Biol. Bull. 209: 204-214. (December 2005)
© 2005 Marine Biological Laboratory
Spermiogenesis in the Hagfish Eptatretus burgeri (Agnatha)
Sachiko Morisawa
Division of Biology, Department of Anatomy, St. Marianna University, School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki 216-8511, Japan
E-mail: s2mori{at}marianna-u.ac.jp
 |
Abstract
|
|---|
The fine structure of spermatid differentiation in a primitive vertebrate, the hagfish, whose spermatozoa bear acrosomes, was investigated. In early round spermatids, the acrosomal vesicles were spherical and located in a shallow nuclear indentation, flanked by the plasma and the nuclear membranes. The vesicle underwent a transition through lens-shaped and cap-shaped stages until it attained the shape of a bell in mature spermatozoa. Electron-dense acrosomal material that appeared as deposits in three portions of the vesicle finally joined in the center region at a late stage. Condensation of chromatin occurred in the anterior region of the nucleus. During transformation of the spermatids, many regularly spaced microtubules appeared beneath the plasma membrane except in the anteriormost region of the cell. The microtubules in a single alignment lay parallel to one another and encased the nucleus diagonally. During an early stage, the centrioles changed their orientation from perpendicular to longitudinal and rotated to become parallel to the long axis of the nucleus. Thus, the flagellum lay nearly straight along the cell axis. A cytoplasmic canal appeared transiently during the early stage. A droplet of cytoplasm was eliminated after descending along the flagella. The features of spermiogenesis in hagfish, which lies between invertebrates and vertebrates, are compared with those of other animals.
|
Introduction
|
|---|
Spermatozoa of the hagfish, a member of one of the most primitive vertebrate groups, consist of a head (acrosomal and nuclear regions), a midpiece, and an endpiece (Jespersen, 1975; Morisawa, 1995). The nucleus and the midpiece are long, as in the modified spermatozoa of internally fertilizing species (Franzen, 1970). As in higher vertebrates, the bell-shaped acrosomal vesicle covers the apical portion of the nucleus, although it covers only a very limited region (Morisawa, 1995). Recent studies on the hagfishes Eptatretus burgeri and E. stouti show that acrosomal exocytosis of hagfish spermatozoa forms an acrosomal process, as is often seen in the spermatozoa of invertebrates, and that it produces numerous vesicles through membrane fusion at many points, which is also observed in the spermatozoa of many vertebrates (Morisawa, 1999a; Morisawa and Cherr, 2002). The hybrid structure and function of hagfish spermatozoa is interesting because of the phylogenetic position of the group. However, investigations that are helpful in understanding those phenomena are limited.
Fertilization in hagfish has not yet been described. Gonadal development has been studied histologically in Myxine glutinosa, Eptatretus stouti, and E. burgeri (Walvig, 1963; Patzner, 1977; Gorbman, 1997; Nozaki et al., 2000). The ultrastructure of late stages of oogenesis has been reported (Morisawa, 1999b), but the fine structure of spermiogenesis in Myxine glutinosa and Eptatretus stouti (Alvestad-Graevner and Adam, 1977; Jespersen, 1975) was studied before papers about the acrosome reaction (Morisawa, 1999a; Morisawa and Cherr, 2002) were published.
The present paper describes the fine structure of hagfish spermiogenesis in detail, using the Japanese hagfish Eptatretus burgeri. The new findings—such as the appearance of a simple manchette, cytoplasmic canal, and cytoplasmic droplet, and formation of the acrosome—are interesting and helpful for understanding a species that occupies the border between invertebrates and vertebrates.
|
Materials and Methods
|
|---|
Hagfish (Eptatretus burgeri Girard) were caught from April to July at a depth of about 50 m near the Misaki Marine Biological Station of the University of Tokyo, in Kanagawa prefecture, Japan, and kept in a seawater laboratory tank at 15 °C. At an appropriate time between April and October of the same year, males were anesthetized with 3-aminobenzoic acid ethyl ester (MS222, Sankyo Pharmaceuticals, Tokyo, Japan, or Sigma Chemical, St. Louis, MO, USA) (0.05% in seawater), and the testes were removed.
For transmission electron microscopy (TEM), small pieces of testis were fixed for 1 h with either 2% glutaraldehyde or 2% glutaraldehyde-0.5% formaldehyde, both in 0.1 M phosphate buffer (pH 7.4), and postfixed with 1% osmium tetroxide in the same buffer. After dehydration in a graded alcohol series, followed by propylene oxide, samples were embedded in Epon. Thin sections, obtained using diamond knives, were stained with uranyl acetate and lead citrate. The samples were examined under a transmission electron microscope (JEOL 100B or JEM 1200EX, JEOL Ltd., Tokyo). For scanning electron microscopy (SEM), samples were prepared as described previously (Morisawa, 1995) and examined using a scanning electron microscope (US4, JEOL Ltd., Tokyo).
|
Results
|
|---|
Fine structure of spermatozoa (SEM)
Figure 1 is a whole view of a spermatozoon of Eptatretus burgeri. The head, bearing the acrosome at the apex, gradually leads to a thin, ribbon-like tail. The thin side of the twisting portion of the tail was about one-third the width of the thick side. This observation agrees with images of a cross section of the midpiece in which the axoneme is flanked by two long mitochondria (cf. Fig. 6 and Fig. 8w).
View larger version (53K):
[in this window]
[in a new window]
|
Figure 1. A scanning electron micrograph of an Eptatretus burgeri spermatozoon. Abbreviations: A, acrosome; arrowhead, a twisting portion; E, endpiece; MP, midpiece; N, nucleus. Bar = 5 µm.
|
|
View larger version (153K):
[in this window]
[in a new window]
|
Figure 6. Late stages of spermiogenesis. (a) The head and anterior portion of the flagellum. The axoneme is flanked by long mitochondria in the tail. Cytoplasmic droplet is seen. (b) The anterior region of the head. Note bell-shaped acrosomal vesicle, thin projection of the nucleus through the subacrosomal material, and microtubules beneath the plasma membrane. (c) The anterior region of the head at an off-center level, showing diagonally and regularly arranged microtubules. (d) The posterior region of the nucleus and the anterior region of the midpiece. Note microtubules (arrow) and a portion of the nucleus (arrowhead). (e) The posterior region of the nucleus, and the flagellum bearing a cytoplasmic droplet. Thin cytoplasm connects the cytoplasmic droplet to the flagellum. (f) A longitudinal section of the acrosomal region. Note microtubules. The acrosomal contents are tightly packed in the anterior portion of the acrosomal vesicle and loosely packed in the posterior region. (g) A cross section of the posterior portion of the acrosomal region, showing microtubules surrounding the acrosomal vesicle. (h-n) Cross sections at various levels: at the level of the nucleus (h); of the centriole (arrow) (i, j); and of the midpiece with 4, 3, and 2 mitochondria (k, l, and m, respectively); and with two mitochondria and a few microtubules (arrow) (n). Note the distribution of microtubules. Abbreviations: Ax, axoneme; M, mitochondria. For other labels, see legend to Figure 5. Bar = 2 µm (a), 200 nm (b, f–n), 500 nm (c, d), and 1 µm (e).
|
|
View larger version (36K):
[in this window]
[in a new window]
|
Figure 8. Schematic drawings of spermiogenesis in the hagfish Eptatretus burgeri. (a) An early spermatid. (b–f) and (g–i) Formation of head region. (*f) Region behind the nucleus in f, showing a centriole in the nuclear fossa. (j) Head and the anterior region of flagellum at a late stage. (k) A whole view of a mature spermatozoon. (l–r) Formation of acrosomal region. (s) Acrosomal region of mature spermatozoa. (t) and (u) Formation of longitudinal alignment of centrioles and flagellum during early stages with transient appearance of cytoplasmic canal. (v) Base of the flagellum in mature spermatozoa. (w) Cross section of a main portion of the flagellum in mature spermatozoa. (x) and (y) Cross sections at the levels of the nucleus and of the flagellum, respectively, during late stages such as j. Microtubules of the manchette are illustrated. Abbreviations: AV, acrosomal vesicle; Ax, axoneme; C, centriole; CC, cytoplasmic canal; CD, cytoplasmic droplet; F, flagellum; M, mitochondria; Mt, microtubule; N, nucleus; Nl, nucleolus; S, subacrosomal material.
|
|
Fine structure of spermiogenesis (TEM)
In April and May, spermatocytes containing numerous mitochondria and exhibiting a small intercellular space were observed in the follicle (not shown). At the end of June, differentiating spermatids were free from the follicle wall (Fig. 2), but were linked by cytoplasmic bridges. In the cells, radial microtubules were observed (Fig. 2b). Spermatocytes were about 17 µm in diameter with a nucleus of over 10 µm, whereas spermatids were spherical and about 10 µm in diameter, possessing a nucleus about 6 µm in diameter with a distinct nucleolus.
View larger version (88K):
[in this window]
[in a new window]
|
Figure 2. Spermatocytes and spermatids in a follicle. (a) A section of the follicular lumen. Sc, spermatocyte; St, spermatid. Bar = 5 µm. (b) The spermatid in the upper left of a. Abbreviations: F, flagella; M, mitochondria; Mt, microtubule; N, nucleus; Nl, nucleolus. Bar = 1 µm.
|
|
At an early stage of spermiogenesis, spherical acrosomal vesicles were found in a shallow depression of the nucleus (Fig. 3). Various cytoplasmic components were observed near the vesicles (Fig. 3a, b). Golgi vesicles fused with the developing acrosomal vesicles, as is seen in acrosomal vesicle formation in many other species. A portion of the nucleolus appeared to be an open circle (Fig. 3c, e). Acrosomal vesicles were gradually pressed into the shape of a lens (Fig. 3c–f). They were tightly flanked by the plasma membrane and the nuclear envelope. The two membranes of the nuclear envelope were closely associated in this region. Inside the acrosomal vesicle, accumulation of electron-dense material was apparent in three regions (Fig. 3d, f): a layer underlining the outer acrosomal membrane and two deposits on the opposite side of the vesicle—that is, a disk-like deposit and a small deposit on the disk. In the apical region, the plasma and outer acrosomal membranes were in close association. Subacrosomal material occupied a narrow space between the inner acrosomal membrane and the nuclear envelope (Fig. 3d, f).
View larger version (155K):
[in this window]
[in a new window]
|
Figure 3. Early stages of spermiogenesis. (a) A section of a spermatid head. (b) An acrosomal region. (c) A spermatid head showing a round acrosomal vesicle and the nucleolus. (d) The acrosomal region in c, showing close association between the plasma and outer acrosomal membranes and accumulations of acrosomal material (arrow). Subacrosomal material occupies the narrow space between the inner acrosomal membrane and the nuclear envelope (arrowhead). (e) A spermatid head, showing a lens-shaped acrosomal vesicle. (f) The acrosomal region in e. Abbreviations: AV, acrosomal vesicle; G, Golgi apparatus; N, nucleus; NE, nuclear envelope; Nl, nucleolus; O, outer acrosomal membrane; P, plasma membrane. Bar = 1 µm (a, c, e), 500 nm (b), and 200 nm (d, f).
|
|
Accumulations of the dense acrosomal material became distinct as the anterior portion of the nucleus became a little thinner (Fig. 4). The center portion of the nuclear depression projected forward, thrusting the inner acrosomal membrane upward (Fig. 4a–d, f). Microtubules were regularly and diagonally arranged in the periphery near the acrosomal vesicles (Fig. 4b). The nucleolus appeared to progressively diminish and disappear (Fig. 4a, c, f; cf. Fig. 3c, e). In the thick region behind the nucleus, the centriole was perpendicular to the acrosome-nucleus axis and located in a shallow indentation of the nucleus (Fig. 4f–g). Some mitochondria were aligned longitudinally along the axoneme (Fig. 4e), which probably extended into the flagellum. Mitochondrial fusion, which produces long mitochondria, might not occur in the tail but might occur in the thick region. Sections of flagella revealed that the axoneme was surrounded by cytoplasm and 0–4 mitochondria. Cytoplasmic droplets connected to the cell body by thin cytoplasmic bridges were observed behind the nucleus (Fig. 4e; see also Fig. 6a and e).
View larger version (141K):
[in this window]
[in a new window]
|
Figure 4. Advanced stages of spermiogenesis. (a) The head of a spermatid exhibiting a flattened acrosomal vesicle. Note a flagellum with two mitochondria (lower right). (b) Acrosomal region in a. Note the vesicle shape, deposits of acrosomal material, regularly aligned microtubules (arrow), and nuclear projection. Arrowhead, subacrosomal space. (c) The head region with a more flattened acrosomal vesicle. (d) The acrosomal region in c. (e) The region behind the nucleus in c. Note the axis of the acrosome-nucleus against the axoneme, mitochondria along the axoneme, and cytoplasmic droplet being connected through thin cytoplasm (lower left). (f) The head region, exhibiting the thick region behind the nucleus. (g) The thick regions in the bottom of f. Note the direction of the centriole in the nuclear indentation (left) and the axoneme surrounded by mitochondria (right). Abbreviations: A, acrosome; AV, acrosomal vesicle; Ax, axoneme; C, centriole; CD, cytoplasmic droplet; F, flagella; M, mitochondria; N, nucleus; Nl, nucleolus. Bar = 1 µm (a, c, f), 200 nm (b, d) and 500 nm (e, g).
|
|
At advanced stages (Fig. 5), the head region did not seem to be rigid. A cap-shaped acrosomal vesicle with condensing acrosomal material covered its apical portion. The inner acrosomal membrane formed an anterior pit in the center portion, while remarkably, the anterior portion of the nucleus protruded through the homogeneous subacrosomal material (Fig. 5b, f, h, i). Thinning of the nucleus occurred earlier in the anterior region than in the posterior region (Fig. 5a, c, e, g). Microtubules could be seen in the space between the acrosomal vesicle and the plasma membrane, except in the apical region where the plasma and the outer acrosomal membranes maintained a close association (Fig. 5b, d, f, j). The microtubules, which were aligned singly, regularly, and diagonally, encased and almost twisted the nucleus (Fig. 5h). Centrioles were oriented longitudinally in the nuclear fossa (Fig. 5g). Spermatid cells formed an almost straight line from the head to the tail.
View larger version (151K):
[in this window]
[in a new window]
|
Figure 5. More advanced stages of spermiogenesis. (a) Head region of a spermatid. (b) The acrosomal region in a. The acrosomal vesicle covers the apical portion of the nucleus projecting through subacrosomal material. Note the distribution of microtubules. (c) An off-center image of a head region. (d) The acrosomal region in c. (e) An elongating head region. (f) The acrosomal region in e. Note the distribution of acrosomal material, the close association of the plasma and outer acrosomal membranes, the anterior area lacking microtubules, and protrusion of the nucleus. (g) The head region. Note the flexible head, centrioles in longitudinal orientation, and the cytoplasmic droplet behind the nucleus. (h) The anterior portion of the head in g. Note the diagonally aligned microtubules (arrows). (i) A nearly longitudinal section of the acrosomal region. Labels -j and -k indicate the levels of the sections shown in j and k. (j) An oblique section of the acrosomal region through the apical portion of the acrosome (small arrow) and the nucleus (arrow) surrounded by the subacrosomal material. (k) An oblique section of the acrosomal region. Arrow, nucleus. Abbreviations: A, acrosome; AV, acrosomal vesicle; CD, cytoplasmic droplet; DC, distal centriole; Mt, microtubule; N, nucleus; O, outer acrosomal membrane; P, plasma membrane; PC, proximal centriole; S, subacrosomal material; V, vesicle. Bar = 1 µm (a, c, e, g), 200 nm (b, f, h, i, j, k), and 500 nm (d).
|
|
At late stages of spermiogenesis (Fig. 6), the acrosomal vesicles acquired a mature bell shape (Fig. 6b, f). The acrosomal contents were more tightly packed in the anterior region of the acrosome than in the posterior region. The nuclear protrusion through the subacrosomal material was less than in Figure 5. These features are similar to those of the mature acrosome (Morisawa, 1995). Chromatin was condensed in the anterior portion of the nucleus (Fig. 6d, e; cf. Fig. 5), but the posterior portion extended into the flagellum (Fig. 6d, e, k–m). Microtubules tightly encased the nucleus (Fig. 6c, d, h–j) and the posterior portion of the acrosomal vesicle (Fig. 6g), but were absent from the anteriormost region of the cell where the plasma and outer acrosomal membranes lay parallel (Fig. 6b, f). In the flagella, microtubules were regularly distributed under the plasma membrane (Fig. 6k–m). Irregular membranes in the tail were targeted for discarding. Mitochondria became identical in number and location to those seen in mature spermatozoa (Fig. 6k–n; Morisawa, 1995). Cytoplasmic droplets adhered to flagella through thin connections of cytoplasm (Fig. 6e).
Flagella had begun budding from the distal centriole that was perpendicular to the cell periphery during the stages when the nucleus was spherical and the centrioles lay perpendicular to each other (Fig. 7a). While the flagellum elongated, the centrioles gradually became longitudinal and moved inward toward the nucleus (Fig. 7b–e). The plasma membrane followed the distal centriole and formed a thin gulf, or cytoplasmic canal, that surrounded the base of the flagellum (Figs. 7b–e, arrows and arrowheads). The bottom of the canal was enlarged (Fig. 7c–e, arrowhead), as is seen in many animals. Near the posterior end of the distal centriole, the nucleus formed a corner (Fig. 7c–e), presumably containing an opening to the nuclear fossa. Distinct nuclear pores were rarely observed in the fossa. During these events, flagellar sections revealed that the axoneme was surrounded only by the plasma membrane (Fig. 7a–d). In Figure 4, however, spermatids lacked a cytoplasmic canal and the flagellar axoneme was flanked by mitochondria, suggesting that the events in Figure 7 had finished before the events in Figure 4.
View larger version (159K):
[in this window]
[in a new window]
|
Figure 7. Formation of flagella and orientation of centrioles. (a) An early flagellum and centrioles in perpendicular orientation. (b) Centrioles in a less perpendicular orientation than a. Cytoplasmic canal follows the distal centriole. (c) Centrioles in the nuclear fossa. Cytoplasmic canal and its bottom. (d) The cytoplasmic canal and its enlarged bottom. (e) Centrioles in a longitudinal orientation. (f) A cross section of centriole in the periphery. Abbreviations: Arrow, cytoplasmic canal; arrowhead, bottom of cytoplasmic canal; C, centriole; F, flagella; M, mitochondria. For other labels, see legend to Figure 5. Bar = 500 nm (a), common in (a-e), and 200 nm (f).
|
|
Figure 8 is a diagram of hagfish spermiogenesis. Formation of the head region, acrosomal region, and flagella is illustrated, together with an overview of a mature spermatozoon and sperm structure.
|
Discussion
|
|---|
A manchette—an array of microtubules that appears during transformation of spermatids in spermiogenesis—is observed in many animals. The features of the manchette and the fact that inhibition of microtubular maintenance causes deformation of spermatid nuclei (Abe and Uno, 1984; Russell et al., 1991) suggest that the manchette participates in shaping the sperm head. Despite the expected roles of the manchette, orientation of the microtubules varies among species; for example, they may be present as longitudinal bundles or as both circular and longitudinal bundles (Fawcett et al., 1971; Sousa et al., 1989; Soley, 1997). In higher vertebrates, a linkage between the nuclear envelope and several microtubules is evident (Russell et al., 1991; Soley, 1997). Spermatids of the protochordate Branchiostoma lanceolatum, in which the sperm nucleus is almost spherical, do not contain microtubules, and thus microtubules are not involved in the spermiogenesis (Baccetti et al., 1972). In the hagfish studied here, Eptatretus burgeri, the manchette, whose features suggest its participation in shaping the sperm, is simple and unique: single microtubules align diagonally along the long axis of the cell (Figs. 5, 6). The microtubules are maintained until the very late stages of spermiogenesis, even when the potential for an acrosomal exocytosis has been acquired (Morisawa and Cherr, 2002). They completely disappear in mature spermatozoa (Morisawa, 1995), although the mechanism behind their disappearance at completion of spermiogenesis in hagfish is unknown.
A cytoplasmic canal occurs in the spermatids and mature spermatozoa of many fish species (Mattei, 1970; Stanley, 1971; Afzelius, 1978; Cherr and Clark, 1984; Sprando et al., 1988; Jamieson, 1991; Hara and Okiyama, 1998). Similar features, including flagellar canals, are observed during spermiogenesis in higher vertebrates. On the other hand, spermatozoa of living agnathans—lampreys (Stanley, 1967) and hagfish (Morisawa, 1995)—do not exhibit such a structure. However, a cytoplasmic canal does appear in the spermatids of hagfish, although it is quite short-lived and limited to the early stages of spermiogenesis (Fig. 7).
The extent and timing of cytoplasmic reduction appear to vary among species. Cytoplasmic droplets move downward along flagella after spermiation and are eliminated in the course of sperm maturation in the epididymis in mammals. In a teleost, the bluegill sunfish, residual cytoplasm is reduced around the midpiece (Sprando et al., 1988), whereas during in vitro spermiogenesis in the medaka, some flagella carry residual bodies (Saiki et al., 1997). In quail, the residual bodies are released from the nuclear region at spermiation, leaving no cytoplasmic droplets (Lin and Jones, 1993). In hagfish, motile spermatozoa, which are available from the testis, have already lost such droplets (Morisawa, 1995), although it is evident that a cytoplasmic residual body descends along the flagellum during spermiogenesis (Figs. 4–6). Cytoplasmic reduction during spermiogenesis of the primitive vertebrate, hagfish, appears to progress as the sperm become motile.
The acrosome reaction occurs at the beginning of fertilization. In many invertebrates, acrosomal exocytosis of spermatozoa starts with membrane fusion at the apical point and produces an acrosomal process (Dan, 1967; Morisawa et al., 2004). However, during the mammalian acrosome reaction, the outer acrosomal membrane fuses at many points with the overlying plasma membrane, leaving vesicles (Yanagimachi and Noda, 1970) in the region where the manchette is not observed during spermiogenesis (Russell et al., 1991). The acrosome reaction in hagfish produces an acrosomal process and leaves many vesicles in front of reacted spermatozoa (Morisawa, 1999a; Morisawa and Cherr, 2002). Fusion of the plasma and outer acrosomal membranes occurs at many points in the anterior region of sperm cells, which is not invaded by any microtubules during spermiogenesis. There remains, however, a question of whether acrosomal exocytosis is initiated at just the apical point or at multiple points (Figs. 3–6; Morisawa, 1999a; Morisawa and Cherr, 2002). It seems possible that formation of this region, which has such potential for acrosomal exocytosis and subsequently plays an important role in the interaction between eggs and spermatozoa at fertilization, might include similar processes in both the higher vertebrates and a primitive vertebrate such as the hagfish. The formation and characteristics of the areas for the different modes of acrosomal exocytosis remain to be analyzed in invertebrates and vertebrates, including hagfish.
Acrosomes have been distinguished in the spermatozoa of fish species such as hagfishes (Jespersen, 1975; Alvestad-Graevner and Adam, 1977; Morisawa, 1995), lampreys (Stanley, 1967; Nicander and Szoden, 1971; Jaana and Yamamoto, 1981), elasmobranchs (Stanley, 1971), sturgeons (Cherr and Clark, 1984), lungfishes (Jespersen, 1971), and coelacanths (Mattei et al., 1988). However, investigations of the acrosome reaction and spermiogenesis in these species are few. The present study would thus be useful not only for understanding hagfish spermatozoa but also for improving our understanding of acrosomes and spermiogenesis in organisms that are of phylogenetic interest.
|
Acknowledgments
|
|---|
The author thanks Dr. M. Morisawa, Misaki Marine Biological Station (MMBS), for his encouragement; Mr. M. Sekimoto and Mr. M. Sekifuji, MMBS, for providing the material; and Ms. C. Sasaki and the staff of the Institute for Ultrastructural Morphology, St. Marianna University, for technical assistance. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, and Japan Society for the Promotion of Science, nos. 06839024, 08833013, and 13839016.
|
Footnotes
|
|---|
Received 19 January 2005; accepted 13 October 2005.
|
Literature Cited
|
|---|
Abe, S.-I., and S. Uno. 1984. Nuclear elongation of dissociated newt spermatids in vitro and their nuclear shortening by antimicrotubule agents. Exp. Cell Res. 154: 243–255.[Medline]
Afzelius, B. A. 1978. Fine structure of the garfish spermatozoon. J. Ultrastruct. Res. 64: 309–314.[Medline]
Alvestad-Graevner, I., and H. Adam. 1977. Zur Feinstruktur der spermatogenetischen Stadien von Myxine glutinosa L. (Cyclostomata). Zool. Scr. 6: 113–126.
Baccetti, B., A. G. Burrini, and R. Dallai. 1972. The spermatozoon of Branchiostoma lanceolatum L. J. Morphol. 136: 211–226.[Medline]
Cherr, G. N., and W. H. Clark. 1984. An acrosome reaction in sperm from the white sturgeon, Acipenser transmontanus. J. Exp. Zool. 232: 129–139.
Dan, J. C. 1967. Acrosome reaction and lysins. Pp. 237–293 in Fertilization, Vol. 1, C. B. Metz and A. Monroy, eds. Academic Press, New York.
Fawcett, D. W., W. A. Anderson, and D. M. Phillips. 1971. Morphogenetic factors influencing the shape of the sperm head. Dev. Biol. 26(2): 220–251.
Franzen, A. 1970. Phylogenetic aspects of the morphology of spermatozoa and spermiogenesis. Pp. 29–46 in Comparative Spermatology, B. Baccetti, ed. Academic Press, New York.
Gorbman, A. 1997. Hagfish development. Zool. Sci. 14: 375–390.
Hara, M., and M. Okiyama. 1998. An ultrastructural review of the spermatozoa of Japanese fishes. Bull. Ocean Res. Inst. Univ. Tokyo 33: 1–138.
Jaana, H., and T. S. Yamamoto. 1981. The ultrastructure of spermatozoa with a note on the formation of the acrosomal filament in the lamprey, Lampetra japonica. Jpn. J. Ichthyol. 28: 135–147.
Jamieson, B. G. M. 1991. Fish Evolution and Systematics: Evidence from Spermatozoa. Cambridge University Press, Cambridge.
Jespersen, A. 1971. Fine structure of the spermatozoon of the Australian lungfish Neoceratodus forsteri(Krefft). J. Ultrastruct. Res. 37: 178–185.[Medline]
Jespersen, A. 1975. Fine structure of spermiogenesis in eastern Pacific species of hagfish (Myxinidae). Acta Zool. (Stockholm) 56: 189–198.
Lin, M., and R. C. Jones. 1993. Spermiogenesis and spermiation in the Japanese quail (Coturnix coturnix japonica). J. Anat. 183(3): 526–535.
Mattei, X. 1970. Spermiogenese comparee des poissons. Pp. 57–69 in Comparative Spermatology, B. Baccetti, ed. Academic Press, New York.
Mattei, X., Y. Siau, and B. Seret. 1988. Étude ultrastructurale du spermatozoide du coelacanthe: Latimeria chalumnae. J. Ultrastruct. Mol. Struct. Res. 101: 243–251.
Morisawa, S. 1995. Fine structure of spermatozoa of the hagfish Eptatretus burgeri (Agnatha). Biol. Bull. 189: 6–12.[Abstract]
Morisawa, S. 1999a. Acrosome reaction in spermatozoa of the hagfish Eptatretus burgeri (Agnatha). Dev. Growth Differ. 41: 109–112.[Medline]
Morisawa, S. 1999b. Fine structure of micropylar region during late oogenesis of eggs in the hagfish, Eptatretus burgeri (Agnatha). Dev. Growth Differ. 41: 611–618.[Medline]
Morisawa, S., and G. N. Cherr. 2002. Acrosome reaction in spermatozoa from hagfish (Agnatha) Eptatretus burgeri and Eptatretus stouti: acrosomal exocytosis and identification of filamentous actin. Dev. Growth Differ. 44: 337–344.[Medline]
Morisawa, S., T. Mizuta, K. Kubokawa, H. Tanaka, and M. Morisawa. 2004. Acrosome reaction in spermatozoa from the amphioxus Branchiostoma bulgeri (Cephalochordata, Chordata). Zool. Sci. 21: 1079–1084.[Medline]
Nicander, L., and I. Szoden. 1971. An electron microscopical study on the acrosomal complex and its role in fertilization in the river lamprey, Lampetra fluviatilis. J. Submicrosc. Cytol. 3: 309–317.
Nozaki, M., T. Ichikawa, K. Tsuneki, and H. Kobayashi. 2000. Seasonal development of gonads of the hagfish, Eptatretus burgeri, correlated with their seasonal migration. Zool. Sci. 17: 225–232.
Patzner, R. 1977. Cyclic changes in the testis of the hagfish Eptatretus burgeri (Cyclostomata). Acta Zool. (Stockh.) 58: 223–226.
Russell, L. D., J. A. Russell, G. R. MacGregor, and M. L. Meistrich. 1991. Linkage of manchette microtubules to the nuclear envelope and observations of the role of the manchette in nuclear shaping during spermiogenesis in rodents. Am. J. Anat. 192(2): 97–120.
Saiki, A., M. Tamura, M. Matsumoto, J. Katowgi, A. Watanabe, and K. Onitake. 1997. Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes. Dev. Growth Differ. 39(3): 337–344.
Soley, J. T. 1997. Nuclear morphogenesis and the role of the manchette during spermiogenesis in the ostrich (Struthio camelus). J. Anat. 190: 563–576.
Sousa, M., L. Corral, and C. Azevedo. 1989. Ultrastructural and cytochemical study of spermatogenesis in Scrobicularia plana (Mollusca, Bivalvia). Gamete Res. 24(4): 393–401.
Sprando, R. L., R. C. Heidinger, and L. D. Russell. 1988. Spermiogenesis in the bluegill (Lepomis macrochirus): a study of cytoplasmic events including cell volume changes and cytoplasmic elimination. J. Morphol. 198(2): 165–177.
Stanley, H. P. 1967. The fine structure of spermatozoa in the lamprey Lampetra planeri. J. Ultrastruct. Res. 58: 14–154.
Stanley, H. P. 1971. Fine structure of spermiogenesis in the elasmobranch fish Squalus suckleyi. I. Acrosome formation, nuclear elongation and differentiation of the midpiece axis. J. Ultrastruct. Res. 36: 86–102.[Medline]
Walvig, F. 1963. The gonad and the formation of the sexual cells. Pp. 530–580 in The Biology of Myxine, A. Brodal and R. Fänge, eds. Universitetsforlaget, Oslo.
Yanagimachi, R., and Y. K. Noda. 1970. Ultrastructural change in the hamster sperm head during fertilization. J. Ultrastruct. Res. 31: 465–485.[ISI][Medline]
TOP
Abstract
Introduction
