Histochemistry (1992) 98:365-371

Histochemistry © Springer-Verlag 1992

Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera, Culicidae) S6nia N. Bfio 1,2 Wanderley de Souza 1

1 Laborat6rio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundfio, 21.949-590 Rio de Janeiro, Brasil 2 Departamento de Gen6tica e Morfologia, Instituto de Biologia, Universidade de Brasilia, Brasilia, Brasil Accepted: 14 September 1992

Abstract. The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on L R White-embedded cells. The nuclear c o m p a r t m e n t was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifoIia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzymecontaining structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by P N A and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. C a r b o h y d r a t e residues in the nuclear c o m p a r t m e n t m a y be involved with the process of chromatin condensation. In the acrosomal region these residues m a y play a role in the process of spermoocyte interaction.

Introduction

Glycoconjugates are widely distributed in different cellular compartments. Biological processes such as intercellular interactions, intracellular transport and enzymatic reactions involve the participation of carbohydrate-rich molecules (reviewed in Sharon 1984). Lectins have been largely used for the identification, characterization and localization of carbohydrate-containing molecules (reviewed in Nicolson 1974; Roth 1983; Alroy et al. 1984). In the last few years, numerous studies have suggested the existence of nuclear glycoconjugates in a variety of

Correspondence to : W. de Souza

cell types (reviewed in H a r t etal. 1988; Hubert etal. 1989). Nuclear glycoproteins were biochemically analysed (Stein et al. 1975; Ferraro et al. 1988; H a r t et al. 1989; Kinoshita et al. 1988) and cytologically localized in different nuclear structures (Roth 1983; Seve etal. 1984; Kan and Pinto da Silva 1986; Vannier-Santos et al. 1991). Some studies analysed the distribution of carbohydrates on the spermatozoal surface, which m a y be involved in the processes of molecular recognition and gamete interaction (Nicolson and Yanagimachi 1972; Aketa 1975; Perotti and Riva 1988; Ahluwalia et al. 1990). However, there is a scarcity of studies on the localization of spermatozoal intracellular carbohydrates. The composition and distribution of glycoproteins on the acrosome of bovine and rat spermatozoa have been demonstrated using lectin histochemistry (Ertl and Wrobel 1992; Martinez-Menfirguez et al. 1992). The aim of the present study is to determine the localization of glycoconjugates in the male gamete of the mosquito Culex quinquefasciatus employing direct and indirect lectingold techniques on L R White-embedded cells. Materials and methods

The insects utilized were male and female adults of the mosquito C. quinquefasciatus obtained from a colony maintained in the Department of Entomology of the [nstituto Oswaldo Cruz, Rio de Janeiro, Brasil. The lectins used (Table 1) were obtained from E-Y Laboratories (San Mateo, Calif., USA) or Calbiochem (San Diego, Calif., USA). Horseradish peroxidase (type II) and fetuin were obtained from Sigma Chemical Company. The various glycoproteins were labelled with colloidal gold particles (8 10 nm or 15 nm in diameter), according to Roth (1983). Testis, seminal vesicles and spermatheca were dissected and fixed for 3 h at 4° C in a mixture containing 4% paraformaldehyde, 1% glutaraldehyde, 0.2% picric acid, 5% sucrose and 5 mM calcium chloride in 0.1 M cacodylate buffer, pH 7.2. After washing the specimens with several changes of the same buffer for 2 h, free aldehyde groups were quenched with 50 mM ammonium chloride in buffer, for 1 h at 4° C, followed by block-staining in 2% uranyl acetate in 15% acetone for 2 h at 4° C (Berryman and Rodewald 1990). Specimens were dehydrated in 3(~90% acetone. In-

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Table 1. Lectins used as histochemical probes

Table 2. Summary of the labelling pattern" of ultrathin sections of LR White-embedded spermatids and spermatozoa

Source of lectin

Abbreviation

Sugar specificity

Concanavalia ensiformis Arachys hypogaea Griffonia simplicifolia IB4 Rieinus eommunis Triticum vulgaris

Con A PNA GS-IB4 RCA WGA

Griffonia simplicifolia II

GS II

Wisteria yToribunda

WFA

Ulex europaeus I Limax flavus

UEA-I LFA

D-mannose D-galactose D-galactose D-galactose N-acetyl-Dglucosamine N-acetyl-Dglucosamine N-acetyl-Dgalactosamine L-fucose Neuraminic acid

Lectins b

filtration was performed in LR White resin for 48 h at 4° C, and specimens were embedded in the same resin. Samples were polymerized in vacuum for 72 h at 37° C. Ultrathin sections, collected on 400-mesh nickel grids, were treated for 60 min at room temperature with saturated sodium metaperiodate in order to release resin mesh and unmask some binding sites of sugar residues sufficiently to enable localization. Then the sections were pre-incubated for 60 min at room temperature in phosphate-buffered saline, containing 1% bovine serum albumin (PBS-BSA) and 0.01% Tween 20, and subsequently incubated for 60 min at room temperature in the presence of different gold-labelled lectins in PBS-BSA. For wheat germ agglutinin (WGA), Ulex europaeus agglutinin-I (UEA-I), Wisteria floribunda agglutinin (WFA), peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4), and G. simplicifolia II (GS II), incubation was carried out at pH 7.3. For Ricinus communis agglutinin (RCA), incubation was carried out at pH 8.0 (Bendayan et al. 1990). For localization of concanavalin A (Con A) binding sites, the sections were first incubated with 100 gg/ml Con A and thereafter with goldlabelled horseradish peroxidase dissolved in PBS-BSA, pH 7.3, at a dilution of 1 : 5. For detection of neuraminic acid residues, the sections were initially incubated in the presence of 100 gg/ml Limax flavus agglutinin (LFA) and then with fetuin-gold complex in PBSBSA, pH 6.0, at a dilution of 1 : 5. After incubation, the grids were washed with PBS and distilled water, stained with uranyl acetate and lead citrate, and observed in a Zeiss 900 transmission electron microscope. Controls consisted of the addition to the incubation medium of 200-300 mM of the corresponding monosaccharides (Table 1). Some grids were also incubated in the presence of gold-labelled peroxidase or fetuin without previous lectin incubation. The intensity of labelling was evaluated by analysis of a large number of micrographs, and was considered as one of three situations : high intensity labelling, represented by + + (more than 1000 gold particles/pm2); moderate intensity labelling, represented by + (10-1000 gold particles/pm2); and low intensity labelling, represented by _+ (less than 10 gold particles/gm2).

Results U l t r a t h i n sections o f L R W h i t e - e m b e d d e d s p e r m a t i d s a n d s p e r m a t o z o a were u s e d for the l o c a l i z a t i o n o f b i n d ing sites specific for D - m a n n o s e ( C o n A), D - g a l a c t o s e (RCA, GS-IB4 and PNA), N-acetyl-D-glucosamine (GS II a n d W G A ) , N - a c e t y l - D - g a l a c t o s a m i n e ( W F A ) , L-fucose ( U E A - I ) a n d n e u r a m i n i c a c i d ( L F A ) . A l l b i n d i n g r e a c t i o n s c o u l d be i n h i b i t e d b y a d d i t i o n o f the a p p r o p r i a t e s u g a r to the i n c u b a t i o n m e d i u m . I n c u b a t i o n in the p r e s e n c e o f g o l d - l a b e l l e d fetuin o r h o r s e r a d i s h p e r o x -

Con A PNA GS-IB4 RCA WGA GS II WFA UEA I LFA

Spermatids

Spermatozoa

N

N

_+ + + + _+ _ _+ _+ _+

+ + + + + + + + +

+ + + + + + + + +

AAR

PAR

+ --

+ + + --

AAR, Anterior acrosomal region; PAR, proximal acrosomal region; N, nucleus + + , High intensity labelling (more than 1000 gold particles/ ~tm2); + , moderate intensity labelling (10-1000 gold particles/ gm2); _+, low intensity labelling (less than 10 gold particles/gm2); - , no labelling b The lectin derivations are given in Table 1

idase d i d n o t label the cells. D i f f e r e n t labelling p a t t e r n s were o b s e r v e d for the lectins tested. T h e nucleus was the s t r u c t u r e m o s t f r e q u e n t l y a n d i n t e n s e l y labelled. T h e a c r o s o m e was o n l y labelled b y s o m e lectins, while g o l d particles were n o t seen o n the flagellum. T h e results are s u m m a r i z e d in Table 2. E l e c t r o n - d e n s e a r e a s were o b s e r v e d at s o m e r e g i o n s o f the c h r o m a t i n o f the nucleus o f early s p e r m a t i d s (Figs. 1, 4, 7 a n d 12) while a high degree o f c o n d e n s a t i o n o f n u c l e a r c h r o m a t i n was seen in the nucleus o f the sperm a t o z o a (Figs. 2 3, 5-6, 8-11 a n d 13-16). I n the a c r o s o m e two d i s t i n c t r e g i o n s c o u l d be i d e n t i f i e d : the p r o x i m a l r e g i o n l o c a t e d close to the nucleus a n d c o n t a i n i n g a m o r p h o u s m a t e r i a l , a n d the a n t e r i o r p o r t i o n , w h i c h h a d a g r a n u l a r a p p e a r a n c e (Figs. 6, 9 a n d 13-14). T h i n sections o f e a r l y s p e r m a t i d s i n c u b a t e d in the presence of Con A-horseradish peroxidase-colloidal gold, r e v e a l e d a light labelling in the dense c h r o m a t i n regions o f the nucleus (Fig. 1). H o w e v e r , an intense labelling o f the nucleus was o b s e r v e d in the last stages o f the p r o c e s s o f s p e r m m a t u r a t i o n (Fig. 2). T h e nuclei o f s p e r m a t o z o a were labelled in u l t r a t h i n sections, w h i c h were i n c u b a t e d in the presence o f R C A

Figs. 1-15. Spermatids and spermatozoa obtained from testis and seminal vesicles of Culex quinquefasciatus Fig. 1. Spermatid nucleus (N) lightly labelled by concanavalin A (Con A)-peroxidase-gold. Note that the particles are found in the dense chromatin region (arrowheads). Bar, 0.25 pm

Fig. 2. Sperm nucleus (N) exhibiting Con A-peroxidase-gold labelling. Bar, 0.25 Ixm Fig. 3. Griffonia simplieifolia IB4 (GS-IB4) lectin-labelled sperm nucleus (N). The labelling is uniform over the entire nuclear section. Bar, 0.25 pm Fig. 4. Spermatid nucleus (N) labelling by GS-IB4 lectin. Gold particles are mainly found in the dense chromatin region (arrowheads). Bar, 0.5 pm

!ii ~ii!iiI~~

il)i i!ii~:!::;!:

i:!i!i !i

Fig. 5. Longitudinal section of the proximal region of the flagellum of spermatozoa incubated with GS-IB4 lectin, in which gold particles are distributed over the nucleus (N). The axoneme (Ax) is completely devoid of labelling. Bar, 0.25 g m

i

"i :'if!i!

ii !

!

Fig. 6. Peanut agglutinin (PNA)-labelled s p e r m a t o z o o n exhibiting gold particles over the whole nucleus (N). The labelling is also observed over the anterior (asterisk) a n d proximal (arrow) acrosomal regions. Some particles can be seen in the plasma m e m b r a n e (arrowhead). A, A c r o s o m e ; bar, 0.25 gm

Fig. 7. Wheat germ agglutinin (WGA)-labelled spermatid nucleus (N). Note that the particles are found in the dense chromatin region (arrowheads). Ax, Axoneme; Ca, centriole adjunct; bar, 0.25 gm Fig. 8. Transverse section of the head region of spermatozoa incubated with WGA. The nucleus (N) is intensely labelled. A, Acrosome; bar, 0.25 gm

Fig. 9. Longitudinal section of the head region of spermatozoon incubated with G. simplicifolia II (GS II) lectin. The nucleus (N) is intensely labelled. Moderate labelling is observed over the acrosome proximal region (arrow). A, Acrosome; bar, 0.25 gm Fig. 10. Transverse section of spermatozoa incubated with GS II lectin. The labelling is uniform over the whole nucleus (N). Gold particles are observed in the plasma membrane (arrowheads). Bar, 0.25 pm

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Fig. 11. Spermatozoon nucleus (N) labelled by Wisteriafloribunda agglutinin (WFA). Bar, 0.25 gm Fig. 12. Spermatid nucleus (N) lightly labelled by Ulex europaeus agglutinin I (UEA I). Note that the particles are found in the dense chromatin region (arrowheads). Bar, 0.25 Ixm

Figs. 13, 14. Sperm nucleus (N) intensely labelled by UEA I. Gold particles are also observed in the proximal acrosomal region (arrow). Some particles can be seen in the plasma membrane (arrowheads). A, Acrosome; bar, 0.1 lain (Fig. 13) and 0.25 gm (Fig. 14) Fig. 15. Sperm nucleus (N) intensely labelled by LirnaxJTavus agglutinin (LFA). F, Flagellum; bar, 0.25 gm Fig. 16. Sperm nucleus (N) obtained from spermatheca, intensely labelled by PNA. A, Acrosome; bar, 0.1 gm

(not shown), GS-IB4 (Figs. 3 and 5) and PNA (Fig. 6). In all cases, a diffuse labelling was found over the dense chromatin. The nucleus of the early spermatids showed a moderate affinity for GS-IB4 (Fig. 4) and for PNA (not shown). Again, labelling preferentially occurred in the dense chromatin region. The anterior and proximal acrosomal regions were labelled with PNA, a lectin that recognizes D-galactose residues (Fig. 6). The condensed chromatin regions of the nucleus of early spermatids always showed light labelling by W G A (Fig. 7). In the sperm nucleus this labelling pattern was

more intense and observed thoughout the whole of the chromatin (Fig. 8). Similar observations were made in sections labelled with GS II, which like W G A binds to N-acetylglucosamine residues (Figs. 9 and 10). This lectin also labelled the acrosomal content of the proximal region (Fig. 9). The sperm nucleus was also labelled with W F A (Fig. 11). The lectin UEA-I, which binds to L-fucose residues, labelled the dense chromatin region of the spermatid nucleus (Fig. 12) and intensely labelled the whole nucleus of the spermatozoa (Figs. 13 and 14). The proximal re-

370 gion of the acrosome was also labelled by this lectin (Figs. 13 and 14). The lectin LFA, which binds to neuraminic acid, labelled the condensed chromatin of the spermatozoa nucleus (Fig. 15). For all lectins tested, the pattern of labelling of the spermatozoa that were stored in the spermatheca was basically the same as the labelling observed in spermatozoa localized in the testis and seminal vesicles (Fig. 16). In relation to the spermatozoal surface, very light labelling was observed when ultrathin sections were incubated in the presence of PNA (Fig. 6), GS II (Fig. 10) and UEA-I (Figs. 13 and 14).

Discussion

In the present work we have probed C. quinquefasciatus spermatids and spermatozoa with gold-labelled lectins of different sugar specificities. Most binding sites were found in the nuclear compartment. However, distinct regions of the acrosome were labelled by some lectins. Recent biochemical and cytochemical studies have shown the presence of sugar residues in intracellular compartments, mainly in the nucleus, associated with the dense chromatin (Stein et al. 1975; Kan and Pinto da Silva 1986; Londono and Bendayan 1987; VannierSantos et al. 1991). Nuclear glycoproteins such as H M G high mobility groups (Weisbrod et al. 1980), and also glycosaminoglycans and glycosylated non-histone proteins present in the nucleus of eukaryotic cells (Stein et al. 1975; Rizzo and Bustin 1977; Furukawa and Terayama 1979) might be the sites labelled by the lectins. These nuclear glycoproteins are composed of oligosaccaride side-chains containing glucose, mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine and fucose residues (Hart et al. 1989). The role of nuclear glycoproteins remains unclear, but may be to modulate the physicochemical environment of the nucleoplasm, and/or participate directly in localized molecular interactions at specific sites of the genome (Kan and Pinto da Silva 1986). However, the data reported show that within the nucleoplasm, sugar residues are mainly associated with proteins located in euchromatin regions, in domains where transcription and replication take place (Fakan and Nobis 1987; Hubert et al. 1989). Our present observations show that all the lectins tested with specificities for various carbohydrate residues labelled the nucleus of spermatids and spermatozoa. In the early spermatids, nuclear labelling is less intense and preferentially located in regions of dense chromatin. However with the process of chromatin condensation, labelling of the nuclear compartment gradually increases so that intense labelling is observed in the spermatozoa. This suggests the involvement of sugar-containing macromolecules in the process of chromatin condensation. Previous studies on the process of chromatin condensation during spermatid differentation have shown that substitution of histones for protamine takes place (McMaster-Kayer and Kaye 1976; Loir and Lanneau 1984; Martinage et al. 1985; Mello 1987; Quagio-Grassiotto and Dolder 1988). Thereafter, it is possible that the protamines are glycoproteins and that the

carbohydrate residues are involved in the high degree of chromatin condensation in the spermatozoa. Previous studies using histones isolated from the macronucleus of the protozoan Tetrahyn~,na therrnopila, have demonstrated fucose and mannose residues based upon their specific binding to UEA-I and to Con A, respectively (Levy-Wilson 1983). In Leishmania rnexicana amazonensis it was shown that the nucleus is the compartment most frequently labelled by lectins with specificities for various sugar residues (Vannier-Santos et al. 1991). Similar observations were made in three members of the genus Crithidia, which present bacterium-like endosymbionts in their cytoplasm (Motta MCM et al., to be published). The acrosome is a large secretory vesicle localized in the anterior head region of the spermatozoon and carries a variety of hydrolytic enzymes, stored in the form of proenzymes. The acrosomal enzymes are responsible for the digestion of the oocyte envelopes in an event that leads to spermatozoon penetration allowing fertilization to occur (reviewed in Yanagimachi 1988). In previous studies we have shown that the acrosome of C. quinquefasciatus spermatozoon represents two regions (Bfio SN, De Souza W, to be published). Our present observations using lectins also show that the acrosome is not a uniform compartment in terms of the distribution of sugar residues. D-galactose residues were observed in the anterior and proximal acrosomal regions. However UEA-I, a lectin that recognizes fucose residues and GS II, a lectin that recognizes N-acetylglucosamine residues, labelled the proximal region only. There are no previous studies on the distribution of carbohydrate residues in the acrosomal content of insect spermatozoa. Our present cytochemical observations suggest the presence of glycosylated macromolecules, occupying the proximal acrosoma! region principally, which may play a key role in sperm-oocyte recognition and fertilization. Boar and bull spermatozoa show in the postacrosomal region a prominent UEA staining (Ahluwalia et al. 1990). It has been shown, using lectin horseradish peroxidase conjugates, that some lectins label the acrosomal membrane of cells in various stage of the process of bovine spermiogenesis. However, shortly prior to spermiation no labelling is observed (Ertl and Wrobel 1992). In addition using lectin histochemistry and enzymic and chemical deglycosylation, it has been shown that glycoproteins of rat acrosome contain both N- and O-linked oligosaccharides (Martinez-Menarquez et al. 1992). Recent studies have shown that carbohydrate binding sites of acrosin, a trypsin-like protease, seem to be involved in the complex events that occur during sperm-oocyte interactions (Friess etal. 1987; Topfer-Peterson et al. 1990; Gallo et al. 1991). A lectin-like affinity of acrosin to fucose may also be involved in this process (Tesarik et al. 1990). The lectin-labelling pattern of spermatozoa stored in the spermatheca was the same as that observed in spermatozoa obtained in testis and seminal vesicles, suggesting that the nature and the distribution of sugar residues in the nucleus and the acrosomal matrix do not change during the sperm capacitation process. It is possible that sugar residues located in the head region of the C. quin-

371

quefasciatus s p e r m a t o z o o n m a y be i n v o l v e d in two distinct events. I n the n u c l e u s the c a r b o h y d r a t e - c o n t a i n i n g m a c r o m o l e c u l e s m a y be i n v o l v e d in the process of chrom a t i n c o n d e n s a t i o n , p r o t e c t i n g the s p e r m a t o z o a l gen o m e . I n the a c r o s o m a l region, however, the sugar residues m a y f u n c t i o n as a n a d d i t i o n a l c o n t r o l in the process of s p e r m - o o c y t e i n t e r a c t i o n . Acknowledgements. We wish to thank Dr. R.L. de Oliveira for supplying the mosquitoes and Mr. A. Lisboa for assistance in preparing the micrographs. This work has been supported by Financladora de Estudos e Projetos (FINEP), Conselho Naclonal de Desenvolvimento Cientifico e Tecnol6gico (CNPq) and Coordenaqao de Aperfeigoamento de Pessoal de Nivel Superior (CAPES).

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