Ultrastructure and silver-staining analysis of spermatogenesis in the sea urchin Paracentrotus lividus (Echinodermata, Echinoidea)

JOURNAL OF MORPHOLOGY 195:177-188 (1988)

Ultrastructure and Silver-Staining Analysis of Spermatogenesis in the Sea Urchin Paracentrotus lividus (Echinodermata, Echinoidea)

MARIO SOUSA AND CARLOS AZEVEDO Department o f Cell Bwbgy, Institute of Biomedical Sciences and Center of Experimental Morphology, University of Oporto, 4000 Porto, Portugal

ABSTRACT Spermatogenesis of the sea urchin Paracentrotus lividus has been studied at the ultrastructural level after conventional staining of thin sections and after en bloc silver staining. Cytoplasmic dense bodies are present in all steps of spermatogenesis except in late spermatids and spermatozoa. These bodies are closely associated with the development of centrioles and of the acrosomal vesicle during spermiogenesis. After it first appears, acrosomal vesicle is linked to the nuclear envelope by electron-dense material and subse- quently acquires a dense core. Later the acrosomal vesicle moves to the apical pole of the cell while maintaining its connection to the nucleus. Although chromatin was highly condensed in the head of spermatozoa, one or more nuclear vacuoles within the nucleus were found to contain uncondensed chromatin fibers. Silver nitrate stains several nuclear and cytoplasmic structures. In the nucleus it stains the nucleolus, specific regions at the periphery of the chromatin, the synaptonemal complexes, the nuclear basal fossa, and the nuclear vacuoles of spermatozoa. In the cytoplasm, silver stains the cytoplasmic dense bodies, the material that connects the acrosomal vesicle to the spermatid nucleus, the spermatozoan subacrosomal and periacrosomal materials, the intercellular bridges of spermatids, and the centrioles. Silver staining is abolished by pretreatment with pronase E, suggesting that silver staining is due to protein.

Sea urchin spermatogenesis has been studied ultrastructurally in two species, Arbacia and Strongylocentrotus (Longo and Ander- son, '69), but acrosomogenesis has not been well characterized. Cytoplasmic dense bodies of three types (goniosomes, amorphous dense bodies, and bipartite bodies) in the germ cells of both sexes of the sea urchin have been described by Houk and Hinegardner ('81). Silver nitrate has been used to demonstrate the nucleolar organizer region of chromosomes (Goodpasture and Bloom, '751, and histochemical tests have indicated that the staining reaction is due to acidic proteins (Schwarzacher et al., '78). Recently, the silver nitrate technique has also been shown to stain cytoplasmic structures of male germ cells observed at the ultrastructural level (Krimer and Esponda, '78; Azevedo et al., '85; Czaker, '85a,b).

In this ultrastructural study we describe the spermatogenesis of Paracentrotus lividus with emphasis on nuclear and cytoplasmic differentiation during spermiogenesis. De- velopment of the acrosome is followed in de- tail. We show that silver nitrate stains proteins and labels cytoplasmic dense bodies as well as the periphery of the acrosomal vesicle, the periphery of condensing chromosomes, and some components of the develop- ing flagellum. A possible relationship between the silver-labeled cytoplasmic dense bodies and the other stained cytoplasmic structures is discussed.

MATERIALS AND METHODS

Sea urchins, Paracentrotus lividus, were collected from the intertidal zone of the Por- tuguese Atlantic northern coast. For fine structural studies, small pieces of testis were

0 1988 ALAN R. LISS, INC.

178 M. SOUSA AND C. AZEVEDO

fixed with 2.5% glutaraldehyde in 0.2 M sodium cacodylate, pH 7.4, at 4°C for 2 hr, washed in the same buffer at 4°C for 2 hr, and postfixed with 1% OsO4 in sodium cacodylate buffer under the same conditions. All pieces were dehydrated in a graded ethanol series, transferred to propylene oxide, and embedded in Epon (Azevedo et al., '85). For cytochemical studies the silver-technique of Goodpasture and Bloom ('75) was modified as follows: After fixation and washing as above, the pieces of testis were postfixed in Carnoy's solution (acetic acidmethanol, I:3) for 12 min at room temperature, rapidly rehydrated in a descending ethanol series, and rinsed in distilled water for 30 min. They were then immersed in 70% AgN03 under a photoflood lamp (2,800 K" bulb) for 20 min, so that they were heated to 55"C, rinsed in distilled water, and incubated in ammoniacal silver-formalin solution for 6 min. After rinsing in distilled water, they were again dehydrated and embedded as above. Some pieces of testis, after fixation, were immersed in 0.5% pronase E (70,000 PUWg, Merck) aqueous solution at 40°C for 2 hr, washed in distilled water, and processed by the silver nitrate technique as described.

For fine structural observations, ultrathin sections were routinely double-stained with aqueous 3% uranyl acetate for 20 min and lead citrate for 15 min. Ultrathin sections of silver-stained blocks were observed both stained (10 min in uranyl acetate and 5 min in lead citrate) and unstained. Briefer staining periods did not alter the staining specific- ity but did improve contrast; hence, all micrographs from silver-stained material are from sections stained with these brief periods. Grids were observed in a JEOL I00 B electron microscope operated at 80 kV.

RESULTS Morphological observations

Spermatogonia contain a typical nucleus (Fig. l), and in the cytoplasm we have observed some areas containing several dense bodies (Fig. 2). In the spermatid, cytoplasmic dense bodies of this type were observed near centrioles (Fig. 3) and in association with the forming acrosomal vesicle. Dense bodies related to development of acrosomal vesicles appear connected to the nuclear envelope, and sometimes portions of dense material seem to be detached from them (Fig. 4). Once formed, the acrosomal vesicle is linked to the nuclear envelope by dense material and ac-

quires a dense core (Fig. 5). No more acroso- ma1 vesicle-related dense bodies were seen in spermatids subsequent to this phase. During the next step of spermiogenesis, the acroso- ma1 vesicle moves lateral to the nucleus and in this location is connected both to the nuclear envelope (Fig. 6) and to the cytoplasmic membrane (Fig. 7) by some bridges of electron-dense material. When the acrosomal vesicle reaches the apical region of the cell in its migration, the nuclear apex invagi- nates to create the subacrosomal fossa (Fig. 8). At this stage, a fibrous complex radiates between the proximal centriole and the outer nuclear membrane through the centriolar fossa, and the inner nuclear membrane around the fossa becomes coated with a band of dense material (Fig. 9). In a mature spermatozoon the acrosome is composed of the acrosomal vesicle plus associated perioacro- soma1 and subacrosomal materials (Fig. 10). Within the nucleus in the sperm head, there remains an electron-lucent nuclear vacuole, which has a thick chromatin strand in its center and thinner fibers radiating from this center (Fig. 11).

Cytochemical observations In spermatogonia, silver deposits were

found in three principal locations: on the nu-

Fig. 1. Nucleus and adjacent cytoplasm, separated by perinuclear cisterna, in a spermatogonium. Fibrillar @') and granular (G) components of the nucleolus are seen adjacent to a dense zone of heterochromatin. Ch, chromatin; Cy, cytoplasm. x 31,000.

Fig. 2. Ribosome-rich area (Ri) of cytoplasm in a spermatogonium, containing dense bodies (arrows). X98,300.

Fig. 3. Cytoplasmic dense bodies (arrows) near centriole (C) in a spermatid. GC, Golgi complex showing Golgi-derived vesicles (*I; pAV, proacrosomal vesicle; N, nucleus. x 38,000.

Fig. 4. Nucleus (N) and Golgi region of an early spermatid. The cytoplasmic dense body (*) near proacrosomal vesicle (pAV) and Golgi complex (GC) is connected to the nuclear envelope WE) by fibrillar material (arrows). Smaller portions of this dense body seem to be detached from it (arrowhead). ~96,000.

Fig. 5. Nucleus (N) and Golgi region of an early spermatid. Proacrosomal vesicle (pAV) at this stage has a dense core (*I, is linked to the nuclear envelope (NE) by some dense material (arrow), and is closely associated at its apical face with Golgi vesicles (arrowheads). GC, Golgi complex. X73,800.

Figures 1-5


Figures 6-11

SPERMATOGENESIS IN THE SEA URCHIN 181

Figs. 6, 7. Midspermatids. The acrosomal vesicle (AV) is connected to the nuclear envelope (NE) (Fig. 6) and to the plasmmalema (PI) (Fig. 7) by some dense bridges (arrows). The dense core of the acrosomal vesicle (AV) is linked to the vesicle membrane by thin strands (arrowheads). Figure 6, x 120,000; Figure 7, ~96 ,000 .

Fig. 8. Late spermatid. The acrosomal vesicle (AV) is observed at the apical pole of the cell. The subacrosomal (arrow) and periacrosomal (arrowheads) materials have begun to differentiate. The nucleus (N) is beginning to elongate but still has uncondensed chromatin in its lateral nuclear zones (*) Cy, cytoplasm. X 34,000.

Fig. 9. Midpiece and adjoining regions of the nucleus and tail piece in a spermatid. The nuclear dense coat at the centriolar fossa is seen at arrowheads. Dense material of the pericentriolar complex has become localized (arrows). NE, nuclear envelope; Ch, chromatin strands; PC, proximal centriole; DC, distal centriole; FC, fibrous complex; M. Mitochondria; F1, flagellum. ~48,100.

Fig. 10. Spermatozoon acrosome, showing acrosomal vesicle (AV), subacrosomal (arrow), and periacrosomal (arrowheads) materials. N, nucleus. x 36,000.

Fig. 11. Nuclear vacuole in head of spermatozoon. In its center there i s a thick chromatin strand (arrow) from which thinner fibers emanate (arrowheads) N, nucleus. X 141,800.


Figures 12-15


Fig. 12. Silver-stained nucleus and adjacent cytoplasm of a spermatogonium, showing localization of the stain. The fibrillar (F) component of the nucleolus stains more heavily than the granular component (GI. At the periphery of the sparsely stained regions of heterochromatin (Ch) there are several heavily argyrophilic patches (arrows). A silver-stained cytoplasmic dense body is seen at arrowhead. X36,OOO.

Fig. 13. Prophase of a primary spermatocyte at the pachytene stage, showing a synaptonemal complex. Sil- ver stains the inner faces (arrows) of paired chromosomes (Ch) and silver grains accumulate at a connection between the complex and the nuclear envelope (arrowhead). Argyrophilic patches are localized at the periphery of heterochromatin (double arrows). Cy, cytoplasm; CF, central filament. ~54 ,000 .

Fig. 14. Spermatids. Argyrophilic patches (arrows) at periphery of chromatin (Ch) are seen over those areas not yet condensed. Successive stages in condensation are seen in upper, lower, and right spermatids, respectively. M, mitochondrion; CF, stained centriolar fossa; Cy, cytoplasm. ~27,000.

Fig, 15. Silver-stained dense bodies (arrows) in spermatid cytoplasm. The dense cytoplasmic coat of intercellular bridges is stained (arrowheads). N, nucleus. x90,000.

Figures 16-19


cleolus, as argyrophilic patches scattered along the periphery of the heterochromatin, and on the cytoplasmic dense bodies Wig. 12). At the pachytene stage in primary spermatocytes, the inner face of each chromosome in synaptonemal complexes was also selectively stained (Fig. 13). As spermiogenesis pro- gresses, chromatin condenses, and argyrophilic granules at the chromatin periphery disappear (Figs. 14, 16, 17). Meanwhile, silver granules accumulate at the basal inden- tation of the nucleus (Figs. 16, 17). In the cytoplasm, silver stains the dense bodies (Fig. 15), the cytoplasmic dense coat of intercellular bridges (Figs. 15,21), midpiece structures (Figs. 16, 171, and the material between the acrosomal vesicle and the nucleus (Fig. 17). In the mature spermatozoon, silver stains the periacrosomal material and the lower zone of the subacrosomal region (Figs. 18.191, the nuclear vacuole (Fig. 181, and midpiece structures (Fig. 20). None of these structures are stained in spermatids or spermatozoa in pieces of testis treated with pronase and then with silver nitrate (Figs. 22-24).

DISCUSSION Morphology

In r! lividus, a prominent early feature of acrosomogenesis is that the acrosomal vesicle is precociously linked to the nucleus by some electron-dense material, which may be incorporated into or may already represent a precursor of the subacrosomal material of the differentiated acrosome. Precocious forma-

Fig. 16. Midspermatid. Silver grains are found at heterochromatin periphery (arrows), over the nuclear coat at the centriolar fossa (arrowheads), on the proximal (PC) and distal (DC) centrioles, and in the pericentriolar complex (double arrows). N, nucleus; FC, fibrous complex; FI, flagellum. ~37,500.

Fig, 17. Midspermatid. The acrosomal vesicle (AV) is connected to the nuclear envelope by argyrophilic material (arrowheads). The nuclear coat at the centriolar fossa (arrow) is heavily stained. FC, fibrous complex; PC, DC, proximal and distal centrioles. X 45,000.

Fig. 18. Mature spermatozoon. Silver stains the acrosome (Ac), nuclear vacuole (arrow) and midpiece (Mp). The axoneme (Ax) is lightly stained. N, nucleus. X24,OOO. Inset shows the silver-stained nuclear vacuole (arrow). ~36,000.

Fig. 19. Spermatozoon acrosome. Silver stains the periacrosomal material (arrowheads) and the lower zone of the subacrosomal fossa (arrow). N, nucleus; AV, acro- soma1 vesicle. X 96,000.

tion of subacrosomal materials has been re- ported in other echinoderms (Dan and Sirakami, '71; Atwood, '74; Yamashita and Iwata, '83; Buckland-Nicks et al., '841, in molluscs (Longo and Dornfeld, '67; Takaichi and Dan, '77), and in annelids (Potswald, '67; Eckelbarger, '84). Migration of the acrosomal vesicle while maintaining close association with the nucleus has been described in other echinoderms (Atwood, '74; Bickell et al., '80; Yamashita and Iwata, '831, but the vesicle may also migrate in contact with the cytoplasmic membrane as in some molluscs (Buckland-Nicks and Chia, '86) and annelids (Potswald, '67; Eckelbarger, '84) or without any attachment to cell structures as in some echinoderms (Dan and Sirakami, '71; Buck- land-Nicks et al., '84) and molluscs (Longo and Dornfeld, '67; Takaichi and Dan, '77).

In stages of spermatogenesis in l? lividus from spermatogonia to spermatids, cytoplasmic dense bodies were observed, and they seem to develop in close association with centrioles and acrosomal vesicle. Similar dense bodies have been described in germ cells of echinoids (Longo and Anderson, '69; Houk and Hinegardner, 'Sl), holothuroids (Atwood, '74), and ophiuroids (Yamashita and Iwata, '831, but their fate was not followed. The ori- gin of the cytoplasmic dense bodies is un- known, but several studies refer to them as nuages of electron-dense material (Houk and Hinegardner, '81; Rice, '81; Eckelbarger, '84). In Annelida, cytoplasmic dense bodies were observed near centrioles and intercellular bridges (Bertout, '76; Rice, '81; Eckelbarger, '84).

Sperm nuclear vacuoles are a characteris- tic of some species, but their structure has not been described in echinoderms. Mature sperm of I? lividus contain one or more nuclear vacuoles, and we have shown that they form at sites where chromatin fibers remain uncondensed. In mammals, nuclear vacuoles were shown to arise from randomly occur- ring defects during the process of chromatin condensation or from degeneration of the nu- cleoli (Ohtomo, '81).

Cytochemistry Electron microscopic Ag-staining has pre-

viously been used for demonstrating the argyrophilia of nucleolar components and their counterparts a t metaphase (Hernandez-Ver- dun et al., '78; Hernandez-Verdun and Der- enzini, '83). Histochemical tests on both mammalian and invertebrate cells have

Fig. 20. Spermatozoon midpiece. Silver stains the dense nuclear band that surrounds the centriolar fossa (arrowheads), the periphery of centriolar microtubules (small arrows), and the pericentriolar complex (large arrows). The material connecting the distal centriole (DC) to the nuclear envelope is formed by an initial branch (a), an intermediary knob (b), and peripheral strands (c). Silver stains the knob intensely. The axoneme (Ax) is lightly stained. N, nucleus; PC, proximal centriole. x90,000.

Fig. 21. Spermatid intercellular bridge, internally reinforced by silver-stained material (arrowheads). An argyrophilic cytoplasmic dense body (*) is seen within the bridge. A tangential section of the pericentriolar cornpIex in one cell is heavily stained (arrow). Cy, cytoplasm; N, nucleus. ~90,000.

Figs. 22-24. Pronase- and silver nitrate-treated cells, showing absence of silver staining.

Fig. 22. Spermatids, with condensed nucleus (N) and unstained intercellular bridges (arrowheads). x 24,000.

Fig. 23. Spermatozoon, showing acrosomal vesicle (AV), periacrosomal (arrowheads), and subacrosomal (arrow) regions. N, nucleus. X24,OOO.

Fig. 24. Spermatozoon midpiece and centriolar fossa (arrowheads), showing lack of silver staining. Material connecting centriole to nucleus (large arrow) and pericentriolar complex (small arrows) likewise is unstained. N, nucleus; DC, distal centriole; Ax, axoneme. ~96,000.


shown that acidic nonhistone proteins are responsible for Ag-staining (Goodpasture and Bloom, '75; Schwarzacher et al., '78; Ami- kura and Ihnuma, '84; Hubbell, '85). In so- matic mammalian cells an acidic, highly phosphorylated protein, C23, was isolated and was proven to be responsible for Ag-NOR staining (Lischwe et al., '81; Ochs and Busch, '84; Spector et al., '84). It has been suggested that argyrophilic proteins are associated with chromatin decondensation (Hernandez-Ver- dun et al., '82); hence, our demonstration that silver nitrate stains the periphery of hetero- chromatic regions is confirmatory of this view. As chromatin condenses during spermiogenesis in P. lividus, the peripheral label- ing disappears except in nuclear vacuoles where uncondensed chromatin persists. When chromatin condenses in mammals, argyrophilic proteins are degraded or other- wise modified (possibly dephosphorylated) (Ochs et al., '83). Similar modification may explain why fully condensed chromatin in the sea urchin sperm is no longer argyrophilic.

Recent ultrastructural studies have shown that silver solutions can selectively stain cer- tain nuclear regions, such as the chromosome core (Bertout, '86), and some cytoplasmic regions of spermatozoa, namely, the periacrosomal material and midpiece structures (Krimer and Esponda, '78; Azev- edo et al., '85; Czaker, '85a,b). The shared property of argyrophilia of the nucleolar and the other nuclear and cytoplasmic structures suggest similarity though not necessarily identity in the proteins associated with these structures.

In I? lividus, Ag-staining of cytoplasmic structures may actually be due to a common set of argyrophilic proteins. The morphological association between cytoplasmic dense bodies and other cytoplasmic structures in the acrosome and midpiece suggests that dense bodies may be primordial storage de- pots containing the argyrophilic proteins that will be needed for the formation of other argyrophilic organelles. To explain both nuclear and cytoplasmic Ag-staining, Czaker ('85b) has suggested that proteins associated with the cytoskeleton were responsible for the argyrophilia. Such proteins may account for the silver staining we have observed in the cytoplasm as well as in the synaptonemal complexes and at the centriolar fossa of the nucleus. The fact that silver staining of these structures is inhibited by pronase pretreatment indicates that they are protein in nature.

ACKNOWLEDGMENTS

This work was supported by the University of Oporto under contract No. 4/86, by the Eng. A. Almeida Foundation, and by "DGQA"-Lisbon.

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Ultrastructure and silver-staining analysis of spermatogenesis in the sea urchin Paracentrotus lividus (Echinodermata, Echinoidea)