J. Anat. (2001) 198, pp. 47–56, with 9 figures Printed in the United Kingdom 47

Carbohydrate moieties of the interstitial and glandular tissues

of the amphibian Pleurodeles waltl testis shown by lectin

histochemistry

FRANCISCO JOSE! SA! EZ1, JUAN FRANCISCO MADRID1, RAQUEL APARICIO1,

FRANCISCO HERNA! NDEZ2 AND EDURNE ALONSO1

"Department of Cell Biology and Morphological Sciences, University of the Basque Country. Leioa (Vizcaya), and

#Department of Cell Biology, University of Murcia, Espinardo (Murcia), Spain

(Accepted 4 July 2000)

The amphibian testis is a useful model because of its zonal organisation in lobules, distributed along the

cephalocaudal axis, each containing a unique germ cell type. Sperm empty lobules form the so-called

glandular tissue at the posterior region of the gonad. Androgen production is limited to the cells of the

interstitial tissue surrounding lobules with spermatozoa bundles and to the cells of the glandular tissue. In

this work, we have studied the distribution of terminal carbohydrate moieties of N- and O-linked

oligosaccharides in the interstitial and glandular tissue of the Pleurodeles waltl testis, by means of 14 lectins

combined with chemical and enzymatic deglycosylation pretreatment. Some differences in glycan

composition between the interstitial and the glandular tissue have been detected. Thus in both tissues, N-

linked oligosaccharides contained mannose, Gal(β1,4)GlcNAc, and Neu5Ac(α2,3)Gal(β1,4)GlcNAc, while

O-linked oligosaccharides contained Con A-positive mannose, Gal(β1,3)GalNAc, Gal(β1,4)GlcNAc,

Neu5Ac(α2,3)Gal(β1,4)GlcNAc, and WGA-positive GlcNAc. Fucose was also detected in both tissues.

However, GlcNAc on N-linked oligosaccharides and GalNAc and Neu5Ac(α2,6)Gal}GalNAc on both N-

and O-linked oligosaccharides were found only in the interstitial tissue. As glandular tissue cells arise from

the innermost cells of interstitial tissue that surround lobules, the differences in the glycan composition of

interstitial and glandular tissue shown in this work may be related to the start of androgen synthesis when

steroid hormone (SH)-secreting cells develop.

Key words : Glycoconjugates ; Leydig cells ; testosterone; steroid hormones.

The zonal organisation of the testis of the lower

vertebrates (Loir et al. 1995; Pudney, 1995) makes it

easier to study the complex relationships between

somatic tissues and germ cells than in mammals, since

the somatic cells are close to germ cells that are at the

same stage of development. The urodele testis has

been used as a useful model to study germ cell-somatic

cell interactions (Picheral, 1966, 1968, 1970, 1972;

Hardy et al. 1992; Ji & Abe! , 1994; Del Rio-Tsonis et

al. 1996). The testis of Pleurodeles waltl, like other

urodeles, is composed of transient structures called

lobules. New lobules develop in every testicular cycle

Correspondence to Dr Francisco Jose! Sa! ez. Universidad del Paı!s Vasco, Departamento de Biologı!a Celular y Ciencias Morfolo! gicas,

Facultad de Medicina y Odontologı!a, B° Sarriena s}n, E-48940 Leioa (Vizcaya), Spain. Tel : ­34 946 015 791; fax: ­34 944 648 966; e-

mail : gcpsacrf!lg. ehu.es

in the most anterior tip of the testis, and degenerate at

the end of the cycle in the posterior region of the

gonad (Lofts, 1974; Sa! ez et al. 1989; Pudney, 1995).

All lobules are embedded in a poorly developed

connective tissue, the interstitial tissue, which also

surrounds the efferent ducts. Cells of the interstitial

tissue are fibroblast-like cells but, at the periphery of

the spermatozoa-containing lobules, some of these

mesenchymal cells develop into a cell layer that

surrounds every lobule, forming the lobule boundary

cells, which start to synthesise sex hormones (Berg-

mann et al. 1982; Pudney et al. 1983; Fraile et al.

1989; Hardy et al. 1992). Eventually, when sperm are

liberated to the efferent ducts in the most posterior

region of the testis, lobules develop in a glandular

tissue. In the developing glandular tissue, hypertro-

phied lobule boundary cells surround the follicle cells

occluding the lobule lumen. The follicle cells de-

generate and the mature glandular tissue is formed by

a parenchymal strand of cells originating from the

lobule boundary cells, embedded by the vascularised

connective interstitial tissue (Lofts, 1974; Bergmann

et al. 1983; Fraile et al. 1990).

According to this testicular organisation, androgen

production in urodeles is confined to the interstitial

tissue surrounding lobules with mature spermatozoa

and to the glandular tissue, but no androgen synthetic

activity has been reported in the interstitial tissue that

embeds earlier germ cell developmental stages (Lofts,

1974; Bergman et al. 1982, 1983; Pudney et al. 1983;

Fraile et al. 1989, 1990; Hardy et al. 1992). The

implication of the glycoconjugates in these processes

is an unknown matter. The oligosaccharide chains of

glycoconjugates are known to mediate many im-

portant biological roles including cell differentiation

(Gagneux & Varki, 1999). Specific carbohydrate

expression patterns may exhibit striking changes

related to cell differentiation (Roth, 1996). The

important role of glycan composition of some glyco-

conjugates is shown, for example, by the fact that loss-

of-function mutations of some proteoglycans are

subtle or undetectable, because of compensation or

functional redundancy among glycosaminoglycans,

showing that the active role resides in the sugar chains

(Lander & Selleck, 2000).

Recent advances in glycan research have shown

that cell surface proteoglycans are implicated in cell

development and differentiation through a role in cell

signalling by paracrine and autocrine factors, such as

growth factors, chemokines and cytokines (Iozzo,

1998; Bernfield et al. 1999; Lander & Selleck, 2000).

In addition, SH-secreting cells in mammalian testis,

the Leydig cells, are under endocrine, paracrine and

autocrine control mediated by hormones and growth

factors (Benton et al. 1995; Mendis-Handagama,

1997; Abney, 1999; Le Roy et al. 1999; Hedger et al.

2000), supporting the hypothesis that glycoconjugates

could play a role in this process and, probably, glycan

composition of SH-secreting tissues could be modified

during differentiation.

Modern lectin histochemical methods have become

invaluable tools to analyse glycan composition and

modifications at the cellular level (Spicer & Schulte,

1992; Roth, 1996). Some works have attempted to

study the presence of glycans in the testis of several

vertebrates, including mammals (Malmi et al. 1990;

Jones et al. 1992a, b), birds (Ballesta et al. 1991),

reptiles (Labate & Desantis, 1995) and anurans

(Ballesta et al. 1991). In some previous studies, we

have studied the glycan composition of urodele male

germ cells by means of lectin histochemistry (Sa! ez et

al. 1999, 2000), but no report exists about the glycan

composition of the interstitial and glandular tissue.

The aim of this work is to study the distribution of N-

and O-linked oligosaccharides in the interstitium and

the glandular tissue of P. waltl testis by means of lectin

histochemistry and several chemical and enzymatic

deglycosylation procedures. This will contribute to

the knowledgment of oligosaccharide chain modifi-

cations during the differentiation of the glandular

tissue.

Tissue preparation

Fourteen male adult specimens of Spanish newt,

Pleurodeles waltl, were employed for this study. The

newts were from our stock, developed from animals

provided from the laboratory of Prof. Boucaut

(University of Paris VI), and reared in our laboratory.

The animals were killed throughout the annual cycle

(Sa! ez et al. 1989) to obtain glandular tissue at different

rates of development. They were anaesthetised with 3-

aminobenzoic acid ethyl ester (MS-222, Sigma

Quı!mica, Alcobendas, Madrid, Spain), and perfused

with Bouin’s solution. After 20 min, the testes were

removed and immersed in the same fixative for 12 h.

The samples were washed in 30%(v}v) ethanol,

dehydrated in ethanol and embedded in paraffin.

Sections (5 µm), were obtained.

Lectin histochemistry

For this study, 14 lectins that bind fucose (Fuc),

galactosamine (GalNAc), galactose (Gal), sialic acid

(Neu5Ac), glucosamine (GlcNAc) and mannose

(Man) were used. The lectins used in this work, their

carbohydrate binding specificity and working dilution

are shown in Table 1.

Histochemical lectin labelling was performed using

horseradish peroxidase (HRP) and digoxigenin

(DIG)-conjugated lectins (Table 1). Lectin binding

pattern was established on paraffin sections in 3 ways:

(1) without previous treatment; (2) after β-elim-

ination, a chemical deglycosylation method that

removes O-linked oligosaccharides ; and (3) after

enzymatic deglycosylation with endoglycosidase

F}peptide N-glycosidase F (PNGase F) to remove

N-linked oligosaccharides. For Canavalia ensiformis

48 F. J. SaU ez and others

Table 1. Carbohydrate binding specificity of lectins (Spicer & Schulte, 1992)

Lectin Abbreviation Labelled to*

Carbohydrate

binding

specificity

Working

dilution

(µg}ml) Supplier

Lotus tetragonolobus

agglutinin

LTA HRP Fuc 50 Sigma

Ulex europaeus

agglutinin-I

UEA-I HRP Fuc 20 Sigma

Aleuria aurantia

agglutinin

AAA DIG Fuc 20 Boehringer–Mannheim

Canavalia ensiformis

agglutinin (Concanavalin A)

Con-A HRP Man"Glc 20 Sigma

Galanthus nivalis agglutinin GNA DIG (Man)n

60 Boehringer–Mannheim

Arachis hypogea

(peanut) agglutinin

PNA HRP Gal β1,3 GalNAc 50 Sigma

Datura stramonium

agglutinin

DSA DIG Gal β1,4 GlcNAc 10 Boehringer–Mannheim

Glycine max

(soybean) agglutinin

SBA HRP GalNAc 18 Sigma

Dolichos biflorus

agglutinin

DBA HRP GalNAc 30 Sigma

Helix pomalia

agglutinin

HPA HRP GalNAc"Gal 6 Sigma

Limax flavus

agglutinin

LFA HRP Neu5Ac 25 EY

Maackia amurensis

agglutinin

MAA DIG Neu5Ac α2,3

Gal β1,4 GlcNAc

10 Boehringer–Mannheim

Sambucus nigra

agglutinin

SNA DIG Neu5Ac α2,6

Gal}GalNAc

30 Boehringer–Mannheim

Triticum vulgaris

(wheat germ) agglutinin

WGA HRP (GlcNAc)n"Neu5Ac 10 Sigma

* HRP, horseradish peroxidase; DIG, digoxigenin.

agglutinin (Con A), lectin histochemistry was also

done after glucose-oxidase treatment, which converts

glucose (Glc) into gluconic acid. After this treatment

staining was due to Man instead of Glc. For wheat

germ agglutinin (WGA), all treatments were repeated

after acid hydrolysis, which removes sialic acid. After

this treatment, WGA staining was due to GlcNAc

instead of sialic acid.

The staining intensity was classified by 2 inde-

pendent observers into 4 arbitrary categories : no

labelling (0), weak (1), moderate (2) and strong (3).

This allowed us to evaluate the effects of the

pretreatments.

Histochemical staining using HRP-conjugated lec-

tins was performed by the method previously reported

by Madrid et al. (1990). Briefly, endogenous per-

oxidase was blocked with 0.3%(v}v) hydrogen per-

oxide in Tris-buffered saline (TBS). The sections were

then incubated for 2 h at room temperature with the

HRP-conjugated lectins. After brief washing with the

buffer, peroxidase was developed with 0.05% 3,3«-diaminobenzidine and 0.015%(v}v) hydrogen per-

oxide in TBS. Finally, sections were counterstained

with haematoxylin.

Histochemical staining using DIG-labelled lectins

was performed by the technique previously reported

by Sata et al. (1990). Briefly, after endogenous

peroxidase blocking with 0.3%(v}v) hydrogen per-

oxide in TBS, the sections were incubated with 1%

BSA in TBS for 10 min. and then for 1.5 h at room

temperature with the DIG-conjugated lectin. After 2

rinses of 5 min each with TBS, sections were incubated

with HRP-conjugated anti-DIG-antibodies (Boeh-

ringer–Mannheim Biochemica, Barcelona, Spain) for

1 h at 0.6 U}ml in TBS supplemented with 1% BSA.

Peroxidase was developed as described above and the

sections were counterstained with haematoxylin.

Chemical deglycosylation pretreatments (β-

elimination and acid hydrolysis)

Paraffin sections were treated with 0.5 N sodium

hydroxide in 70% ethanol at 4 °C for 10 d according

to Ono et al. (1983). This procedure removes the O-

linked oligosaccharides.

Acid hydrolysis, which eliminates sialic acid res-

idues (Schauer, 1982; Madrid et al. 1994) was

performed by immersing the sections in 0.1 hydro-

Steroid hormone-secreting tissues in the urodele testis 49

Figs. 1–9. For legend see opposite.

50 F. J. SaU ez and others

chloric acid for 2–3 h at 82 °C. This technique was

used in combination with WGA lectin histochemistry.

Enzymatic deglycosylation (peptide N-glycosidase F

treatment)

The buffer used for this pretreatment was 0.1 Tris,

150 m NaCl, 2.5 m EDTA, pH 9. Paraffin sections

were incubated in the buffer containing 1% triton X-

100 for 1 h and then in the same buffer with 1% BSA

for 10 min. After brief washing in the buffer, the

sections were incubated in the enzyme endo-

glycosidase F}peptide N-glycosidase F (Endo F}PNGase F, Boehringer–Mannheim Biochemica, Bar-

celona, Spain) at 6 U}ml for 3 d. At this pH, PNGase

F activity predominated over Endo F activity so that

removal of the N-linked oligosaccharides was per-

formed (Lucocq et al. 1987).

Glucose oxidase treatment

Paraffin sections were treated by the method pre-

viously described (Sa! ez et al. 1999). Sections were

washed in sodium acetate buffer at pH 5.0, incubated

overnight with 50 U}ml type VII glucose oxidase

from Aspergillus niger (Sigma, Alcobendas, Madrid,

Spain) at 37 °C in a moist chamber, washed in TBS 3

times for 5 min each and stained with Con A lectin as

usual.

Fig. 1. Developing glandular tissue after LTA alone (a) and in combination with β-elimination (b). A newly formed lobule of glandular tissue

originating after spermiation can be seen in a. Inner cells (black star) arise from the follicle (Sertoli) cells after sperm release. Outer cells,

the lobule boundary cells (black asterisk), originate from the interstitial tissue surrounding the lobules. Between both cell types, there is a

basal lamina (white arrowhead ). Note the lobules with spermatozoa near the developing glandular tissue. In these lobules, spermatozoa

bundles (black arrow) are attached to follicle cells (black arrowhead ), and at the periphery immature lobule boundary cells (white arrow) can

be seen. The interstitial tissue is positive (white star). An efferent duct with released sperm can be seen (white asterisk). ¬225.

Fig. 2. GNA histochemical staining alone (a) and after PNGase F procedure (b) of developing glandular tissue. The inner follicle cells (black

star) of the early glandular tissue shown in a are more stained than the lobule boundary cells (asterisk). The interstitial tissue is slightly

positive (white star). All tissues are negative after PNGase F pretreatment (b). ¬225.

Fig. 3. Mature glandular tissue after β-elimination and Con A histochemistry. Mature glandular tissue is formed by parenchymal strands

of cells arising from the lobule boundary cells. There are no derivatives from the follicle cells, which are degenerated. Strongly stained

cytoplasmic granules can be seen close to nuclei (arrows). ¬225.

Fig. 4. Maturating glandular tissue after DSA histochemistry. Lobule boundary cells are stained at the plasma membrane level and in a

cytoplasmic granule (arrows). Degenerating follicle cells are strongly labelled (black star). Interstitial tissue between lobules is also stained

(white star). ¬225.

Fig. 5. PNA histochemistry alone (a) and in combination with β-elimination (b). Degenerating follicle cells (black star) show stronger labelling

than outer lobule boundary cells (asterisk) (a). Interstitial tissue is also positive to PNA (white star). After β-elimination procedure, which

removes O-linked oligosaccharides, no staining can be seen (b). ¬225.

Fig. 6. DBA staining of early glandular tissue. Basal lamina between follicle and lobule boundary cells is labelled (arrow). Labelling of

interstitial tissue is shown (asterisk). Note the connection between the lobule and an efferent duct (arrowhead ). ¬225.

Fig. 7. HPA histochemistry of early glandular tissue. No staining can be seen except for slight labelling of the interstitial tissue (asterisk)

and the basal lamina between follicle and lobule boundary cells (arrow). ¬225.

Fig. 8. LFA staining of early glandular tissue. The glandular (star) and the interstitial tissue (asterisk) are both strongly labelled. Note the

strongly stained cytoplasmic granule close to the nucleus (black arrows). A lobule with spermatozoa bundles (white arrow) can be seen.

¬225.

Fig. 9. SNA histochemistry after PNGase F pretreatment. The lobule boundary cells (black asterisk) are negative. The inner (follicle) cells

of the lobule are strongly stained (star). The interstitial tissue (white asterisk) is also positive. Note the lobule with spermatozoa bundles

(arrow) at the bottom of the figure. ¬225.

Controls

The following controls were used: (1) substitution of

the lectins and the antibodies by the buffer alone; (2)

preincubation of the lectins with the corresponding

hapten sugar inhibitor (Fuc for AAA, UEA-1 and

LTA; GlcNAc for WGA; GalNAc for HPA, SBA

and DBA; α-methyl-mannopyranoside for Con A and

GNA; Neu5Ac, for LFA; lactosamine for DSA; Gal

for PNA; α2,3 sialyllactosamine for MAA and

α2,6 sialyllactosamine for SNA; from Sigma, Spain)

at a concentration of 0.2 ; and (3) preabsorption of

the antibodies with the corresponding antigen.

Histology of SH-secreting cells

Interstitial tissue was observed around the lobules and

between strands of parenchymal cells of the glandular

tissue. Usually the staining observed in the interstitial

tissue did not allow differentiation between the cells

and the extracellular matrix; occasionally, however,

the staining was clearly restricted to the perinuclear

region of the cells, suggesting that the staining was

due to the cells, while the extracellular matrix was

negative.

Newly-formed glandular tissue could be seen as

lobules showing 2 cell layers. The external layer was

constituted by the cells that originally formed the wall

Steroid hormone-secreting tissues in the urodele testis 51

of spermatogenetic lobules, the so-called lobule

boundary cells. The internal layer was formed by the

follicle cells of the modified cysts. The limit between

both layers was stained with some lectins. This

staining could be due to the basal lamina of the

follicular cells. The lobule boundary cells progres-

sively enlarged while the internal follicle cells quickly

degenerated (Figs 1, 2). In most of the glandular

tissue, the follicle cells degenerated and they could not

be detected; only the layers of the lobule boundary

cells remained. These were the components of the

mature glandular tissue. Eventually, mature glandular

tissue was constituted by a vascularised parenchyma

arranged in strands of cells separated by a connective

interstitial tissue (Figs 2b, 3).

Fucose-binding lectins (Fuc)

Histochemical results are summarised in Table 2. The

labelling of the interstitial tissue with LTA and UEA-

I disappeared after PNGase F and β-elimination pre-

treatment (Fig. 1). Conversely, interstitial tissue was

positive for AAA in all cases.

For LTA (Fig. 1a) and UEA-1, the lobules of newly

formed glandular tissue were more strongly stained at

the limit between lobule boundary cell and the inner

follicle cells. This labelling disappeared after both β-

elimination and PNGase F pretreatment (Fig. 1b). A

more intense staining was clearly observed in the

cytoplasmic juxtanuclear region of the glandular

tissue cells after AAA staining. Labelling with AAA

was removed after deglycosylation pretreatment.

Mannose-binding lectins (Man)

The interstitial tissue was positive for Con A, a

staining pattern that was not significantly modified

after any of the pretreatments employed, i.e. glucose

oxidase, β-elimination or PNGase F. For GNA, only

the cells were slightly positive, the extracellular matrix

being negative. After removal of N-linked oligo-

saccharides with PNGase F pretreatment, there was

no labelling with GNA (Fig. 2).

As for the interstitial tissue, glandular tissue

labelling with Con A was not modified by any

pretreatment. Glandular tissue was positive for GNA

in a similar pattern as for Con A, but staining

disappeared after PNGase F pretreatment (Fig. 2).

With GNA and Con A, the central lobular cells in the

early glandular tissue that arose from the follicle

cells were more stained that the cells that arose from

the lobule boundary cells (Fig. 2a). In the latter cells,

after β-elimination and PNGase F a juxtanuclear

Table 2. Lectin labelling pattern of the interstitial and the

glandular tissues in the Pleurodeles waltl testis. A scale from

0 to 3 indicates label intensity.

Intestitial

tissue

Glandular

tissue*

Fuc-binding lectins

LTA 2 1

β-elimination 0 0

PNGase F 0 0

AAA 2 1–2 ­β-elimination 2 0

PNGase F 1 0

UEA-I 2 1 ­β-elimination 0–1 0

PNGase F 0 0

Man-binding lectins

Con A 2 2

Glucose-oxidase 2 2

β-elimination 1–2 2 ­PNGase F 1–2 1 ­

GNA 0–1** 1–2

β-elimination 0–1** 1 ­PNGase F 0 0

Gal-binding lectins

DSA 2 1 ­β-elimination 2 1 ­PNGase F 1–2 1 ­

PNA 2 1

β-elimination 0–1 0

PNGase-F 2 1 ­GalNAc-binding lectins

HPA 0–1 0

β-elimination 0–1 0

PNGase F 0–1 0

DBA 2 1

β-elimination 2 0–1

PNGase F 0 0

SBA 2 0

β-elimination 3 1–2

PNGase F 2 0–1

Neu5Ac-binding lectins

SNA 2 0–1

β-elimination 2 0

PNGase F 2 0

LFA 2 1 ­β-elimination 2 0–1 ­PNGase F 2 1 ­

MAA 2 1 ­β-elimination 2 1 ­PNGase F 1 1 ­

WGA-binding lectin

WGA 3 2 ­β-elimination 3 1 ­PNGase F 2 1 ­

Acid hydrolysis ­ WGA 1 0–1

β-elimination 2 0

PNGase F 2 1 ­

* The existence of a cytoplasmic granular material with the most

intense labelling in the glandular tissue cells is indicated by ­.

** Only the interstitial cells were labelled, the intercellular matrix

being negative.

52 F. J. SaU ez and others

region that stained more intensely was observed after

Con A staining (Fig. 3).

Galactose-binding lectin

Both DSA and PNA stained interstitial tissue (Figs 4,

5). This tissue was positive for DSA after the 2

deglycosylation pretreatments employed. There was

no PNA labelling after β-elimination (Fig. 5b).

Glandular tissue was always positive for DSA (Fig.

4). However, this tissue was not stained or only

slightly stained with PNA, the labelling in the remnant

follicle cells of the early glandular tissue lobules only

being higher (Fig. 5a). After β-elimination pretreat-

ment, glandular tissue was negative for this lectin

(Fig. 5b). The juxtanuclear granular material was

positive for DSA; with PNA it was only stained after

PNGase F pretreatment.

N-acetylgalactosamine-binding lectins

The 3 GalNAc-binding lectins showed different

patterns of staining in the interstitial tissue. While this

tissue was positive for DBA (Fig. 6) and SBA, it was

only slightly positive for HPA (Fig. 7). Moreover,

there was no staining with DBA after PNGase F

procedure, but this pretreatment did not modify the

labelling pattern of HPA and SBA. Glandular tissue

was positive only for DBA, with a more intense

labelling of the luminal surface of the developing

glandular cells (Fig. 6). With this lectin, staining

diminished with β-elimination and there was no

labelling after PNGase F pretreatment. Usually, the

tissue was negative for the other 2 GalNAc-labelling

lectins, HPA (Fig. 7) and SBA.

N-acetylneuraminic acid-binding lectins

With the 3 lectins, SNA, LFA and MAA, intense

labelling of the interstitium was always seen (Figs 8,

9). The same was true for the glandular tissue with

LFA and MAA, the intensity of staining of the inner

follicle cells being higher in the early glandular tissue

(Fig. 8). Juxtanuclear material of some cells was

labelled for both lectins (Fig. 8), but glandular tissue

was almost negative after SNA staining, only the

inner follicle cells of the early glandular lobules being

positive (Fig. 9).

N-acetylglucosamine binding lectin

WGA labelled the interstitial tissue alone or after the

2 deglycosylation pretreatments, β-elimination and

PNGase F. Removal of Neu5Ac with acid hydrolysis

produced a lower intensity of staining.

In the glandular tissue, a similar pattern of staining

was observed, the main difference being that after acid

hydrolysis in combination with β-elimination the

glandular tissue cells were negative. The juxtanuclear

material was positive to WGA, but the labelling

disappeared after removal of sialic acid.

In the present histochemical study, 14 different lectins

combined with the chemical and enzymatic deglyco-

sylation pretreatment were used to establish the

carbohydrate moieties of the androgen-producing

tissues in the Pleurodeles waltl testis. Usually, cell

types of the glandular tissue, i.e. follicle cells and

lobule boundary cells, show a different pattern of

staining. As a general rule, staining of the paren-

chymal cells was higher at the plasma membrane level.

Sometimes, a granular juxtanuclear region, probably

the Golgi apparatus and}or the associated smooth

endoplasmic reticulum, was more strongly stained

with some lectins. The results in the interstitial tissue

suggest the presence of Fuc in N and O-linked

oligosaccharides. In the glandular tissue, two distri-

bution patterns of Fuc residues were shown: the

first, demonstrated by LTA and UEA-I, indicated the

presence of Fuc in oligosaccharides of the glandular

tissue, glycans that were removed by β-elimination or

PNGase F. These sugars were mostly at the limit

between the lobule boundary and the follicle cells at

the early glandular tissue and, only with UEA-I, in the

Golgi zone. The other one, shown by means of AAA,

discovered Fuc at all the glandular tissue cells,

especially in the plasma membranes and the Golgi

zone. Previous lectin histochemical studies about

testis of other vertebrates have failed to identify Fuc

in the Leydig cells (Arya & Vanha-Perttula, 1984,

1985; Ballesta et al. 1991; Labate & Desantis, 1995) ;

the only exception being reported by Arya & Vanha-

Perttula (1986) in some mammalian species.

Since Con A staining was not abolished after

glucose oxidase pretreatment, the labelling of the

lectin was due to Man. Moreover, the findings with

GNA suggest that cells of the interstitial tissue, but

not the intercellular matrix, contain Man in N-linked

oligosaccharides. Man residues demonstrated by

GNA in the glandular tissue were always located on

N-linked oligosaccharides. Man has also been shown

in the Leydig cells and interstitium of the testis in

several species, including man (Malmi et al. 1987;

Wollina et al. 1989), mouse, gerbil, guinea pig and

Steroid hormone-secreting tissues in the urodele testis 53

nutria (Arya & Vanha-Perttula, 1986), bull (Arya &

Vanha-Perttula, 1985; Ertl & Wrobel, 1992), lizard

(Labate & Desantis, 1995), toad, turtle, pigeon and

hamster (Ballesta et al. 1991).

Our results indicate that the interstitial and the

glandular tissues contain O-linked glycans with

Gal(β1,3)GalNAc, as demonstrated by PNA histo-

chemistry. However, the Gal(β1,4)GlcNAc sequence

seems to be on both N and O-linked glycans of both

tissues, because DSA-staining was not modified by

deglycosylation pretreatments. PNA has also been

reported as negative in the human testicular inter-

stitium, and in the Leydig cells and interstitium of the

bull, hamster, pigeon, turtle and toad (Arya & Vanha-

Perttula, 1985; Wollina et al. 1989; Ballesta et al.

1991; Ertl & Wrobel, 1992). In the lizard Podarcis

campestris, Leydig cells were positive for PNA only

after neuraminidase treatment (Labate & Desantis,

1995). In the rat testis, contradictory results have been

reported. Thus while Leydig cells are positive for both

RCA and PNA in one paper (Arya & Vanha-Perttula,

1984), other reports indicate that PNA was negative

(So$ derstro$ m et al. 1984; Malmi et al. 1990), RCA

staining being variable with sexual maturity (Malmi

et al. 1990).

Terminal GalNAc residues were demonstrated in

N-linked oligosaccharides of the interstitial tissue by

both DBA and SBA lectins. The same sugar moiety

was localised in O-linked glycans by SBA histo-

chemistry. However, GalNAc moieties in the glandu-

lar tissue cells are not clearly demonstrated, because

staining with all the GalNAc-binding lectins was

negative or faint. DBA histochemical results suggest

that few GalNAc moieties could be present on N-

linked glycans in the glandular tissue. Different results

have been shown for these lectins in several tetrapod

vertebrate testes. Most of the cases have shown that

SBA and DBA do not label Leydig cells (Arya &

Vanha-Perttula, 1984, 1985, 1986; Malmi et al. 1990;

Ballesta et al. 1991; Ertl & Wrobel, 1992; Labate &

Desantis, 1995). However, in some species, such as

man (Malmi et al. 1987; Wollina et al. 1989), turtle

(Ballesta et al. 1991), mouse and nutria (Arya &

Vanha-Perttula, 1986), the Leydig cells have shown

reactivity either for SBA or for both lectins. No

previous report exists about HPA staining.

All Neu5Ac-binding lectins employed, including

WGA, stained the interstitial tissue, showing that this

moiety was located in N- and O-linked glycans. As

acid hydrolysis diminished but not abolished WGA-

labelling, the presence of N- and O-linked oligo-

saccharides with GlcNAc was also inferred. The

existence of the terminal Neu5Ac(α2,3)Gal(β1,

4)GlcNAc sequence on N- and O-linked glycans of

the glandular tissue cells, especially in the Golgi zone,

is shown by MAA histochemical treatment. LFA also

stains these cells, which support this conclusion.

Glandular tissue cells were also positive for WGA, but

most WGA labelling disappeared after acid hydroly-

sis, indicating that the major labelling was due to

Neu5Ac, GlcNAc only being on O-linked oligo-

saccharides.

A relationship between androgen secretory activity

and cellular glycan composition has previously been

proposed. Malmi et al. (1990) reported a decreasing

reaction intensity of RCA referring to a modification

of carbohydrate-containing substances just before

sexual maturity, i.e. when androgen synthesis in-

creases. Most of the lower vertebrates, such as P.

waltl, have a cyclic seasonal spermatogenesis and

androgen production (Fraile et al. 1989) and, as

occurs in the lizard Podarcis campestris, previous

results suggest that cyclic variations in androgen

concentration can be related to lectin-binding sites

(Labate & Desantis, 1995). Furthermore, slight stain-

ing differences between early and mature glandular

tissue shown in the present work, as well as the fact

that some but not all SH-secreting cells possess

prominent granular material, can reflect a structural

and functional difference between parenchymal cells,

as has been suggested by other authors (Callard et al.

1980; Bergmann et al. 1982). This granular material

observed in the cells of the glandular tissue contained

Fuc, Man, Gal(β1-4)GlcNAc and Neu5Ac(α2-3)Gal.

It is widely accepted that glycoconjugates are

involved in cell differentiation (Roth, 1996; Ziak et al.

1996). Recently it has been shown that glycosamino-

glycans of the cell surface proteoglycans are involved

in controlling cell differentiation and proliferation by

binding to local mediators. For example, fibroblast

growth factors (FGFs), hepatocyte growth factor

(HGF), epithelial growth factors (EGFs) and platelet

derived growth factor (PDGF) require heparan

sulphate for high affinity binding to their receptors

(Bernfield et al. 1999; Lander & Selleck, 2000). In

addition, binding of transforming growth factors

(TGF-β) to heparan sulphate prevents inactivation.

Mammalian Leydig cells are under endocrine, para-

crine and autocrine regulation (Huhtaniemi &

Toppari, 1995; Carreau, 1996; Mendis-Handagama,

1997; Hedger & de Kretser, 2000). Recently it has

been shown that some growth factors activate (insulin-

like growth factor-1, IGF-1) or inhibit (TGF-β1)

testosterone synthesis and Leydig cell differentiation

(Benton et al. 1995; Le Roy et al. 1999). Thus it is

possible to speculate about some possible roles for

54 F. J. SaU ez and others

carbohydrate chains in the differentiation of SH-

secreting cells of amphibians.

In summary, some differences in glycan compo-

sition between the interstitial and glandular tissue

have been detected by means of lectin histochemistry.

These differences in the glycan composition of these

tissues may be related to SH-secreting cell devel-

opment and the onset of androgen synthesis.

The authors thank Mrs M. Portuondo and Mrs C.

Otamendi for technical assistance. This work was

supported by grants from the Autonomous Basque

Government (PI 1997-48) and the University of the

Basque Country (G10}99). R. A. and E. A. were

supported by fellowships from the University of

the Basque Country.

ABNEY TO (1999) The potential roles of estrogens in regulating

Leydig cell development and function: a review. Steroids 64,

610–617.

ARYA M, VANHA-PERTTULA T (1984) Distribution of lectin-

binding in rat testis and epididymis. Andrologia 16, 495–508.

ARYA M, VANHA-PERTTULA T (1985) Lectin-binding pattern

of bull testis and epididymis. Journal of Andrology 6, 230–232.

ARYA M, VANHA-PERTTULA T (1986) Comparison of lectin-

binding pattern in testis and epididymis of gerbil, guinea pig,

mouse, and nutria. American Journal of Anatomy 175, 449–469.

BALLESTA J, MARTI!NEZ-MENA! RGUEZ JA, PASTOR LM,

AVILE! S M, MADRID JF, CASTELLS MT (1991) Lectin

binding pattern in the testes of several tetrapod vertebrates.

European Journal of Basic and Applied Histochemistry 35,

107–117.

BENTON L, SHAN LX, HARDY MP (1995) Differentiation of

adult Leydig cells. Journal of Steroid Biochemistry and Molecular

Biology 53, 61–68.

BERGMANN M, SCHINDELMEISER J, GREVEN H (1982)

The zone of mature spermatozoa in the testis of Salamandra

salamandra (L.) (Amphibia, Urodela). Zeitschrift fuX r mikro-

skopisch-anatomische Forschung 96, 221–234.

BERGMANN M, SCHINDELMEISER J, GREVEN H (1983)

The glandular tissue in the testis of Salamandra salamandra (L.)

(Amphibia, Urodela). Acta Zoologica 64, 123–130.

BERNFIELD M, GO$ TTE M, PARK PW, REIZES O,

FITZGERALD ML, LINCECUM J et al. (1999) Functions of

cell surface heparan sulfate proteoglycans. Annual Reviews of

Biochemistry 68, 729–777.

CALLARD GV, CANIK JA, PUDNEY J (1980) Estrogen

synthesis in Leydig cells : structural-functional correlations in

Necturus testis. Biology of Reproduction 28, 461–471.

CARREAU S (1996) Paracrine control of human Leydig cell and

Sertoli cell functions. Folia Histochemica et Cytobiologica 34,

111–119.

DEL RIO-TSONIS K, COVARRUBIAS L, KENT J, HASTIE

ND, TSONIS PA (1996) Regulation of the Wilms’ tumor gene

during spermatogenesis. Developmental Dynamics 207, 372–381.

ERTL C, WROBEL KH (1992) Distribution of sugar residues in

the bovine testis during postnatal ontogenesis demonstrated with

lectin-horseradish peroxidase conjugates. Histochemistry 97,

161–171.

FRAILE B, PANIAGUA R, RODRI!GUEZ MC, SA! EZ FJ,

JIME! NEZ A (1989) Annual changes in the number, testosterone

content and ultrastructure of glandular tissue cells of the testis in

the marbled newt Triturus marmoratus. Journal of Anatomy 167,

85–94.

FRAILE B, SA! EZ FJ, PANIAGUA R (1990) The cycle of

follicular and interstitial cells (Leydig cells) in the testis of the

marbled newt, Triturus marmoratus (Caudata, Salamandridae).

Journal of Morphology 204, 89–101.

GAGNEUX P, VARKI A (1999) Evolutionary considerations in

relating oligosaccharide diversity to biological function. Glyco-

biology 9, 747–755.

HARDY MP, SPRANDO RL, EWING LL (1992) Leydig cell

renewal in testis of seasonally breeding animals. Journal of

Experimental Zoology 261, 161–172.

HEDGER MP, DE KRETSER DM (2000) Leydig cell function

and its regulation. Results and Problems in Cell Differentiation 28,

69–110.

HUHTANIEMI I, TOPPARI J (1995) Endocrine, paracrine and

autocrine regulation of testicular steroidogenesis. Advances in

Experimental Medicine and Biology 377, 33–54.

IOZZO RV (1998) Matrix proteoglycans: from molecular design to

cellular function. Annual Review of Biochemistry 67, 609–652.

JI Z-S, ABE! S-I (1994) Mammalian follicle-stimulating hormone

stimulates DNA synthesis in secondary spermatogonia and

Sertoli cells in organ culture of testes fragments from the newt

Cynops pyrrhogaster. Zygote 2, 53–61.

JONES CJ, MORRISON CA, STODDART RW (1992a)

Histochemical analysis of rat testicular glycoconjugates. 1.

Subsets of N-linked saccharides in seminiferous tubules. Histo-

chemical Journal 24, 319–326.

JONES CJ, MORRISON CA, STODDART RW (1992b) Histo-

chemical analysis of rat testicular glycoconjugates. 2. Beta-

galactosyl residues in O- and N-linked glycans in seminiferous

tubules. Histochemical Journal 24, 327–336.

LABATE M, DESANTIS S (1995) Histochemical analysis of lizard

testicular glycoconjugates during the annual spermatogenetic

cycle. European Journal of Histochemistry 39, 201–212.

LANDER AD, SELLECK SB (2000) The elusive functions of

proteoglycans: in vivo veritas. Journal of Cell Biology 148,

227–232.

LE ROY C, LEJEUNE H, CHUZEL F, SAEZ JM, LANGLOIS

D (1999) Autocrine regulation of Leydig cell differentiation

functions by insulin-like growth factor I and transforming

growth factor beta. Journal of Steroid Biochemistry and Molecular

Biology 69, 379–384.

LOFTS B (1974) Reproduction. In Physiology of the Amphibia (ed.

Lofts B), vol.1, pp. 197–218. New York: Academic Press.

LOIR M, SOURDAINE P, MENDISH-HANDAGAMA SMLC,

JE! GOU B (1995) Cell-cell interactions in the testis of teleosts and

elasmobranchs. Microscopy Research and Technique 32, 533–552.

LUCOCQ JM, BERGER GE, ROTH J (1987) Detection of

terminal N-linked N-acetylglucosamine residues in the Golgi

apparatus using galactosyltransferase and endoglucosaminidase

F}peptide N-glycosidase F: adaptation of a biochemical ap-

proach to electron microscopy. Journal of Histochemistry and

Cytochemistry 35, 67–74.

MADRID JF, BALLESTA J, CASTELLS MT, HERNA! NDEZ F

(1990) Glycoconjugate distribution in the human fundic mucosa

revealed by lectin- and glycoprotein-gold cytochemistry. Histo-

chemistry 95, 179–187.

MADRID JF, CASTELLS MT, MARTI!NEZ-MENA! RGUEZ

JA, AVILE! S M, HERNA! NDEZ F, BALLESTA J (1994)

Subcellular characterization of glycoproteins in the principal cells

of human gallbladder. A lectin cytochemical study. Histo-

chemistry 101, 195–204.

MALMI R, KALLAJOKI M, SUOMINEN J (1987) Distribution

of glycoconjugates in human testis. A histochemical study using

fluorescein- and rhodamine- conjugated lectins. Andrologia 19,

322–332.

Steroid hormone-secreting tissues in the urodele testis 55

MALMI R, FRO$ JDMAN K, SO$ DERSTRO$ M K-O (1990)

Differentiation-related changes in the distribution of glyco-

conjugates in rat testis. Histochemistry 94, 387–395.

MENDIS-HANDAGAMA SM (1997) Luteinizing hormone on

Leydig cell structure and function. Histology and Histopathology

12, 869–882.

ONO K, KATSUYAMA T, HOTCHI M (1983) Histochemical

applications of mild alkaline hydrolysis for selective elimination

of O-glycosidically linked glycoproteins. Stain Technology 58,

309–312.

PICHERAL B (1966) Sur l’ultrastructure des mitochondries des

cellules du tissu glandulaire du testicule de Pleurodeles waltlii

Michah (Amphibien Urode' le). Comptes Rendus de la AcadeUmie

de Sciences de Paris, SeU rie D 262, 1769–1772.

PICHERAL B (1968) Les tissues e! laborateurs d’hormones ste! roı$des

chez les amphibiens urode' les I. Ultrastructure des cellules du

tissu glandulaire du testicule de Pleurodeles waltl Michah. Journal

de Microscopie 7, 115–134.

PICHERAL B (1970) Les tissus e! laborateurs d’hormones ste! roı$des

chez les amphibiens urode' les. IV. E! tude en microscopie e! lec-tronique et photonique du tissu glandulaire du testicule et de la

glande interre!nale apre' s hypophysectomie, chez Pleurodeles

waltlii Michah. Zeitschrift fuX r Zellforschung 107, 68–86.

PICHERAL B (1972) Les tissus e! laborateurs d’hormones ste! roı$des

chez les urode' les. V. Mise en e! vidence et localisation cyto-

chimique d’une activite! de type peroxydasique endoge' ne dans la

cellule du tissu glandulaire du testicule de Pleurodeles waltlii

Michah. Comparaison avec d’autres types cellulaires. Journal de

Microscopie 13, 247–262.

PUDNEY J (1995) Spermatogenesis in nonmammalian vertebrates.

Microscopy Research and Technique 32, 459–497.

PUDNEY J, CANICK JA, MAK P, CALLARD GV (1983) The

differentiation of Leydig cells, steroidogenesis, and the spermato-

genetic wave in the testis of Necturus maculosus. General and

Comparative Endocrinology 50, 43–66.

ROTH J (1996) Protein glycosylation in the endoplasmic reticulum

and the Golgi apparatus and cell type-specificity of cell surface

glycoconjugate expression: analysis by the protein A-gold and

lectin-gold techniques. Histochemistry and Cell Biology 106,

79–92.

SA! EZ FJ, FRAILE B, PANIAGUA R (1989) Histological and

quantitative changes in the annual testicular cycle of Triturus

marmoratus marmoratus. Canadian Journal of Zoology 68, 63–72.

SA! EZ FJ, MADRID JF, APARICIO R, LEIS O, OPORTO B

(1999) Lectin histochemical localization of N- and O-linked

oligosaccharides during the spermiogenesis of the urodele

amphibian Pleurodeles waltl. Glycoconjugate Journal 16, 639–648.

SA! EZ FJ, MADRID JF, APARICIO R, ALONSO E,

HERNA! NDEZ F (2000) Glycan residues of N- and O-linked

oligosaccharides in the premeiotic spermatogenetic cells of the

urodele amphibian Pleurodeles waltl characterized by means of

lectin histochemistry. Tissue & Cell, in press.

SATA T, ZUBER C, ROTH J (1990) Lectin-digoxigenin con-

jugates : a new hapten system for glycoconjugate cytochemistry.

Histochemistry 94, 1–11.

SCHAUER R (1982) Chemistry, metabolism and biological

functions of sialic acid. Advances in Carbohydrate Chemistry and

Biochemistry 40, 131–234.

SO$ DERSTRO$ M KO, MALMI R, KARJALAINEN K (1984)

Binding of fluorescein isothiocyanate conjugated lectins to rat

spermatogenic cells in tissue sections. Histochemistry 80, 475–579.

SPICER SS, SCHULTE BA (1992) Diversity of cell glycocon-

jugates shown histochemically : a perspective. Journal of Histo-

chemistry and Cytochemistry 40, 1–38.

WOLLINA U, SCHREIBER G, ZOLLMANN C, HIPLER C,

GU$ NTHER E (1989) Lectin-binding sites in normal human

testis. Andrologia 21, 127–130.

ZIAK M, QU B, ZUO X, ZUBER C, KANAMORI K,

KITAJIMA K et al. (1996) Occurrence of poly α-2,8-de-

aminoneuraminic acid (KDN) in mammalian tissues : widespread

and developmentally regulated but highly selective expression on

glycoproteins. Proceedings of the National Academy of Sciences

of the USA 93, 2759–2763.

56 F. J. SaU ez and others