,D). Further in the direction confined to cysts located within the seminiferous of the mitochondrial pole, microtubules form a single row tubules. Earlier stages of spermatogenesis in M. barberi approaching the nuclear membrane and reach the do not show any peculiarities; spermatogonia and sper- cytoplasm of the mitochondrial tip of the spermatid (Fig. 4 matocytes are very similar to those described in other E,F). The spermatids are still connected by the teleosts (Billard 1983; Selman and Wallace 1986). Sper- cytoplasmic bridges (Fig. 4G).

q 1999 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 80: 35±45 (January 1999) Pecio and RafinÂski . Spermiogenesis in the teleost Mimagoniates barberi

Fig. 1.ÐThe inner surface of the spermatogenic cysts in SEM. Ð A, The primary spermatocytes (sc) situated in depressions of Sertoli cells (Sc). Ð B, Spherical (ss), ovoid (so) and strongly elongated (se) spermatids in separate cysts during spermiogenesis. Scale bar: 10 mm.

At the next stage, the nucleus of the spermatid The flagellar canal opens and becomes shorter. This elongates further and becomes compressed laterally. process begins at the mitochondrial pole and continues in Elongated spermatids within the cysts have the same the direction of the centriolar pole (Fig. 5D). Flagella orientation (Fig. 5A). The centriolar tips of most freed from the canal are often each surrounded by spermatids are in close contact with the Sertoli cells cytoplasm droplets (Fig. 5E). Condensation of the which line the cysts (Fig. 5B). Free flagella are chromatin is not finished by the spermiation (i.e. when the immersed in finely granular electron dense material cysts open and spermatozoa are shifted along tubules) and (Fig. 5C). In the centriolar region the nucleus forms a continues within the lumen of the tubules. The chromatin flat plate with a reduced number of chromatin granules granules fuse together and the nucleus becomes more (Fig. 5D). In the rest of the nucleus chromatin granules homogenous with only small uncondensed areas left. are regularly spaced and have a diameter of 110± The nucleus of a mature spermatozoon assumes the 120 nm. At the mitochondrial pole the nucleus is also form of a long plate,  18 mm in length. Nuclear strongly compressed laterally and almost reaches the flattening is always at right angles to the plane of posterior tip of the spermatid. axonemal singlets. The nucleus is very flat and narrow at

Fig. 2.ÐA secondary spermatocyte and the early stages of with round nucleus and a wide zone of cytoplasm, mitochondria spermiogenesis. Ð A, A secondary spermatocyte at the beginning shifted to one pole. Proximal centriole (pc) close to nuclear of cell division. Chromatin mottled, Golgi apparatus (Ga) is close membrane. The flagellum situated in a short cytoplasmic canal to divided centrioles (arrows). Scale bar: 1 mm. Ð B, Spermatids (arrow). Scale bar: 1 mm. Ð E, One side of the spermatid nucleus with round nuclei connected by intercellular bridges (arrows). already slightly flattened with cytoplasmic cisternae (asterisks). Scale bar: 1 mm. Ð C, Spaces among spermatids partly filled with Flagellum located in elongating cytoplasmic canal. Scale bar: finely granular material. Scale bar: 0.5 mm. Ð D, Early spermatid 1 mm.

q 1999 The Royal Swedish Academy of Sciences Spermiogenesis in the teleost Mimagoniates barberi . Pecio and RafinÂski Acta Zoologica (Stockholm) 80: 35±45 (January 1999)

Fig. 2

q 1999 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 80: 35±45 (January 1999) Pecio and RafinÂski . Spermiogenesis in the teleost Mimagoniates barberi

Fig. 3.ÐSagittal and cross sections of the slightly elongated elongated spermatids. Flagellum located in cytoplasmic canal (f). spermatids. Ð A, Spermatid polarized by the position of the Mitochondria located in the more distal region. In both sections centrioles situated at one pole and the position of mitochondria at large flat cisternae (c and arrow) situated close to nuclear the opposite pole. Elongated cytoplasmic canal situated at the membrane on one side of nucleus. Scale bar: 1 mm. Ð C, The flattened surface of spermatid nucleus. A large flat cisterna microtubules (mt) associated with a cisterna and nuclear situated parallel to the flattened surface of the nucleus (c and membrane (small arrows), a large vesicle visible (v). Ð D, Flat arrow). The cytoplasm shifted to the flagellar side and cisternae (arrows and c), dictyosomes (Ga) and vesicles (v) mitochondrial pole. Scale bar: 1 mm. Ð B, Cross sections of situated in the cytoplasm. Scale bar: 0.5 mm.

q 1999 The Royal Swedish Academy of Sciences Spermiogenesis in the teleost Mimagoniates barberi . Pecio and RafinÂski Acta Zoologica (Stockholm) 80: 35±45 (January 1999) the centriolar apex of the spermatozoon, approximately zoon and the flagellum in live spermatozoa runs almost oval shaped in a cross-section in the middle and again parallel to the long axis of the head. In several fish flat and laterally compressed at the mitochondrial pole species the flagellum in a mature spermatozoon is asym- (Fig. 6A-C). Mitochondria are situated along the metrically located. In Paracheirodon innesi,theonly posterior part of the spermatozoon on one side of the characid for which the ultrastructure of the spermato- nucleus and at the tip of the mitochondrial pole zoon has been published, the head is situated slightly (Fig. 6C,D). The nucleus does not reach the very tip of oblique to the flagellar axis (Jamieson 1991). Such a the mitochondrial pole (Fig. 6D). situation has also been described for the carp (Cyprinus Numerous microtubules are found in the cytoplasm of carpio, Cyprinidae), the pike (Esox lucius, Esocidae) the centriolar pole of the spermatozoon in two groups at (Billard 1986), and a cichlid (Oreochromis niloticus) (Lou the sides of the flagellar canal. In the middle part of the and Takahashi 1989). nucleus they are found in many layers on one side of the In several fish species the diplosome is first situated nucleus (Fig. 6B). Further, in the direction of the mito- eccentrically in relation to nucleus, later the nucleus chondrial pole they are found close to the plasmalemma rotates taking up its position in front of the diplosome. and mitochondria but usually they form a single layer only This type of spermiogenesis in teleosts was described as (Fig. 6C). A few microtubules reach the mitochondrial tip type I by Mattei (1970). Rotation of the nucleus during of the spermatozoon (Fig. 6D). spermiogenesis has been found in several unrelated In a mature spermatozoon, the flagellar canal is very teleosts, for example in Lepomis macrochirus (Centrarchi- short (Fig. 6A,F). The spermatozoon head is  20.6 mm dae) (Sprando et al. 1988) and Acanthopagrus schlegeli and the flagellum  54.0 mm in length. The centriolar pole (Sparidae) (Gwo and Gwo 1993). Even in species with of the head is round, whereas at the mitochondrial pole elongated spermatozoon head, like Poeciliidae and Ze- the head tapers to a short tip. The axoneme of the narchopteridae, the long axis of the sperm head is flagellum has a typical ‡ pattern with A tubules continuous with the flagellar axis (Jamieson and Grier occluded in all doublets (Fig. 6E). 1993). In type II of Mattei there is no nuclear rotation during spermiogenesis and the flagellum in a mature spermatozoon is asymmetrically located. This type of Discussion spermiogenesis has been found for example in Liza The nucleus of the spermatozoon of M. barberi has a aurata (Mugilidae) (Brusle 1981). Thus, spermiogenesis form of a long flat plate. Elongation of the sperm head in M. barberi should be classified as the extreme case of has been observed in many internally fertilized species, type II spermiogenesis of Mattei (1970). and is therefore deduced to give some advantage to the The spermatozoon with apically situated centrioles spermatozoon in the internal environment within the and backward elongated nucleus, which in general female (Jamieson 1991). The flattening of the M. barberi structure resembles the spermatozoon of M. barberi,has nuclus is at the right angles to the plane of axonemal also been described in a scorpaenid fish Dactylopterus singlets. Quite similar orientation of the axoneme and volitans (Boissin et al. 1968) and in a primitive bony fish the flat nucleus has been observed in viviparous fishes Polypterus senegalus (Mattei 1970). The two latter species belonging to the family Zenarchopteridae (Jamieson and are externally fertilized. To provide functional Grier 1993). The most characteristic feature of the sper- explanation for such spermatozoon structure we have matozoon of M. barberi is the backward position of the undertaken studies on movement of M. barberi head relative to the diplosome. The present study shows spermatozoa. that in M. barberi the eccentric position of the diplosome Another characteristic and derived feature of the sper- in relation to the nucleus in early stages of spermatogen- matozoon of M. barberi is the dislocation of mitochondria esis is later augmented by the backward elongation of to the posterior tip of the head. In the majority of fish the nucleus. In this way the proximal centriole becomes spermatozoa mitochondria are located in the neck region situated at the advancing, apical pole of the spermato- next to the diplosome beneath the nucleus. However, in

Fig. 4.ÐSagittal and cross sections of the strongly elongated Ð D, The microtubules scattered in the cytoplasm around the spermatids. Ð A, The nucleus attenuated at the centriolar pole and nucleus. Ð E, A single row of microtubules around the nuclear obtuse and round at the mitochondrial pole. The flagellar side of membrane. Ð F, The cytoplasmic canal containing the flagellum at the nucleus is flat. Scale bar: 1 mm. Ð B-F, Asequenceofcross the level of the mitochondrial pole. A few microtubules situated sections of the elongated spermatids from the centiolar tip to the mainly beneath the plasmalemma. In spaces between spermatids mitochondrial pole. Scale bar: 0.5 mm. Ð B, Two bunches of granular material still present. Ð G, Oblique section trough microtubules present on two sides of the distal centriole (dc). Ð C, elongated spermatids still connected by the intercellular bridges The microtubules distributed in the cytoplasm around the nucleus (arrow). All microtubules parallel to the long axis of the nucleus. in many rows and densly packed alongside the cytoplasmic canal. Scale bar: 0.5 mm.

q 1999 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 80: 35±45 (January 1999) Pecio and RafinÂski . Spermiogenesis in the teleost Mimagoniates barberi

q 1999 The Royal Swedish Academy of Sciences Spermiogenesis in the teleost Mimagoniates barberi . Pecio and RafinÂski Acta Zoologica (Stockholm) 80: 35±45 (January 1999)

Fig. 5.ÐSpermatids before spermiation. Ð A, Strongly the laterally compressed nucleus. Flagella already emergent from elongated spermatids organized in a parachute-like structure the spermatid. Cytoplasmic canal present only at the very tip of within a cyst before spermiation. Fractured fragment of the testis the spermatid (arrow). The proximal part of the nucleus in a in SEM. Scale bar: 20 mm. Ð B, Strongly vacuolated cytoplasm form of a flat blade. The microtubules are present in two of the Sertoli cell (Sc). The centriolar tip of the spermatid bunches at the level of the cytoplasmic canal (D). Further inserted in the depresion of Sertoli cell within a cyst. Ð C, A towards the mitochondrial pole microtubules located around the cross section of the bunch of flagella in a cyst immersed in a nucleus (E). No microtubules at the flattened nuclear surface, fine granular material. The axonemal A-microtubules occluded cross section approximately at the midlength of the nucleus (D, (black arrow) but in some not (empty arrow). Ð D-E, Cross blank arrow). A cytoplasmic droplet with small vesicles present sections of the elongated spermatids, the cytoplasm reduced and on one of the flagella (asterisk). Scale bar: 0.5 mm.

q 1999 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 80: 35±45 (January 1999) Pecio and RafinÂski . Spermiogenesis in the teleost Mimagoniates barberi

Fig. 6

q 1999 The Royal Swedish Academy of Sciences Spermiogenesis in the teleost Mimagoniates barberi . Pecio and RafinÂski Acta Zoologica (Stockholm) 80: 35±45 (January 1999) several fish species mitochondria are situated excentrically. during spermiogenesis indicate that the microtubular In the spermatozoon of the loach (Misgurnus anguillicauda- manchette is involved in the shaping of the spermatid tus), which together with M. barberi is a member of Ostar- nucleus (Meistrich et al. 1990; Russell et al. 1991). This iophysi, cytoplasmic components of the midpiece such as hypothesis is also supported by the finding that mitochondria are shifted to one side, therefore the other manchette microtubules are associated with the side of the midpiece is formed by a thin layer of cytoplasm cytoplasmic dynein in rat spermatids (Yoshida et al. only (Ohta et al. 1993). In M. barberi, the midpiece 1994). It suggests that the manchette may play a role in disapears as a result of the extreme elongation of the intracellular movements. nucleus without rotation and the reduction of the So far M. barberi is the only fish species in which mi- cytoplasmic sheath on the opposite side. A location of mi- crotubules are retained in mature spermatozoa, although tochondria similar to that in spermatozoa of M. barberi has recently, microtubules have also been found in the evolved independently in Anguilla anguilla (Elopomorpha) spermatozoa of another Mimagoniates species (J. Burns, (Gibbons et al. 1983) and in Blennius pholis (Percomorpha) personal communication). Microtubules form a multi- (Silveira et al. 1990). layered scaffolding on the one side of the nucleus and A unique feature of the late spermatid in M. barberi possiblylendrigiditytoaverylongspermhead.This is the presence of a single row of microtubules located might be important both in the movement of close the nuclear envelope. Shifted to one side of the spermatozoa within the female and during insemination. nucleus they are also present in a mature spermato- We have observed that the spermatozoa of M. barberi zoon. Microtubules in association with the centriolar deform very quickly when mixed with water. complex are present in spermatids of several viviparous The deformation is restricted to the distal portion of the poeciliid teleosts, these microtubules disappear by the spermatozoon where only a few microtubules are end of the spermiogenesis (Billard 1970; Grier 1973, present. Since no intromittent organ is present in males 1975). In poeciliids the formation of microtubules of M. barberi the spermatozoa of this species must coincides with nuclear elongation and therefore one can experience a transient osmotic stress during insemina- conclude that the microtubules in these species are tion.Unexpectedlynomicrotubuleswerefoundinother involved somehow in the shape change of the nucleus Glandulocaudinae genera which have even more of spermatids. However, the presence of microtubles elongated sperm heads (J.Burns, personal communica- during fish spermiogenesis has been also reported for tion). However, unlike in Mimagoniates in all these species with rounded, unmodified sperm heads species the flagellum runs in a long cytoplasmic canal (Yasuzumi 1971). along the whole length of the head which perhaps Circumnuclear microtubules are commonly known as provides some support for the long head. the manchette and have been described in several groups of both vertebrates and invertebrates (Kessel 1970; Acknowledgements Ferraguti and Lanzavecchia 1971; Stanley et al. 1972). In fishes the manchette has been found only during sper- We thank Professor W. Kilarski (the Electron Microscopy miogenesis of Polypterus senegalus (Mattei 1970). Since Centre, Jagiellonian University, KrakoÂw, Poland) and Dr no electron micrographs for this species have so far been F. Lahnsteiner (University of Salzburg, Austria) for access published, a direct comparison with M. barberi is to the facilities and the staff for technical assistance. We difficult. The function of the microtubules in spermio- thank also Dr J. Szymura (Jagiellonian University) for genesis of M. barberi is not known but quite probably reading the manuscript and his suggestions, Dr John the microtubules could play an active role in elongation Burns (George Washington University, Washington, D.C.) and shaping of the sperm head. Several data indicate for providing unpublished data and an anonymous that this is the function of microtubules in mammalian reviewer for many helpful comments which improved the spermiogenesis. Observations on mice bearing a paper. mutation which affects the distribution of microtubules This research was supported by a grant IZ/BW/14/97.

Fig. 6.ÐA series of cross sections of mature spermatozoa from plasmalemma on one side of the nucleus. Ð C, Distal portion of the centriolar to the mitochondrial tip (A-E). Scale bar: 0.2 mm. the spermatozoon. Mitochondria located alongside the flat Ð A, The centriolar tip with strongly compressed nucleus, nucleus. The microtubules located between plasmalemma and the microtubules scattered in the cytoplasm on two sides of distal mitochondria. Ð D, Tips of the spermatozoa with mitochondria centriole (a), and along the cytoplasmic canal (b). c across and a few microtubules. Ð E, Cross section through flagella and section just below the opening of the cytoplasmic canal. All an endpiece. All A-tubules occluded. Ð F, Schematic diagram of axonemal microtubules empty. Ð B, Cross sections through the spermatozoon structure. Microtubules omitted. The capital middle portion of the head of the spermatozoon. 1±2 rows of letters A-E correspond to the numbers of the micrographs to the microtubules located between the nucleus membrane and left. Scale bar 1 mm.

q 1999 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 80: 35±45 (January 1999) Pecio and RafinÂski . Spermiogenesis in the teleost Mimagoniates barberi

References Mattei, X. 1970. SpermiogeneÂse compareÂe des poissons. In Baccetti B. (Ed.): Comparative Spermatology, pp. 57±69. Billard, R. 1970. La spermatogeneÁse chez Poecilia reticulata.IVLa Academic Press, New York. spermiogeneÁse. E tude structurale. ± Annales de Biologie Animale, Mattei, X. 1991. Spermatozoon structure and its systematic Biochimie, Biophysique 10: 493±510. implications in fishes. ± Canadian Journal of Zoology 69: 3038± Billard, R. 1983. Spermiogenesis in rainbow trout (Salmo 3055. gairdneri). ± Cell and Tissue Research 233: 265±284. Mattei, C. and Mattei, X. 1974. Spermiogenesis and spermatozoa Billard, R. 1986. Spermiogenesis and spermatology of some of the elopomorpha (teleost fish). In Afzelius B. A. (Ed.): The teleost fish species. ± Reproduction, Nutrition, DeÂveloppment 26: Functional Anatomy of the Spermatozoon, pp. 211±221. 877±920. Pergamon Press, Oxford. Boisson, C., Mattei, X. and Mattei, C. 1968. Le spermatozoõÈde Meistrich, M. L., Trostle-Weige, P. and Russell, L. D. 1990. de Dactylopterus volitans, Linne (Poisson Cephalacanthidae), Abnormal manchette development in spermatids of azh/azh eÂtudie au microscope eÂlectronique. ± Comptes Rendus des SeÂances mutant mice. ± American Journal of Anatomy 188: 74±86. de la SocieÂte de Biologie de l'Ouest Africain 162: 820±823. Ohta, T., Kato, K. H., Abe, T. and Takeuchi, T. 1993. Sperm Breder, C. M. and Rosen, D. E. 1966. Modes of reproduction in morphology and distribution of intramembranous particles in fishes. The American Museum of Natural History, The Natural the sperm heads of selected freshwater teleosts. ± Tissue and Cell History Press, New York. 25: 725±735. Brusle, S. 1981. Ultrastructure of spermiogenesis in Liza aurata Pecio, A. and RafinÂski, J. 1994. Structure of the testis, Risso, 1810 (Teleostei, Mugilidae). ± Cell and Tissue Research spermatozoon and spermatozeugmata of Mimagoniates barberi 217: 415±424. Regan 1907 (Teleostei: Characidae: Glandulocaudinae), an Burns, J. R., Weitzman, S. H., Grier, H. J. and Menezes, N. A. internally fertilizing, oviparous fish. ± Acta Zoologica (Stockholm) 1995. Internal fertilization, testis and sperm morphology in 75: 179±185. glandulocaudine fishes (Teleostei: Characidae: Russell, L. D., Russell, J. A., MacGregor, G. R. and Meistrich, Glandulocaudinae). ± Journal of Morphology 224: 131±145. M. L. 1991. Linkage of manchette microtubules to the nuclear Ferraguti, M. and Lanzavecchia, G. 1971. Morphogenetic aspects envelope and observations of the role of the manchette in of microtubules. ± Journal of Submicroscopic Cytology 3: 121±137. nuclear shaping during spermiogenesis in rodents. ± American Gibbons, B. H., Gibbons, I. R. and Baccetti, B. 1983. Structure Journal of Anatomy 192: 97±120. and motility of the 9+0 flagellum of eel spermatozoa. ± Journal Selman, K. and Wallace, R. A. 1986. Gametogenesis in Fundulus of Submicroscopic Cytology 15: 15±20. heteroclitus.±American Zoologist 26: 173±192. Grier, H. J. 1973. Ultrastructure of the testis in the teleost Poecilia Silveira, H., Rodrigues, P. and Azevedo, C. 1990. Fine structure latipinna. Spermiogenesis with reference to the intercentriolar of the spermatogenesis of Blennius pholis (Pisces, Blennidae). ± lamellated body. ± Journal of Ultrastructural Research 45: 82±92. Journal of Submicroscopic Cytology and Pathology 22: 103±108. Grier, H. J. 1975. Spermiogenesis in the teleost Gambusia affinis Sprando, R. L., Heidinger, R. C. and Russell, L. D. 1988. with patricular reference to the role played by microtubules. ± Spermiogenesis in the bluegill (Lepomis macrochirus): a study Cell and Tissue Research 165: 89±102. of cytoplasmic events including cell volume changes and Gwo, J. C. and Gwo, H. H. 1993. Spermatogenesis in the black cytoplasmic elimination. ± Journal of Morphology 198:165± porgy, Acanthopagrus schlegeli (Teleostei: Perciformes: Sparidae). 177. ± Molecular Reproduction and Development 36: 75±83. Stanley, H. P., Bowman, J. T., Romrell, L. J., Reed, S. C. and Jamieson, B. G. M. 1991. Fish Evolution and Systematics: Evidence Wilkinson, R. F. 1972. Fine structure of normal spermatid from Spermatozoa. Cambridge University Press, Cambridge. differentiation in Drosophila melanogaster.±Journal of Jamieson, B. G. M. and Grier, H. J. 1993. Influences of Ultrastructural Research 41: 433±466. phylogenetic position and fertilization biology on spermatozoal Weitzman, S. H. and Fink, S. V. 1985. Xenurobryconin ultrastructure exemplified by exocoetoid and poeciliid fish. ± phylogeny and putative pheromone pumps in glandulocaudine Hydrobiologia 271: 11±25. fishes (Teleostei: Characidae). ± Smithsonian Contributions to Kessel, R. G. 1970. Spermiogenesis in the dragonfly with special Zoology 421: 1±121. reference to a consideration of the mechanisms involved in the Yasuzumi, F. 1971. Electron microscope study of the fish development of the cellular asymetry. In Baccetti B. (Ed.): spermiogenesis. ± Journal of Nara Medical Association 22: 343± Comparative Spermatology, pp. 531±549. Academic Press, New 355. York. Yoshida, T., Ioshi, S. O., Imanaka-Yoshida. K. and Izutsu, K. Lou, Y. H. and Takahashi, H. 1989. Spermiogenesis in the nile 1994. Association of cytoplasmic dynein with manchette tilapia Oreochromis niloticus with notes on a unique pattern of microtubules and spermatid nuclear envelope during nuclear chromatin condensation. ± Journal of Morphology 200: spermiogenesis in rats. ± Journal of Cell Science 107:625± 321±330. 633.

q 1999 The Royal Swedish Academy of Sciences