THE ANATOMICAL RECORD 294:363–371 (2011)

Identification of N-Acetylgalactosaminein Carbohydrates of Xenopus

laevis TestisGALDER VALBUENA,1 EDURNE ALONSO,1 LUCIO DIAZ-FLORES, JR,2

JUAN FRANCISCO MADRID,3 AND FRANCISCO JOSE SAEZ1*1Department of Cell Biology and Histology, School of Medicine and Dentistry,

University of the Basque Country (UPV/EHU), Leioa, Spain2Department of Anatomy, Pathological Anatomy and Histology, School of Medicine,

University of La Laguna, La Laguna, Spain3Department of Cell Biology and Histology, School of Medicine,

University of Murcia, Espinardo, Spain

ABSTRACTIdentification of glycans in amphibian testis has shown the existence of

N-acetylgalactosamine (GalNAc)-containing carbohydrates. Labeling of thesperm acrosome with GalNAc-binding lectins has allowed the identificationof GalNAc-containing glycans in this organelle. Futhermore, this specificlabeling of the acrosome has allowed the study of acrosomal biogenesis bylectin histochemistry. However, the testis of Xenopus laevis has never beenanalyzed by lectin histochemistry to locate GalNAc-containing glycoconju-gates. The aim of this work was to elucidate the expression of GalNAc inglycoconjugates of Xenopus testis using five specific lectins. The resultsshowed that most of the lectins labeled the interstitium with variable inten-sity. However, labeling of the different spermatogenetic germ cell typesshowed different labeling patterns. Some lectins produced weak or veryweak staining in germ cells, for example, horse gram Dolichos biflorusagglutinin, which labeled most of the germ cell types, and lima bean Pha-seolus lunatus agglutinin, which weakly labeled only spermatogonia, butdid not stain other germ cells. By contrast, Maclura pomifera lectin (MPL)moderately labeled all germ cell types, except mature sperm. Labeling withother lectins was seen only at later stages, suggesting variations involvedin the spermatogenetic development. Thus, snail Helix pomatia agglutininlabeled spermatids, but neither spermatogonia nor spermatocytes, whilesoybean Glycine max agglutinin (SBA) labeled from preleptotene spermato-cytes to later stages. The periphery of the acrosome was labeled with MPLand SBA, but no specific labeling of the acrosomal content was seen withany lectin. Thus, the GalNAc-binding lectins that have been used as acroso-mal markers in some amphibians cannot be used in Xenopus testis, sug-gesting that acrosomal glycoconjugates in amphibians are species specific.Anat Rec, 294:363–371, 2011. VVC 2010 Wiley-Liss, Inc.

Keywords: lectin histochemistry; glycoconjugates; oligo-saccharides; spermatogenesis; acrosome

Grant sponsor: UPV/EHU; Grant number: 1/UPV00075.310-E-14847/2002, 1/UPV00077.310-E-15927/2004; Grant sponsor: Funda-cion Seneca (Comunidad Autonoma de la Region de Murcia); Grantnumber: 04542/GERM/06. GVwas a fellowship from theUPV/EHU.

*Correspondence to: Francisco Jose Saez, Departamento deBiologıa Celular e Histologıa, Facultad de Medicina y Odontolo-gıa, Universidad del Paıs Vasco/Euskal Herriko Unibertsitatea,

B� Sarriena s/n, E-48940 Leioa (Vizcaya), Spain. Fax:þ34946013266. E-mail: francisco.saez@ehu.es

Received 17 June 2010; Accepted 3 November 2010

DOI 10.1002/ar.21316Published online 16 December 2010 in Wiley Online Library(wileyonlinelibrary.com).

VVC 2010 WILEY-LISS, INC.

INTRODUCTION

Spermatogenesis is a complex process involved inmale gamete production, in genetic transmission, and inthe perpetuation of the species (Hess and de Franca,2008). Several strategies have been designed to under-stand the complex regulatory mechanisms involved inspermatogenesis (Sharpe, 1994; Zhao and Garbers,2002). Advances in molecular biology and genomics,transcriptomics, and proteomics have been applied tounderstand spermatogenesis (de Rooij and de Boer,2003; Rolland et al., 2008). The focus on proteomics isnot only based on protein synthesis but also on thestructural alterations undergone by the proteins (Aeber-sold and Mann, 2003).

One of the main post-translational modifications ofproteins is glycosylation. In general, glycoproteins, glyco-lipids, glycosaminoglycans, and other carbohydrate com-pounds are known as glycoconjugates (Gabius, 2000).Glycoproteins and other glycoconjugates may be involvedin many biological functions, including cell signaling(Etzler and Esko, 2009), embryonic development (Varkiet al., 2009), and diseases (Gheri et al., 2004; Nizet andEsko, 2009; Varki and Freeze, 2009). The importance ofglycoconjugates in fertilization is well established(Tanghe et al., 2004; Shur, 2008; Wassarman andLitscher, 2008), and it has been shown that spermsurface proteins recognize specific oocyte surface carbo-hydrates, including N-acetylgalactosamine (GalNAc)-con-taining glycans both in mammals and amphibians(Gougoulidis et al., 1999; Ueda et al., 2007). Mammaliansperm surface glycans, including GalNAc moieties,which are modified during maturation and capacitation,have also been studied (Retamal et al., 2000; Yudinet al., 2005). Recently, a role for some glycoconjugates inmammalian spermatogenesis has been stated (Takamiyaet al., 1998; Fujimoto et al., 2000; Muramatsu, 2002;Akama et al., 2002; Sandhoff et al., 2005).

As in mammals, amphibian spermatogenesis takesplace in seminiferous tubules. There the germ cells syn-chronously develop forming clusters, called cysts orfollicles, each one being enclosed by a capsule formed byfollicle (Sertoli) cells, which resemble fibroblasts (Lofts,1974). In recent studies, we have described that someamphibians show a strong expression of GalNAc-contain-ing glycoconjugates in the acrosome of the developingspermatids (Saez et al., 1999, 2004; Valbuena et al.,2008). However, Xenopus laevis is the amphibian modelmost extensively used in biological research, but noreport exists about the expression of GalNAc-containingglycans in the testis of this species. The aim of this workwas to elucidate for the first time the expression ofGalNAc in glycoconjugates of Xenopus testis by means oflectin histochemistry and to test the possible useof these lectins as an acrosomal marker tool for thisspecies.

MATERIALS AND METHODSMaterials

Adult Xenopus laevis were supplied by Harlan Inter-fauna Iberica (Sant Feliu de Codines, Barcelona, Spain).As control for deglycosylation pretreatments, adult malerats were supplied by the Animal Facility Service-SGIker of the University of the Basque Country (Leioa,

Vizcaya, Spain). Bovine serum albumin (BSA), GalNAc,and lectins from snail Helix pomatia (HPA) and Maclurapomifera (MPA/MPL) were supplied by Sigma-AldrichQuımica (Tres Cantos, Madrid, Spain). Lectins fromhorse gram Dolichos biflorus (DBA), lima bean Phaseo-lus lunatus (LBA), and soybean Glycine max (SBA) werefrom EY Laboratories (San Mateo, CA). Vectastain ABCKit was from Vector Laboratories (Peterborough, Eng-land). Recombinant peptide-N-glycosidase F (PNGase F)was supplied by Roche Diagnostics (San Cugat delValles, Barcelona, Spain).

Histochemical Procedures

Xenopus were reared in the Animal Facility Service-SGIker of the University of the Basque Country untilnecessary, then the testis were removed and fixed byimmersion in Bouin’s solution for 2 hr, embedded inparaffin wax, and 5-lm-thick sections were obtained. Forcontrol treatments, testes of male rats were processed bythe same procedure.

Histochemical methods were carried out using Gal-NAc-specific biotin-labeled lectins HPA, DBA, LBA andSBA, and horseradish peroxidase (HRP)-labeled lectinMPL. Specificity of each lectin is described in Table 1.

Histochemistry with biotinilated lectins was carried outas previously described (Valbuena et al., 2010). Briefly,sections were sequentially deparaffinized and rehydrated,immersed in 1% H2O2 in PBS for 30 min, washed in PBS,incubated in 1% BSA, and then incubated with the lectinat working dilution in Tris-buffered saline (TBS) in amoist chamber at room temperature for 1.5 hr. Afterwashing in PBS, the sections were incubated for 1 hr inAvidin Biotin complex (ABC) obtained from VectastainABC Kit, and finally developed with 0.25 mg/mL 3,30-dia-minobenzidine and 0.1% H2O2 and counterstained withhematoxylin. Working dilutions were 60 lg/mL for LBA,50 lg/mL for DBA and SBA, and 6 lg/mL for HPA.

Histochemistry with HRP-labeled MPL was carried outas previously described (Madrid et al., 1998; Saez et al.,1999), using MPL at 40 lg/mL working dilution. In sum-mary, the main difference between both the methods is theabsence of incubation with ABC for HRP-labeled lectins.

The following controls were used: the substitution ofthe lectin by the buffer alone, and the preincubationof the lectin with 0.2 M GalNAc to verify specificity oflabeling.

Deglycosylation Pretreatments

In addition to the procedures described above, thehistochemical techniques were carried out on other tis-sue sections after deglycosylation procedures, independ-ently performed, to remove O- or N-linked oligosaccharides,respectively: (1) chemical b-elimination (Ono et al., 1983)and (2) incubation with recombinant PNGase F.

b-Elimination was carried out following the techniquepreviously described (Saez et al., 2000a). The deglycosy-lation pretreatment was assayed for 5, 7, and 12 days aspreviously reported (Valbuena et al., 2010). As control totest, the complete removal of O-linked glycans by b-elim-ination, HPA staining of rat testis sections was carriedout. If the pretreatment has been successful, the rat tes-tis should not be labeled by this lectin (Martınez-Menar-guez et al., 1993; Valbuena et al., 2010).

364 VALBUENA ET AL.

To remove N-linked glycans, incubation with 40 U/mLrecombinant PNGase F in PBS at 37�C for 72 h was per-formed as previously described (Saez et al., 2000a; Gomez-Santos et al., 2007). As control for the complete N-glycanremoval, rat testis sections were stained with Galanthusnivalis agglutinin (GNA, EY Laboratories, 60 lg/mL work-ing dilution). Sections should be negative after incubationwith PNGase F (Martınez-Menarguez et al., 1993).

Analysis of Results

The staining intensity was always evaluated by threeindependent researchers and classified into five catego-ries: no labeling (0), very weak (1), weak (2), moderate(3), and strong (4).

RESULTS

In the present work, the GalNAc-containing glycocon-jugates of Xenopus laevis testis have been studied forthe first time by means of lectin histochemistry. The Gal-

NAc-binding lectin labeling in Xenopus laevis testis isshown in Table 2, and some representative results areshown in Figures 1–5.

HPA labeled Sertoli cells, spermatids, and sperm tails,but neither spermatogonia nor spermatocytes (Fig. 1a,b).The interstitium was strongly labeled, but the duct cellswere negative (Fig. 1a). Sections were unstained whennegative controls were performed for this and the otherlectins (Fig. 1c). After PNGase F pretreatment, thestaining in the sperm tails increased, while that in earlyspermatids and follicle cells decreased (Fig. 1d). Rattestis sections stained with GNA, which were negativeafter PNGase F incubation, were used as control for thecomplete N-glycan removal (Fig. 1e,f). b-Eliminationturned most of the testis negative, except sperm tails(Fig. 1g). Rat testis sections stained with HPA, whichwere negative after the b-elimination pretreatment,were used as control of the procedure (Fig. 1h,i).

DBA histochemistry produced a very weak labeling ofgerm cell types, while Sertoli and duct cells were nega-tive and the interstitium showed a strong labeling

TABLE 1. GalNAc binding lectins employed in this work

Lectin Abbreviation Binding specificity Reference

Snail (Helix pomatia)agglutinin

HPA Baker et al., 1983; Pilleret al., 1990; Wu andSugii, 1991; Spicer andSchulte, 1992; Sanchezet al., 2006

Horse gram (Dolichosbiflorus) agglutinin

DBA Baker et al., 1983; Pilleret al., 1990; Wu andSugii, 1991; Spicer andSchulte, 1992

Soybean (Glycine max)agglutinin

SBA Pereira and Kabat, 1974;Piller et al., 1990; Wuand Sugii, 1991; Spicerand Schulte, 1992

Osage orange tree(Maclura pomifera)lectin

MPA/MPL Sarkar et al., 1981; Younget al., 1991; Lee et al.,1998; Wu, 2005

Lima bean (Phaseoluslunatus) agglutinin

LBA Roberts et al., 1982; Basuand Appukuttan, 1983;Roberts and Goldstein,1984; Sikder et al., 1986;Wu and Sugii, 1991

GalNAc IN Xenopus laevis TESTIS 365

Fig. 1. HPA histochemistry of Xenopus testis (a, b, c, d, and g) andrat testis controls (e, f, h, and i). (a) HPA labeled the interstitium (in)but not the spermatic ducts (sd). Premeiotic germ cells, that is, pri-mary (not showed) and secondary (g2) spermatogonia, preleptotene(plc), and primary (c1) spermatocytes were not labeled. The cyst wallthat consisted of the cytoplasm of the follicle (Sertoli) cells was posi-tive (white arrows). (b) Early spermatids (es) were weakly labeled. (c)Lectin negative control, showing a Xenopus testis section incubatedwith the buffer alone. (d) After PNGase F pretreatment, the labeling

pattern was similar, except for the fact that spermatozoa tails were la-beled (sp). (e) To test incubation with PNGase F, rat testis was labeledwith GNA (agglutinin of Galanthus nivalis), showing labeling of mostcells. (f) After PNGase F incubation, rat testis was negative for GNA.(g) b-elimination turned the entire testis negative, but labeling wasseen at the spermatozoa tails. (h) To test removal of O-glycans, rattestis was labeled with HPA lectin. (i) After b-elimination for 7 days, rattestis was negative to HPA. g1, primary spermatogonia; ls, late sper-matids. Scale bars: 20 lm.

TABLE 2. Evaluation of GalNAc-binding lectin labelling of Xenopus laevis testis

HPA DBA SBA MPL LBA

W.pt. PNG b-Elim W.pt. PNG b-Elim W.pt. PNG b-Elim W.pt. PNG b-Elim W.pt. PNG b-Elim

g1 0 0 0 1 0 0 0 0 0 3 3 2 0–2 0 0g2 0 0 0 1 0 0 0 0 0 3 3 2 2 0 0plc 0 0 0 1 0 0 3 2 3 3 3 2 0 0 0c1 0 0 0 1 0 0 3 2 3 3 3 2 0 0 0es 2 1 0 1 0 0 3 2 3 3 3 2 0 0 0ms 1 1 0 1 0 0 3 2 3 3 3 2 0 0 0ls 1 1 0 1 0 0 3 2 3 2 2 2 0 0 0sp 1a 3a 2a 0 0 1a 3 2 3 0 0 2 0 0 0fc 2 1 0 0 0 0 0 0 0 3 3 2 0 0 0in 2–4 2 0 2–4 2–4 0–2 1 0 0 3 3 2 1 0 0sd 0 0 0 0 0 0 1 0 1 1 1 0–1 2 0 0

The staining intensity was evaluated and classified into five categories: no labelling (0), very weak (1), weak (2), moderate(3), and strong (4). When there was a variation in staining of the same structure, the range of staining is indicated withthe minimum and maximum values separated by a dash.b-elim, b-elimination prepreteatment; c1, primary spermatocytes; es, early spermatids; fc, follicle (Sertoli) cells, g1: primaryspermatogonia, g2: secondary spermatogonia, in: interstitium, ls: late spermatids, ms: midstage spermatids; plc, prelepto-tene spermatocytes; PNG, incubation with PNGase F; sd, spermatic ducts; sp, spermatozoa; w.pt., without pretreatment.aOnly sperm tails.

366 VALBUENA ET AL.

(Fig. 2a,b). PNGase F pretreatment did not modify thestaining pattern of the interstitium but annulled label-ing of germ cells (Fig. 2c). The b-elimination pretreat-ment turned most of the structures negative, but spermtails were weakly labeled (Fig. 2d).

SBA showed a moderate staining of germ cells (frompreleptotene spermatocytes to spermatozoa), with astronger labeling at the cell periphery and the peripheryof the acrosome of early spermatids. The interstitiumand spermatic ducts were very weakly stained (Fig. 3a–d). After PNGase F incubation, the interstitium becamenegative, and the staining of the germ cells was weaker,

without a distinguishable stronger staining of the cellperiphery (Fig. 3e). The b-elimination procedure did notmodify the labeling pattern of germ cells by SBA, exceptin the interstitium, which became negative (Fig. 3f).Finally, SBA did not label the Sertoli cells.

MPL moderately labeled the interstitium and all thegerm cells in the seminiferous tubules but not the tailsof elongated spermatids and spermatozoa. The peripheryof the acrosome of spermatids was labeled. Follicle (Ser-toli) cells were moderately positive (Fig. 4a,b) and theduct cells were weakly labeled (data not shown). AfterPNGase F, the only modification was that the periphery

Fig. 2. DBA labeling of Xenopus testis. (a) Low-magnification viewof the Xenopus testis labeled with DBA lectin. The germ cells wereweakly labeled, the interstitium (in) was labeled moderately to stronglybut spermatic ducts were negative (sd). (b) Spermatozoa (sp) were notlabeled, while early spermatids (es) were weakly labeled. The Sertolicells were negative (white arrows). (c) Labeling of the interstitium (in)

remained after PNGase F pretreatment; while no labeling of the germcells was observed. (d) After b-elimination, most of the labeling disap-peared but sperm tails were weakly labeled (sp, see inset). g2, sec-ondary spermatogonia; plc, preleptotene spermatocytes; c1, primaryspermatocytes; ls, late spermatids. Scale bars: 20 lm.

Fig. 3. SBA histochemical labeling of Xenopus testis. (a) A lowmaginification view showing that only the primary (g1) and secondary(g2) spermatogonia were not labeled. The interstitium showed a weakstaining (in). (b) Labeling of preleptotene spermatocytes (plc), early(es), and late (ls) spermatids. The Sertoli cells were negative (whitearrow). (c) Labeling of primary spermatocytes was stronger at the cell

periphery (c1). (d) The periphery of the acrosome at early spermatidswas labeled (arrows, see inset). (e) The PNGase F pretreatmentshowed a weaker staining. (f) b-Elimination turned the interstitium neg-ative (in) but most of the labeling at the germ cells remained. ms, mid-stage spermatids. Scale bars: 20 lm, inset 10 lm.

GalNAc IN Xenopus laevis TESTIS 367

of germ cells became negative; the staining in other loca-tions remained unaltered (Fig. 4c). On the contrary, af-ter b-elimination staining was generally weaker, whilespermatozoa became positive (Fig. 4d).

LBA only labeled primary and secondary spermatogo-nia and the interstitium. Other germ cell types and Ser-toli cells were negative (Fig. 5a–c). Duct cells wereweakly stained. The PNGase and b-elimination pretreat-ments turned the entire testis negative (Fig. 5d,e).

DISCUSSION

Five GalNAc binding lectins have been employed forthe first time to identify GalNAc moieties in glycoconju-

gates of Xenopus laevis testis. Although all the lectinsbind to GalNAc, they show different and complex specif-icities, which are related to several poorly understoodfactors (Table 1). The affinity for each lectin usuallydepends on the sugar linked to GalNAc. HPA and DBAhave a higher affinity for the Forssman antigen than forthe blood group A antigen; by contrast, LBA specificallyrecognizes A antigen. In addition to GalNAc, some lec-tins also have affinity for Gal, as happens for SBA andMPL and, with a minor affinity, HPA, which also recog-nizes the T antigen (see Table 1 for more details andreferences). Thus, the peculiar characteristics of eachlectin must be carefully considered to analyze the stain-ing pattern observed in Xenopus laevis testis.

Fig. 4. MPL histochemistry of Xenopus laevis testis. (a) The follicle(Sertoli) cells (white arrows), interstitium (in), and premeiotic spermato-genetic cells, that is, primary (g1) and secondary (not showed) sper-matogonia and preleptotene (plc) and primary (c1) spermatocytes,were labeled. (b) Postmieotic cells were also positive. In early sperma-tids (es), the periphery of the acrosome was labeled (arrows, see

inset). (c) After incubation with PNGase F, which removes N-linked gly-cans, labeling pattern was not modified. (d) b-Elimination pretreat-ment, which removes O-linked oligosaccharides, reduced the intensityof labeling in Xenopus testis. G2, secondary spermatogonia; ls, latespermatids; sp, spermatozoa. Scale bars: 20 lm, inset 5 lm.

Fig. 5. LBA histochemistry of Xenopus testis. (a) LBA lectin only la-beled the interstitium (in), and the primary (g1) and secondary (g2)spermatogonia. The Sertoli cells were negative (white arrows). (b) Pri-mary spermatocytes (c1), midstage spermatids (ms), and spermatozoa

(sp) showed no labeling. (c) Late spermatids (ls) were negative. (d) Af-ter PNGase F pretreatment the entire testis was not labeled. (e) The b-elimination procedure turned all the testis negative. Scale bars: 20lm.

368 VALBUENA ET AL.

The staining pattern of HPA and DBA, two lectinswith a high affinity for Forssman antigen and A glyco-topes, was similar. HPA labeled interstitium, but notafter b-elimination, suggesting that it is identifying For-ssman and/or A glycotopes on O-linked glycans. HPAlabeling in germ cells also turned negative after b-elimi-nation, suggesting that the carbohydrates are on O-gly-cans. Sperm tails were weakly labeled by HPA, butlabeling increased after both deglycosylation pre-treat-ments. Two possibilities are inferred: GalNAc containingoligosaccharides are either in both N- and O-linked oli-gosaccharides or are in glycolipds.

DBA labeled the interstitium and, in a similar way toHPA-labeling, removal of labeling after b-elimination sug-gests that the lectin is localizing Forssman and/or A gly-cotopes on O-linked glycans. By contrast, most DBAlabeling in germ cells disappeared after each deglycosyla-tion procedure, suggesting that the presence of some ofthese carbohydrates on N-linked oligosaccharides cannotbe discarded. Sperm tails were weakly labeled by DBAonly after the b-elimination procedure. This could beexplained by the unmasking of GalNAc moieties in N-gly-cans, which are initially inaccessible to the lectin, andthe removal of O-glycans allows the labeling by DBA. Theunmasking of carbohydrates by deglycosylation techni-ques has been described previously (Alonso et al., 2001;Saez et al., 2001; Gomez-Santos et al., 2007).

As indicated above, SBA labels both GalNAc and Galmoieties (Pereira and Kabat, 1974; Wu and Sugii, 1991).Labeling of spermatocytes and spermatids could beattributed to GalNAc or Gal containing glycans, mostlyin N-linked oligosaccharides, because PNGase F incuba-tion produced a weaker staining. On the other hand,most of the MPL staining in the interstitium and germi-nal cysts could be due to GalNAc or Gal in O-glycans,because labeling diminished, but did not disappear, afterb-elimination pretreatment.

LBA weakly labeled only the interstitium and sperma-togonia; the staining disappeared after both deglycosyla-tion pretreatments, suggesting that some GalNAcmoieties are in both N- and O-oligosaccharides. It can behypothesized that after elimination of some glycans byany pretreatments, the remaining carbohydrates areinsufficient to be labeled by the lectin.

Previous works have shown the importance of glyco-conjugates in premeiotic cells (Ertl and Wrobel, 1992;Koshimizu et al., 1993; Maylie-Pfenninger, 1994; Sudaet al., 1998), but only a few glycans with a known rolehave been identified, including a glycolipid that regu-lates differentiation and cell interactions in mouse sper-matocytes (Fujimoto et al., 2000), and an N-linkedoligosaccharide which regulates the spermatogonia–Ser-toli cell relationship (Akama et al., 2002). In the presentwork, spermatogonia were labeled by MPL, DBA, andLBA, while spermatocytes were positive for MPL, DBA,and SBA. In previous works using other amphibians, thepresence of GalNAc in spermatogonia has been shown(Saez et al., 2000b, 2004).

Glycoconjugates in postmiotic cells have beenreported, with an emphasis on acrosomal-related carbo-hydrates (Martınez-Menarguez et al., 1992, 1993; Labateand Desantis, 1995; Saez et al., 1999, 2004; Valbuenaet al., 2008). In the amphibian Pleurodeles waltl, HPAhas been used as an acrosomal marker, which hasallowed the analysis of the biosynthesis of the acrosome

(Valbuena et al., 2008). The presence of GalNAc in acro-somal carbohydrates is not exclusive to this amphibianspecies; it has been reported in mammals and insects(Yamamoto, 1982; Lee and Damjanov, 1984; Arya andVanha-Perttula, 1984, 1985, 1986; Malmi and Soder-strom, 1987; Malmi et al., 1987, 1990; Kurohmaru et al.,1991, 1995, 1996; Ertl and Wrobel, 1992; Craveiro andBao, 1995). However, no lectin showed a significantlabeling of the Xenopus acrosome, only MPL and SBAlabeled the periphery of the acrosome. Hence, althoughthe presence of GalNAc in the Xenopus acrosome is notrejected, our data suggest that there is not a specific seg-regation of GalNAc-containing glycoconjugates, as occursin other amphibians.

Finally, the present work shows that the carbohydratecomposition of Xenopus germ cells is modified duringspermatogenesis. This can be inferred from the labelingwith HPA and SBA, the first one only labeling sperma-tids and the second one labeling spermatids as well asspermatocytes. These results show that some GalNAc-containing glycoconjugates appear during spermatoge-netic development. Modification of glycoconjugates ingerm cells of other species has been previously reported(Yamamoto, 1982; Soderstrom et al., 1984; Arya andVanha-Perttula, 1984, 1986; Ballesta et al., 1991; Joneset al., 1992; Kurohmaru et al., 1995, 1996; Saez et al.,1999, 2000b; 2004; Desantis et al., 2010).

In conclusion, GalNAc-containing glycoconjugates inboth N- and O-linked glycans have been reported for thefirst time by lectin histochemistry in the testis of Xeno-pus laevis. Contrary to the acrosomal labeling reportedin other amphibian species, none of the five GalNAc-binding lectins can be used as an acrosomal marker inXenopus. This suggests that the acrosomal glycoconju-gate content in amphibians is species specific. Inaddition, some glycans detected in germ cells of Xenopusare present in later spermatogenetic stages, but not inearlier stages, suggesting that glycoconjugates areinvolved in spermatogenetic maturation and spermdevelopment. On the other hand, the scarce number oflectin histochemical studies on amphibian speciesreported in the literature makes it impossible to estab-lish more conclusions.

ACKNOWLEDGMENTS

Mrs. M. Portuondo and Mrs. C. Otamendi contributedto sample preparation. The authors thank Mrs. M. J.Aldasoro for her support in the office work.

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