GENERAL AND COMPARATIVE ENDOCRINOLOGY 59, 192-198 (1985)

Identification by lmmunofluorescence of ACTH-Producing Cells in the Pituitary Gland of the Tree Frog Phyla arboreal

EZIO CAMPANTICO, ALDAGUASTALLA, AND ELENA PATRIARCA

Dipartimento di Biologia Animale dell’llniversitri di Torino, Via Accademia Albertina, 17-10123, Turin, Italy Accepted September 14, 1984

The indirect immunofluorescence procedure was used to localize ACTH-producing cells in the pituitary distal lobe from Hy/a arborea adult specimens; the antiserum employed was rabbit anti-synthetic ACTH (l-24) conjugated with BSA. Immunohistochemical staining was suppressed by solid-phase absorption of the anti-ACTH with the specific antigen. In the distal lobe one cell population is immunoreactive; these cells are predominantly localized in the anterior half, both ventrally and dorsally; they exclusively correspond to type-3 basophils. In the intermediate lobe the total cell population is immunoreactive. Parallel inspections carried out on the pituitary of Xenopus laevis specimens gave results fully consistent with those reported by other authors. 0 1985 Academic Press. Inc.

Since in the literature information on the cytology of Hyla arborea pituitary is lacking, we have undertaken the study of this gland in this anuran species. The PRL- and STH-producing cells had already been immunohistochemically identified (Cam- pantico et al., 1985).

The purpose of the present investigation was to study the distribution of cortico- tropic cells in the pars distalis of H. arborea by means of the immunofluorescence tech- nique and to study their staining character- istics with classical histological methods. In addition, in order to get some profitable hint on the accuracy and sensitivity of the methods employed, parallel inspections were carried out on the pituitary of Xen- opus laevis specimens, in which identifi- cation and location of ACTH-producing cells are already available.

MATERIALS AND METHODS Animals. In the present study 11 H. arboreu adult

specimens (7 males and 4 females) were used. The animals were captured in October and sacrificed in March, after being maintained at 4” under dormant conditions. A lot of 6 Xenopus luevis adult specimens

’ This work was supported in part by a grant from the Italian Ministry of Education.

(3 males and 3 females) maintained at room tempera- ture (ZOO) in our laboratory was also employed.

Histological procedures. All the animals were sac- rificed under MS 222 (Sandoz) 0.5% anesthesia. Their skulls were fixed in Bouin-Hollande fluid, then the brains were isolated, dehydrated, embedded in par- affin, and cut sagittally at 5 urn. For each animal eight corresponding slides were prepared. each containing four pairs of adjacent sections, i.e., two pairs from the pituitary mid region and two from the lateral region: these slides were employed for immunohistochemical techniques. The remaining sections were stained with Cleveland-Wolfe or aldehyde-thionine-PAS-orange methods.

Immunofik~rescence technique. For fluorescence staining the indirect method was employed. After ap- plication of the primary antiserum, the sections were incubated in a moisture chamber for 26 hr at 6”. After rinsing in PBS, sections were incubated with FITC (fluorescein isothiocyanate)-conjugated swine anti- rabbit IgG at l/SO dilution for 1 hr in a moisture chamber (room temperature). The sections were then mounted in glycerol-PBS 1:2 and viewed and photo- graphed with a Leitz Ortholux II fluorescence micro- scope using epi-illumination (Ploemopak). The slides were then demounted and stained with Galgano’s method (modified Mallory) or PAS-orange.

The antiserum. The antiserum anti-ACTH was a gift from Dr. D. V. Sofroniew, Oxford, England, and pro- duced by immunization of rabbits with synthetic ACTH (l-24) conjugated with BSA. It was employed routinely at the dilution of 1:600.

Immunohistochemical controls. The method and serum specificity tests included incubation of adjacent paraffin sections with normal rabbit serum (NRS) or

192 0016~6480/85 $1.50 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

ACTH CELLS IN Hyh arboreu 193

with anti-ACTH serum preadsorbed on solid phase In the intermediate lobe the immunoreac- with ACTH covalently bound to agarose (Sepharose 4B CNBr-activated) beads, according to the procedure

tive cells correspond to the total cell pop- of Swaab and Pool (1975). ulation of the lobe (Fig. la).

Xenopus luevis. In the pituitary from X.

RESULTS laevis, -the (l-24)ACTH antibodies react with the totality of the cell population of

Hyla arborea. In the pituitary from H. the intermediate lobe and with one cell pop-

arborea the (l-24)ACTH antibodies consis- ulation in the distal lobe, which in the sec-

tently stain one cell population in the distal tions stained by classical cytological tech-

lobe and one in the intermediate lobe. niques corresponds to type-3 basophils. No

In the mediosagittal sections of the distal other cell type ever reacted with the anti-

lobe the immunoreactive cells are very nu- ACTH serum.

merous in the anterior half, both ventrally, Immunohistochemical controls. No reac- near the median eminence along the portal tion was observed when sections were in- vessels entering the rostra1 end of the cubated with normal rabbit serum in place gland, and dorsally beneath the interme- of primary antibodies. Absorption of the diate lobe (Fig. la). Although predomi- antiserum on synthetic (l-24)ACTH com-

nantly localized in the anterior part, some pletely abolished the immunoreaction. immunoreactive cells are scattered in the ventral region of the posterior third of the distal lobe (Fig. 2a). These cells, average in size, are round or oval, with a generally spheroid nucleus. The intensity of the im- munoreaction was uniform and very pro- nounced. The anti-( l-24)ACTH immuno- reactive cells of the distal lobe in the sec- tions stained by classical cytological techniques show definite staining charac- teristics. They are PAS-positive and weakly orange-positive with aldehyde-thionine- PAS-orange or PAS-orange techniques (Fig. lb). With Cleveland and Wolfe’s tri- chrome they stain violet; with Galgano’s method their cytoplasm stains bluish- green (Fig. 2b). These cells correspond to type-3 basophils described in the distal lobe of the amphibians hitherto examined. Nei- ther type-l nor type-2 basophils, which also occur in the pituitary of H. arborea, re- acted with anti-( l-24)ACTH serum. Anal- ogously, the cells identified by us as PRL- producing cells (type- 1 acidophils) and STH-producing cells (type-2 acidophils) in a previous study, never stained with anti- (l-24)ACTH serum, even though the distri- bution of PRL-producing cells is very sim- ilar to that of the anti-(1-24)ACTH immu- noreactive cells.

DISCUSSION

Our observations show that in the pitu- itary distal lobe of H. arborea the anti-(l- 24)ACTH immunoreactive cells exclusively correspond to type-3 basophils; neither type-l nor type-2 basophils reacted with (l- 24)ACTH antiserum.

The identification of ACTH-producing cells as type-3 basophils in the tree frog is in agreement with both cytophysiological and immunohistochemical data obtained in other anurans and urodeles. In the distal lobe of the pituitary of Amphibia, ACTH function was first attributed to type-3 ba- sophilic cells by van Kemenade and van Dongen (1967) and van Kemenade (1969). Their assumption was based on evidence gained from changes observed in this cell type in Rana temporaria specimens treated with metopirone, drug which inhibits the 11-p hydroxylation in the synthesis of cor- ticosteroids in the interrenal tissue, or with aldactone, an aldosterone antagonist. In R. esculenta uni- or bilateral interrenalectomy and metopirone administration were re- ported by DuPont (1967, 1968) and DuPont and Gaudray (1969) to induce some changes in cells whose location and morphological

194 CAMPANTICO, GUASTALLA, AND PATRIARCA

FIG. 1. Nearly midsagittal section through the pituitary of Hyla arborea (rostra] two thirds); rostra1 to right. Mag. x 330. (a) lmmunofluorescent localization of ACTH-like containing cells in the distal and intermediate lobe (IL).Section treated with anti-synthetic (I-24)ACTH serum diluted 1:600. (b) Same section as in (a) after staining according to PAS-orange method: type-3 basophils (PAS-positive cells) appear black; corresponding cells in (a) and (b) are indicated by arrows. Green filter.

features corresponded to those of type-3 treated specimens in R. temporaria (van basophilic cells by van Kemenade. How- Kemenade, 1971; Larsen et al., 1971) and ever, the staining affinities of these cells led in R. pipiens (Ortman et al., 1972). Within the above workers to interpret them as acid- the urodeles, the experimental approach ophils. Within the anura, further evidence led to the identification of type-3 basophilic of the corticotropic nature of type-3 baso- cells as ACTH-producing cells in Taricha philic cells was obtained by cytophysio- torosa (Bunt, 1969) and in Triturus cristatus logical investigations on experimentally carnifex (Mazzi, 1971; Mazzi et al., 1973).

ACTH CELLS IN Hyla urborea

FIG. 2. Nearly midsagittal section through the pituitary of Hyla arborea (caudal half); rostra1 to right; x 330. (a) Immunofluorescent localization of ACTH-containing cells in the ventral region of the posterior third. Section treated with anti-synthetic (l-24)ACTH serum diluted 1:600. (b) Same section as in (a) after staining according to Galgano’s method: type-3 basophils (bluish-green cells) appear black; corresponding cells in (a) and (b) are indicated by arrows. Red filter.

By means of immunohistochemical tech- niques alone, the same conclusions were reached, within the anura, in R. esculenta (Vaudry et al., 1972), R. temporaria (Doerr- Schott and Dubois, 1972; Doerr-Schott, 1974a, b, 1976), Rana ridibunda (Gracia- Navarro and Doerr-Schott , 1982)) Bufo vul- garis (Doerr-Schott and Dubois, 1972; Doerr-Schott, 1974a, b, 1976), Bombina variegata (Doerr-Schott, 1976), X. faevis

(Doerr-Schott and Dubois, 1972; Doerr- Schott, 1974a, 1976), and, within the uro- deles, in Triturus marmoratus (Doerr- Schott and Dubois, 1970; Doerr-Schott, 1974a, 1976), Ambystoma mexicanum (Hauser-Gunsbourg et al., 1973; Doerr- Schott, 1976), Salamandra salamandra (Doerr-Schott, 1976), Pleurodeles waftlii (Olivereau et al., 1976), and Triturus cris- tutus (Gaudino et al., 1983).

196 CAMPANTICO, GUASTALLA, AND PATRIARCA

Additional evidence for the production of ACTH in type-3 basophilic cells comes from parallel cytophysiological and immu- nohistochemical studies performed in R. escdenta by Doerr-Schott (1972) and Vaudry et al. (1977).

The method and serum specificity tests carried on by us, as well as the results ob- tained in Xenupus, fully consistent with those reported by Doerr-Schott and Dubois (1972) and Doerr-Schott (1974b, 1976), con- firm the reliability of the methods used here.

As to the localization, in the median sag- ittal sections of H. arborea distal lobe the corticotropic cells line the blood vessels en- tering the pars distalis from the median em- inence: this means that, although they are mostly concentrated in the rostra1 region, small groups may occur scattered in the central and ventral region of the posterior third (Fig. 3). From a comparative view- point, this distribution is very similar to that observed in some Bufonidae (van Oordt, 1968; Doerr-Schott and Dubois, 1972; Doerr-Schott, 1974a, b, 1976; Zuber et al., 1975; Zuber-Vogeli, 1983) and in Rana species (van Kemenade and van Dongen, 1967; van Kemenade, 1969, 1971; Larsen et al., 1971; Ortman et al., 1972; Doerr-Schott, 1972, 1974a, b, 1976; Doerr- Schott and Dubois, 1972; Vaudry et. al., 1972, 1977; Gracia-Navarro and Doerr- Schott, 1982).

On the contrary, in X. laevis pituitary the ACTH-producing cells are concentrated in a crescent-shaped zone around the rostra1 border of the pars distalis, beneath the me- dian eminence (Fig. 4). They are much fewer in number than in H. arboreu and less stainable histologically: they often ap- pear chromophobe-like after Galgano’s staining method.

As to the reactivity of the cells of the intermediate lobe to the anti-( l-24)ACTH serum, it should be recalled that ACTH and MSH are known to share a relevant part of

FIGS. 3 AND 4. Midsagittal diagrammatic sections showing distribution of ACTH containing cells (stars) in the distal lobe (DL) of pituitary of Hyla nrboreu (Fig. 3) and Xenopus laevis (Fig. 4). IL intermediate lobe; ME median eminence: NL neural lobe.

their molecules: thus also MSH cells may be recognized by the anti-ACTH serum (Hansen et al., 1980). It has to be stressed, however, that:

(1) According to Lowry and Scott (1975) in mammals ACTH appears to be synthe- sized in both the pars distalis and in the pars intermedia. In the pars intermedia ACTH acts as the precursor of MSH and CLIP.

(2) In rat pituitary gland, several workers, by means of immunohistochemical tech- niques, were able to demonstrate ACTH in both pars distalis corticotropic cells and pars intermedia cells (see, among others, Phifer and Spicer, 1970; Kraicer et al., 1973; Cantin et al., 1983).

(3) In the amphibians, the cells of the in- termediate lobe positively react with a-(17- 39)ACTH antiserum, sequence which is not common with a- or l3-MSH (Doerr-Schott, 1976; Vaudry et al., 1977).

REFERENCES Bunt, A. H. (1969). Fine structure of the pars distalis

and interrenals of Turicha torosa after adminis- tration of Metopirone (SU-4885). Gen. Camp. En- docrinol. 12, 134- 147.

ACTH CELLS IN Hyla arborea 197

Campantico, E., Guastalla, A., and Patriarca, E. (1985). Identification by immunofluorescence of prolactin- and somatotrophin-producing cells in the pituitary gland of the tree frog Hyla arborea. Gen. Camp. Endocrinol. 57, 110-116.

Cantin, M., Ballak, M., Lu, C. L., Seidah, N. G., and Chretien, M. (1983). Corticotropin (ACTH) and the N-terminal fragment of pro-opiomelanocortin are located in the same granules in cells of rat pituitary gland. J. Histochem. Cytochem. 31, 479-487.

Doerr-Schott, J. (1972). Les cellules corticotropes de l’hypophyse d’un amphibien apres interrenalec- tomie. Etude cytoimmunochimique et ultrastruc- turale. Z. Zellforsch. 132, 333-346.

Doerr-Schott, J. (1974a). Ctyo-immunochemical study of the hypophysial cells of amphibians by light- and electron-microscopy. Fortschr. Zool. 22, 245-267.

Doerr-Schott, J. (1974b). Localisation submicrosco- pique par cytoimmunoenzymologie de differents principes hormonaux de l’hypophyse de Rana temporaria L. J. Microsc. (Paris) 20, 151-164.

Doerr-Schott, J. (1976). Immunohistochemical detec- tion, by light and electron microscopy, of pituitary hormones in cold-blooded vertebrates. Gen. Comp. Endocrinol. 28, 487-512.

Doerr-Schott, J., and Dubois, M. P. (1970). Les cel- lules corticotropes de l’hypophyse de Triton (Tri- turus marmoratus Latr.). Mise en evidence par immunofluorescence dune secretion apparentee a l’hormone corticotrope [B-(1-24) corticotro- pine]. C.R. Acad. Sci. (Paris) 271, 1534-1536.

Doerr-Schott, J., and Dubois, M. P. (1972). Identifi- cation par immunofluorescence des cellules cor- ticotropes et melanotropes de l’hypophyse des Amphibiens. Z. Zellforsch. 132, 1323-331.

DuPont, W. (1967). Effects de la surrenalectomie bi- laterale sur la cytologic du lobe distal de l’hypo- physe de la grenouille verte (Rana escu- /enra):essai d’identification des cellules respon- sables de la secretion de l’hormone corticotrope. C.R. Sot. Biol. (Paris) 161, 2374-2376.

DuPont, W. (1968). Action de la Metopirone sur la cytologic du lobe distal de I’hypophyse de la gren- ouille verte, Rana esculenta (L.). C.R. Sot. Biol. (Paris) 162, 383-385.

DuPont, W., and Gaudray, A. (1969). Modifications cytologiques de l’hypophyse et de la glande in- terrenale de la grenouille verte, Rana esculenta L. aprts surrenalectomie unilaterale. C.R. Sot. Biol. (Paris) 163, 34-39.

Gaudino, Cl., Mazzi, V., and Fasolo, A. (1983). Im- munocytochemical identification of ACTH con- taining cells in the hypophysis of the crested newt. Monitore Zool. Ital. (N.S.) 17, 409-413.

Gracia-Navarro, F., and Doerr-Schott, J. (1982). Im- munohistochemical detection of adenohypoph-

yseal cells containing hormones in Rana ridi- bunda. Cell. Tissue Res. 222, 687-690.

Hansen, G. N., Hansen, B. L., and Hummer, L. (1980). The cell types in the adenohypophysis of the south-american lungfish, Lepidosiren para- doxa, with special reference to immunocytochem- ical identification of the corticotropin-containing cells. Cell Tissue Res. 209, 147-160.

Hauser-Gunsbourg, N., Doerr-Schott, J., and Dubois, M. P. (1973). Localisation par immunocytologie de secretions apparentees aux hormones thyreo- tropes, gonadotropes et corticotropes dans l’hy- pophyse distale de l’axolotl (Ambystoma mexi- canum, Shaw.). Z. Zellforsch. 142, 539-548.

Kraicer, J., Gosbee, J. L., and Bencosme, S. A. (1973). Pars intermedia and pars distalis: Two sites of ACTH production in the rat hypophysis. Neuroendocrkology 11, 156-176.

Larsen, L. O., van Kemenade, J. A. M., and van Dongen, W. J. (1971). Localization and identifi- cation of corticotropin-producing cells in the distal lobe of the Rana temporaria pituitary gland. Gen. Comp. Endocrinol. 16, 165- 168.

Lowry, P. J., and Scott, A. P. (1975). The evolution of vertebrate corticotrophin and melanocyte stimu- lating hormone. Gen. Comp. Endocrinol. 26, 16-23.

Mazzi, V. (1971). Preliminary observations on the ef- fect of subtotal adenohypophysectomy on some endocrine glands in the crested newt. Contribu- tion to the pituitary histophysiology. Atti. Accad. Sci. (Torino), I. Cl. Sci. Mat. Fis. Nat. 105, 639- 645.

Mazzi, V., Vellano, C., Giunta, C., and Bona, A. (1973). Localization of corticotropic activity in the pituitary of the crested newt. Monitore Zool. Zral. (NJ.) 7, 97-l 16.

Olivereau, M., Bugnon, C., Fellmann, D., Olivereau, J. M., and Aimar, C. (1976). Etude cytoimmuno- logique de l’hypophyse du Pleurodele. C.R. Sot. Biol. 170, 1147-l 151.

Ortman, R., Sirett, N. E., Cohen, K. H., and Druker, M. (1972). Bioassay studies of rostra1 and caudal halves of the pars distalis of Rana pipiens for ad- renocorticotropin and gonadotropin. Gen. Comp. Endocrinol. 19, 325-330.

Phifer, R. F., and Spicer, S. S. (1970). Immunologic and immunopathologic demonstration of adreno- corticotropic hormone in the pars intermedia of the adenohypophysis. Lab. Invest. 23, 545-550.

Swaab, D., and Pool, C. W. (1975). Specificity of ox- itocin and vasopressin immunofluorescence. J. Endocrinol. 66, 263-272.

van Kemenade, J. A. M. (1969). The effects of Me- topirone and Aldactone on the pars distalis of the pituitary, the interrenal tissue and interstitial tissue of the testis in the common frog, Rana fem- poraria. Z. Zelvorsch. 96, 466-477.

198 CAMPANTICO, GUASTALLA, AND PATRIARCA

van Kemenade, J. A. M. (1971). The localization of corticotropic function in the pars distalis of the pituitary in the common frog, Rana temporariu. J. Endocrinol. 49, 349-350.

van Kemenade, J. A. M., and van Dongen, W. J. (1967). The corticotropic cells in the common frog, Rana temporaria. Gen. Comp. Endocrinol. 9, 512.

van Oordt, P. G. W. J. (1968). The analysis and iden- tification of the hormone producing cells of the adenohypophysis. In “Perspectives in endocri- nology” (E. J. W. Barrington and C. Jorgensens, eds.), pp. 405-467. Academic Press, New York.

Vaudry, H., DuPont, W., and Leboulenger, F. (1972). Mise en evidence des cellules corticotropes de I’hypophyse de la grenouille verte, Rana escu- lenta L., par la technique d’immunofluorescence. C.R. Sot. Bio/. 166, 1362-1365.

Vaudry, H., Leboulenger, F., Trochard, M. C., and Vaillant, R. (1977). Changes in corticotropin pro- ducing cells in the pituitary of Rana esculenta L. following interrenalectomy and Metopirone treat- ment. An immunohistochemical study. Gen. Comp. Endocrinol. 32, 78-88.

Zuber, M., Doerr-Schott, J., and Dubois, M. P. (1975). Localisation par immunofluorescence des secre- tions apparentees aux hormones gonadotrope, corticotrope, mtlanotrope et somatotrope dans l’hypophyse de Nectophrynoides occidentalis. C.R. Acad. Sci. (Paris) 280, 1595-1598.

Zuber-Vogeli, M. (1983). L’hypophyse de Necfophry- noides occident& Angel (Amphibien anoure vi- vipare). Arch. Biol. (Bruxelles) 94. 109- 154.