Identification of N-Acetylgalactosamine in Carbohydrates of Xenopus laevis Testis.код для вставкиСкачать
THE ANATOMICAL RECORD 294:363–371 (2011) Identification of N-Acetylgalactosamine in Carbohydrates of Xenopus laevis Testis GALDER VALBUENA,1 EDURNE ALONSO,1 LUCIO DÍAZ-FLORES, JR,2 JUAN FRANCISCO MADRID,3 AND FRANCISCO JOSÉ SÁEZ1* 1 Department of Cell Biology and Histology, School of Medicine and Dentistry, University of the Basque Country (UPV/EHU), Leioa, Spain 2 Department of Anatomy, Pathological Anatomy and Histology, School of Medicine, University of La Laguna, La Laguna, Spain 3 Department of Cell Biology and Histology, School of Medicine, University of Murcia, Espinardo, Spain ABSTRACT Identiﬁcation of glycans in amphibian testis has shown the existence of N-acetylgalactosamine (GalNAc)-containing carbohydrates. Labeling of the sperm acrosome with GalNAc-binding lectins has allowed the identiﬁcation of GalNAc-containing glycans in this organelle. Futhermore, this speciﬁc labeling of the acrosome has allowed the study of acrosomal biogenesis by lectin histochemistry. However, the testis of Xenopus laevis has never been analyzed by lectin histochemistry to locate GalNAc-containing glycoconjugates. The aim of this work was to elucidate the expression of GalNAc in glycoconjugates of Xenopus testis using ﬁve speciﬁc lectins. The results showed that most of the lectins labeled the interstitium with variable intensity. However, labeling of the different spermatogenetic germ cell types showed different labeling patterns. Some lectins produced weak or very weak staining in germ cells, for example, horse gram Dolichos biﬂorus agglutinin, which labeled most of the germ cell types, and lima bean Phaseolus lunatus agglutinin, which weakly labeled only spermatogonia, but did not stain other germ cells. By contrast, Maclura pomifera lectin (MPL) moderately labeled all germ cell types, except mature sperm. Labeling with other lectins was seen only at later stages, suggesting variations involved in the spermatogenetic development. Thus, snail Helix pomatia agglutinin labeled spermatids, but neither spermatogonia nor spermatocytes, while soybean Glycine max agglutinin (SBA) labeled from preleptotene spermatocytes to later stages. The periphery of the acrosome was labeled with MPL and SBA, but no speciﬁc labeling of the acrosomal content was seen with any lectin. Thus, the GalNAc-binding lectins that have been used as acrosomal markers in some amphibians cannot be used in Xenopus testis, suggesting that acrosomal glycoconjugates in amphibians are species speciﬁc. C 2010 Wiley-Liss, Inc. Anat Rec, 294:363–371, 2011. V Key words: lectin histochemistry; glycoconjugates; saccharides; spermatogenesis; acrosome Grant sponsor: UPV/EHU; Grant number: 1/UPV00075.310-E14847/2002, 1/UPV00077.310-E-15927/2004; Grant sponsor: Fundación Séneca (Comunidad Autónoma de la Región de Murcia); Grant number: 04542/GERM/06. GV was a fellowship from the UPV/EHU. *Correspondence to: Francisco José Sáez, Departamento de Biologı́a Celular e Histologı́a, Facultad de Medicina y Odontologı́a, Universidad del Paı́s Vasco/Euskal Herriko Unibertsitatea, C 2010 WILEY-LISS, INC. V oligo- B Sarriena s/n, E-48940 Leioa (Vizcaya), Spain. Fax: þ34946013266. E-mail: firstname.lastname@example.org Received 17 June 2010; Accepted 3 November 2010 DOI 10.1002/ar.21316 Published online 16 December 2010 in Wiley Online Library (wileyonlinelibrary.com). 364 VALBUENA ET AL. INTRODUCTION Spermatogenesis is a complex process involved in male gamete production, in genetic transmission, and in the perpetuation of the species (Hess and de Franca, 2008). Several strategies have been designed to understand the complex regulatory mechanisms involved in spermatogenesis (Sharpe, 1994; Zhao and Garbers, 2002). Advances in molecular biology and genomics, transcriptomics, and proteomics have been applied to understand spermatogenesis (de Rooij and de Boer, 2003; Rolland et al., 2008). The focus on proteomics is not only based on protein synthesis but also on the structural alterations undergone by the proteins (Aebersold and Mann, 2003). One of the main post-translational modiﬁcations of proteins is glycosylation. In general, glycoproteins, glycolipids, glycosaminoglycans, and other carbohydrate compounds are known as glycoconjugates (Gabius, 2000). Glycoproteins and other glycoconjugates may be involved in many biological functions, including cell signaling (Etzler and Esko, 2009), embryonic development (Varki et al., 2009), and diseases (Gheri et al., 2004; Nizet and Esko, 2009; Varki and Freeze, 2009). The importance of glycoconjugates in fertilization is well established (Tanghe et al., 2004; Shur, 2008; Wassarman and Litscher, 2008), and it has been shown that sperm surface proteins recognize speciﬁc oocyte surface carbohydrates, including N-acetylgalactosamine (GalNAc)-containing glycans both in mammals and amphibians (Gougoulidis et al., 1999; Ueda et al., 2007). Mammalian sperm surface glycans, including GalNAc moieties, which are modiﬁed during maturation and capacitation, have also been studied (Retamal et al., 2000; Yudin et al., 2005). Recently, a role for some glycoconjugates in mammalian spermatogenesis has been stated (Takamiya et al., 1998; Fujimoto et al., 2000; Muramatsu, 2002; Akama et al., 2002; Sandhoff et al., 2005). As in mammals, amphibian spermatogenesis takes place in seminiferous tubules. There the germ cells synchronously develop forming clusters, called cysts or follicles, each one being enclosed by a capsule formed by follicle (Sertoli) cells, which resemble ﬁbroblasts (Lofts, 1974). In recent studies, we have described that some amphibians show a strong expression of GalNAc-containing glycoconjugates in the acrosome of the developing spermatids (Sáez et al., 1999, 2004; Valbuena et al., 2008). However, Xenopus laevis is the amphibian model most extensively used in biological research, but no report exists about the expression of GalNAc-containing glycans in the testis of this species. The aim of this work was to elucidate for the ﬁrst time the expression of GalNAc in glycoconjugates of Xenopus testis by means of lectin histochemistry and to test the possible use of these lectins as an acrosomal marker tool for this species. MATERIALS AND METHODS Materials Adult Xenopus laevis were supplied by Harlan Interfauna Ibérica (Sant Feliu de Codines, Barcelona, Spain). As control for deglycosylation pretreatments, adult male rats were supplied by the Animal Facility ServiceSGIker of the University of the Basque Country (Leioa, Vizcaya, Spain). Bovine serum albumin (BSA), GalNAc, and lectins from snail Helix pomatia (HPA) and Maclura pomifera (MPA/MPL) were supplied by Sigma-Aldrich Quı́mica (Tres Cantos, Madrid, Spain). Lectins from horse gram Dolichos biﬂorus (DBA), lima bean Phaseolus lunatus (LBA), and soybean Glycine max (SBA) were from EY Laboratories (San Mateo, CA). Vectastain ABC Kit was from Vector Laboratories (Peterborough, England). Recombinant peptide-N-glycosidase F (PNGase F) was supplied by Roche Diagnostics (San Cugat del Vallés, Barcelona, Spain). Histochemical Procedures Xenopus were reared in the Animal Facility ServiceSGIker of the University of the Basque Country until necessary, then the testis were removed and ﬁxed by immersion in Bouin’s solution for 2 hr, embedded in parafﬁn wax, and 5-lm-thick sections were obtained. For control treatments, testes of male rats were processed by the same procedure. Histochemical methods were carried out using GalNAc-speciﬁc biotin-labeled lectins HPA, DBA, LBA and SBA, and horseradish peroxidase (HRP)-labeled lectin MPL. Speciﬁcity of each lectin is described in Table 1. Histochemistry with biotinilated lectins was carried out as 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 lectin at working dilution in Tris-buffered saline (TBS) in a moist chamber at room temperature for 1.5 hr. After washing in PBS, the sections were incubated for 1 hr in Avidin Biotin complex (ABC) obtained from Vectastain ABC Kit, and ﬁnally developed with 0.25 mg/mL 3,30 -diaminobenzidine and 0.1% H2O2 and counterstained with hematoxylin. 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 out as previously described (Madrid et al., 1998; Sáez et al., 1999), using MPL at 40 lg/mL working dilution. In summary, the main difference between both the methods is the absence of incubation with ABC for HRP-labeled lectins. The following controls were used: the substitution of the lectin by the buffer alone, and the preincubation of the lectin with 0.2 M GalNAc to verify speciﬁcity of labeling. Deglycosylation Pretreatments In addition to the procedures described above, the histochemical techniques were carried out on other tissue sections after deglycosylation procedures, independently 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 technique previously described (Sáez et al., 2000a). The deglycosylation pretreatment was assayed for 5, 7, and 12 days as previously reported (Valbuena et al., 2010). As control to test, the complete removal of O-linked glycans by b-elimination, HPA staining of rat testis sections was carried out. If the pretreatment has been successful, the rat testis should not be labeled by this lectin (Martı́nez-Menárguez et al., 1993; Valbuena et al., 2010). 365 GalNAc IN Xenopus laevis TESTIS TABLE 1. GalNAc binding lectins employed in this work Lectin Abbreviation Binding speciﬁcity Reference Snail (Helix pomatia) agglutinin HPA Baker et al., 1983; Piller et al., 1990; Wu and Sugii, 1991; Spicer and Schulte, 1992; Sánchez et al., 2006 Horse gram (Dolichos biﬂorus) agglutinin DBA Baker et al., 1983; Piller et al., 1990; Wu and Sugii, 1991; Spicer and Schulte, 1992 Soybean (Glycine max) agglutinin SBA Pereira and Kabat, 1974; Piller et al., 1990; Wu and Sugii, 1991; Spicer and Schulte, 1992 Osage orange tree (Maclura pomifera) lectin MPA/MPL Sarkar et al., 1981; Young et al., 1991; Lee et al., 1998; Wu, 2005 Lima bean (Phaseolus lunatus) agglutinin LBA Roberts et al., 1982; Basu and Appukuttan, 1983; Roberts and Goldstein, 1984; Sikder et al., 1986; Wu and Sugii, 1991 To remove N-linked glycans, incubation with 40 U/mL recombinant PNGase F in PBS at 37 C for 72 h was performed as previously described (Sáez et al., 2000a; GómezSantos et al., 2007). As control for the complete N-glycan removal, rat testis sections were stained with Galanthus nivalis agglutinin (GNA, EY Laboratories, 60 lg/mL working dilution). Sections should be negative after incubation with PNGase F (Martı́nez-Menárguez et al., 1993). Analysis of Results The staining intensity was always evaluated by three independent researchers and classiﬁed into ﬁve categories: no labeling (0), very weak (1), weak (2), moderate (3), and strong (4). RESULTS In the present work, the GalNAc-containing glycoconjugates of Xenopus laevis testis have been studied for the ﬁrst time by means of lectin histochemistry. The Gal- NAc-binding lectin labeling in Xenopus laevis testis is shown in Table 2, and some representative results are shown 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 cells were negative (Fig. 1a). Sections were unstained when negative controls were performed for this and the other lectins (Fig. 1c). After PNGase F pretreatment, the staining in the sperm tails increased, while that in early spermatids and follicle cells decreased (Fig. 1d). Rat testis sections stained with GNA, which were negative after PNGase F incubation, were used as control for the complete N-glycan removal (Fig. 1e,f). b-Elimination turned most of the testis negative, except sperm tails (Fig. 1g). Rat testis sections stained with HPA, which were negative after the b-elimination pretreatment, were used as control of the procedure (Fig. 1h,i). DBA histochemistry produced a very weak labeling of germ cell types, while Sertoli and duct cells were negative and the interstitium showed a strong labeling 366 VALBUENA ET AL. TABLE 2. Evaluation of GalNAc-binding lectin labelling of Xenopus laevis testis HPA g1 g2 plc c1 es ms ls sp fc in sd 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 0 0 0 0 2 1 1 1a 2 2–4 0 0 0 0 0 1 1 1 3a 1 2 0 0 0 0 0 0 0 0 2a 0 0 0 1 1 1 1 1 1 1 0 0 2–4 0 0 0 0 0 0 0 0 0 0 2–4 0 0 0 0 0 0 0 0 1a 0 0–2 0 0 0 3 3 3 3 3 3 0 1 1 0 0 2 2 2 2 2 2 0 0 0 0 0 3 3 3 3 3 3 0 0 1 3 3 3 3 3 3 2 0 3 3 1 3 3 3 3 3 3 2 0 3 3 1 2 2 2 2 2 2 2 2 2 2 0–1 0–2 2 0 0 0 0 0 0 0 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 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 with the minimum and maximum values separated by a dash. b-elim, b-elimination prepreteatment; c1, primary spermatocytes; es, early spermatids; fc, follicle (Sertoli) cells, g1: primary spermatogonia, g2: secondary spermatogonia, in: interstitium, ls: late spermatids, ms: midstage spermatids; plc, preleptotene spermatocytes; PNG, incubation with PNGase F; sd, spermatic ducts; sp, spermatozoa; w.pt., without pretreatment. a Only sperm tails. Fig. 1. HPA histochemistry of Xenopus testis (a, b, c, d, and g) and rat testis controls (e, f, h, and i). (a) HPA labeled the interstitium (in) but not the spermatic ducts (sd). Premeiotic germ cells, that is, primary (not showed) and secondary (g2) spermatogonia, preleptotene (plc), and primary (c1) spermatocytes were not labeled. The cyst wall that consisted of the cytoplasm of the follicle (Sertoli) cells was positive (white arrows). (b) Early spermatids (es) were weakly labeled. (c) Lectin negative control, showing a Xenopus testis section incubated with the buffer alone. (d) After PNGase F pretreatment, the labeling pattern was similar, except for the fact that spermatozoa tails were labeled (sp). (e) To test incubation with PNGase F, rat testis was labeled with GNA (agglutinin of Galanthus nivalis), showing labeling of most cells. (f) After PNGase F incubation, rat testis was negative for GNA. (g) b-elimination turned the entire testis negative, but labeling was seen at the spermatozoa tails. (h) To test removal of O-glycans, rat testis was labeled with HPA lectin. (i) After b-elimination for 7 days, rat testis was negative to HPA. g1, primary spermatogonia; ls, late spermatids. Scale bars: 20 lm. GalNAc IN Xenopus laevis TESTIS 367 Fig. 2. DBA labeling of Xenopus testis. (a) Low-magniﬁcation view of the Xenopus testis labeled with DBA lectin. The germ cells were weakly labeled, the interstitium (in) was labeled moderately to strongly but spermatic ducts were negative (sd). (b) Spermatozoa (sp) were not labeled, while early spermatids (es) were weakly labeled. The Sertoli cells were negative (white arrows). (c) Labeling of the interstitium (in) remained after PNGase F pretreatment; while no labeling of the germ cells was observed. (d) After b-elimination, most of the labeling disappeared but sperm tails were weakly labeled (sp, see inset). g2, secondary spermatogonia; plc, preleptotene spermatocytes; c1, primary spermatocytes; ls, late spermatids. Scale bars: 20 lm. Fig. 3. SBA histochemical labeling of Xenopus testis. (a) A low maginiﬁcation view showing that only the primary (g1) and secondary (g2) spermatogonia were not labeled. The interstitium showed a weak staining (in). (b) Labeling of preleptotene spermatocytes (plc), early (es), and late (ls) spermatids. The Sertoli cells were negative (white arrow). (c) Labeling of primary spermatocytes was stronger at the cell periphery (c1). (d) The periphery of the acrosome at early spermatids was labeled (arrows, see inset). (e) The PNGase F pretreatment showed a weaker staining. (f) b-Elimination turned the interstitium negative (in) but most of the labeling at the germ cells remained. ms, midstage spermatids. Scale bars: 20 lm, inset 10 lm. (Fig. 2a,b). PNGase F pretreatment did not modify the staining pattern of the interstitium but annulled labeling of germ cells (Fig. 2c). The b-elimination pretreatment turned most of the structures negative, but sperm tails were weakly labeled (Fig. 2d). SBA showed a moderate staining of germ cells (from preleptotene spermatocytes to spermatozoa), with a stronger labeling at the cell periphery and the periphery of the acrosome of early spermatids. The interstitium and spermatic ducts were very weakly stained (Fig. 3a– d). After PNGase F incubation, the interstitium became negative, and the staining of the germ cells was weaker, without a distinguishable stronger staining of the cell periphery (Fig. 3e). The b-elimination procedure did not modify the labeling pattern of germ cells by SBA, except in the interstitium, which became negative (Fig. 3f). Finally, SBA did not label the Sertoli cells. MPL moderately labeled the interstitium and all the germ cells in the seminiferous tubules but not the tails of elongated spermatids and spermatozoa. The periphery of the acrosome of spermatids was labeled. Follicle (Sertoli) cells were moderately positive (Fig. 4a,b) and the duct cells were weakly labeled (data not shown). After PNGase F, the only modiﬁcation was that the periphery 368 VALBUENA ET AL. Fig. 4. MPL histochemistry of Xenopus laevis testis. (a) The follicle (Sertoli) cells (white arrows), interstitium (in), and premeiotic spermatogenetic cells, that is, primary (g1) and secondary (not showed) spermatogonia and preleptotene (plc) and primary (c1) spermatocytes, were labeled. (b) Postmieotic cells were also positive. In early spermatids (es), the periphery of the acrosome was labeled (arrows, see inset). (c) After incubation with PNGase F, which removes N-linked glycans, labeling pattern was not modiﬁed. (d) b-Elimination pretreatment, which removes O-linked oligosaccharides, reduced the intensity of labeling in Xenopus testis. G2, secondary spermatogonia; ls, late spermatids; sp, spermatozoa. Scale bars: 20 lm, inset 5 lm. Fig. 5. LBA histochemistry of Xenopus testis. (a) LBA lectin only labeled the interstitium (in), and the primary (g1) and secondary (g2) spermatogonia. The Sertoli cells were negative (white arrows). (b) Primary spermatocytes (c1), midstage spermatids (ms), and spermatozoa (sp) showed no labeling. (c) Late spermatids (ls) were negative. (d) After PNGase F pretreatment the entire testis was not labeled. (e) The belimination procedure turned all the testis negative. Scale bars: 20 lm. of germ cells became negative; the staining in other locations remained unaltered (Fig. 4c). On the contrary, after b-elimination staining was generally weaker, while spermatozoa became positive (Fig. 4d). LBA only labeled primary and secondary spermatogonia and the interstitium. Other germ cell types and Sertoli cells were negative (Fig. 5a–c). Duct cells were weakly stained. The PNGase and b-elimination pretreatments turned the entire testis negative (Fig. 5d,e). gates of Xenopus laevis testis. Although all the lectins bind to GalNAc, they show different and complex specificities, which are related to several poorly understood factors (Table 1). The afﬁnity for each lectin usually depends on the sugar linked to GalNAc. HPA and DBA have a higher afﬁnity for the Forssman antigen than for the blood group A antigen; by contrast, LBA speciﬁcally recognizes A antigen. In addition to GalNAc, some lectins also have afﬁnity for Gal, as happens for SBA and MPL and, with a minor afﬁnity, HPA, which also recognizes the T antigen (see Table 1 for more details and references). Thus, the peculiar characteristics of each lectin must be carefully considered to analyze the staining pattern observed in Xenopus laevis testis. DISCUSSION Five GalNAc binding lectins have been employed for the ﬁrst time to identify GalNAc moieties in glycoconju- 369 GalNAc IN Xenopus laevis TESTIS The staining pattern of HPA and DBA, two lectins with a high afﬁnity for Forssman antigen and A glycotopes, was similar. HPA labeled interstitium, but not after b-elimination, suggesting that it is identifying Forssman and/or A glycotopes on O-linked glycans. HPA labeling in germ cells also turned negative after b-elimination, suggesting that the carbohydrates are on O-glycans. Sperm tails were weakly labeled by HPA, but labeling increased after both deglycosylation pre-treatments. Two possibilities are inferred: GalNAc containing oligosaccharides are either in both N- and O-linked oligosaccharides or are in glycolipds. DBA labeled the interstitium and, in a similar way to HPA-labeling, removal of labeling after b-elimination suggests that the lectin is localizing Forssman and/or A glycotopes on O-linked glycans. By contrast, most DBA labeling in germ cells disappeared after each deglycosylation procedure, suggesting that the presence of some of these carbohydrates on N-linked oligosaccharides cannot be discarded. Sperm tails were weakly labeled by DBA only after the b-elimination procedure. This could be explained by the unmasking of GalNAc moieties in N-glycans, which are initially inaccessible to the lectin, and the removal of O-glycans allows the labeling by DBA. The unmasking of carbohydrates by deglycosylation techniques has been described previously (Alonso et al., 2001; Sáez et al., 2001; Gómez-Santos et al., 2007). As indicated above, SBA labels both GalNAc and Gal moieties (Pereira and Kabat, 1974; Wu and Sugii, 1991). Labeling of spermatocytes and spermatids could be attributed to GalNAc or Gal containing glycans, mostly in N-linked oligosaccharides, because PNGase F incubation produced a weaker staining. On the other hand, most of the MPL staining in the interstitium and germinal cysts could be due to GalNAc or Gal in O-glycans, because labeling diminished, but did not disappear, after b-elimination pretreatment. LBA weakly labeled only the interstitium and spermatogonia; the staining disappeared after both deglycosylation pretreatments, suggesting that some GalNAc moieties are in both N- and O-oligosaccharides. It can be hypothesized that after elimination of some glycans by any pretreatments, the remaining carbohydrates are insufﬁcient to be labeled by the lectin. Previous works have shown the importance of glycoconjugates in premeiotic cells (Ertl and Wrobel, 1992; Koshimizu et al., 1993; Maylie-Pfenninger, 1994; Suda et al., 1998), but only a few glycans with a known role have been identiﬁed, including a glycolipid that regulates differentiation and cell interactions in mouse spermatocytes (Fujimoto et al., 2000), and an N-linked oligosaccharide which regulates the spermatogonia–Sertoli cell relationship (Akama et al., 2002). In the present work, spermatogonia were labeled by MPL, DBA, and LBA, while spermatocytes were positive for MPL, DBA, and SBA. In previous works using other amphibians, the presence of GalNAc in spermatogonia has been shown (Sáez et al., 2000b, 2004). Glycoconjugates in postmiotic cells have been reported, with an emphasis on acrosomal-related carbohydrates (Martı́nez-Menárguez et al., 1992, 1993; Labate and Desantis, 1995; Sáez et al., 1999, 2004; Valbuena et al., 2008). In the amphibian Pleurodeles waltl, HPA has been used as an acrosomal marker, which has allowed the analysis of the biosynthesis of the acrosome (Valbuena et al., 2008). The presence of GalNAc in acrosomal carbohydrates is not exclusive to this amphibian species; it has been reported in mammals and insects (Yamamoto, 1982; Lee and Damjanov, 1984; Arya and Vanha-Perttula, 1984, 1985, 1986; Malmi and Soderstrom, 1987; Malmi et al., 1987, 1990; Kurohmaru et al., 1991, 1995, 1996; Ertl and Wrobel, 1992; Craveiro and Bao, 1995). However, no lectin showed a signiﬁcant labeling of the Xenopus acrosome, only MPL and SBA labeled the periphery of the acrosome. Hence, although the presence of GalNAc in the Xenopus acrosome is not rejected, our data suggest that there is not a speciﬁc segregation of GalNAc-containing glycoconjugates, as occurs in other amphibians. Finally, the present work shows that the carbohydrate composition of Xenopus germ cells is modiﬁed during spermatogenesis. This can be inferred from the labeling with HPA and SBA, the ﬁrst one only labeling spermatids and the second one labeling spermatids as well as spermatocytes. These results show that some GalNAccontaining glycoconjugates appear during spermatogenetic development. Modiﬁcation of glycoconjugates in germ cells of other species has been previously reported (Yamamoto, 1982; Soderstrom et al., 1984; Arya and Vanha-Perttula, 1984, 1986; Ballesta et al., 1991; Jones et al., 1992; Kurohmaru et al., 1995, 1996; Sáez et al., 1999, 2000b; 2004; Desantis et al., 2010). In conclusion, GalNAc-containing glycoconjugates in both N- and O-linked glycans have been reported for the ﬁrst time by lectin histochemistry in the testis of Xenopus laevis. Contrary to the acrosomal labeling reported in other amphibian species, none of the ﬁve GalNAcbinding lectins can be used as an acrosomal marker in Xenopus. This suggests that the acrosomal glycoconjugate content in amphibians is species speciﬁc. In addition, some glycans detected in germ cells of Xenopus are present in later spermatogenetic stages, but not in earlier stages, suggesting that glycoconjugates are involved in spermatogenetic maturation and sperm development. 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