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Acta Histochemica 114 (2012) 626– 635

Contents lists available at SciVerse ScienceDirect

Acta Histochemica

j o ur nal homepage: www.elsev ier .de /ac th is

istochemical analysis of glycoproteins in the gill epithelium of an Indian majorarp, Cirrhinus mrigala

idhi Srivastava, Usha Kumari, Amita Kumari Rai, Swati Mittal ∗, Ajay Kumar Mittal1

kin Physiology Laboratory, Centre of Advanced Study, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India

r t i c l e i n f o

rticle history:eceived 2 September 2011eceived in revised form5 November 2011ccepted 16 November 2011

eywords:

a b s t r a c t

Glycoproteins were analyzed by a range of histochemical methods in the epithelium of gills of Cirrhinusmrigala, a valuable food fish of great economic importance cultured extensively in India. The gills consistof gill arches, gill rakers, gill filaments and secondary lamellae. Major components of the epithelium ofgill arches and gill rakers are epithelial cells, mucous goblet cells, rodlet cells, lymphocytes, eosinophilicgranular cells and taste buds. In contrast, in the gill filament epithelium, rodlet cells and taste buds, and insecondary lamellae epithelium, rodlet cells, lymphocytes, eosinophilic granular cells and taste buds arenot discernible. The epithelial cells, the mucous goblet cells and the eosinophilic granular cells elaborate

istologyistochemistrylycoproteinsillsirrhinus mrigala

glycoproteins with oxidizable vicinal diols and glycoproteins with sialic acid residues without O-acylsubstitution. In addition, glycoproteins with O-sulphate esters are secreted by the mucous goblet cells.The rodlet cells elaborate glycoproteins with oxidizable vicinal diols. Different types of glycoproteinselaborated on the epithelial surface of gills are discussed in relation to physiological significance of gly-coprotein classes with special reference to their roles in lubrication, protection and inhibition of invasion

geni

and proliferation of patho

ntroduction

The epithelium of gills is a complex tissue and forms the firstarrier between the external and the internal environment. It isquipped with different cell types which are involved in the elab-ration of surface mucus, which is a gel-like viscous substance.lycoproteins (GPs), one of the major components of the mucus,ave a wide range of functions including protection, respiration,isease resistance, ionic and osmotic regulation, excretion, feeding,eproduction, communication and nest building (Shephard, 1994).

In the literature, there are extensive reports on fish gills withpecial reference to their role in respiration. There are severalmportant reviews on the structure of gills of several fish species inelation to their respiratory function (Hughes, 1984; Laurent anderry, 1995; Wilson and Laurent, 2002; Evans et al., 2005). Nev-rtheless, studies on the histological organization along with theistochemical nature of GPs secreted by various cell types in thepithelium in different regions of fish gills are scarce. Previous stud-

es mainly dealt with the histochemical analysis of mucous gobletells in different fish species using conventional methods of peri-dic acid–Schiff (PAS) procedures and the affinity of the anionic

∗ Corresponding author.E-mail addresses: swatimittal73@gmail.com (S. Mittal), profakmittal@gmail.com

A.K. Mittal).1 Present address: Retired Professor of Zoology (Banaras Hindu University), 9,ani Nagar, Kandawa, Near Chitaipur Crossing, Varanasi 221106, India.

065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.acthis.2011.11.009

c micro-organisms.© 2011 Elsevier GmbH. All rights reserved.

groups towards cationic dyes (Díaz et al., 2001; Arellano et al., 2004;Cinar et al., 2008, 2009; Diler and C inar, 2009). Díaz et al. (2005)demonstrated the presence of sialic acids and neutral glycoconju-gates in the mucous goblet cells of gill filaments and secondarylamellae of Micropogonias furnieri. Kumari et al. (2009) made a his-tochemical analysis of glycoproteins (GPs) in the mucus elaboratedby the secretory cells in the gill epithelium of Rita rita.

In the present investigation, histological and histochemicalmethods were employed to understand the structural organiza-tion and to identify the different classes of GPs elaborated by theepithelium in different regions of gills of an Indian major carp,Cirrhinus mrigala. Structural organization further supported by his-tochemical visualization of GPs possibly will reveal the functionalsignificance and physiological adjustment of gills in relation to thehabits and habitat of the fish.

C. mrigala is an Indian major carp belonging to the familyCyprinidae of the order Cypriniformes (Taxonomic Serial Number163679, retrieved from Integrated Taxonomy Information System,2008). It is an herbivorous bottom feeder fish feeding mainly onblue-green algae, diatoms and pieces of higher plants (Hora andPillay, 1962). It is a valuable quick growing fish and cultivatedwidely in India.

Materials and methods

Live specimens of C. mrigala (mean ± SD, Standard length, Ls,93 ± 9 mm; weight 12.63 ± 3.05 g; n = 10) were collected from local

N. Srivastava et al. / Acta Histochemica 114 (2012) 626– 635 627

Table 1Summary of single-dye histochemical methods employed for the visualization of glycoproteins (GPs) in the superficial layer (SL), the middle layer (ML) and the basal layer(BL) epithelial cells, the mucous goblet cells, the rodlet cells, the eosinophilic granular cells and the taste buds in the gill arch epithelium of Cirrhinus mrigala.

Histochemicalmethods

Reactions Interpretation ofreactions

Epithelial cells Mucousgoblet cells

Rodletcells

Eosinophilicgranular cells

Taste buds

SL ML and BL

1. WO/S M Free aldehydes 0 0 0 0 0 02. PAS M GPs with oxidizable

vicinal diols and/orglycogen

1–2M 1M 4M 1M 2M 1M

3. Acetylation/PAS 0 (M) Same as ‘2’ 0 0 0 0 0 04. Acetylation/deacetylation/PAS M Same as ‘2’ 1M 0–1M 3M 0–1M 1M 1M5. �-Amylase/PAS M GPs with oxidizable

vicinal diols0–1M 0–1M 3M 0–1M 1M 1M

0 (M) Glycogen6. AB2.5 T GPs with carboxyl

groups and/or GPs withO-sulphate esters

0–1T 0 4T 0 1T 0

7. AM/AB2.5 0 (T) Same as ‘6’ 0 0 0 0 0 08. AM/KOH/AB2.5 T GPs with carboxyl

groups0–1T 0 2T 0 1T 0

0 (T) GPs with O-sulphateesters

9. AB1.0 T GPs with O-sulphateesters

0 0 2T 0 0 0

10. AM/AB1.0 0 (T) Same as ‘9’ 0 0 0 0 0 011. AM/KOH/AB1.0 0 (T) Same as ‘9’ 0 0 0 0 0 012. LID PBr GPs with carboxyl

groups and/or GPs withO-sulphate esters

0–1Br 0 2PBr 0 0 0

13. LD 0 (PBr) Same as ‘12’ 0 0 0 0 0 014. HID PBr GPs with O-sulphate

esters0 0 1PBr 0 0 0

15. HD 0 (PBr) Same as ‘14’ 0 0 0 0 0 0

A ive ms cid/Scr

pataTCtfiipfiatwwaiwa2waiBaltlvtdtT

bbreviations: AB2.5, alcian blue at pH 2.5; AB1.0, alcian blue at pH 1.0; AM, actaponification; LD, low diamine; LID, low iron diamine; M, magenta; PAS, periodic aeaction; 1–4, weak to very strong reaction.

onds at Varanasi, India. The fishes were maintained in a laboratoryquarium with a layer of sand on the bottom for 24–48 h at con-rolled room temperature (25 ± 2 ◦C) and were fed daily with algaend minced yolk of boiled egg, plankton and commercial fish food,okyu® brand baby pellets (M.C.T. Aquarium, Import and Export,hangwat Nakhon Pathom, Thailand). The fishes were cold anes-hetized following the method of Mittal and Whitear (1978). Theshes were kept in water at room temperature (25 ± 2 ◦C); crushed

ce was added to the water gradually at such a rate that the tem-erature fell a few degrees every few minutes. At 10 ± 1 ◦C theshes became immobile and unresponsive to touching or prickingnd the fishes were taken out of water without struggle to excisehe gills. The fishes were euthanized after sampling. Pieces of gillsere excised and rinsed in physiological saline. Thereafter, gillsere fixed in aqueous Bouin’s fluid or in Carnoy’s fluid (Bancroft

nd Gamble, 2002). Following fixation, the tissues were dehydratedn an ethanol series of ascending concentrations, cleared in cedar

ood oil and embedded in paraffin wax. Serial sections were cut at thickness of 6 �m using a Leica Rotary Microtome (Model RM125RT, Leica Microsystems, Bensheim, Germany). The sectionsere mounted on ethanol cleaned glass slides and were kept in

n oven at 37 ◦C overnight to dry. Sections were deparaffinizedn xylene and hydrated in a descending ethanol series. Sections ofouin’s fluid-fixed tissues were stained with Ehrlich’s hematoxylinnd eosin (H/E) to study the histological organization of the epithe-ium in different regions of the gills. Sections of Carnoy’s fluid-fixedissues were subjected to a series of histochemical methods, fol-owing Yashpal et al. (2007), for the identification and simultaneousisualization of different classes of glycoproteins (GPs) in the secre-

ory cells (Tables 1–3). These include GPs with oxidizable vicinaliols, GPs with sialic acid residue with or without O-acyl substi-ution, GPs with O-sulphate esters and GPs with O-acyl sugars.he stained sections were dehydrated through a series of ethanol

ethylation; GPs, glycoproteins; HD, high diamine; HID, high iron diamine; KOH,hiff; PBr, purplish brown; S, Schiff; T, turquoise; WO, without oxidation; 0, negative

in ascending concentration, cleared in xylene, and mounted inDPX (Lendrum’s Distrene Dibutylphthalate Xylene) (Qualigens FineChemicals, GlaxoSmithKline Pharmaceuticals Ltd., Mumbai, India).

Observations were made on a Leitz “Laborlux S” microscope(Ernst Leitz, Wetzlar, Germany). The results were recorded usinga digital camera system Leica DFC 290 (Leica Microsystems, Wet-zlar, Germany) and an Intel® Pentium® D computer (Model dx2280MT, Hp Compaq, CA, USA).

Results

In C. mrigala, like in other teleosts, the gills are located on bothsides at the boundary of the pharynx and the opercular cham-ber. The first four pairs of gill arches are respiratory in function.Each gill arch bears two rows of gill rakers on its pharyngeal side,and two rows of gill filaments on its opercular side (Fig. 1a). Thedorsal and ventral surfaces of each gill filament are distinguishedinto a series of thin, leaf-like structures, the secondary lamellae.In general, at the major proximal region of each gill filament, thesecondary lamellae, are arranged parallel to each other in closeproximity, evenly spaced and appear similar in length (Fig. 1b).Nevertheless, at the small distal region of each gill filament, the sec-ondary lamellae, decrease in length towards the distal tip (Fig. 1c).As a result, the gill filament in this portion appears as an inter-lamellar cell mass (Fig. 1c). The epithelium covering the gill archesand the gill rakers, for convenience may arbitrarily be divided intothree principal layers: the superficial layer, the middle layer andthe basal layer (Fig. 2a and b). The basal layer lies on a thin acellu-lar basement membrane, which separates the epithelium from the

underlying tissues.

The epithelium covering the gill arches and the gill rakers con-sists of the cellular components – the epithelial cells, the mucousgoblet cells, the rodlet cells, the lymphocytes and the eosinophilic

628 N. Srivastava et al. / Acta Histochemica 114 (2012) 626– 635

Table 2Summary of double or triple-dye histochemical methods employed for the visualization of GPs in the superficial layer (SL), the middle layer (ML) and the basal layer (BL)epithelial cells, the mucous goblet cells, the rodlet cells, the eosinophilic granular cells and the taste buds at the gill arch epithelium of Cirrhinus mrigala.

Histochemicalmethods

Reactions Interpretation of reactions Epithelial cells Mucousgoblet cells

Rodlet cells Eosinophilicgranular cells

Tastebuds

SL ML and BL

16. AB2.5/PAS M GPs with oxidizable vicinal diols and/orglycogen

1–2MP 0–1M 4BlP 1M 2M 1M

T GPs with carboxyl groups and/or withO-sulphate esters

P GPs with oxidizable vicinal diols and/orglycogen and GPs with carboxyl groups and/orwith O-sulphate esters

17. AM/AB2.5/PAS M GPs with oxidizable vicinal diols and/orglycogen

1M 0–1M 3M 1M 2M 1M

0 (T) GPs with carboxyl groups and/or withO-sulphate esters

18. AM/KOH/AB2.5/PAS M GPs with oxidizable vicinal diols and/orglycogen

1MP 0–1M 3MP 1M 2M 1M

T GPs with carboxyl groups0 (T) GPs with O-sulphate esters

19. AB1.0/PAS M GPs with oxidizable vicinal diols and/orglycogen

1M 0–1M 2MP 1M 2M 1M

T GPs with O-sulphate estersP GPs with carboxyl groups and/or with

O-sulphate esters20. AM/AB1.0/PAS M GPs with oxidizable vicinal diols and/or

glycogen1M 0–1M 1M 1M 2M 1M

0 (T) GPs with O-sulphate esters

21. AM/KOH/AB1.0/PAS M GPs with oxidizable vicinal diols and/orglycogen

1M 0–1M 1M 1M 2M 1M

0 (T) GPs with O-sulphate esters

22. HID/PAS M GPs with oxidizable vicinal diols and/orglycogen

1MP 0–1M 3MPBr 1M 2M 1M

PBr, B GPs with O-sulphate esters

23. HID/AB2.5 T GPs with carboxyl groups 0–1T 0 3T 0 1T 0PBr, B GPs with O-sulphate esters

24. HID/AB2.5/PAS M GPs with oxidizable vicinal diols and/orglycogen

1MP 0–1M 4Bl 1M 2M 1M

T GPs with carboxyl groupsPBr, B GPs with O-sulphate estersP, Bl GPs with oxidizable vicinal diols and/or

glycogen and GPs with carboxyl groupsPBrM GPs with oxidizable vicinal diols and/or

A urplish

gbsilrlt(gscegt(acacr(l

glycogen and GPs with O-sulphate esters

bbreviations: B, black; Bl, blue; MP, magenta with purple tinge; P, purple; PBrM, p

ranular cells (Fig. 2a–d). In addition, sensory structures, the tasteuds, are also located. The epithelial cells, in general, appear wedge-haped or vertically flattened in the superficial layer, polygonaln the middle layer and low columnar or cuboidal in the basalayer. The nuclei of these cells are centrally placed and appearounded, ovoid or flattened (Fig. 2a). In contrast, in the epithe-ium covering the gill rakers the epithelial cells in different layers ofhe epithelium, in general, appear flattened with flattened nucleiFig. 2b). The mucous goblet cells in the epithelium covering theill arches and the inner surface of the gill rakers are small, goblethaped or rounded and are observed at irregular intervals oftenonfined in the outer layers (Fig. 2a and b). In contrast, in thepithelium on the outer surface of the gill rakers, the mucousoblet cells, in general, appear large, sac-like and extend up tohe deeper layers. The nuclei are rounded and basal in positionFig. 3a and b). The rodlet cells are small, oval or goblet-shapednd, in general, are located interspersed between the epithelialells in the outer layer of the epithelium of both the gill archesnd the gill rakers (Fig. 2a and b). The contents of these cells

haracteristically show slightly eosinophilic rod-like inclusions, theodlets, and the nuclei are basal, rounded and strongly basophilicFig. 2b and c). The lymphocytes are small, rounded or irregu-ar shaped and could be located at intervals in the intercellular

brown with magenta tinge. Rest of the symbols are as same as in Table 1.

spaces in between the epithelial cells in the basal layer and themiddle layer. Each lymphocyte has a thin rim of cytoplasm and arounded nucleus, which is strongly basophilic in H/E (Fig. 2a). Theeosinophilic granular cells are oval or rounded and in general, areirregularly distributed in the deeper layers of the epithelium cov-ering the gill arches and the gill rakers (Fig. 2a, b and d). In theH/E preparations the cytoplasmic contents appear granular andbrightly eosinophilic. The nuclei are eccentric, rounded or ovaland strongly basophilic. The taste buds are located at intermit-tent intervals in the epithelium covering the general surface ofthe gill arches. In the epithelium of the gill rakers, the taste budsare frequently located at their apical ends and on the inner sur-faces. Characteristically, the taste buds projected well above thegeneral surface of the epithelium are often arranged close to eachother on the inner surface of each gill raker (Fig. 3a and c). Thetaste buds could not be located on the outer surfaces of the gillrakers.

The main cellular components of the epithelium of the gill fila-ments, in contrast to that of the gill arches and the gill rakers, consist

of epithelial cells and mucous goblet cells. In addition, eosinophilicgranular cells are discernible at intermittent intervals. The epithe-lium covering the secondary lamellae, in general, is representedby a superficial layer and a basal layer lying on a thin acellular

N.

Srivastava et

al. /

Acta

Histochem

ica 114 (2012) 626– 635

629

Table 3Summary of histochemical methods employed to differentiate GPs involving oxidizable vicinal diols, GPs with O-sulphate esters, GPs with O-acyl sugars, GPs containing sialic acid residues with or without side chain O-acylsubstituent at C7 C8 and C9 in the superficial layer (SL), the middle layer (ML) and the basal layer (BL) epithelial cells, the mucous goblet cells, the rodlet cells, the eosinophilic granular cells and the taste buds at the gill archepithelium of Cirrhinus mrigala.

Histochemicalmethods

GPs withOxidizablevicinaldiols

GPs withO-acylsugars

GPs withO-sulphateesters

GPs with sialic acid side chainO-acyl substitution

Epithelial cells Mucous gobletcells

Rodletcells

Eosinophilicgranular cells

Tastebuds

None C7 C8** C9 SL ML and BL

25. PA/P/S 0 0 0 M M 0 0 1M 0 2M 0 1M 026. AB1.0/PA/P/S 0 0 T M M 0 0 1M 0 2MP 0 1M 027. KOH/AB1.0/PA/P/S 0 0 T M M M M 1M 0 2MP 0 1M 028. PA/Bh/KOH/AB1.0/PAS 0 M T 0 M M M 0 0 1T 0 0 029. PA/Bh/KOH/AB2.5/PAS 0 M T T P P P 1T 0 3T 0 1T 030. PA/Bh/KOH/PAS 0 M 0 0 M M M 0 0 0 0 0 031. PA/T/KOH/PAS Bl 0 0 Bl Bl M M 1Bl 0–1Bl 3Bl 1Bl 2Bl 1Bl32. PA/T/KOH/Bh/PAS Bl M 0 Bl Bl M M 1Bl 0–1Bl 3Bl 1Bl 2Bl 1Bl33. KOH/PA/DNPH/Az/KOH Y Y 0 Bl Bl Bl Bl 1GBlY 1Y 4 GBlY 1Y 2GBlY 1Y34. PA/DNPH/Az/KOH Y 0 0 Bl Bl 0 0 1GBlY 1Y 4 GBlY 1Y 2GBlY 1Y35. PA/DNPH/Az/KOH/Bh/PAS Y M 0 Bl Bl M M 1GBlY 1Y 4 GBlY 1Y 2GBlY 1Y36. PA/P/T/KOH/Bh/PAS 0 M 0 Bl Bl M M 1Bl 0 3Bl 0 2Bl 037. PA/Bh/KOH/PA*/Bh/PAS 0 M 0 0 0 0 0 0 0 0 0 0 0

Abbreviations: Az, azure A Schiff; Bh, borohydride; Bl, blue; DNPH, 2,4-dinitrophenylhydrazine; GBlY, greenish blue with yellow tinge P, phenylhydrazine; PA, periodic acid; PA*, selective periodate oxidation; T, thionin Schiff; Y,yellow; YG, yellow with greenish tinge; **, this group of sialic acid includes those with di-(C7 C8, C7 C9, C8 C9) or tri-(C7 C8 C9) side chain O-acyl substituents as well. Rest of the symbols are same as in Tables 1 and 2.

Fig. 1.

(a) Ph

otograph

of th

e gill

arch of

C. m

rigala. N

ote gill

rakers (barred

arrows)

on p

haryn

geal sid

e an

d gill

filam

ents

(arrows)

on op

ercular

side.

Scale bar

= 5

mm

. (b

and

c) Ph

otomicrograp

hs

of tran

sverse section

s of

gills of

C. m

rigala (H

/E). (b)

Part of

gill fi

lamen

t. N

ote secon

dary

lamellae

(arrows)

app

ear even

ly sp

aced an

d sim

ilar in

length

, on

dorsal

and

ventral

surfaces

of gill

filam

ent

(asterisks). Scale

bar =

30 �

m.

(c) D

istal region

of gill

filam

ent.

Note

second

ary lam

ellae (arrow

s) d

ecrease in

length

toward

s d

istal tip

of gill

filam

ent

and

the

region ap

pears

like an

inter-lam

ellar cell

mass

(asterisks). Scale

bar =

50 �

m.

630 N. Srivastava et al. / Acta Histoche

Fig. 2. Photomicrographs of transverse sections of gills of C. mrigala (H/E). (a) Gillarch with two adjacent gill rakers. The epithelium showing epithelial cells in super-ficial layer (SL), middle layer (ML) and basal layer (BL). Note distribution of mucousgoblet cells (arrows), rodlet cell (arrow head), eosinophilic granular cell (wingedarrow) and lymphocytes (barred arrows) in different layers. Scale bar = 20 �m. (b)Gill raker. The epithelium showing epithelial cells in superficial layer (SL), middlelayer (ML) and basal layer (BL). Note distribution of mucous goblet cells (arrows),rodlet cell (arrow head) and eosinophilic granular cell (winged arrow) in differentlayers. Scale bar = 20 �m. (c) Gill arch epithelium similar as (a) in higher magnifica-tion. Note in outer layers of epithelium, a rodlet cell (arrow head) with eosinophilicreb

bla

G

eeseocte

M

t

od-like inclusions. Scale bar = 10 �m. (d) Gill arch epithelium similar as (a). Noteosinophilic granular cells (winged arrows) in deeper layers of epithelium. Scalear = 20 �m.

asement membrane (Fig. 3d). The central core of the secondaryamellae consists of blood spaces, separated at regular intervals by

series of pillar cells (Fig. 3d).

lycoprotein histochemistry

The histochemical staining properties of the glycoproteins (GPs)laborated by different cellular components in the epithelium cov-ring the gill arches of C. mrigala are summarized in Tables 1–3. Thetaining properties of the corresponding cellular constituents in thepithelium covering the gill rakers, the gill filaments and the sec-ndary lamellae, in general, are similar to those in the epitheliumovering the gill arches. Negative reaction with method 1 indicateshe absence of free aldehydes in various cellular components of thepithelium in different regions of the gills.

ucous goblet cells

In the mucous goblet cells, reactions with methods 2–4, indicatehe possibility of the presence of GPs with oxidizable vicinal diols

mica 114 (2012) 626– 635

and/or glycogen (Fig. 4a and b). A slight decline in the intensity ofthe reaction with prior �-amylase digestion (method 5) confirmsthe presence of GPs with oxidizable vicinal diols in high concentra-tions together with small amounts of glycogen.

The reactions with methods 6–8 and 12 (Fig. 4c and d) indicatethat these cells may also contain GPs with carboxyl groups and/orGPs with O-sulphate esters. The possible presence of GPs with car-boxyl groups and/or GPs with O-sulphate esters together with GPswith oxidizable vicinal diols is further indicated by reactions withmethods 16–18 (Fig. 4e–g). The reactions with methods 19–22 alsoindicate that GPs with O-sulphate esters may be present togetherwith GPs with oxidizable vicinal diols. The reactions with methods9–11 and 14 further suggest the possibility of the presence of GPswith O-sulphate esters. Turquoise reaction with method 23 (Fig. 4h)and blue reaction with method 24 (Fig. 4i) indicates that GPs withO-sulphate esters are present in relatively low amounts that mayhave been masked by the presence of GPs with carboxyl groupsand/or GPs with oxidizable vicinal diols in higher concentrations.Weak turquoise reaction with method 28 confirms the presence ofGPs with O-sulphate esters in low moieties.

Magenta with purple tinge reaction with method 27 (Fig. 4j)indicates the possibility of presence of GPs with sialic acid residueswithout O-acyl substitution or with O-acyl substitution at C7,C8 and C9 as well as GPs with O-sulphate esters. Nevertheless,magenta purple reaction with method 26 indicates the presenceof GPs with O-sulphate esters possibly with GPs with sialic acidresidues without O-acyl substitution or with O-acyl substitutionat C7. The possible presence of GPs with sialic acid residues with-out O-acyl substitution or with O-acyl substitution at C7 is furtherindicated by magenta reaction with method 25.

Greenish blue with yellow tinge reaction with method 33indicates, in addition to the presence of GPs with oxidizable vic-inal diols, the possible presence of GPs with sialic acid residueswithout O-acyl substitution or with O-acyl substitution at C7, C8,C9 and GPs with O-acyl sugars as well. Reactions with methods 31,32, 34–36 (Fig. 4k) indicate the possibility of the presence of GPswith sialic acid residues without O-acyl substitution or with O-acylsubstitution at C7 and rule out the possibility of the presence of GPswith sialic acid residues with O-acyl substitution at C8 and C9 andGPs with O-acyl sugars. The reactions, turquoise with method 29(Fig. 4l) and negative with method 30 indicate that GPs with sialicacid residues present in these cells are without O-acyl substitution.Moreover, these reactions exclude the possibility of the presenceof GPs with sialic acid residues with O-acyl substitution at C7 andGPs with O-acyl sugars. The absence of GPs with O-acyl sugars isfurther confirmed by a negative reaction with method 37.

Analysis of the results obtained by a combination of these meth-ods show that the mucous goblet cells contain high amounts of GPswith oxidizable vicinal diols, GPs with sialic acid residues withoutO-acyl substitution, small amounts of GPs with O-sulphate estersand traces of glycogen (Table 4).

Epithelial cells

In the superficial layer epithelial cells, reactions with most his-tochemical methods employed in this study are, in general, similarto those in the mucous goblet cells. In contrast, the reactions areweak. Further negative reactions with methods 9, 14 and 28 andmagenta reactions with methods 19, 26 and 27 rule out the possi-bility of the presence of GPs with O-sulphate esters in these cells(Tables 1–3). Analysis of the results thus obtained shows that thesuperficial layer epithelial cells contain low moieties of GPs with

oxidizable vicinal diols, GPs with sialic acid residues without O-acylsubstitution and traces of glycogen (Table 4).

In the middle layer and the basal layer epithelial cells, the reac-tions, in general, with most of the histochemical methods used are

N. Srivastava et al. / Acta Histochemica 114 (2012) 626– 635 631

Fig. 3. Photomicrographs of transverse sections of gills of C. mrigala (H/E). (a) Distal region of gill raker. Note a series of taste buds (arrows) in epithelium on the inner surfaceand a large number of mucous goblet cells (arrow heads) in epithelium on the outer surface. Scale bar = 50 �m. (b) Similar as (a) in higher magnification. Note voluminousmucous goblet cells (arrows) in the epithelium on outer surface of gill raker. Scale bar = 10 �m. (c) Similar as (a) in higher magnification. Note taste buds (arrows) close toeach other in the epithelium on inner surface. Scale bar = 10 �m. (d) Similar as (Fig. 1b) in higher magnification showing a part of gill filament (asterisks) with secondarylamellae (arrows). The core of secondary lamellae consists of pillar cells (winged arrows) and blood spaces (arrow heads) between them. Note epithelial cells are arrangedin 1–2 layers in the epithelium of secondary lamellae and in several layers in the epithelium of gill filament. Scale bar = 20 �m.

Table 4Summary of glycoprotein classes secreted by the secretory cells in the epithelium covering the gills of Cirrhinus mrigala.

Secretory cells Glycoproteins Concentrations

Superficial layer epithelial cells GPs with oxidizable vicinal diols Small amountsGPs with sialic acid residues without O-acyl substitution Small amounts

Middle and basal layer epithelial cells GPs with oxidizable vicinal diols Small amounts

Mucous goblet cells GPs with oxidizable vicinal diols High amountsGPs with O-sulphate esters Small amountsGPs with sialic acid residues without O-acyl substitution High amounts

Rodlet cells GPs with oxidizable vicinal diols Small amounts

Eosinophilic granular cells GPs with oxidizable vicinal diols Moderate amountsGPs with sialic acid residues without O-Acyl substitution Moderate amounts

632 N. Srivastava et al. / Acta Histochemica 114 (2012) 626– 635

Fig. 4. Photomicrographs of cross sections of gills of C. mrigala, showing histochemical reactions for glycoproteins. (a) Gill arch epithelium. Note strong magenta reactionin mucous goblet cells (arrows), weak to moderate in superficial layer epithelial cells (arrow heads) and moderate to strong in eosinophilic granular cells (winged arrows)(PAS). Scale bar = 20 �m. (b) Gill raker. Note strong magenta reaction in mucous goblet cells (arrows) and weak to moderate in superficial layer epithelial cells (arrow heads).A taste bud (open arrow) stained weak magenta is discernible at the apical end of gill raker (PAS). Scale bar = 20 �m. (c) Gill filament (asterisks) with secondary lamellae(dotted arrows). Note strong turquoise reaction in mucous goblet cells (arrows) and weak in superficial layer epithelial cells (arrow heads) (AB2.5). Scale bar = 20 �m. (d)Gill raker epithelium. Note strong turquoise reaction in mucous goblet cells (arrows) and weak in superficial layer epithelial cells (arrow heads) (AB2.5). Scale bar = 20 �m.

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egative. Nevertheless, the reactions with methods 2–5 indicateresence of GPs with oxidizable vicinal diols in low moieties asell as glycogen in traces. Moreover, weak magenta reactions withethods 16–22 and 24, weak blue with methods 31 and 32 and

ellow with methods 33–35 confirm the presence of GPs with oxi-izable vicinal diols in low moieties and rules out the possibility ofhe presence of GPs with O-sulphate esters and GPs with sialic acidesidues (Tables 1–3).

osinophilic granular cells

In eosinophilic granular cells, reactions with most histochemicalethods employed in this study are, in general, similar to those in

he mucous goblet cells. In contrast, the reactions are moderate.urther negative reactions with methods 9, 14 and 28 and magentaeactions with methods 19, 26 and 27 rule out the possibility of theresence of GPs with O-sulphate esters in these cells (Tables 1–3).nalysis of the results show that the eosinophilic granular cellsontain moderate amounts of GPs with oxidizable vicinal diols, GPsith sialic acid residues without O-acyl substitution and traces of

lycogen (Table 4).

odlet cells, lymphocytes and taste buds

In the rodlet cells and the taste buds, reactions with the series ofistochemical methods (Tables 1–3) indicate the presence of smallmounts of GPs with oxidizable vicinal diols and traces of glycogen.egative reactions with the histochemical methods employed in

he study indicate that the GPs are absent in the lymphocytes.

iscussion

The epithelium covering different regions of the gills: the gillrch, the gill raker, the gill filament and the secondary lamellae of. mrigala, revealed differential distribution of various cell typesnd their glycoprotein composition.

The presence of mucous goblet cells on the surface epithelium ofhe gill is a common characteristic of fishes, but their density mayary in different regions of the gill. The high density of mucous gob-et cells on the outer surface of gill raker epithelium in C. mrigala isnteresting. Copious mucus secretion in this region could providerotection on the surface. In addition, this could be regarded as andaptation, following Ojha and Hughes (1988), in the removal ofediments from ventilating water and help maintain the epithe-ium free from deposits, which could interfere with their efficientunctioning. Munshi and Singh (1968) also reported that the mucusecreted by the mucous goblet cells of gills helps to keep their fila-ents clear of mud and fine sediments, which will otherwise clog

hem up with deposits.Analysis of the histochemical results revealed that the various

ellular components elaborate a mixture of different classes of GPsTable 4). Release of GPs with oxidizable vicinal diols on the sur-ace of the epithelium in small amounts by the superficial layerpithelial cells and in high concentration by the mucous goblet

e) Gill raker epithelium. Note strong blue with purple tinge reaction in mucous goblet cearrow heads) (AB 2.5/PAS). Scale bar = 20 �m. (f) Gill filament (asterisks) with secondaryarrows) and weak magenta with purple tinge in superficial layer epithelial cells (arrow hlue reaction in mucous goblet cells (arrows) and moderate magenta in eosinophilic granote strong turquoise reaction in mucous goblet cells (arrows) and weak in superficialote strong blue reaction in mucous goblet cells (arrows) and weak magenta with purar = 20 �m. (j) Gill raker. Note strong magenta with purple tinge reaction in mucous gobleKOH/AB1.0/PAP/S). Scale bar = 50 �m. (k) Distal region of gill filament. Note strong greeniosinophilic granular cells (winged arrows) and weak in superficial layer epithelial cells (trong turquoise reaction in mucous goblet cells (arrows) and weak to moderate in superfi

mica 114 (2012) 626– 635 633

cells may be concerned with the diversified physiological activi-ties. These GPs may control the acidity of acidic GPs (Tsukise andYamada, 1981; Mittal et al., 1994), have a buffering effect on thehigh acidity of the stomach (Smith, 1989; Scocco et al., 1996),assist in the transport of macromolecules (Stroband et al., 1979;Northcott and Beveridge, 1988), in the digestion of food, its trans-formation into chyme and in absorptive functions (Domeneghiniet al., 2005; Khojasteh et al., 2009) and protect the mucosa againstproteolytic degradation.

Secretion of small amounts of GPs with O-sulphate esters onthe surface of the epithelium by mucous goblet cells in differentregions of gills in C. mrigala could function in lubrication to protectthe gills against mechanical damage. In addition, the lubrication ofthe pharyngeal cavity may assist in smooth transport and swallow-ing of food. Tibbetts (1997) reported that the GPs with O-sulphateesters in mucus are considered to increase the viscosity of mucus.Thus, it may be surmised that the mucus is viscous in nature owingto the presence of GPs with O-sulphate esters, and it may play acrucial role in trapping food particles in the gills. Sibbing and Uribe(1985) also suggested that highly viscous mucous sulphomucins aidin trapping small particles and the less viscous sialomucins main-tain a laminar flow during the lubrication and particle handling inthe pharynx of Cyprinus carpio.

The primary role of GPs with sialic acid residues has been impli-cated in the protection of the tissues against bacterial degradation(Culling et al., 1974; Al-Suhail et al., 1984a,b) and with the inhibi-tion of the invasion of viruses (Rosenberg and Schengrund, 1976;Herrler et al., 1985; Wharton et al., 1989). In their studies onthe human respiratory tract, Ramphal and Pyle (1983) suggestedthat sialic acids in mucus act as receptors for bacteria, bindingtightly to them and thus preventing them from adhering to theunderlying epithelial cells. Schulte and Spicer (1985) postulatedthat sialic acids and related saccharide residues could serve asreceptor sites for binding of exogenous macromolecules such asthose of bacterial or viral etiology, thus playing a role in the organ-ism’s host defence mechanisms. The presence of GPs with sialicacid residues in the mucus secretions that are in high amounts onthe surface of the gills of C. mrigala may be associated with theadherence of micro-organisms and could be defensive, i.e., thesesaccharide residues could protect the underlying cell surfaces frommicrobial attachment.

The presence of the rodlet cells in the epithelium covering thegill arches and the gill rakers and their absence in the gill filamentsand secondary lamellae of C. mrigala is interesting. However, thesecell were reported in the gill filaments and secondary lamellaeof several fish species, e.g., Micropogonias furnieri (Mattey et al.,1979; Díaz et al., 2005), Abramis brama (Dezfuli et al., 2003), C.carpio (Mazon et al., 2007), Dicentrarchus labrax (Poltronieri et al.,2009) and in the gill filaments Cathorops spixii (Eiras-Stofella andFank-de-Carvalho, 2002); Brachydanio rerio (Karlsson, 1983). Sucha distribution of rodlet cells in different regions of the gill in the

fish has yet to be investigated.

In the gills of C. mrigala the rodlet cells secrete their contentson the surface of the epithelium. These cells are considered to beinvolved in non-specific defence mechanism of the skin or other

lls (arrows) and weak magenta with purple tinge in superficial layer epithelial cells lamellae (dotted arrows). Note strong blue/purple reaction in mucous goblet cellseads) (AB2.5/PAS). Scale bar = 20 �m. (g) Distal region of gill filament. Note strongular cells (winged arrows) (AB2.5/PAS). Scale bar = 50 �m. (h) Gill arch epithelium.

layer epithelial cells (arrow heads) (HID/AB2.5). Scale bar = 20 �m. (i) Gill raker.ple tinge in superficial layer epithelial cells (arrow heads) (HID/AB2.5/PAS). Scalet cells (arrows) and weak magenta in superficial layer epithelial cells (arrow heads)sh blue with yellow tinge reaction in mucous goblet cells (arrows), moderate in thearrow heads) (PA/DNPH/Az/KOH). Scale bar = 50 �m. (l) Gill raker epithelium. Notecial layer epithelial cells (arrow heads) (PA/Bh/KOH/AB2.5/PAS). Scale bar = 50 �m.

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sh tissues (Mazon et al., 2007). There seems to be a relationshipetween the presence of rodlet cells and the likelihood of stresso the fish (Leino, 1996; Dezfuli et al., 2000). Their number risesn response to diverse types of pathogens and also as consequencef other types of exogenous stressors (Schmachtenberg, 2007). Thetructure and functions of the rodlet cells are well documented.evertheless, the literature regarding the histochemical aspects of

hese cells is fragmentary. Analysis of the results obtained by theistochemical methods employed in this investigation has revealedhat the rodlet cells secrete GPs with oxidizable vicinal diols. Hirjind Courtney (1979) reported that the rodlet cells stain faint pinktriations with PAS and fail to stain with Alcian blue at pH 2.5. Thectivities of alkaline phosphatases at the periphery of the rodletsnd peroxidise activity at their cores have been reported by Igernd Abraham (1997).

The eosinophilic granular cells, a type of intrusive or wanderingell, are observed in the epithelium of the gill arches, the gill rak-rs and the gill filaments in C. mrigala. Analysis of results obtainedrom a series of other histochemical reactions in the present inves-igation show that eosinophilic granular cells elaborate GPs withxidizable vicinal diols (= neutral mucopolysaccharides) in mod-rate amounts and GPs with sialic acid residues without O-acylubstitution (= acidic mucopolysaccharides) in low amounts. Theseells, however, do not contain GPs with O-sulphate esters, one ofhe essential components of the mammalian mast cells. The presentbservation corroborates with the reports that similar granulareucocytes in blood and connective tissues of Catostomus commer-oni contain non-sulphated acid mucopolysaccharides and neutralucopolysaccharides (Barber and Westermann, 1975). Further-ore, such cells were PAS positive in the gills of Sparus aurata

Noya and Lamas, 1996) and in blood and connective tissues ofeveral fish species, C. commersoni, Notropis atherinoides, Notropishotogenis, C. carpio, Carassius auratus, B. rerio, Salmo gairdneri androtopterus (Barber and Westermann, 1978a,b). Ezeasor and Stokoe1980) and Holland and Rowley (1998), on the contrary, reportedhat similar cells in S. gairdneri and Oncorhynchus mykiss respec-ively were PAS negative. Eosinophilic granular cells, reported inariety of tissues of different fish species, are thought to be involvedn the non-specific innate defence systems of fish and have beenhown to degranulate and induce neutrophil migration in responseo numerous immunological challenges (Reite, 1998; Matsuyamand Iida, 1999). Recently, Murray et al. (2007) provided the firstvidence of an antimicrobial peptide, pleurocidin, in the cyto-lasmic granules of an eosinophilic granule cell from the winterounder gill.

The pattern of distribution of the taste buds varies among fishpecies according to the difference in their feeding habits. Tasteuds, in general, are involved in chemical reception, which mighte used to help in food selection at swallowing. In C. mrigala, theresence of a series of taste buds on the inner surface of the gillakers projecting well above the general surface of the epithelium,s well as at intervals on the surface of gill arches, may possiblylay a vital role to increase the efficiency of the fish in sorting andelection of food items in the pharynx before they are transferredowards the esophagus.

We conclude that the superficial epithelial cells, the mucousoblet cells and the rodlet cells present in the gill epithelium ofirrhinus mrigala contribute to mucus glycoproteins on the surface.he distribution of taste buds on the pharyngeal side of the gillakers assists in feeding activities and the mucous goblet cells onhe outer surface of the gill rakers are concerned with the profuseecretion of the mucus to provide protection and to remove sed-

ments from ventilating water and keep the epithelium free fromeposits, preventing clogging of gills. This could play important role

n the maintenance of the structural and functional integrity, andaptation of the fish related to its behavior and habitat.

mica 114 (2012) 626– 635

Acknowledgements

Ms. Nidhi Srivastava was supported as Project Fellow underthe Major Research Project (Principal Investigator: Dr. Swati Mit-tal) sponsored by the University Grants Commission, Governmentof India. Dr. Usha Kumari was supported as Research Associatesponsored by the Council of Scientific and Industrial Research, Gov-ernment of India.

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