Immunocytological localization and identification of the MSH-producing cells in the pituitary of the stickleback (Gasterosteus aculeatus L.)

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

Immunocytological Producing

24,

203207

Localization Cells

in the

and Pituitary

(Gasterosteus ERNEST Laboratoire du CNRS

FOLLENIUS

(1974)

Identification

MAURICE

L.) P. DUBOIS

de Cytologie animale, Universite’ Louis Pasteur et E.RA. No. b7 000 Strasbourg and Station de Physiologie de la Reproduction de 1’INRA d Nouzilly SY380, France

Received

April

MSH-

of the Stickleback

aculeatus AND

of the

492

11, 1974

The cellular origin of MSH in the pituitary of Gasterosteus aculeatus has been demonstrated with a bovine ant.i-cuMSH serum. In the intermediate lobe only the lead hematoxvlin-positive cells become fluorescent and may hence be considered the source of MSH. However, with bovine anti+MSH serum both cell types are negative.

fied at the level of the intermediate lobe by biological tests (Baker, 1965), has recently been made by cytophysiological observations (Baker, 1972). The study of the histological changes occurring in correlation with the adaptation to black and white backgrounds and the corresponding variations of the MSH content of the pituitary point toward the participation of the PbH + cells. Other arguments in favour of the origin of MSH from the PbH+ cells have been obtained by treatments with metopirone (Olivereau, 1965) or with reserpine (Olivereau, 1972). Both treatments induce a darkening of the skin and corresponding degranulation of the ibH+ cells. In all these experiments, the second cell type (PAS+ ) seems not to be involved; however, as Olivereau (1967) and Baker (1972) have suggested for the eel, this cell type may play a role in osmoregulation in connection with the change from salt to fresh water. Leatherland (1970) ascribes this function to the chromophobic cells which according to his data change after the migration of the stickleback to fresh water. In several ultrastructural studies (Folldnius, 1963-1968; Jasinski et al., 1970) on fish pituitaries, two more or less different

The problem of the cellular origin of the melanocyte-stimulating hormone (MSH) in the fish pituitary has been analyzed in several different ways. Histological studies have shown that in many different species (Cyprinodonts: FolGnius, 1959, 1963; Leuciscus rutilus: Kerr, 1948, Olivereau, 1959; Perca fluviatilis: Bugnon, 1960, FolGnius, 1963; Anoptichthys jorduni: Mattheij et al., 1968; Anguilla anguilla: Oliverau and Herlant, 1960, Olivereau, 1964 ; Mugilidae : Stahl, 1958, Olivereau, 1968; Cichlasoma biocellatum: Mattheij et al., 1971; Gasterosteus aculeatus: Van Mullem, 1959, Zoarces viviparus: Oztan, 1966; Fugu niphobles: Chiba and Honma, 1973; Mystus: Prasad, 1972) the intermediate lobe contains two cell types. In this connection the lead hematoxylin-PAS technique introduced by Stahl (1958) for the differentiation of the two cell types of the intermediate lobe often has been used. One cell type stains with the Pb hematoxylin (PbH), whereas the other displays a positive PAS reaction. Our aim was to determine whether one or both cell types participate in the melanophore-regulatory process. An attempt to localize the melanophore-stimulating function, which had previously been identi203 Copyright All rights

@ 1974 by Academic Press, of reproduction in any form

Inc. reserved.

204

FOLLENIUS

cell populations have been described, but with ultrastructural analysis, it could not be decided whether their cytological differences were due to different stages in the secretory cycle of a single cell type or to the presence of two cell types. In some species such as Phoxinus laevis and Perca @viatiZis, the possibility of a gradual differentiation of the cells from an agranular state to a state characterized by numerous granules has been considered (FolEnius, 1963). This possibility seemed to fit well with the fact that cell renewal in the intermediate lobe is more important than in the other parts of the pituitary. Radioautographic studies after administration of tritiated thymidine had shown heavy labeling in this region of the pituitary in Lebistes reticulatus (Follinius, 1964). Itj appears, therefore, that the question of the cellular origin of MSH in te1eost.s needs reconsideration. To date, any direct proof of the presence of an MSH-like substance in certain cells of the metaadenohypophysis is lacking. Immunocytological techniqlles were employed to solve this problem. Several authors had used this technique with success for the detection of different hypophyseal hormones in fish; the celh~lar source of ACTH, prolactin, growth hormone, TSH, and gonadotropin have so far been identified in several species (Emmart et al.. 1966, 1967; Emmart,. 19F9; Aler, 1970; hlattheij, et al., 1971; MrKeown et al, 1971) but localization of MSH has not, yet been attempted. MATERIALS

AND

METHODS

The pituitaries of eight mature sticklebacks were fixed for 2 hr in Bouin-Hollande solution with sublimate according to Herlant’s method. After embedding in paraplast, sections of 5-pm thickness were treated in the following manner: first, rabbit anti-cuMSH or anti+MSH serum is applied, then the sites of fixation of this antibodv are revealed by a fluorescent sheep anti-rabbit globulin (Institut Pasteur, Paris). On the same slide three sections are treated the same way ; the fourth is subjected only to the second step and serves as control. Svnthetic bovine MSH (Ciba-Geigy BLle) has been used as antigen. For the induction of immune response in rabbit. aMSH linked to ovalbumin by glutaraldehyde (Vance, 19%) has

AND

DUBOIS

been administered intrasplenically (Dubois and Renoux, 1971) by a single injection containing 0.5 mg rvMSH mixed with an equal volume of Freund’s adjuvant (total volume, 0.5 ml). The rabbits were bled 1 mo after this injection. The antiserum is treated with ovalbumin to eliminate nonspecific antibodies. Absorption tests with specific antigen have been performed by one of us (P. Dubois) on mammalian material. The serum treated by (uMSH before the immunocytological reaction is inactive. The antiserum to synthetic bovine /3MSH (Ciba?Geigy BBle) was produced by several injections under the dorsal skin of rabbits (Dubois, 1972). The antigen linked to an albumin(bovine sel’urn albumin or ovalbumin). as indicated above, is emulsified with Freund’s complete adjuvant. Four injections at. IO-da> intervals were given. One month after thr last injection a final intravenous injection of the antigenic complex was administered. The animals were bled on the 5th and 7th days after the final injcxction. For the identification of the fluorescrnt, ~11s thcl sam(’ field is photographed in blue light, and then wiih phasr caontrast. Some preparations havr been colored after the immunoc+ytological treatment, with 111~ PbH-PAS method (M&onaill, 1947) to ascertain whi& c>ell type is displaying the imrmlnofluor~scent reaction RECWLTY

The treatment of the sections by the double immunocytological technique for (uMSH reveals only some cells in the fish adenohypophysis. They are all located in the intermediate lobe. A great number of them are grouped in this part of the pituitary. They display a strong fluorescence (Fig. 1). The fluorescence is evident throughout, the cytoplasm; the nucleus is devoid of fluorescent material and aDpears as a dark spot, in the center of eack cell. At higher magnificat,ion the heterogeneity of the cell population which constitutes the intermediate lobe becomes evident. Nonfluorescent, cells are scattered among the reactive cells, sometimes occurring in small clusters. The same field observed bv phase contrast (Fig. 2) reveals that the difference between the reactive cells and those which are nonreactive corresponds to the two celI types constituting the intermediate lobe. The fluorescent cells are not very prominent under phase contrast, whereas the negative cells correspond to the densely

MSH

CELLS

IN

&&?rOStt?U-S

FIG. 1. Immunocytological reaction after applicatic of Gasterosteus aculeatus. Three negative cells located by arrows. X 1300. FIG. 2. The same field photographed under phase rows) appear dark in this picture. X 1300.

granular cells which, therefore, give a rather dark picture. Further identification is achieved by staining the same section with PbH-PAS. With this staining, the fluorescent light cells take the lead hematoxylin whereas the nonreactive cells are PAS positive. On the same material immunocytological reactions with /?MSH have been made. The resu1t.s are negative for both cell types. A faint nonspecific fluorescence occurs over the caudal neurohypophysis. DISCUSSION

The results obtained by the immunocytological study of the fish intermediate lobe lead to several important conclusions. They consit,ute a strong argument in favour of the existence of two different cell types in this part of the pituitary. The problem of the origin of the MSH-like secretion which has been discussed in a great number of experimental studies (cf. Ball and Baker, 1969) seems resolved. According to our immllnocytological observations, the PbH+ cells secrete an MSH-like sub-

UdmtUs

m of bovine anti MSH serum near a mass of neurohypophyseal contrast.

The

three

nonfluorescent

to the intermediate lobe tissue are indicated cells

of Fig.

1 (ar-

stance. At present there is no indication of participation of the second cell type in this function. Study of other teleosts is needed to determine whether the conclusions reached in Gasterosteus aculeatus will hold for all species where the duality of cell types in the intermediate lobe has so far been demonstrated. In species like the sockeye salmon (Oncorhynchus nerka) where only one cell type has been described (McKeown et al., 1973)) it will be especially useful to examine the homogeneity of the cell population. The second conclusion concerns the fact that the immunocytological reaction has been obtained by an antibody directed against synthetic mammalian &SH. The MSH of teleost fishes and bovine &MSH seem to have common immuno-reactive sites. This fact is of great practical importance for the detection of arMSH-like substances existing in the intermediate lobe of the pituitaiies from species of the different classes of vertebrates. Though immunological specificity does not neces-

206

FOLLENIUR

sarily parallel hormonal specificity the hormonal polypeptrdes detected by immunocytological means used seem to be closely related and may, therefore, have similar biological activities. It remains to be established how far the MSH of fish, whose chemical composition is not yet known, is related to the aMSH of certain mammals. Previous work (Burgers, 1963) has only shown that a substance with the electrophoretic behaviour of (YMSH exists in the intermediate lobe of several vertebrates, including one teleost species (Gadus morhua). It remains to be explained why the results are negative with anti$MSH serum. The presence of /3MSH in the teleost pituitary was not clearly demonstrated by Burgers (1963). The electrophoretic analysis yields a strong peak for an ~YMSHlike substance but a very weak one for an ,8MSH-like substance. Furthermore, Lee et al. (1963) have shown that the composition of /3MSH varies in different mammals. Only anti-bovine ,8MSH has been used in our immunocytological reactions. Further studies with other anti MSH’s are needed before one may conclude whether PMSH is present in the teleost pituitary. ACKNOWLEDGMENTS The authors are indebted to Mrs. Doerr-Schott for very helpful advice and to Miss Meunicr and Miss Moritz for skilled technical assistance. We thank also Prof. H. A. Bern for his critical reading of the manuscript. REFERENCES ALER, G. M. (1970). Prolactin cells: study of the pituitary gland of the roach (Leucbcus rut&s) by immuno-histochemical methods. Acta Zool. 51,

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