the fine structure of the spermatozoa of siphonaria algesirae (gastropoda, pulmonata)

JOURNAL OF MORPHOLOGY 186:107-117 (1985)

The Fine Structure of the Spermatozoa of Siphonaria algesirae (Gastropoda, Pulmonata)

CARLOS AZEVEDO AND LAURA CORRAL Department of Cell Biology, Institute ofBiomedical Sciences (C.A.) and Center of Experimental Morphology (L. C.), University of Oporto, 4000 Porto, Portugal

ABSTRACT The sperm of Siphonaria algesirae (Gastropoda, Pulmonata), a species with internal fertilization, was studied by light and electron microscopy. The spermatozoon is a very long, uniflagellate cell composed of a conical head with an apical acrosome, a midpiece with a helically coiled external sheath containing a complex mitochondria1 derivative with a wavelength of - 5.5 pm, and an endpiece. There are no axonemal microtubules. Instead, nine homogeneous coarse fibers with transverse striations in the apical zone project toward the anterior section of the midpiece. In the posterior zone of the midpiece the coarse fibers are differentiated in a common microtubular axoneme. The complex omitochondrial derivative of the midpiece shows an organized group of 100 A diameter spherical particles. Externally the midpiece is surrounded along its length by a cylinder formed by two membranes. A complex structure separates the transitional zone between the midpiece and the endpiece.

The numerous investigations available on spermatozoon structure (see Baccetti and Afzelius, '76; Maxwell, '83) suggest the sig- nificance of the spermatozoan for taxonomic classification (Hinsch, '74). Despite the fact that the Mollusca is probably the second largest phylum (Maxwell, '83), there are few morphological studies of this group, and these are mainly on marine pulmonate species. Two recent works review sperm biology. One is a comparative study of the hermaphroditic gonads, spermatogenesis, and diversity of sperm morphology with special emphasis on cytochemistry (Maxwell, '83); the other makes an extensive revision of the comparative morphology of pulmonate reproductive anatomy, including sperm morphology and fertilization biology (Tompa, '84).

The present study deals with morphological aspects of the spermatozoa with special reference to the unusual composition of the head, midpiece, transitional zone, and endpiece. The ultrastructural studies of these specializations may contribute to a better un- derstanding of the phylogenetic relation- ships among other pulmonate species having internal fertilization (Bayne, '70; Thompson, '73), for which relatively few fine structural observations have been reported.

MATERIALS AND METHODS

Several specimens of the hermaphroditic marine plate limpet Siphonaria algesirae (Gastropoda, Pulmonata) were collected during a period of 2 years in the intertidal zone of the Portuguese Atlantic coast.

The hermaphroditic gonads (ovotestis and hermaphroditic duct or vesicula seminalis) were dissected and fixed in Bouin's fluid for light microscopic studies. Live spermatozoa were observed by various methods of light and phase-contrast microscopy.

For scanning electron microscopy (SEM), the fixed and isolated spermatozoa were de- hydrated in an ascending series of ethanol and were then gold coated and examined in a JEOL JSM 35C at 6 Kv. For transmission electron microscopy, free spermatozoa and small pieces of the hermaphroditic gonads were fixed in one of several ways: 1) in 2.5% glutaraldehyde buffered with 0.2 M sodium cacodylate (pH 7.6) at 4°C for 2 hours, rinsed for about 4 hours with the same buffer, and postfixed with buffered 2% OsO4 at 4°C for 2 hours; or 2) in 2.5% glutaraldehyde with 8% tannic acid, 0.05 M phosphate buffer, 0.015 M CaClz (Tilney et al., '731, then rinsed and postfixed as previously indicated. After de-

0 1985 ALAN R. LISS. INC.

108 C. AZEVEDO AND L. CORRAL

hydration in the ethanol series they were embedded in Epon. The semithin sections were stained in methylene blue-Azure I1 and the ultrathin sections were double stained with uranyl acetate and lead citrate, then examined in a JEOL lOOB electron micro- scope, operated at 80 Kv.

RESULTS Light microscopy

The mature spermatozoa are arranged irregularly in the hermaphroditic duct. They are typically aggregated by the apical zone and occupy the lumen of the duct. At the peripheral zone (in contact with an epithelial layer), different developmental stages of sperm and oocytes are present.

Live spermatozoa from the hermaphroditic duct appear identical when observed by nor- mal light and phase-contrast microscopy. Sperm are only motile through the duct's anterior portion; they move with lashing mo- tions. The head and anterior zone of the midpiece have a higher wave frequency than those of the midpiece and endpiece (which seem immobile). The spermatozoon is a very long, uniflagellated cell 756 f 8.2 pm (50 measurements) in total length.

Electron microscopy (EM) Owing to the great total length of the sper-

matozoon it was not possible to observe it in its entirety by EM. By scanning electron microscopy (SEM) all spermatozoa show a similar morphology (Fig. 1) in that they are divided into the head, midpiece, and endpiece. Head

The head is occupied by the conical nucleus with an apical sharp point (Figs. 1,2). Includ- ing the 1.1 pm long tapering conical acrosome, it measures -6.5 pm in length (Figs. 1, 2). The acrosome contains dense material in a spherical vesicle in the apical zone and lacks a subacrosomal space (Fig. 3). Exter- nally, the head is surrounded by the plasmalemma in close contact with the nuclear envelope (Figs. 2,3), which shows an infolded basal fossa (Figs. 2,4). The nuclear matrix is occupied by light chrqmatin composed of several filaments -65 A thick, which are longitudinally oriented (Figs. 2, 4). This chromatin is much denser in the apical nuclear zone near the acrosome (Fig. 3). Lat- erally, the long fibrous core - 1.2 pm long is in close contact with the acrosome and with

the anterior zone of the nucleus (Fig. 3). The surface of the mature sperm head plasmalemma (collected in the hermaphroditic duct) presents an irregular membranous myelin- like appearance with its irregularly distributed lamellae (Figs. 5, 6). These structures are formed by several concentric curved dense lines alternating With light spaces with a pe- rjodicity of - 60 A . The dense lines are - 35 A and the light spaces are -25 A thick (Fig. 6).

Midpiece Observed by SEM (Fig. 11, the 650 pm long

midpiece contains a prominent spiral ridge with a 5.5 pm wavelength (Fig. 1; see also Fig. 9). The midpiece begins in the infolded basal zone of the nuclear fossa, which itself contains a central amorphous mass in close contact with the basal nuclear envelope (Figs. 4, 5, 7, 8). In transverse section this amorphous mass shows a homogeneous contour with nine prominent semicircular zones (Fig. 7) giving rise to nine longitudinal columns (Fig. 4). These nine columns occupy a regular circular arrangement around a central mass (Figs. 7, 8). In longitudinal sections, these columns show a tranFverse striation with a periodicity of -500 A (Fig. 4). The columns are in continuity with the nine coarse fibers that surround the single central mass (Fig. 8). The coarse fibers are all alike and are arranged radially. No centrioles or microtubular axonemes occur in the anterior zone of the midpiece (Figs. 7,s).

The external region of the midpiece is occupied by a very complicated mitochondria1 derivative (Thompson, '73) that surrounds the central coarse fibers (Figs. 9, 10). In longitudinal (Figs. 9, 10) and transverse (Fig. 12) sections, the spiral ridge appears to be

Abbreviations A, A c r o s o m e Ax, Axoneme C, Double membranous cape CF. Coarse fibers EP; Endpiece H, Head IM, Internal membrane ISP, Internal space M, Mitochondrial complex Md, Mitochondrial derivative MP, Midpiece N, Nucleus PI, Plasmalemma TZ, Transitional zone

ULTRASTRUCTURE OF SIPHONARIA SPERM 109

Fig. 1. SEM of the head region of the spermatozoon of S. algesirae. Left side is the apical end containing the nucleus. ~6,350.

Fig. 2. Longitudinal ultrathin section GUS) of the head. x 18,800

Fig. 3. LUS of the apical zone of the nucleus, containing dense chromatin and the acrosome. Laterally, a longitudinal fibrillar core (arrows) is situated in close contact with the nucleus, acrosome, and plasmalemma. The apical zone of the acrosome contains a small vesicle (arrowhead). ~57,400.

Fig. 4. Detail of the basal zone of the nucleus and the subnuclear fossa showing a complex arrangement of the longitudinal coarse fibers, which begin in a homogeneous mass (*I and longitudinally project to the midpiece. ~53,300.

eccentric in relation to the external zone of the midpiece.

A vesicular structure, containing floccu- lent material (Figs. 10, 12), begins in close association with the nuclear basal region (Fig. 4), and 200-220 p m away its spiral ridge gradually decreases. In the posterior zone of the midpiece the mitochondrial derivative is less prominent and can be detected only in transversely sectioned material (Fig. 12). In favorable longitudinal serial sections (Figs.

9, lo), we see that this vesicle is a continuous mitochondrial derivative possessing a vesicular structure 0.30 pm wide at its maxi- mum, surrounded by a limiting membrane (Figs. 10,12).

With tannic acid fixation we observed the presence of spherical arrangements forming several longitudinal layers with two or three ranks of protofilaments in the inner mitochondrial derivative (Fig. 13). In favorable cross sections they appear to have a diameter


of -100 A (Fig. 13). These structures touch the mitochondrial derivative membranes. They are parallel structures that run obliquely in relation to the longitudinal axis. The final part of the midpiece does not con- tain the mitochondrial vesicular derivative. In this zone also the coarse fibers reside in a common microtubular axoneme (Figs. 11,141.

The midpiece is surrounded externally by a longitudinal and cylindrical sheath formed by two adherent membranes (Figs. 10, 12, 14). The external plasmalemma is continuous with the head and the endpiece (Figs. 10-12, 15-21) and completely surrounds the entire spermatozoon. The inner membrane forms a very long space along the spermatozoon with the external membrane of the mitochondrial derivative. This space seems to be mostly empty (Figs. 10-12, 15-19). In serial transverse sections it was possible to draw the arrangement of the mitochondrial derivative and endpiece (Fig. 22).

Endpiece The endpiece, -100 pm long, begins with

a very complicated structure in the transitional zone between the mid- and the endpiece (Figs. 15-19). The plasmalemma seems to penetrate this transitional zone, forming a 3 pm long tapering dense ring (Figs. 15-19). The axoneme of the midpiece reaches to the endpiece, passing through the central zone of the dense ring (Figs. 15-19).

In favorable longitudinal sections it was possible to see that the first external space is the little annulus communicating with the second internal space in the terminal zone of the midpiece (Figs. 15-17). From serial lon-

Fig. 5. A transverse ultrathin section (TUS) of the nucleus and different midpiece levels showing the external double-membranous cape and mitochondrial derivative. The periphery of the spermatozoon head plasmalemma shows some myelin-like structures (arrowheads). In the central zone of the nucleus can be seen the top of the subnuclear fossa (*). ~29,000.

Fig. 6. Higher magnification of a myelin-like structure irregularly distributed at the periphery of the nucleus. x 170,000.

Fig. 7. TUS of the nucleus showing coarse fibers (*) radially distributed and externally surrounded by the nuclear envelope (arrows). ~83,000.

Fig. 8. TUS of the nucleus at subnuclear fossa level. The central core containing the nine single coarse fibers and the central mass (*I surrounded by a distinct limiting membrane (arrows) can be observed. X61,OOO.

gitudinal sections (Figs. 15-19) it was possible to make a three-dimensional model of the complex annular tapering structure, illus- trated in Figure 23.

The endpiece is occupied by a granular material that surrounds the axoneme (Figs. 19, 20). The final portion of the endpiece with a tapering morphology shows the absence of microtubular arrangements (Fig. 21).

DISCUSSION

The ultrastructure of the marine pulmonate Siphonaria algesirae sperm has the ultrastructural organization of the "modified" sperm type, and its similarity to sperm of the Opistobranchia and Pulmonata is indicative of its phylogenetic affinity (Franzen, '55, '70). The very long midpiece, containing the complex mitochondrial derivative with the prominent spiralling ridge, seems to be the main evidence of systematic affinities. The presence of the nine peripheral coarse fibers in the anterior portion of the midpiece and mitochondrial derivative, as described in other pulmonates (Anderson and Personne, '67, '69; Bayne, '70; Maxwell, '76, '831, is also in agreement with Franzen's theory of relatedness.

The presence of a myelin-like structure in contact with the external head plasmalemma, never described in this spermatozoon before, is thought to be the result of a secon- dary segregation of the duct cells between the ovotestis and the hermaphroditic duct, when the sperm attains maturation just prior to fertilization. A similar structure, named lamellar body, was described inside the neck of the sperm of the pulmonate Agriolimax reticulatus, but its function is not known (Bayne, '70).

The extra acrosomal layer between the plasmalemma and the acrosomal vesicle (in continuity with the lateral zone of the nuclear apical region) is occupied by a fibrillar dense core. It only appears during the final stage of sperm maturation, probably owing to the formation of a microfilamentous structure. It seems to protect the acrosome (Bac- cetti and Afzelius, '76).

Besides, having some similarities with other pulmonate species, the spermatozoon of S. algesirae does present some unique fea- tures. The arrangement of the sperm nucleus in an organized network of chromatin fibers suggests that to attain maturation it was not necessary to complete dehydration, contrary to what is described in several other gastro-


Fig. 9. LUS of the midpiece showing the vesicle of the mitochondrial derivative (*I, with a wavelength of - 5.5 pm, and coarse fibers (CF). X20,OOO.

Fig. 10. LUS of the midpiece in the zone of the mitochondrial derivative (*) demonstrates its arrangement with respect to mitochondrion. The midpiece is totally surrounded by a complex system of external double-membranous cape. The thinness of the central zone (**) is not favorable to the observation of the coarse fibers. Between the mitochondrial derivative and central zone of the coarse fibers a small light space is visible (arrowheads). ~90 ,000 .

Fig. 11. LUS of the last zone of the midpiece containing the mitochondrial complex without vesicles. The central zone is occupied by the longitudinal sections of the axonemal microtubules. The external double-membranous cape surrounding the spermatozoon and an internal small space (arrowheads) are visible. x 54,000.

Fig. 12. Detail of the TUS of the midpiece at the mitochondrial derivative (*I level. The nine single coarse fibers in the radial position are separated from the mitochondrial complex by a light space (arrowheads). This material, as well as that of Figure 13, was fixed by glutaraldehyde and tannic acid. X 100,000.

Fig. 13. Higher magnification of the boxed area in Figure 12-external zone of the mitochondrial derivative (*), Between the two membranes (arrowheads) a complex system of the spherical bodies -100 A in diameter are regularly distributed in two layers. x 160,000.

Fig. 14. TUS of the midpiece shown in Figure 11. Here the mitochondrial vesicle is not present. ~90 ,000 .


d Md

Fig. 22. Schematic drawing of the spermatozoon and of four different transverse sections at different levels.

pods (Buckland-Nicks, '73; Thompson, '73; Azevedo, '81; Giusti and Selmi, '82), including pulmonates (Starke, '71; Takaichi, '78; Reger and Fitzgerald, '82; Maxwell, '83).

The external double-membranous sheath surrounding the midpiece of S. algesirae spermatozoa (Fig. 22), seems to represent a characteristic structure previously observed in some pulmonate sperm but never fully described (Anderson and Personne, '69). In our material this sheath undergoes autolysis and disappears after fertilization (unpub- lished results). The most recent review of this subject is by Tompa ('84), who describes morphological differences among reproductive organs and the spermatozoa of pulmonate species.

The sperm of this species is similar to that described in other species of pulmonates (An- derson and Personne, '67; Anderson et al., '68; Bayne, '70; Thompson, '73; Bogitsh, '74; Takaichi, '78; Reger and Fitzgerald, '82;

Tompa, '84) and several species of other gas- tropods (Thompson, '73; Maxwell, '83). In the highest form of the Gastropoda, simultane- ous hermaphroditism is accompanied by a radical change in the fine structure of the spermatozoa (Thompson, '73). The mitochondrial derivative in this and other pulmonate species has a very complicated organization (Rousset-Galangau, '72; Thompson, '73; Bog- itsh, '74; Reger and Fitzgerald, '82; Maxwell, '83) containing several enzyme complexes (Anderson et al., '68; Anderson and Per- sonne, '70; Bogitsh, '74; Baccetti and Afzel- ius, '76) and a food reserve of ripe autosperm during fertilization (Anderson and Personne, '70; Bogitsh, '74).

As we have observed in S. algesirae, the midpiece in pulmonate species is very long and the mitochondria1 derivative represents about 95% of the total cell volume (Favard and Andre, '70). Most of this is occupied by globular proteinaceous material. These char-

Fig. 15. LUS at a sagittal plane of the transitional zone between midpiece and endpiece. The plasmalemma seems to penetrate the endpiece, becoming a dense complex annular structure (large arrowheads). The internal membrane of the cape is in continuity with the membrane that surrounds the axoneme (small arrowheads) (see Fig. 16). The anterior zone of the endpiece is occupied by the projection of the axoneme surrounded by a granular material (9 (as in Figs. 16-20). ~49,000.

Fig. 16. Detail of the transitional zone in box in Figure 15. ~75,000.

Figs. 17,18. Sequential LUS of the complex annular structure situated in the transitional zone shown in Figure 15. Fig. 17, ~54,000. Fig. 18, ~53,000.

Fig. 19. LUS of the transitional zone of the midpiece and endpiece. The longitudinal section of the axoneme (arrows) is present. (In TUS, see Fig. 20). ~38,700.

Fig. 20. TUS of the middle zone of the endpiece showing a common axoneme (arrows). ~56,600

Fig. 21. TUS of the two consecutive tapering zones of the endpiece ('#) without any microtubular arrangement. x 60,000.

dW


Fig. 23. Schematic drawing of the transitional zone of the spermatozoon.

acteristics, which have apparently not been observed in any other taxonomic group, cor- respond generally to that of sperm with internal fertilization (Favard and Andre, '70).

The most important difference in relation to other pulmonate spermatozoa is the complex annular tapering structure (=annulus) (Fig. 23). This structure, never observed in this group before, represents an ultrastructural feature whose function we have been unable to determine. The structure found in Littorina sperm, called a joint (Buckland- Nicks, '73), and in Fusitriton, called an annulus, seem to represent a similar structure; the origin of these structures is controversial

(Buckland-Nicks et al., '82). The results described in this paper suggest

that the spermatozoon of S. algesirae may serve as a taxonomic link between marine and terrestrial pulmonate species.

ACKNOWLEDGMENTS

This work was partially supported by Uni- versity of Oporto, under contract No. 66/84, and by Eng. A. Almeida Foundation. We thank Mr. J. Carvalheiro for reproduction of photographs.


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the fine structure of the spermatozoa of siphonaria algesirae (gastropoda, pulmonata)

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