Bulletin of the Japanese Society of Scientific Fisheries 52(11), 1895-1899 (1986)

Cytochemistry of Basophil Granulocyte in Carp and Puffer

Yuzuru Suzuki*

(Accepted April 15, 1986)

Basophil granulocytes of carp and puffer were studied cytochemically by light microscope. The basoghil cells were round with an eccentric nucleus and many coarse granules. These

granules were found to be extremely water soluble, aud fixed with basic dyes or fourth amines. They were stained by basophilic procedures and showed metachromasia with basic dyes. These findings indicate that the granules contain acid mucopolysaccharide, and"basophil"is a rational name for the granulocyte in fishes. However, these granules did not stain by astra blue or alcian blue procedures. Furthermore, metachromasia was not seen at pH 1.5 in puffer, and 3.0 in carp. In addition, basophilic material combined with basic dye was extruded easily even in extremely diluted electrolyte solution. These results indicate that the rnucapolysacchazide in fishes does not contain many suffate radicals in the molecule. This indication is in contrast to heparin in mam-malian basophils.

In mammals, basophil granulocytes are the

fe west cellular elements in the blood, and their functional features are not fully understood.

However, in some fishes such as carp and puffers, basophils are rather abundant, even though a few

studies were carried out. This inadequate study

could be due to unsettled nomenclature and dassification of fish leucocytes,1) and even some

investigators believe that the basophils do notexist in fishes. These converse results might be

due to the fact that the granules in fish basophils

are extremely water soluble. Therefore, they have been overlooked in most of the past studies.

In the present investigations, the morphological,

and cytochemical characteristics of the basophils were carefully studied.

Materials and Methods

Blood Preparation

Puffer Takifugu vermicularis, weighing 130-180g, were collected at Misaki, Kanagawa and

carp Cyprinus carpio, 170-350g, were purchased

from a local fish dealer. A toad Bufo vulgaris,

and a rabbit were also used for comparison.Blood was taken from dorsal aorta in carp and

from caudal vessels in puffer, by heparinized

syringes. Blood smears were made for cyto-

chemical observation.

Observations

For general observation, May-Grunwald

Giemsa (MGG) method of staining was employed.

Techniques for histochemical study of poly-

saccharide in the basophilic granules were as

follows: 1) fixation by basic dyes or fourth

amines.2) 2) PAS reaction, and 3) metachromatic

reaction. For metachromasia study, smears were

fixed and stained for 5 min with 1% methanolic

solutions of azure A or toluidin blue, then rinsed

in aquaous solution of hydrochloric acid of pH

1.5, 3.0 and 4.5.

Following enzymes were analyzed histochemical-

l y; peroxidase,3) acid phosphatase,4) alkalin phos-

phatase,5) β-gulucuronidase6) and esterase.7) For

analysis of esterase, two methods were adopted:

a-naphthyl butyrate and naphthol AS-D chloro-

acetate.

Solubility of the granules in electrolyte solution

was estimated by revised method of Scott and

Dorling.8) Smear preparations were fixed and

stained with 1% methylene blue methanolic solu-

tion for 5 min, then rinsed in various concentra-

tions of MgCl2 solutions. Maximum MgCl2

concentrations at which the basophilic granules

did not extruded out were determined.

Living basophils were also observed by a

Nomarsky differential interference contrast sys-

tem.

Results

General Observations

The basophil granulocytes were found to be

* Department of Fisheries, Faculty of Agriculture, The University of Tokyo, Bunkyo, Tokyo 113, Japan

(鈴 木 譲:東 京 大 学 農 学 部 水 産 学 科).

round with an eccentric nucleus. Their dia-

meters were 10 to 12μm in puffer and 13 to 15μm

in carp. In both species, coarse granules were

stained reddish purple (Fig. 1-a, b), which were

similar to rabbit (Fig. 1-c) and frog basophils.

Cytoplasm was stained weakly, and it was grayish

white colour. These coarse granules were highly

soluble in water. Therefore, they were extruded

out by Giemsa stain (fixed with methanol) or by

MGG stain with M/15 phosphate buffer. But

fine reticular structure with vacuoles remained in

the cytoplasm (Fig. 1-d, e). Dilution of dyes with

distilled water was essential for retention of the

granules in MGG stain. In carp, sometimes

degranulation was incomplete and the granules

became eosinophilic after extrusion of the baso-

philic material (Fig. 1-f).

1896 Suzuki

In addition, neutrophil granulocytes were also found in these species. The nuclei of these cell swere in different shapes, i.e. kidney shaped or seg-mented. Their unstained cytoplasm was filled

with fine and slightly eosinophilic granules. Eosinophils were not observed in both species.

CytochemistryThe granules were fixed, clearly stained, and

became water insoluble by 1% methandic solu-tion of basic dyes, such as methylen blue and safranin O. But the granules could not be stained with astra blue or alcian blue, and further de-granulation occured. The granules were also fixed well by methanolic solutions of fourth amines such as cetyl pyridinium chloride and cetyl tri-methyl ammonium bromide. The granules could be stained by Giemsa staining procedure.

Routine methods for metachromatic reactionswere not suitable for this leucocyte, because ofhighly solubility. of the granules. By revised

method, metachromasia was recognized as red-dish purple granules by azure A or toluidin blue, at pH 3.0 and 4.5 in puffer (Fig. 1-g), and at pH 4.5 in carp (Fig. 1-h).

The granules in carp basophils were clearly PAS positive, and the reaction was diastase re-sistant. However, degranulation occured in puffer basophils when methanol, Carnoy's fluid, methylenblue-methanol, and fburth amines-methanol were used for fixation. Therefore, PAS reaction was not recognized. The cytoplasm was weakly PAS positive.

The results of enzymatic cytochemical observa-tions are shown in Table 1. Due to degranula-tion, basophils reactions occured only in the

cytoplasm. In contrast to neutrophils which have active peroxidase and many hydrolases, the baso-

phils showed weak enzymatic activities except- with acid phosphatase.

Degranulation by ElectrolyteDegranulation occured at high MgCl2 con-

centration. Maximum concentration at which the granules did not extruded out was 0.01M in

puffer, 0.001M in carp 0.5M in toada and rabbit basophils.

Observation in the Living StateBasophii granulocytes of both carp.and puffer

had very active amoeboid movements (Fig. 2-a, b). They crawled on glass with lamellipods, com-

parable to those of neutrophils.

Discussion

Cytochemically, granules of basophils in carp, and puffer are characterized as follows: 1) ex-

tremely water soluble,2) insoluble in water after fixation with basic dyes or fourth amines, 3) basophilic, and 4) stain metachnomaticallq. These results indicate that the granules contain acid mucopolysaccharide. Similar results were

ported in basophils in mammals.9) Therefore, "basophil" is a rational name for that granulocyte in fishes.

In carp, the basophil granules were PAS posi-tive, but they do not contain glycogen, because

their reaction was diastase resistance. This find-ing indicates that the granules contain neutral

mucopolysaccharide or glycoprotein. These sub-stances can stain with eosin, when partial de-

granulation occurs. In contrast, the granules were not PAS positive in puffer. And the de-granulation was always complete. Therefore,

they have neither neutral mucopolysaccharide nor glycoprotein.

The basophilic granules showed following natures.1) They did not stain with astra bluc or alcian blue.2) Metachromasia was not seen at

pH 1.5 in puffer and 3.0 in carp.3) Basophilic material combined with basic dye was extruded out easily even in extremely diluted electrolyte solution. These findings indicate that acid muscopolysaccharide in fishes does not have maany sulfate radicals in the molecule. This is in con-trast to heparin in mammalian basopils.9)

"Basophils" have been seen in many species of fish, but they are called under different names.

Basophil in Carp and Puffer 1897

Fig. 1. Light micrographs of basophils. ×1700. a) Basophil of puffer, MGG stain. b)

Basophil of carp, MGG stain. c) Basophil of rabbit, MGG stain. d) Degranulated basophil

of puffer, methanol fixed Giemsa stain. e) Degranulated basophil of carp, methanol fixed

Giemsa stain. f) Partially degranulated basophil of carp, MGG stain. Granules are stained

eosinophilic. g) Basophils of puffer. The granules show methachromasia with azure A

(pH 3.0). h) Basophils of carp. The granules show methachromasia with azure A (pH 4.5).

That is because many workers did not pay enough attention to the nature of the cell as the granules are highly soluble in water. Kelenyi and Nemeth,10) who worked with carp, river bleak andtench, called them neutrophil. Chlebeck andphillips11) called the cell "secretory granulocyte" in two buffalofishes. They thought it was the advanced stage of neutrophil with vacuoles con-taining undissolved particles of the coalesced

granules. On the contrary, Yasuda et al.12) reported that the cells in carp were young neutro-

phils which stayed in the peripheral blood for shortperiod. Hines and Spira13) observed the ce

ll without granules, in carp. Therefore they called it finely reticuiar ceil. Lester and Daniels14) agreed to them and thought it is analogous to eosinophil as were observed in tissue preparations in white sucker. Sabins and Ramgneker25) and others 16-18) called it eosinophil. So far, no one

gave concrete reasons for naming this cell. How-ever in my study, I found that the granules in carp basophils become eosinophilic by partial de-

granulation. Incorrect use of the name "eosino-

Phil" is probably due to this phenomenon.

1898 Suzuki

Table 1. Enzymatic histochemical reactions of basophil (Baso) and neutrophil (Neu) of puffer

and carp

Puffer CarpEnzyme

Baso Neu Baso Neu

Peroxidase - + - +

Acid Phosphatase + + + +

Alkaline Phosphatase - - - -

β-Glucuronidase ± + ± +

Esterase

α-Naphthyl butyrate - + 士 +

Naphthol AS-D chloroacetate 士 - 士 +

Fig. 2. Living basophils of puffer (a) and carp (b) viewed by Nomarsky differential interference

contrast system. They crawl on glass with lamellipods (arrows). ×1800.

Barber and Westerman19,20) observed PAS

positive reaction of the granules of this type of cell in white sucker and named PAS positive

granular leukocyte. They concluded that this cell had no acid mucopolysaccharide from its negative reactions for metachromasia, and had no histamin because it was not affected by histamine liberator 48/80. They thought it may represent an evolutionally precursor of basophil. But most of the figures of light rnicroscopy presented in their reports showed degranulated forms. There-fore, I think reexamination is needed about cyto-chemical reactions.On the other hand, the term of basophil had

been used by Weinreb,21) and Watson et al,22) in

goldfish, and by Fey23) in many species. Fey studied cytochemically in detail and mentioned that the cells were positive for metachromasia, PAS positive, peroxidase negative, and esterase

positive. These results are almost equal to my findings. But in puffer I could not see PAS posi-tive reaction. The basophil of puffer may be

exceptional in fishes.With the exception of acid phosphatase, the

enzymatic activities of basophils were weak. Therefore they have weak ability in sterilization

and digestion. In contrast to neutrophils, the basophils of carp and puffer are not the scanvenger

cells, even both have enough motility.

Basophil in Carp and Puffer 1899

Acknowledgement

I wish to thank Professor I. Hanyu and Asso-ciate Professor K. Aida, Faculty of Agriculture,

The University of Tokyo, for reading the manu-script. This work was supported in part by

Grant-in-Aid for Encouragement of Young Scientist from the Ministry of Education, Science and Clilture.

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