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Identification of N-Acetylgalactosamine in Carbohydrates of Xenopus laevis Testis.

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THE ANATOMICAL RECORD 294:363–371 (2011)
Identification of N-Acetylgalactosamine
in Carbohydrates of Xenopus
laevis Testis
Department of Cell Biology and Histology, School of Medicine and Dentistry,
University of the Basque Country (UPV/EHU), Leioa, Spain
Department of Anatomy, Pathological Anatomy and Histology, School of Medicine,
University of La Laguna, La Laguna, Spain
Department of Cell Biology and Histology, School of Medicine,
University of Murcia, Espinardo, Spain
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,
B Sarriena s/n, E-48940 Leioa (Vizcaya), Spain. Fax:
þ34946013266. E-mail:
Received 17 June 2010; Accepted 3 November 2010
DOI 10.1002/ar.21316
Published online 16 December 2010 in Wiley Online Library
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
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
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).
GalNAc IN Xenopus laevis TESTIS
TABLE 1. GalNAc binding lectins employed in this work
Binding specificity
Snail (Helix pomatia)
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
Baker et al., 1983; Piller
et al., 1990; Wu and
Sugii, 1991; Spicer and
Schulte, 1992
Soybean (Glycine max)
Pereira and Kabat, 1974;
Piller et al., 1990; Wu
and Sugii, 1991; Spicer
and Schulte, 1992
Osage orange tree
(Maclura pomifera)
Sarkar et al., 1981; Young
et al., 1991; Lee et al.,
1998; Wu, 2005
Lima bean (Phaseolus
lunatus) agglutinin
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).
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
TABLE 2. Evaluation of GalNAc-binding lectin labelling of Xenopus laevis testis
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;, without pretreatment.
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
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
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
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.
Five GalNAc binding lectins have been employed for
the first time to identify GalNAc moieties in glycoconju-
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. On the other hand, the scarce number of
lectin histochemical studies on amphibian species
reported in the literature makes it impossible to establish more conclusions.
Mrs. M. Portuondo and Mrs. C. Otamendi contributed
to sample preparation. The authors thank Mrs. M. J.
Aldasoro for her support in the office work.
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