GENERAL AND COMPARATIVE ENDOCRINOLOGY 59, 149-154 (1985)

lmmunoelectron Microscopic Localization of Growth Hormone in the Pituitary Glands of Two Teleosts, Tilapia (Sarotherodon

mossambicus) and Amago Salmon (Oncorhynchus rhodurus)

HIROSHI UEDA, HIROHIKO KAGAWA, AND SUNAO FUJIMOTO

Department of Anatomy, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807, Japan

Accepted October 22, 1984

Growth hormone (GH) cells were investigated with the protein A-gold technique on the pituitary glands of tilapia (Sarotherodon mossambicus) and amago salmon (Oncorhynchus rhodurus). By the use of specific antiserum against tilapia GH to both species, the immu- noreactive gold particles were demonstrated to be preferentially located on the secretory granules of the GH cells. Specimens tixed only with periodate-lysine-paraformaldehyde (PLP) preserved the hormonal antigenicity well. Osmium posttixation, although consider- ably reducing the antigenicity and thus resulting in a decrease in number of the gold particles on the GH cells, gave much more satisfactory ultrastructural preservation and immuno- reactive localization of immunoreactive material. This investigation demonstrated that, after combined fixation with PLP and PLP-osmium, we could determine the function of a given cell type in various endocrine organs as well as the precise antigenic sites in such Cells. 0 1985 Academic Press, Inc.

Recent advances in immunocytochem- ical techniques have been very useful for identification of pituitary hormone-pro- ducing cells in numerous species of teleosts (Follenius et al., 1978). The availability of antisera to teleost growth hormone (GH) has provided direct identification of GH cells in teleost pituitaries (Ingleton and Stri- bley, 1977; Komourdjian and Idler, 1979; Nagahama et al., 1981). More recently, ul- trastructural localization of GH in goldfish pituitary has been reported using antiserum against carp GH by the peroxidase-anti- peroxidase (PAP) technique (Cook et al., 1983).

The protein A-gold technique, first uti- lized in demonstrating the intracellular lo- calization of antigenic sites by Roth et al. (1978), has been so far used for the identi- fication of various hormone- or protein-pro- ducing cells in several endocrine glands (Garaud et al., 1980; Tanaka et al., 1980; Doerr-Schott and Garaud, 1981; Geuze et al., 1981; Bendayan, 1982; Kobayashi and Uchida, 1983). This technique makes it

easier than the PAP technique to distinguish immunoreactive products from electron- dense intracellular organelles. However, only a few studies have been concerned with the identification of pituitary hor- mone-producing cells (Batten and Hopkins, 1979; Larsson, 1979; Hisano et al., 1983).

The present study describes electron mi- croscopical localization of GH in the pitu- itary glands of tilapia and amago salmon by means of the protein A-gold technique using an antiserum against tilapia GH, with special reference to the effects of osmium postfixation on antigenic and cytoplasmic preservation.

MATERIALS AND METHODS

Animals and tissue preparation. Three adult male tilapia (Sarotherodon mossambicus) weighing 30-40 g, and five adult female amago salmon (Oncorhynchus rhodurus) weighing about 300 g, were killed by rapid decapitation and the pituitary glands were quickly re- moved. The proximal pars distalis (PPD) was sepa- rated from the rostra1 pars distalis and pars intermedia before being cut into smaller pieces. Specimens were

149 0016~6480185 $1.50 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

150 UEDA, KAGAWA, AND FUJIMOTO

fixed with 2% paraformaldehyde in 0.0375 M phos- phate buffer containing 0.01 M sodium metaperiodate and 0.075 M L-lysine monohydrochloride, pH 6.2 (PLP, McLean and Nakane, 1974) for 18 hr at 4”, and washed by 0.1 M phosphate buffer containing 5% su- crose for 18 hr at 4”. Some of specimens were post- fixed with 1% osmium tetroxide in 0.1 M phosphate buffer for 1 hr at 4”. Then all the specimens were de- hydrated by graded concentrations of acetone, and embedded in Epon 812. Ultrathin sections (60-80 nm) were mounted on uncoated 150-mesh nickel grids. The sections were stained with immunocytochemical pro- cedures listed below.

Immunocytochemistry. All procedures were con- ducted at room temperature. Colloidal gold was pre- pared by the method of Frens (1973). The protein A- gold complex was made by the method of Roth et a/. (1978). The nickel grids were placed on 1% egg al- bumin in phosphate-buffered saline (PBS) for 10 min to reduce nonspecific adsorption of protein on the sec- tions, and then incubated for 2 hr with 1: 100, 1500, and I:1000 dilutions of rabbit anti-tilapia GH serum, which was raised from highly purified and well-char- acterized tilapia GH (Farmer et al., 1976). After rinsing with PBS, the grids were immersed in the pro- tein A-gold complex undiluted solution for 1 hr, stained by 1% uranyl acetate for 20 min and Reynolds’ lead citrate for 1 min, and examined with a JEM 100 CX electron microscope. The specificity of the present immunocytochemical labeling was confirmed by the following controls: (1) the anti-tilapia GH serum was absorbed with tilapia GH, (2) normal rabbit serum or PBS was substituted for the anti-tilapia GH serum, and (3) the ultrathin sections were incubated only with the protein A-gold complex solution for 1 hr.

RESULTS

The present protein A-gold technique applied to the PLP-fixed tilapia pituitary re- vealed specific immunolabel on only one cell type-GH cells, as identified by ultra- structural features (Fig. 1). The gold parti- cles, approximately lo-20 nm in diameter, were mainly aggregated on secretory gran- ules, ranging from 200 to 300 nm in size, but a few nonspecific labelings were ob- served on the other cytoplasmic portion of the cells. No other cell types in the PPD of tilapia showed immunoreactivity. In the PLP-osmium-fixed specimens, the number of gold particles on GH cells was substan- tially decreased; they were located more frequently on secretory granules rather

than on rough endoplasmic reticulum or Golgi apparatus (Fig. 2). The preservation of these organelles was much better than that of the PLP-fixed specimens.

In the PPD of amago salmon, immuno- reactive gold particles were also concen- trated on secretory granules (200-350 nm in size) of only one cell type, namely GH cells, in both fixation methods (Figs. 3 and 4). No other immunoreactive cells were ob- served.

At all the dilutions of the antiserum, the immunolabeling was specific for one cell type in both species, but a high level of non- specific labelings was observed at a low di- lution (1: 100). The specific immunoreactive gold particles were almost abolished by the preincubation of the anti-tilapia GH serum with tilapia GH. In the other controls, very few gold particles were located on the cy- toplasmic matrix, nucleus, and mitochon- dria.

DISCUSSION

The present study revealed that the an- tiserum against tilapia GH labeled specifi- cally one cell type in the tilapia and amago salmon pituitaries. These labeled cells cor- respond to the GH cells identified by pre- vious ultrastructural analyses in the tilapia (Bern et al., 1974) and salmonid pituitaries (Nagahama, 1973). Nagahama et al. (1981), using the light microscopic indirect enzyme method, found that this antiserum reacted only with the GH cells in seven species of teleosts including tilapia and salmonids. The present investigation provides addi- tional confirmation that the antiserum reacts mainly with secretory granules of GH cells in both species. The immunoreac- tive gold particles on rough endoplasmic re- ticulum and on Golgi apparatus were very few in number suggesting that these organ- elles contain little or no immunoreactive material in the present fixative conditions.

The protein A-gold technique seems to be fairly useful for immunolabeling of ti- lapia GH on the PLP-fixed pituitaries from

IMMUNOELECTRON MICROSCOPY OF TELEOST GH CELLS 151

FIGS. 1 and 2. Immunoelectron micrographs of the tilapia pituitary glands incubated with anti-tilapia GH serum at a concentration of 1500. The immunoreactive gold particles mainly concentrate on the secretory granules of the GH cells (GH) but not on those of putative gonadotroph (GTH) or thyrotroph (TSH) in the PLP (Fig. 1) and the PLP-osmium fixation (Fig. 2). g, Golgi apparatus. x 15,000

152 UEDA, KAGAWA, AND FUJIMOTO

IMMUNOELECTRON MICROSCOPY OF TELEOST GH CELLS 153

tilapia and amago salmon with or without osmium postfixation. From our electron mi- crographs, it is apparent that immunolog- ical antigen properties are extensively re- duced by osmium fixation. Without os- mium postfixation, more antigenic sites were preserved resulting in heavier label- ings of gold particles on the secretory gran- ules than those in the osmium-fixed speci- mens. However, if we take the preservation of cytoplasmic organelles into consider- ation, the specimens with osmium postfix- ation yield much more satisfactory results for the investigation of antigenic sites of a given cell.

For the immunoelectron microscopic demonstration of GH in osmium-fixed pi- tuitaries of mammals (Li et al., 1977; Baskin et al., 1979; Pelletier et al., 1981) and teleost (Cook et al., 1983), hydrogen peroxide was used for the etching treat- ment. It has been reported that oxidizing agents can restore the hormonal antigen- icity on osmium-fixed specimens (Ben- dayan and Zollinger, 1983). Although we also tested oxidizing treatment with sodium metaperiodate or hydrogen peroxide, no significant improvement of labeling inten- sity was observed in osmium-fixed tilapia pituitaries (H. Ueda, unpublished data). The use of this treatment was therefore omitted.

It can be said from the present study that the antigenicity of the tilapia GH is well preserved in the PLP-fixed specimens and that the good ultrastructural preservation can be obtained in the PLP-osmium-fixed ones without a complete loss of the anti- genicity. Thus, the combination of the PLP and PLP-osmium fixations allows us not only to identify the precise antigenic sites in a given cell type in various endocrine organs but to examine the function of such cells.

ACKNOWLEDGMENTS We thank Dr. Y. Nagahama, National Institute for

Basic Biology, for supplying tilapia GH and its cor-

responding antiserum, and the amago salmon. We also thank Dr. M. Nakamura, Teikyo University, School of Medicine, for providing the tilapia.

REFERENCES Baskin, D. G., Erlandsen, S. L., and Parsons, J. A.

(1979). Influence of hydrogen peroxide or alco- holic sodium hydroxide on the immunocytochem- ical detection of growth hormone and prolactin after osmium fixation. J. Histochem. Cytochem. 27, 1290- 1292.

Batten, T. F. C., and Hopkins, C. R. (1979). Use of protein A-coated colloidal gold particles for im- munoelectronmicroscopic localization of ACTH on ultrathin sections. Histochemistry 60, 317- 320.

Bendayan, M. (1982). Double immunocytochemical la- beling applying the protein A-gold technique. J. Histochem. Cytochem. 30, 81-85.

Bendayan, M., and Zollinger, M. (1983). Ultrastruc- tural localization of antigenic sites on osmium- fixed tissues applying the protein A-gold tech- nique. J. Histochem. Cytochem. 31, 101-109.

Bern, H. A., Nishioka, R. S., and Nagahama, Y. (1974). The relationship between nerve fibers and adenohypophysial cell types in the cichlid teleost Tilapia mossambica. Rech. Biol. Contemp. 1974, 179-194.

Cook, H., Cook, A. F., Peter, R. E., and Wilson, S. W. (1983). Ultrastructural immunocytochem- istry of growth hormone cells in the goldfish pi- tuitary gland. Gen. Comp. Endocrinol. 50, 348- 353.

Doerr-Schott, J., and Garaud, J. C. (1981). Ultrastruc- tural identification of gas&in-like immunoreactive nerve fibres in the brain of Xenopus laevis by means of colloidal gold or ferritin immunocyto- chemical methods. Cell Tissue Res. 216, 581- 589.

Farmer, S. W., Papkoff, H., Hayashida, T., Bewley, T. A., Bern, H. A., and Li, C. H. (1976). Purili- cation and properties of teleost growth hormone. Gen. Comp. Endocrinol. 30, 91-100.

Frens, G. (1973). Controlled nucleation for the regu- lation of the particles size in monodisperse gold suspension. Nature (London) Phys. Sci. 241, 20-22.

Follenius, E., Doerr-Schott, J., and Dubois, M. P. (1978). Immunocytology of pituitary cells from te- leost fishes. Int. Rev. Cytol. 54, 193-223.

Garaud, J. C., Eloy, R., Moody, A. J., Stock, C., and Grenier, J. F. (1980). Glucagon- and glicentin-im- munoreactive cells in the human digestive tract. Cell Tissue Res. 213, 121-136.

Geuze, H. J., Slot, J. W., van der Ley, P. A., and Scheffer, R. C. T. (1981). Use of colloidal gold particles in double-labeling immunoelectron mi-

154 UEDA, KAGAWA, AND FUJIMOTO

croscopy of ultrathin frozen tissue sections. J. Cell Biol. 89, 653-665.

Hisano, S., Adachi, T., and Daikoku, S. (1983). An application of the protein A-gold method to elec- tron microscopic immunohistochemistry. Seitai no Kagaku 34, 233-238.

Ingleton, P. M., and Stribley, M. E (1977). Immuno- fluorescent identification of the cells of origin of eel (Anguilla an&la) pituitary hormones sepa- rated by polyacrylamide gel electrophoresis. Gen. Comp. Endocrinol. 31, 37-44.

Kobayashi, S., and Uchida, T. (1983). Adrenal chro- maflin cells: Immunocytochemical demonstration of catecholamines and opioid peptides. Gunma Symp. Endocrinol. 29, 39-49.

Komourdjian, M. P., and Idler, D. R. (1979). Chum salmon pituitary fractions: Somatotropic activity and cytoimmunofluorescence studies. Gen. Comp. Endocrinol. 37, 343-349.

Larsson, L. I. (1979). Simultaneous ultrastructural demonstration of multiple peptides in endocrine cells by a novel immunocytochemical method. Nature (London) 282, 743-746.

Li, J. Y., Dubois, M. P., and Dubois, P. M. (1977). So- matotrophs in the human fetal anterior pituitary: An electron microscopic-immunocytochemical study. Cell Tissue Res. 181, 545-552.

McLean, I. W., and Nakane, P. K. (1974). Periodate-

lysine-paraformaldehyde fixative: A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077-1083.

Nagahama, Y. (1973). Histophysiological studies on the pituitary glands of some teleost fishes, with special reference to the classification of hormone- producing cells in the adenohypophysis. Mem. Fat. Fish. Hokkaido Univ. 21, l-63.

Nagahama, Y., Olivereau, M., Farmer, S. W., Ni- shioka, R. S., and Bern, H. A. (1981). Immuno- cytochemical identification of the prolactin- and growth hormone-secreting cells in the teleost pi- tuitary with antisera to tilapia prolactin and growth hormone. Gen. Comp. Endocrinol. 44, 389-395.

Pelletier, G., Puviani, R., Bosler, O., and Descarries, L. (1981). Immunocytochemical detection of pep- tides in osmicated and plastic-embedded tissue. J. Histochem. Cytochem. 29, 759-764.

Roth, J., Bendayan, M., and Orci, L. (1978). Ultra- structural localization of intracellular antigens by the use of protein A-gold complex. J. Histochem. Cytochem. 26, 1074- 1081.

Tanaka, T., Gresik, E. W., Michelakis, A. M., and Barka, T. (1980). Immunocytochemical localiza- tion of renin in kidneys and submandibular glands of SWRiJ and C57BLl6J mice. J. Histochem. Cy- tochem. 28, 1113-1118.