Glycan residues of N- and O-linkedoligosaccharides in the premeioticspermatogenetic cells of the urodeleamphibian pleurodeles waltlcharacterizedby means of lectin histochemistry
F. J. Sáez,1 J. F. Madrid,1 R. Aparicio,1 E. Alonso1 and F. Hernández2
1Department of Cell Biology and Morphological Sciences, School of Medicine and Dentistry, University of the Basque Country, B° Sarriena s/n, E-48940 Leioa(Vizcaya), Spain. 2Department of Cell Biology, School of Medicine, University of Murcia, E-30071 Espinardo (Murcia), Spain
Received 7 March 2000Accepted 16 May 2000
Correspondence to: Dr F.J. Sáez, Departamento de Biología Celular y Ciencias Morfológicas, Universidad del País Vasco, Facultad de Medicina y Odontología,B° Sarriena s/n, E-48940 Leioa (Vizcaya), Spain. Tel.: + 34 946 015 791; Fax: + 34 944 604 485; E-mail: [email protected]
istoij
thatausl
99
anamhte &edan8canmcth2zoe7ng ativ
70st95e
n-is of
ndtionWecies,wl-ge-
thylthee fored in
er thero-
lac-c),eened inz et
tins
ngju- onionstiones)se
ove the
LECTIN HISTOCHEMISTRY OFP. WALTLPREMEIOTIC MALE GERM CELLS. 303
Introduction
Spermatogenesis is one of the most interesting mechanof cell differentiation, by means of which diploid spermagonial stem cells generate haploid spermatozoa (De RooGrootegoed, 1998). The oligosaccharide composition ofmammalian testicular germ cells has been investig(Ballesta et al. 1991; Labate & Desantis 1995; Runnebet al., 1995) and a role in the control of spermatogeneincluding spermatogonial proliferation (Ertl & Wrobe1992; Suda et al. 1998) and meiosis (Koshimizu et al. 1Maylie-Pfenninger 1994), has been suggested.
The nature of the link between the oligosaccharide the peptide has been used to distinguish between two flies of sugar chains. The so-called O-linked oligosaccrides comprise the sugar chains linked to hydroxylaamino acid of proteins (serine or threonine) (StrousDekker, 1992). The other group, the named N-linkoligosaccharides are those in which an N-glycosidic bonestablished between N-acetylglucosamine (GlcNAc) the amide nitrogen of asparagine (Staneloni & Leloir 19Kornfeld & Kornfeld, 1985). There are some histochemitechniques that can be employed to study the localizatiothe carbohydrate residues of the oligosaccharides. The interesting ones are those based on the properties of lesugar binding. The lectins are proteins or glycoproteins bind specifically to sugar groups (Spicer & Schulte 199Furthermore, when they are used in combination with enmatic and chemical deglycosylation, the lectins give infmation on the N- or O-linked nature of oligosaccharid(Ono et al. 1983; Plummer et al. 1984; Lucocq et al. 198
Previous works have reported the distribution of N- aO-linked oligosaccharides in the mammalian spermatonetic cells by means of lectin histochemistry (Jones et1992a, 1992b). However, in lower vertebrates the detecof carbohydrates has been carried out by means of contional histochemical techniques (Sandoz 19Schindelmeiser 1986), and rarely by means of lectin hichemistry (Ballesta et al., 1991; Labate & Desantis 19No report exists about lectin histochemistry of the urodtestis.
Table 1 Carbohydrate binding specificity of lectins (S
The aim of this work is the characterization of glycocojugates of premeiotic spermatogenetic cells of the testan urodele amphibian, Pleurodeles waltl, by means of 14different lectins in combination with several chemical aenzymatic procedures, in order to establish the distribuof N- and O-linked oligosaccharides in these cells. compare the results obtained in the testis of other speespecially in mammals. Our results contribute to the knoedge of the glycoconjugate composition during spermatonetic development.
Material and methods
Sample preparationFourteen male adult specimens of Spanish newt, Pleurodeleswaltl, were anaesthetized with 3-aminobenzoic acid eester (MS-222), perfused with Bouin’s solution, and testes were removed and immersed in the same fixativ12 h. The samples were routinely processed, embeddparaffin and 5µm-thick sections were obtained.
Lectin histochemistryThe present study has been made using lectins that coventire spectrum of carbohydrate constituents of glycopteins, i.e. lectins that bind fucose (Fuc), N-acetylgatosamine (GalNAc), galactose (Gal), sialic acid (Neu5AGlcNAc and mannose (Man). These lectins have bchosen because they have been successfully employour previous works (Madrid et al. 1994, 1998, 2000; Sáeal 1999). The carbohydrate binding specificity of the lecused in this work is shown in Table 1.
Histochemical lectin labeling was performed usihorseradish peroxidase (HRP)- or digoxigenin (DIG)-congated lectins. Lectin binding pattern was establishedparaffin sections without previous treatment; other sectwere stained with the lectins after chemical deglycosylaβ-elimination, which removes O-linked oligosaccharidor after enzymatic deglycosylation with EndoglycosidaF/Peptide N-glycosidase F (PNGase F) to remN-oligosaccharides. For wheat germ agglutinin (WGA)
picer and Schulte, 1992)
eviation Carbohydrate binding specificity
GalNAcGalNAcGalNAc > GalFuc
-1 FucFuc
Gal β1, 3 GalNAcGal β1, 4 GlcNAcNeu5AcNeu5Ac α2,3 Gal β1,4 GlcNAcNeu5Ac α2,6 Gal/GalNAc
A (GlcNAc)n > Neu5AcA Man > Glc
(Man)n
at
uds
aidlt
nh9ia
a i)
e:
hSina
o1ePS
idev
Min
v/v)%e
dasel forndo
N-
fer atII
hreesual.
theein-gar
A;
n-ies
: 1.theary
miced.ato-Gaseere
A-
Cs,Fig.
he
SA
304 SÁEZ ET AL.
same three above indicated treatments were repeated acid hydrolysis, which removes Neu5Ac. After this treament staining was due to GlcNAc instead of Neu5Ac. Themploying WGA alone and in combination with acihydrolysis information about Neu5Ac and GlcNAc waobtained. For Canavalia ensiformisagglutinin (ConA), thethree treatments were done again after glucose-oxidtreatment, which converts glucose (Glc) in gluconic acAfter this treatment staining was due to Man instead of GCells and cellular structures were arbitrarily classified infour categories according to their labeling intensity: labeling (–), weak (+), moderate (++) and strong (+++). Tfollowing cell types were analyzed (Sáez et al., 198primordial germ cells (PGCs), primary spermatogonsecondary spermatogonia, and primary spermatocytes.
Histochemical staining using HRP-conjugated lectins wperformed by the method previously reported by Madridal. (1989). Briefly, endogenous peroxidase was blocked w0.3% (v/v) hydrogen peroxide in Tris-buffered saline (TBSThen, the sections were incubated for 2 h at room tempture with the following HRP-conjugated lectinsConcanavalin A (ConA, 20µg/ml), peanut agglutinin (PNA,50µg/ml), wheat germ agglutinin (WGA, 10µg/ml), Helixpomatiaagglutinin (HPA, 6µg/ml), Dolichos biflorusagglu-tinin (DBA, 30µg/ml), soybean agglutinin (SBA, 18µg/ml),Lotus tetragonolobusagglutinin (LTA, 50µg/ml), Ulexeuropaeusagglutinin (UEA-I, 20µg/ml), and Limax flavusagglutinin (LFA, 25µg/ml). Peroxidase was developed wit3,3′-diaminobenzidine and hydrogen peroxide in TBEventually, sections were counterstained with hematoxyl
Histochemical staining using DIG-labeled lectins wperformed with the following lectins: Galanthus nivalisagglutinin (GNA, 60µg/ml), Datura stramoniumagglutinin(DSA, 10µg/ml), Sambucus nigraagglutinin (SNA, 30µg/ml), Maackia amurensisagglutinin (MAA, 10µg/ml)and Aleuria aurantiaagglutinin (AAA, 20µg/ml) as previ-ously reported (Sata et al, 1990). The main differences wthe above described method are that after endogenperoxidase blocking, the sections were incubated with (w/v) BSA in TBS for 10 min and, after incubation with thDIG-labeled lectins, the sections were incubated with HRconjugated anti-DIG-antibodies for 1 h at 0.6 U/ml in TBsupplemented with 1% (w/v) BSA.
Chemical deglycosylation (β-elimination and acidhydrolysis)Paraffin sections were treated with 0.5 N sodium hydroxin 70% ethanol at 4°C for 10 days, a procedure that removthe O-linked oligosaccharides (Ono et al, 1983). To remosialic acid residues the sections were immersed in 0.1hydrochloric acid for 2–3 h at 82°C (Schauer 1982; Madridet al. 1994).
Enzymatic deglycosylation (PNGase F treatment)To perform this technique the buffer 0.1 M Tris, 150 mNaCl, 2.5 mM EDTA (pH 9) was preferred. The paraff
fter-s,
se.
c.ooe):,
setth.ra-
.
.s
ithus
%
-
eseM
sections were incubated in the buffer containing 1% (triton X-100 for 1 h and then in the same buffer with 1(w/v) BSA for 10 min. After brief washing in the buffer, thsections were incubated in the enzyme EndoglycosiF/Peptide N-glycosidase F (Endo F/PNGase F) at 6 U/m3 days. At pH 9, PNGase F activity predominated over EF activity, so that cleavage of β-aspartyl glycosyl linkage(GlcNAcβ1-NAsn), and consequently the removal of theoligosaccharides, was preferred (Lucocq et al., 1987).
Enzymatic oxidation of GlcThe paraffin sections were washed in sodium acetate bufpH 5.0 and incubated overnight with 200 U/ml Type Vglucose-oxidase from Aspergillus nigerat 37°C in a moistchamber. Eventually, sections were washed in TBS ttimes for 5 m each and were stained with ConA lectin as u
ControlsThe following controls were used: 1. substitution of lectins and antibodies by the corresponding buffer, 2. prcubation of the lectins with the corresponding hapten suinhibitor (Fuc for AAA, UEA-I and LTA; GlcNAc forWGA; GalNAc for HPA, SBA and DBA; α-methyl-mannopyranoside for ConA and GNA; Neu5Ac for LFlactosamine for DSA; Gal for PNA; α2,3sialyllactosaminefor MAA and α2,6sialyllactosamine for SNA) at a concetration of 0,2M; 3. preabsortion of the anti-DIG antibodwith the corresponding antigen.
Results
Some subcellular structures were analyzed in detaillabeling in cell cytoplasm and 2. granular structure of PGCs, primary and secondary spermatogonia and primspermatocytes. The results are summarized in Table 2.
N-acetylgalactosamine (GalNAc)It is interesting to note that there was no cytoplaslabeling with any of the three GalNAc-binding lectins usThe staining in the granular structure of PGCs, spermgonia and spermatocytes was heterogeneous. After PNF procedure, new binding sites for SBA and HPA wrevealed, while DBA-labeling disappeared (Fig. 1).
Fucose (Fuc)The cytoplasm of the cells showed a slight labeling with UEI and AAA. The UEA-I-staining usually disappeared with β-elimination pre-treatment. The granular structure of the PGspermatogonia and spermatocytes was positive to AAA (2); UEA-I showed a variable labeling in the different cells.
Galactose (Gal)The cytoplasm of the cells was only stained with DSA. Tgranular structure of PGCs, spermatogonia and spermatocyteswere labeled with both lectins, but more intensely with D
Ae,
nia
cidGA
LECTIN HISTOCHEMISTRY OFP. WALTLPREMEIOTIC MALE GERM CELLS. 305
Table 2 Lectin-labeling pattern in the premeiotic spermatogenetic cells of Pleurodeles waltl. PGC: primordial germ cells or pre-spermatogonia, C: cyto-plasmic labeling, GR: granular structure. Staining intensity: – negative, + weak, ++ moderate, +++ strong. When two symbols are employed, for example–/+, it indicates some variability
1 These labeling are growing according to the maturation of the spermatocytes.2 WGA lectin binds to both Neu5Ac and GlcNAc.
(Fig. 3); the PNA-staining was not observed after β-elimi-nation pre-treatment.
N-acetylneuraminic acid (Neu5Ac)The cytoplasm of the different cells was positive to LFand MAA. LFA and MAA labeled the granular structur
while SNA was only positive in PGCs and spermatogoafter β-elimination pre-treatment (Fig. 4).
N-acetylglucosamine (GlcNAc)As a general rule, it seemed that WGA-staining after ahydrolysis followed the same pattern as normal W
306 SÁEZ ET AL.
Fig 1 DBA lectin histochemistry a and DBA lectin histochemistry after PNGase F b of primordial germ cells (PGCs). A PGC with a labeled granulelocated into an indentation of the polimorphic nucleus can be seen in 1a. After PNGase F, PGCs with polimorphic nuclei have not labeling in the granularstructure 1b. Counterstained with hematoxylin. 650x.
Oro
lsTnott
a
a
ec
ag
tan aveeaeskelrtivelaet
teeAn
re be t
o-, itSBAltil-
enseNoman985;
ueses in theFucd ins ordesioticer, in
uskll
ultsuse &al.
llartin
lkedly in
thethelac-as and
ula,man
an-
nce
LECTIN HISTOCHEMISTRY OFP. WALTLPREMEIOTIC MALE GERM CELLS. 307
Fig 2 AAA lectin histochemistry of spermatogonia. A testicular lobule with follicular cells and spermatogonia. Labeling of granular structure in thecytoplasm can be seen. Counterstained with hematoxylin. 650x.
Fig 3 DSA lectin histochemistry of primary spermatocytes. A granular structure in the cytoplasm is stained. Counterstained with hematoxylin. 650x.
Fig 4 SNA lectin histochemistry a and SNA lectin histochemistry after β-elimination b of primary spermatogonia. Testicular sections showing primaryspermatogonia with their accompanying follicular cells. A stained cytoplasmic granular structure can only be seen after β-elimination. Counterstained withhematoxylin. 650x.
Fig 5 ConA lectin histochemistry a and ConA lectin histochemistry after Glucose-oxidase pre-treatment b of secondary spermatogonia. In a, a labeledspherical granule can be seen in the cytoplasm. No granular structure is labeled in b. follicular cells. Counterstained with hematoxylin. 650x.
labeling, but the intensity of staining could be lower. interest is the absence of staining to WGA after acid hydysis in the granular structure of spermatocytes.
Mannose (Man)To elucidate if ConA-labeling was Man-specific, we apreviously carried out the glucose-oxidase technique. cytoplasm of the cells was positive to both lectins, Coand GNA. The granular structure was positive with blectins; however it is interesting to note that in spermagonia GNA was unreactive and the ConA-labeling dispeared with glucose-oxidase pre-treatment (Fig. 5).
Discussion
In this article, the terminal carbohydrates of the oligoscharides of the Pleurodeles waltlpremeiotic male germcells has been investigated by means of lectin cytochistry, combined with several chemical and enzymatic produres to obtain information about the nature of the linkto the peptide.
The cytoplasm of the different cell types was positivedifferent lectins. Moreover, in the PGCs, spermatogonia spermatocytes a granular structure can be observed icytoplasm. These granules were close to the nucleuswere reactive for several lectins. A few HPA-positiGalNAc residues were found only after PNGase F trment, suggesting the location of this GalNAc in O-linkoligosaccharides. These GalNAc residues would be maby N-linked oligosaccharides, and so, they are not labby the lectin until the N-linked oligosaccharides aremoved. These granules also contained DBA-posiGalNAc residues located in N-linked oligosaccharidDBA-binding GalNAc has been localized in a granustructure of the bull spermatogonia that has been assumbe the Golgi apparatus (Ertl and Wrobel 1992). RecenSuda et al. (1998) have reported that DBA only boundtype A spermatogonia in active testes of Syrian hamsMesocricetus auratus, but it binds to type A, intermediatand type B spermatogonia in inactive testes, making DBuseful marker for this cell. SBA also labels lightly the graular structure, mainly after PNGase F, indicating the pence of GalNAc in O-linked oligosaccharides maskedN-linked oligosaccharides. SBA-binding GalNAc residuhave been previously reported in granular structure of
fl-
oheAho-p-
c-
m-e-e
ondthend
t-dededee
s.rd toly,tors,
a-s-yshe
cricket Gryllus bimaculatusspermatogonia and spermatcytes (Suzuki & Nishimura, 1995). In the last cell typehas been seen at the electron microscopic level that lectin bound to some clusters of dense bodies with a muaminar structure. The nature and function of these dbodies are unknown (Suzuki & Nishimura, 1995). labeling with these lectins has been shown in the huspermatogonia and spermatocytes (Lee & Damjanov, 1Malmi et al., 1987; Wollina et al, 1989).
The granular structure also contained Fuc residmainly visualized with AAA, although in some cell typUEA-I was also positive. LTA did not label any granuleP. waltl testes, but has been shown to label the Golgi ofhamster spermatocytes (Ballesta et al., 1991). The residues shown in the present work were mainly locateN-linked oligosaccharides, since PNGase F decreaseabolishes the staining. Fucosyl N-linked oligosaccharihave also been reported in pre-meiotic and post-metesticular germ cells of the mouse (Maylie-Pfenning1994). Moreover, UEA-I-binding Fuc has been reportedthe lizard Podarcis campestrisspermatogonia (Labate &Desantis, 1995), but this lectin was negative in the mshrew Suncus murinus(Kurohmaru et al., 1995) and the bu(Arya & Vanha-Perttula, 1985) premeiotic cells. The resobtained in the human testis are difficult to explain, becaUEA-I positivity and negativity have been reported (LeeDamjanov 1985; Malmi & Söderström 1987; Malmi et 1987; Wollina et al. 1989).
The findings with PNA suggest the presence of Gaβ1,3GalNAc in O-linked oligosaccharides of the granustructure. On the contrary, the other Gal-binding lec(DSA) shows an unaltered reactivity after β-elimination andPNGase F pre-treatment, indicating that the Gaβ1,4GlcNAc sequence can be located in both O- and N-linoligosaccharides. Gal residues have been described onO-linked oligosaccharides in premeiotic germ cells of testis of the mouse (Maylie-Pfenninger, 1994), but, in rat, no conclusive evidence for glycans terminating in gatosyl residues, either of the N-linked or O-linked type, wshown (Jones et al., 1992b). PNA labels spermatogoniaspermatocytes of the guinea pig (Arya & Vanha-Pertt1986) and a granular structure in these cells in the (Malmi et al., 1987).
With respect to the Neu5Ac-binding lectins, in the grular structure the Neu5Acα2,3Galβ1,4GlcNAc sequencewas found both in N- and O-linked oligosaccharides, si
eee
a
t
t
p
trt
l
t
thn
e
8mrlarino
vthgie
rat,sent
ure is and
ed in a al, the bym,ed tot al.,ensemito-lu-ouy,e ofBArted of
cyto-t in1.5soci-The
Lin, the
ion al., theeralrater theling inor-
al.,and.ystystalesls atike
A-pe,stic et
ke)nd
308 SÁEZ ET AL.
MAA was positive after β-elimination and PNGase F. SincSNA-positivity in the granular structure of PGCs and spmatogonia is only observed after removal of O-linkoligosaccharides, the presence of Neu5Acα2,6Gal/GalNAcin N-linked oligosaccharides masked by O-linked oligosacharides is suggested. N-linked sialylgalactosides present in spermatogonia but not in spermatocytes of thewhile the latter have a population of O-linked sialylgalacsides absent from the former (Jones et al., 1992Moreover, in the spermatocytes of the lizard sialylgalatosyl glycans of the O-linked type have been shown (Lab& Desantis 1995).
In the PGCs and spermatogonia the granular strucshowed a similar WGA-binding with and without Neu5Aremoval by acid hydrolysis, suggesting that the staining wdue to the presence of GlcNAc residues. However, in smatocytes the WGA-binding of the granular structure wnot observed after acid hydrolysis, suggesting that WGA-binding was due to Neu5Ac. A granular structucontaining GlcNAc has been shown in the cytoplasm of spermatocytes of the common tree shrew Tupaia glisbymeans of Griffonia simplicifolia agglutinin II (Kurohmaruet al., 1996). Moreover, WGA-labeling of spermatogoniaspermatocytes was shown in the toad Bufo calamita(Ballesta et al., 1991), the lizard (Labate & Desantis 199the rat (Arya & Vanha-Perttula 1984) and the man (LeeDamjanov 1985; Malmi & Söderström 1987; Malmi et a1987), but not in the bull (Arya & VanhaPerttula 1985).
The ConA labeling in the granular structure of spermagonia and spermatocytes was almost abolished aglucose-oxidase procedure. This would indicate that Cois identifying Glc residues. On the contrary, in PGCs strain remained, indicating the presence of Man. Costaining, usually attributed to Man, has been reportedspermatogonia and spermatocytes of the lizard (LabatDesantis, 1995), the pigeon Columba livia(Ballesta et al.,1991), the guinea pig (Arya & Vanha-Perttula, 1986), the(Söderström et al, 1984; Arya & Vanha-Perttula, 198Jones et al., 1992a), the bull (Arya & Vanha-Perttula, 19and the man (Lee & Damjanov, 1985; Malmi & Söderströ1987; Malmi et al., 1987; Wollina et al., 1989), which avery repetitive results. In the present work, Man is aidentified with GNA, which was reactive in the granulstructure of PGCs and spermatocytes, but not in the gules of both primary and secondary spermatogonia. Sthe labeling disappears after PNGase F pretreatment it cbe concluded that these Man residues are located inlinked oligosaccharides. In the rat spermatogonia, GNbinding Man contained in N-linked oligosaccharides hbeen localized in the granular structure (Jones et al., 199Previous works have attributed a role for ConA-positiglycoproteins in germ cell-Sertoli cell interactions (Millet& Scott, 1984), and for N-linked oligosaccharides of thigh mannose type in the spermatocyte-Sertoli cell recotion (D’Agostino & Stefanini, 1990). Both the ConA-postive glycoproteins and the N-linked oligosaccharides w
r-d
c-re
rat,o-b).c-ate
urecaser-asheehe
or
5), &.,
o-fternAeA
in &
rat4;5),
esoran-ceuld
N-A-as2a).e
eeni--re
exclusively observed in the spermatocytes of mouse andrespectively. The Man residues described in the prework in the spermatocytes of P. waltl could be implied insimilar functions.
The origin and significance of the granular structdescribed in male premeiotic germ cells in this workunknown. In some of these amphibian cell types (PGCsprimary and secondary spermatogonia) the Golgi is formby several arrays of cisternae, closely packed togetherspherical cluster (Ikenishi & Nieuwkoop, 1978, Sáez et1989), making this organelle a candidate. Moreover, inprimary spermatocytes, the Golgi apparatus is formedmultiple small stacks distributed all over the cytoplassome of which are associated to granules that are believbe the precursors of the proacrosomal vesicles (Fraile e1992). On the other hand, several types of electrondmaterials and nuages that are frequently associated to chondria or vesicular bodies, originating cytoplasmic incsions, have been described (Sentein & Temple, 1971; R1974; Sáez, et al. 1989). Furthermore, the existenccluster bodies with a multilaminar structure positive to Sin spermatocytes of the cricket has been previously repo(Suzuki & Nishimura 1995). We should note that somethese organelles could be homologous of the Drosophilaspectrosome (Pepling et al., 1999). The spectrosome, a plasmic organelle implicated in cyst formation presenDrosophilagermline stem cells, is a spherical structure µm diameter that contains membrane vesicles and is asated to a centriole (Cuevas et al, 1997; Lin, 1997). molecular components of the Drosophila spectrosome aremembrane skeletal proteins and regulatory molecules (1997). During spermatogenesis, spectrosome give risefusome, a vesicle-rich organelle with similar compositthat is associated to the intercellular bridges (Hime et1996). The spectrosome and its derived organelle,fusome, are not only present in insect germ cells; sevcytoplasmic structures have been identified in vertebgerm cells that might correspond to the spectrosome ofusome due to their morphology and composition (Pepet al., 1999). For example the mitochondrial cloud foundXenopus oocytes, which give rise the germ plasm of primdial germ cells during embryo development (Kloc et 1998), and the reticular complex reported by Sentein Temple (1971) in the primordial germ cells of newt testis
Fusome-like structures are related with germline cformation during gametogenesis. The similarities in cmorphology of insects and vertebrates males and femsuggest that important functions take place in germ celthe time of meiosis (Pepling et al., 1999). A fusome-lstructure found in mouse germ cells is labeled by the EM1 antibody, which detects a carbohydrate epitosuggesting that glycoconjugates can play a role in cygerm cell development (Hahnel & Eddy, 1986, Pepplingal., 1999).
In summary, we have found a granular (fusome-listructure in the cytoplasm of PGCs, primary a
ytacpsperi
t ath b
moohes
tio
thatntia- and
firsttureatic
likesper-co-s, it may.
LECTIN HISTOCHEMISTRY OFP. WALTLPREMEIOTIC MALE GERM CELLS. 309
Fig 6 Schematic representation showing the carbohydrates located into the granular structure of the premeiotic spermatogenetic cells of Pleurodeleswaltl. Carbohydrates are classified into four groups according with their distribution pattern during the differentiation process, that is represented from theearliest steps (left) to the latest ones (right), as indicated in the top.
secondary spermatogonia and primary spermatocwhich contains several types of N- and O-linked oligoscharides, which may be put together in four grouaccording to their presence during the premeiotic smatogenetic process. These four sets, that are summain the figure 6, are made up of: 1. some glycans thalocated in all the four cell types above mentioned, 2. ocarbohydrate chains that are present in the PGCs andprimary and secondary spermatogonia, but not in the differentiated cell type, i.e. spermatocytes, 3. the carbdrates that are not in the early steps of spermatogenand 4. those that appear at the earliest and latest stagenot in the intermediates ones. This variable distribu
es-,r-zedreerothsty-sis,, butn
indicates i. the presence of some conservative motives are necessary during the premeiotic steps of differetion, ii. the appearance of new terminal carbohydratesthe removal of other as spermatogenesis progresses iii. theexistence of some terminal carbohydrates that are removed and then newly synthesized in the most macell types. These data suggest that, although no dramchanges in the glycosilation pattern of the fusome-structure are observed during the early steps of the matogenetic differentiation process, some cellular glyconjugates are modified in a predetermined way. Thucan be speculated that changes in glycan compositionbe related with cyst formation and the onset of meiosis
h
r
idv.f
u
e-
n
d,
eJ
l
lya
r
a
ic
,,
l
n
t.
edosyl-
9.rte-
lyco-
998.sa.
- in
i- 10,
-
a-11,
sis
of
.L.
éris-s:
g in
ing
e la
w4,
plex
ire
,
wn.
310 SÁEZ ET AL.
ACKNOWLEDGMENTS
The authors thank Mrs M. Portuondo and Mrs. C. Otamefor their technical assistance. This work was supportedgrants from the Autonomous Basque Government (PI-1948) and the University of the Basque Country (G10/9R.A. and E.A. were supported by fellowships from tUniversity of the Basque Country.
REFERENCES
Arya, M., Vanha-Perttula T. 1984. Distribution of lectin-binding in rattestis and epididymis. Andrologia 16, 495–508.
Arya, M., Vanha-Perttula, T. 1985. Lectin-binding pattern of bull testisand epididymis. J. Androl., 6, 230–232.
Arya, M., Vanha-Perttula, T. 1986. Comparison of lectin-binding pattein testis and epididymis of gerbil, guinea pig, mouse, and nutria.Amer. J. Anat., 175, 449–469.
Ballesta, J., Martínez-Menárguez, J.A., Pastor, L.M., Avilés, M., MadrJ.F., Castells, M.T. 1991. Lectin binding pattern in the testes of setetrapode vertebrates. Eur. J. Bas. Appl. Histochem., 35, 107–117
D’Agostino, A., Stefanini, M. 1990. An intercellular adhesion protein orat testis: isolation and preliminary characterization. Mol. Reprod.Dev., 25, 172–176.
De Cuevas, M., Lilly, M.A., Spradling, A.C. 1997. Germline cyst. formation in Drosophila. Annu. Rev. Gen., 31, 405–428.
De Rooij, D.G., Grootegoed, J.A. 1998. Spermatogonial stem cells. COpin. Cell. Biol., 10, 694–701.
Ertl, C., Wrobel, K.H. 1992. Distribution of sugar residues in the bovintestis during postnatal ontogenesis demonstrated with lectin-horseradish peroxidase conjugates. Histochemistry 97, 161–171.
Fraile, B., Sáez, F.J., Codesal, J., Paniagua, R. 1992. Characterizatiosecondary spermatocytes in the marbled newt (Triturus marmoratus).J. Anat., 180, 81–88.
Hahnel, A.C., Eddy, E.M. 1986. Cell surface markers of mouse primorgerm cell defined by two monoclonal antibodies. Gamete Res., 1525–34.
Hime, G.R., Brill, J.A., Fuller, M.T. 1996. Assembly of ring canals in thmale germ line from structural components of the contractile ring. Cell. Sci., 109, 2779–2788.
Ikenishi, K., Nieuwkoop, P.D. 1978. Location and ultrastructure ofprimordial germ cells (PGCs) in Ambystoma mexicanum. Dev.Growth. Differ. 20, 1–9.
Jones, C.J., Morrison, C.A., Stoddart R.W. 1992b. Histochemical anaof rat testicular glycoconjugates. 2. Beta-galactosyl residues in O- N-linked glycans in seminiferous tubules. Histochem J. 24, 327–33
Kloc, M., Larabell, C., Pean, A.P.Y., Etkin, L.D. 1998. Contribution ofMETRO pathway localized molecules to the organization of the gelineage.
Kornfeld, R., Kornfeld, S. 1985. Assembly of asparagine-linked oligoscharides. Annu. Rev. Biochem., 54, 631–664.
Koshimizu, U., Watanabe, D., Sawada, K., Nishimune, Y. 1993. A nostage-specific differentiation antigen is expressed on mouse testular germ cells during early meiotic prophase. Biol. Reprod., 49,875–884.
Kurohmaru, M., Kobayashi, H., Kanai, Y., Hattori, S., Nishida, T.,Hayashi, Y. 1995. Distribution of lectin binding in the testes of themusk shrew, Suncus murinus. J. Anat., 187, 323–329.
Kurohmaru, M., Maeda, S., Suda, A., Hondo, E., Ogawa, K., Endo, H.Kimura, J., Yamada, J., Rerkamnuaychoke, W., ChungsamarnyartHayashi, Y., Nishida, T. 1996. An ultrastructural and lectin-histo-chemical study on the seminiferous epithelium of the common treeshrew (Tupaia glis). J. Anat., 189, 87–95.
Labate, M., Desantis, S. 1995. Histochemical analysis of lizard testicuglycoconjugates during the annual spermatogenetic cycle. Eur. J.Histochem., 39, 201–212.
Lee, M-C., Damjanov, I. 1985. Lectin binding sites on human sperm aspermatogenic cells. Anat. Rec., 212, 282–287.
Lin, H. 1997. The Tao of stem cells in the germline. Annu. Rev. Gene31, 455–491.
ndi by97-9).e
n
,eral
-
rr.
of
ial
.
ysiss in
sisnd
6.
m
c-
vel-
N.,
ar
d
,
Lucocq, J.M., Berger, G.E., Roth, J. 1987. Detection of terminal N-linkN-acetylglucosamine residues in the Golgi apparatus using galacttransferase and endoglucosaminidase F/peptide N-glycosidase F:adaptation of a biochemical approach to electron microscopy. J.Histochem. Cytochem. 35, 67–74.
Madrid, J.F., Ballesta, J., Castells, M.T., Marín, J.A., Pastor, L.M. 198Characterization of glycoconjugates in the intestinal mucosa of vebrates by means of lectin histochemistry. Acta. Histochem.Cytochem., 22, 1–14.
Madrid, J.F., Castells, M.T., Martínez-Menárguez, J.A., Avilés, M.,Hernández, F., Ballesta, J. 1994. Subcellular characterization of gproteins in the principal cells of human gallbladder. A lectin cyto-chemical study. Histochemistry 101, 195–204.
Madrid, J.F., Leis, O., Díaz-Flores, L., Sáez, F.J., Hernánde#alz, F. 1Lectin gold localization of Fucose residues in human gastric mucoJ. Histochem. Cytochem. 46, 1311–1320.
Madrid, J.F., Aparicio, R., Sáez, F.J., Hernández, F. 2000. Lectin cytochemical characterization of the N- and O-linked oligosaccharidesthe human rectum. Histochem. J. (in press).
Malmi, R., Söderström, K-O. 1987. Lectin binding sites in human semniferous epithelium, in CIS cells and in seminomas. Int. J. Androl.,157–162.
Malmi, R., Kallakoki, M., Suominen, J. 1987. Distribution of glycoconjugates in human testis a histochemical study using fluorescein- andrhodamine-conjugated lectins. Andrologia 19, 322–332.
Maylie-Pfenninger, M.F. 1994. Developmentally regulated oligosacchrides in mouse spermatogenetic cells. Arch. Biochem. Biophys., 3469–479.
Millette, C.F., Scott, B.K. 1984. Identification of spermatogenic cellplasma membrane glycoproteins by two-dimensional electrophoreand lectin blotting. J. Cell. Sci., 65, 233–248.
Ono, K., Katsuyama, T., Hotchi, M. 1983. Histochemical applications mild alkaline hydrolysis for selective elimination of O-glycosidicallylinked glycoproteins. Stain Technol., 58, 309–312.
Pepling, M.E., de Cuevas, M., Spradling, A.C. 1999. Germline cysts: aconserved phase of germ cell development? Trends Cell. Biol., 9,257–262.
Plummer, T.H., Edler, J.H., Alexander, S., Phelan, A.W., Tarentino, A1984. Demonstration of peptide-N-glycosidase F activity in endo-β-N-acetylglucosaminidase F preparations. J. Biol. Chem., 259,10700–10704.
Rouy, S. 1974. Observations cytologuiques sur deux éléments caracttiques de la structure du testicule multiple des amphibiens urodèlespermatogonies de réserve et épithélium columnaire. Arch. Anat.Micros. Morphol. Exp., 63, 183–202.
Runnebaum, I.B., Schill, W.B., Topferpetersen, E. 1995. ConA bindinproteins of the sperm surface are conserved through evolution andsperm maturation. Andrologia 27, 81–90.
Sáez, F.J., Fraile, B., Paniagua, R. 1989. Histological and quantitativechanges in the annual testicular cycle of Triturus marmoratusmarmoratus. Can. J. Zool., 68, 63–72.
Sáez, F.J., Madrid, J.F., Aparicio, R., Leis, O., Oporto, B. 1999. Lectinhistochemical localization of N- and O-linked oligosaccharides durthe spermiogenesis of the urodele amphibian Pleurodeles waltl.Glycoconi. J. 16, 639–648.
Sandoz, D. 1970. Etude cytochimique des polysaccharides au cours dspermatogénèse d’un amphibian anoure: Le Discoglosse Discoglossuspictus(OTTH). J. Microso., 9, 243–262.
Sata, T., Zuber, C., Roth, J. 1990. Lectin-digoxigenin conjugates: a nehapten system for glycoconjugate cytochemistry. Histochemistry 91–11.
Schauer, R. 1982. Chemistry, metabolism and biological functions ofsialic acid. Adv. Carbohydrate Chem. Biochem., 40, 131–234.
Schindelmeiser, J. 1986. Light and electron microscopic study on comcarbohydrates in the testis of Salamandra salamandraL (Amphibia,Urodela). Acta. Histochem., 78, 189–195.
Sentein, P., Temple, D. 1971. Développement d’un complexe réticulaau cours de l’evolution des spermatogonies primaires de Triturushelveticus Razoumowsky. Archiv. Anat. Micros. Morphol. Exp., 6083–94.
Söderström, K.O., Malmi, R., Karjalainen, K. 1984. Binding of fluores-cein isothiocyanate conjugated lectins to rat spermatogenic cells intissue sections. Histochemistry 80, 475–579.
Spicer, S.S., Schulte, B.A. 1992. Diversity of cell glycoconjugates shohistochemically: a perspective. J. Histochem. Cytochem. 40, 1–38
LECTIN HISTOCHEMISTRY OFP. WALTLPREMEIOTIC MALE GERM CELLS. 311
ev
ev
98.s in95.
9.30.
Staneloni, R.J., Leloir, L.F. 1982. The biosynthetic pathway of theasparagine-linked oligosaccharides of glycoproteins. CRC Crit. RBiochem., 12, 289–326.
Suda, A., Hashimoto, O., Ogawa, K., Kurohmaru, M., Hayashi, Y. 19Distribution of lectin binding in spermatogonia of Syriam hamstergonadally active and inactive states. J. Vet. Med. Sci., 60, 189–1
.
.
Suzuki, A.C., Nishimura, K. 1995. Soybean agglutinin binding in germcells during spermatogenesis of the cricket, Gryllus bimaculatus. Zool.Sci., 12, 591–597.
Wollina, U., Schereiber, G., Zollmann, C., Hipler, C., Günther, E. 198Lectin-binding sites in normal human testis. Andrologia 21: 127–1
