The Cellular Origins of Corticotropin and Melanotropin as Revealed by Immunochemical Staining ’

BURTON L. BAKER AND SISTER THaRBSE DRUMMOND2 Department of Anatomy, The University of Michigan Medical School, Ann Arbor, Michigan 481 04

ABSTRACT The cellular origins of corticotropin and melanotropin were studied by observing the response of the rat hypophysis to adrenalectomy and administration when stained immunochemically with peroxidase-labeled antibody following application of antiserums to P1-24-corticotropin,pp17-39-cortico- tropin, human p-melanotropin, and a-melanotropin. The presence of corticotropin in the is indicated since this lobe, in addition to corticotropic cells of the pars distalis, stained with antiserum to pp17-39-~orti~otropin.However, the concentration of corticotropin is less in the pars intermedia than in cortico- tropic cells since at higher dilutions of antiserum the latter cells stained intensely while the pars intermedia was unstained. Also, corticotropic cells exhibited strik- ing hypertrophy after adrenalectomy and regression after cortisol administration, while morphological and tinctorial properties of the pars intermedia were min- imally altered. These observations suggest that the pars distalis is primarily responsible for supplying the corticotropin required to meet the changing re- quirements of the body. With antiserum to human P-melanotropin, the pars intermedia stained in- tensely. Since the antiserum was ineffective after absorption the staining re- action appears to be specific for p-melanotropin. Except for a rare , the pars distalis did not stain. Thus p-melanotropin is restricted primarily to the pars intermedia.

Sever a1 observations implicate the pars tropin content. Also in microscopic sections intermedia of the rat hypophysis in the prepared with histological staining pro- secretion of corticotropin. First, both paren- cedures, they observed an increased num- chymal cells of the pars intermedia (Purves ber of “dark cells in the pars intermedia and Bassett, ’63) and many corticotropic after adrenalectomy and no cytological cells (Hess et al., ’68; Baker et al., ’70) change in the pars distalis. Fourth, all in- of the pars distalis stain with aldehyde- vestigators who have utilized immunofluo- fuchsin and the periodic acid-Schiff pro- rescence or immunohistochemical proce- cedure, indicating a similarity in their dures for demonstrating the cellular origin chemical composition. Second, with elec- of corticotropin have observed that the pars tron microscopy two types of cells have intermedia stains in addition to cortico- been described in the pars intermedia tropic cells of the pars distalis. This re- (Kurosumi et al., ’61; Porte et al., ’71) and sult has been obtained with antiserums to Kobayashi (’65) found enlargement and up-corticotropin (Nakane, ’70; Baker et accumulation of vesicles in “light” cells of al., ’70), synthetic ,t?1-24-~~rti~~tr~pin(Hess the pars intermedia after adrenalectomy. et al., ’68), and ppi7-39-~~rti~~tr~pin(Phjfer Third, as revealed by labeling of DNA with and Spicer, ’70). “-thymidine followed by autoradiography, Prior attempts to correlate changes in Gosbee et al. (’70) reported an increase corticotropin content with cytology of the in the labeling index of the pars intermedia hypophysis have been unsuccessful when after adrenalectoniy and a decrease after histological staining techniques were used cortisol administration, these situations be- 1 Supported in part by NIH grant HD-03159-04. ing accompanied by an elevation and a de- 2 Present address: Instituto de Nutriciio, Universi- dade Federal de Pernambuco, Recife, Pernambuco, cline, respectively, in the pituitary cortico- Brazil.

AM. J. ANAT., 134: 393-410. 395 396 BURTON L. BAKER AND SISTER THERRSE DRUMMOND

TABLE 1 The effect of absorption with on the utility of antiserums to corticotropin aizd melanotropin for immuizochemical staining ~-- Antiserum

Hormone ~.- - ~ .. absorbant fl1-24- P,,li-39. a- P- corticotropin 1 corticotropin 1 melanotropin 2 melanotropiil 2 Yi- corticotropin 0 I + t ,pp17-:19- corticotropin 0 0 + + d- inelanotropin i- + t 0 . - - - - ___ ------__~ 0, no staining obtained after absorption -, stdiiiing obtained after absorption although in some cases it WAS reduced over the control level 1 Refer5 to staining of corticotropic cells of pars distalis and of the pars intermedia 2 Refer< to staining of pars intermedia only.

because the corticotropic cell could not be after the operation. At one week, the ad- identified. This difficulty is alleviated renalectomized rats included 12 males and with iminunochemical staining and experi- 10 females with 10 males and 13 fe- mental proof that the procedure is specific males serving as controls; at two weeks, 4 for demonstration of corticotropic cells niale and 4 female adrenalectomized rats when used with anti-a),-corticotropin has with 4 male and 3 female controls; and been presented (Baker, et al., ’70). at 3 weeks, 5 adrenalectomized and 5 con- Reported here is a comparative study of trol male rats. The controls for the one- the responses of the pars intermedia and week group were sham-operated. All ad- pars distalis to adrenalectomy and cortisol renalectomized animals received 1% NaCl administration carried out to secure addi- solution as drinking fluid. For study of the tional .information about the origins of effects of cortisol, five male rats received corticotropiii and melanotropin (MSH). daily for 19 days 5 mg (0.5 ml) of cortisol For this purpose, antiserums to p1-24-acetate suspended in Upjohn vehicle 98 (carboxymethylcellulose, polysorbate, and cor tico tropin , /jr’ :‘“-corticotropin, hum an p- melanotropin. and x-melanotropin were propylparaben) diluted 1.2 with 0.9:; used with the irnmunochernical procedure. NaCl. The control rats received an equi- valent volume of vehicle. MATERIALS AND METHODS Since immunological cross-reaction may be expected between corticotropin and Young adult Sprague-Dawley rats were melanotropin because an identical seven- killed by decapitation and their hypophyses sequence occurs in the two fixed in Bouin’s fluid. After being embedded hormones, immunological controls were re- in paraffin the glands were cut at 3 I*. quired to validate the specificity of the im- Iniinunochernical staining of sections from munochemical procedure. Hence, each hor- various regions of the gland was carried mone antiserum was absorbed with each out with the peroxidase-labeled antibody of the four hormones before use (table 1 ) procedure of‘ N akane and Pierce (’67),3,3’- :jThe nomenclature recommended by Li (’59) for diaminobenzidine being used as substrate corticotropin will be followed in part with a indicat- for the peroxidase reaction. For prepara- ing the natural intact ; p, other molecules having corticotropic activity; superscript numbers, the tion of antiserums, rabbits weighing 2-4 amino acid residues included; and subscript letters, kg received injections of synthetic the species of origin. Thus, Br.“-”-corticotropin con- notes the synthetic fragment of the porcine hormone. corticotropin,“ fipli-Jg-corticotropin,human No subscript is used for pzkorticotropin since this sequence is commoy to sheep, beef, pig and man (Li, pmelanotropin or a-melanotropin sus- ’59; Riniker et a1 72). The term “corticotropic cell” pended in complete Freund’s adjuvant, the will designate c&ticotropin-containing cells of the pars distalis only. rabbits being injected and bled as described 4 We are deeply grateful to Organon, Inc., W. Orange, N. J., for supplying B’-”-corticotropin (Cosyn- by Midgley et al. (’71). tropinn) and to Professor H. J. Bcin and Doctor For study of the influence of adrenal- W. Rittel of Ciba-Geigy Ltd., Bask, Switzerland, for pr”~:’9-corticotropin, B-melanotropin. and a-melano- ectomy. rats were killed at weekly intervals tropin. ORIGIN OF CORTICOTROPLN AND MELANOTROPIN 397 to see if staining could be prevented. In so cell appeared in the pars distalis, not more doing, the antiserum was used at the maxi- than eight ever having been observed in a mal dilution that, without absorption, single section of the gland. The staining would permit strong staining. The absorb- obtained with anti-a-melanotropin varied ing hormone was dissolved in physiological according to the antiserum used. With one buffered saline and employed at several antiserum numerous ovoid cells were de- concentrations. Absorption studies with a- lineated (fig. 6) most of them being located melanotropin were unsatisfactory because near the cephalic and caudal borders of of its low solubility. the lateral wings of the pars distalis. These OBSERVATIONS cells appeared to be included among the gonadotropic cells. Another antiserum to Immunological controls a-melanotropin brought out these cells in The following observations suggest that addition to the rare ones stained with anti- the antiserums to the corticotropins and p- a-melanotropin. melanotropin may be used for immuno- Pars intermedia. Composing the rat chemical, specific intracellular localization pars intermedia is the pars intermedia of these hormones in the rat hypophysis. proper consisting of several layers of poly- The utility of anti-p'~""-corticotropin was gonal cells and a narrow marginal zone lost after absorption with p'-"- and p""- made up of somewhat flattened cells that corticotropins (table 1). This result was faces the residual cleft (Kurosumi et al., unexpected with pr'7~39-~~rti~~tr~pinbut '62). With all antiserums used the mar- could have resulted from the identity of ginal zone and colloid in the residual the 17-24 sequence of amino acids in the cleft failed to stain. two molecules. Although antibodies to Application of the immunochemical pro- p~"'9-corticotropin were removed from the cedure with anti-p1-'4-corticotropin to hy- serum by absorption with the same hor- pophyses of control rats stained almost all mone, they apparently did not couple with parenchymal cells in the pars intermedia molecules of pi-24-corti~otropin.Antiserum proper (figs. 4, 12, 16). However, the apices to each of the corticotropins was unaffected of cells facing the marginal zone on one by absorption with p-melanotropin. Anti-p- side, or the pars nervosa on the other (figs. melanotropin was neutralized by absorp- 12, 16), were more intensely colored. Espe- tion with p-melanotropin, but its effective- cially in the most lateral (Kobayashi, '65) ness was not altered by absorption with external region of the pars intermedia a either pi-'"- or ~~pi7-39-~~rti~~tr~pin.Thus, few nonstaining cells were evident. With cross-reaction be tween the corticotropins anti-pF'7-39-~~rti~~tropin(figs, 1, 9, 15) the and p-melanotropin was not an interfering distribution of stain in the pars intermedia factor. proper was similar to that described for anti-pi-z4-corticotropinexcept that in some The normal hypophysis cases a more clear distinction could often Pars distalis. As revealed by immuno- be made between cells that stained well chemical staining with anti-up-corticotropin and those that did not. (Nakane, '70; Baker, '70; Baker et al., 'TO), If either ar~ti-pl-*~-or anti-pp'7-39-~~rti~~- anti-pp17-39-~~rti~~tr~pin(fig. 1) or anti- tropin was used at the highest dilution that ,P24-corticotropin (fig. 4), corticotropic would permit nearly maximal staining of cells of the rat pars distalis are small, corticotropic cells in the pars distalis, no stellate (fig. 7), constitute a low percentage staining of the pars intermedia occurred. of the cell population, and are distributed This observation indicates clearly that if diffusely through most of the lobe. Com- corticotropin occurs in the pars intermedia, parison of adjacent sections, each of which its concentration there is far less than in was stained with one of these anti- corticotropic cells of the pars distalis. serums, revealed that a single cell type With anti-p-melanotropin, cells of the was demonstrated. pars intermedia proper generally stained In hypophyses from control rats of both quite uniformly (figs. 5, 13); however, in sexcs stained with anti-p-melanotropin the lateral region some cells contained no (fig. 5), a rare intensely stained polygonal brown deposit at all. As with antiserums 398 BURTON L. BAKER AND SISTER THBRBSE DRUMMOND

to corticotropin, cells facing the marginal little change in the pars intermedia. At zone or pars nervosa colored most intensely. two and three weeks a slight cellular en- Thus, the pars intermedia responded simi- largement sometimes seemed evident in larly with anti$ L'-corticotropinand anti-p- the pars intermedia proper but it could melanotropin. In all cells stained with not be demonstrated consistently (figs. 9, anti-~1-24-corticotropin(fig. 16), anti-pp'7-39- 10). A significant modification in staining corticotropin (fig. 15) or anti-p-melano- intensity did not occur with anti-,@"- tropin, some darkly stained cytoplasmic cortico tropin or anti-p-melanotropin while structures were punctate and may cor- a slight decline was sometimes evident if respond to the granules observed by others anti-~p'r-Js-~~rti~~tr~pinwas used. with electron microscopy. Pars nervosa. Following adrenalectomy, With one exception all antiserums to a- the structures staining with anti-p-melano- melanotropin did not permit immuno- tropin in the pars nervosa were somewhat chemical staining of the pars intermedia more densely arranged. (fig. 6). In the pars nervosa some structures The effect of cortisol administration stained with anti-p-melanotropin when Pars distalis. Except for a few cells, ad- used at high concentration. They were most ministration of high doses of cortisol ace- common around small blood vessels and tate obliterated the stainability of most appeared to be nerve endings, with granu- corticotropic cells when demonstrated with lar intracellular components (fig. 14). The either anti-p'-2J-or anti-ppl'-Ds-~~rti~otr~pin significance of this observation is not clear (fig. 3). If some staining capacity was re- because anti-p-melanotropin continued to tained by corticotropic cells one could as- permit staining of the pars nervosa certain that they were as small or smaller after absorption with p-melanotropin even than those in vehicle-treated controls. Con- though no color at all appeared in the clusive demonstration of a loss in cell pars intermedia. volume was difficult because corticotropic cells are so small in normal rats. In those The effect of adrenalectomy cells stained immunochemically with anti- Pars distalis. Previously we (Baker et p-melanotropin or anti-a-melanotropin no al., '70) reported that seven days after ad- significant alterations could be attributed renalectomy of rats some corticotropic cells to cortisol treatment. are enlarged in both sexes. In this study Pars intermedia. Parenchymal cells of at two weeks after adrenalectomy, most the pars intermedia proper either remained corticotropic cells as revealed immuno- unchanged in size after cortisol treatment chemically with or anti-ppl'-Js- (fig. 11) or in certain rats appeared some- corticotropin were markedly hypertrophied what enlarged with "foamier" cytoplasm and possessed enlarged nuclei (figs. 2, 8). and greater dispersion of chromatin in The general cytoplasm was stained, as well the nucleus. With anti-p'-"- and anti-pp17-3s- as granules located peripherally (fig. 8) in corticotropin (figs. 3, I]), as well as with many of the hypertrophied corticotropic anti-p-melanotropin, a slight decrease in cells. Although no mitotic figures were overall staining capacity of the lobe found, the number of corticotropic cells occurred. was clearly increased in most of the ad- renalectomized rats (figs. 1, 2). After ad- DISCUSSION renalectomy there was a rather wide range Correlation of alterations in morphology in the staining intensity of the hypertro- with hormone content in the pars inter- phied corticotropic cells. At three weeks media and pars distalis after adrenalectomy the magnitude of the cytological response or cortisol administration should facilitate to adrenalectomy had regressed somewhat. clarification of the origins of corticotropin At two and three weeks after adrenal- and melanotropin because following ad- ectomy the cells staining with anti-p- renalectomy of rats, the pituitary content melanotropin and anti-a-melanotropin re- of corticotropin is increased manyfold at mained unchanged. 14 and 21 days (Fortier, '59a) while after Purs intermedia. Adrenalectomy elicited chronic treatment with cortisol it is re- ORIGIN OF CORTICOTROPIN AND MELANOTROPIN 399 duced greatly (Fortier, ’59b). On the other understood. The cells that accept anti-p- hand, total melanotropin content of the melanotropin are probably too scarce to af- hypophysis is unaffected by either treat- fect the total melanotropin content of the ment and the concentration is increased gland and the minor cellular changes that by only cortisol treatment (Gosbee et may have occurred following these treat- al., ’70). ments were undoubtedly inconsequential. The pars distalis as a source of cortico- Thus, there is a close parallelism between tropix. Although Kraicer, Herlant and cytological changes in corticotropic cells Duclos (’67) reported enlargement of chro- in the pars distalis and shifting levels of mophobes (a subclass of acidophils as corticotropin in the gland, but following revealed by the Herlant tetrachrome pro- adrenalectomy or administration of cortisol cedure ) after adrenalectomy, and reduction both the content of p-melanotropin (Gosbee in their size after cortisol administra- et al., ’70) in the hypophysis and the pos- tion, later Kraicer and his colleagues (Gos- sible cells of origin in the pars distalis bee et al., ’70) maintained that cytological remain unaffected. changes elicited in the hypophysis by these The pars intermedia as a source of cor- treatments are restricted to the pars inter- ticotropin. Several investigators agree that media. It is now clear from immunochemi- the pars intermedia is altered by adrenal- cal staining (Baker et al., ’70) and studies ectomy and on the basis of this and other with immunofluorescence (Hess et al., ’68) observations have suggested that it also se- that one week after adrenalectomy numer- cretes corticotropin. Kobayashi (’65, 69) ous corticotropic cells in the pars distalis reported an enlargement of “light” cells, are mildly hypertrophied; further at two engorgement of their cytoplasm with both and three weeks after the operation, when clear and dense vesicles, hypertrophy of the the augmentation xn pituitary corticotropic Golgi apparatus and rough endoplasmic content reaches its highest level (Fortier, reticulum and no change in the smaller ’59a), the response of corticotropic cells is “dark’ cells. In contrast, Gosbee et al. (’70) also maximal with respect to hypertrophy reported a marked increase in the relative and number of cells affected. This conclu- number of shrunken, “dark cells after ad- sion is supported by the electron micro- renalectomy as observed with light micros- scopic observations of Nakayama et al. copy and concluded that their “findings (’69), Siperstein and Miller (’70), and are consistent with recent reports [by Pelletier and Racadot (’71). Two of these Kobayashi] of electron microscopic changes groups (Siperstein and Miller, ’70; Pelletier in the pars intermedia associated with al- and Racadot, ’71) also showed that a single tered secretion of ACTH.” In our prepara- injection of into the adrenal- tions from control animals differentiation ectomized rat increases the storage of secre- of parenchymal cells of the pars intermedia tory granules, in accord with enhancement into light and dark varieties was not pos- of pituitary corticotropic content (Fortier, sible on the basis of criteria used by Gosbee ’59b). Continuation of cortisol treatment et al. (’70) or electron microscopists, nor for one week or longer makes most cortico- did “dark” cells accumulate after adrenal- tropic cells unidentifiable by ultrastructural ectomy. The increased number of small (Siperstein and Miller, ’70) or immuno- dark cells described by these investigators chemical characteristics. Thus, predictable after adrenalectomy may have been de- cytological changes do occur in the pars pendent on the type of fixative used or un- distalis in concert with experimental altera- usual treatment of the tissue during ex- tion in the pituitary level of corticotropin. cision. At any rate shrinkage of a cell with Although the rare cells of the pars dis- condensation of cytoplasm and nucleo- talis that stain immunochemically with plasm are not commonly considered in- anti-p-melanotropin, or the more numerous dicative of accelerated synthesis and re- ovoid cells revealed with a-melanotropin, lease of a cell product. Dark cells identified might produce melanotropin, experimental with electron microscopy were interpreted verification of this inference has not been by Kobayashi to be degenerative in nature. possible because the functional role of Gosbee et al. (’70) believe the changes these hormsnzs in mammals is so poorly in cell proliferation elicited in the partes 400 BURTON L. BAKER AND SISTER THeRESE DRUMMOND

intermedia and distalis by adrenalectomy in both hypophysectomized and adrenal- and cortisol administration also indicate ectomized rats (Orias and McCann, '72). that the pars intermedia secretes cortico- Also, the pars intermedia appears to re- tropin. Thus, they found an increase in spond to changes in the volume and osmo- the labeling index of the pars intermedia larity of the blood. For example, provision after administration of "H-thymidine with of 2% NaCl solution to rats as drinking no change in the pars distalis following fluid may increase mitotic activity and the adrenalectomy and a fall after cortisol ad- number of small dark cells in the pars ministration only in the pars intermedia. intermedia (Duchen, '62). Further, when On the contrary, Crane and Loomes ('67) the hypothalmo-neurohypophyseal systcni observed an increased labeling index in is stimulated in certain mammalian species both lobes at one and two weeks after ad- by deprivation of salt or watfr the pars renalectomy and a fall only in the pars intermedia and its constituent cells become distalis after treatment with cortisol. smaller; contrariwise, damage to this sys- As pointed out previously by others, im- tem leads to hyperplasia of the pars inter- munochemical staining of the pars inter- media (Legait, '63 ). media with antiserums to ar-corticotropiii In conclusion, immunxhemical staining and 13' 24-corticotropinmay not be convinc- provides strong evidence that corticotropin ing evidence for the presence of cortico- occurs in the pars intermedia of the rat. &_pin because the molecules of cortico- However, failure of the pars intermedia tropin and 8-melanotropin in that lobe t3 respond to alteration in the lwel of cir- contain an identical seven-amino acid se- culating adrenocortical steroids with more quence (Evans et al,,'66) making probable than minor structural change suggests that cross-reaction between each hormone and production of corticotropin by the pars the antiserum of the other. However, in our intermedia is controlled by a different control studies such cross-reaction was not mechanism than is its secretion by the pars demonstrable. More crucial is the observa- distalis; as a corollary one may infer that tion made by Phifer and Spicer ('70) and the fluctuating demands of the body for by us that the pars intermedia stains with adrenocortical hormone are met by secre- anti-pl.""'-corticotropin because this se- tion of corticotropin from the pars distalis. quence of amino acids does not occur in The pars distalis does not seem to be in- any known type of melanotropin molecule. volved in the production of p-melanotropin. Assuming that rat corticotropin includes a ACKNOWLEDGMENT similar or identical sequence, this finding is a strong indication that the pars inter- We are grateful to Mrs. Ya-Yen Yu for media contains corticotropin. her skillful assistance. Further, Rochefort et al. ('59) and LITERATURE CITED Mialhe-Voloss ('58) have shown that the Baker, B. L. 1970 Studies on hormone localiza- posterior hypophysis (probably including tion with emphasis on the hypophysis. J. Histo- both pars nervosa and pars intermedia) chem., 18: 1-8. contains significant corticotropic activity. Baker, B. L., S. Pek, A. R. Midgley, Jr. and B. E. Gersten 1970 Identification of the cortico- Since the pars intermedia, but not the pars tropin cell in rat hypophyses with peroxidase- nervosa, stains immunochemically with labeled antibody. Anat. Rec., 166: 557-567. antiserum to corticotropin this hormone is Crane, W. A. J., and R. S. Loomes 1967 Effect Frobably restricted to the pars intermedia. of age, sex, and hormonal state on tritiated thymidine uptake by rat pituitary. Brit. J. The structural changes in the pars inter- Cancer, 21: 787-792. media reported by others to follow adrenal- Duchen, L. W. 1962 The effects of ingestion of ectomy or glucocorticoid administration hypertonic saline on the in the might be mediated by the expected dis- rat: A morphological study of the pars inter- media and posterior lobe. J. Endocr., 25: turbance in the metabolism of electrolytes 161-1 68. and water since increasing evidence indi- Evans, H. M., L. L. Sparks and J. S. Dixon 1966 cates that a functional relationship exists The physiology and chemistry of adrenocortico- between the intermediate lobe and the trophin. In: The Pituitary Gland. . Vol. 1. G. W. Harris and B. T. Dono- levels of electrolytes and water in the body. van, eds. Univ. Calif. Press, Berkeley, pp. 317- Thus, x-melanotropin causes natriuresis 373. ORIGIN OF CORTICOTROPIN AND MELANOTROPIN 40 1

Fortier, C. 1959a Pituitary ACTH and plasma Midgley, A. R., Jr., G. D. Niswender, V. L. Gay free corticosteroids following bilateral adrenal- and L. E. Reichert, Jr. 1971 Use of antibodies ectomy in the rat. Proc. SOC.Exp. Biol. Med., for characterization of gonadotropins and ster- 100: 13-16, oids. Rec. Progr. Horm. Res., 27: 235-301. 1959b Effect of hydrocortisone on pitui- Nakane, P. K. 1970 Classifications of anterior pituitary cell types with immunoenzylne histo- tary ACTH and adrenal weight in the rat. Proc. chemistry. J. Histochem. Cytochem., 18. 9-20. SOC.Exp. Biol. Med., 100: 16-19. Nakane, P. K., and G. B. Pierce, Jr. 1967 En- Gosbee, J. L., J. Kraicer, A. J. Kastin and A. V. zyme-labeled antibodies for the light and elec- Schally 1970 Functional relationship between tron microscopic localization of tissue antigens. the pars intermedia and ACTH secretion in the J. Cell Biol., 33: 307-318. rat. , 86: 560-567. Nakayama, I., P. A. Nickerson and F. R. Skelton Hess, R., D. Barratt and J. Gelzer 1968 Immuno- 1969 An ultrastructural study of the adreno- fluorescent localization of p-corticotropin in the corticotrophic hormone-secreting cell in the rat rat pituitary. Experientia, 24: 584-585. adenohypophysis during adrenal cortical regen- Kobayashi, Y. 1965 Functional morphology of eration. Lab. Invest., 21: 169-178. the pars intermedia of the rat hypophysis as Orias, R., and S. M. McCann 1972 Natriuretic revealed with the electron microscope. 11. Cor- effect of alpha stimulating hormone relation of the pars intermedia with the hypo- (a-MSH) in hypophysectomized or adrenal- physeo-adrenal axis. 2. Zellforsch., 68: 155-171. ectomized rats. Proc. SOC.Exp. Biol. Med., 139: 1969 Functional morphology of the 872-8 76. pars intermedia of the rat hypophysis as re- Pelletier, G., and J. Racadot 1971 Identification vealed with the electron microscope. IV. Ef- des cellules hpoohysaires secretant l’ACTH fects of corticosterone on the pars intermedia of chez le rat. 2. Zellforsch., 116: 228-239. intact and adrenalectomized rats. Gunma Symp. Phifer, R. F., and S. S. Spicer 1970 Immuno- Endocrinology, 6: 107-122. histologic and immunopathologic dernonstra- Kraicer, J., M. Herlant and P. Duclos 1967 tion of adrenocorticotropic hormone in the pars Changes in adenolhypophyseal cytology and nu- intermedia of the adenohypophysis. Lab. Invest., cleic acid content in the rat 32 days after bi- 23: 543-550. lateral adrenalectomy and the chronic injection Porte, A., M. J. Klein, M. E. Stoeckel and F. Stutin- of cortisol. Canad. J. Physiol. Pharm., 45: sky 1971 Sur l’existence de cellules de type 947-956. “corticotrope” dans la pars intermedia de I’hy- Kurosumi, K., T. Matsuzawa and S. Shibasaki pophyse du rat. Etude au microscope elec- 1961 Electron microscore studies on the fine tronique. Z. Zellforsch., 115: 60-68. structures of the pars nervosa and pars inter- Purves, H. D., and E. G. Bassett 1963 The stain- media, and their imorphological interrelation in ing reactions of pars intermedia cells and their the normal rat hypophysis. Gen. Comp. Endocr., differentiation from pars anterior cells. In: I: 433-452. Cytologie de L‘Adenohypophyse. J. Benoit and Kurosumi, K., T. Matsuzawa and E. Fujie 1962 C. Da Lage, eds. ‘Oditions du Centre National Histological and histochemical studies on the de la Recherche Scientifique, Paris, pp. 231-238. rat pituitary pars intermedia. Arch. Hist. Jap., Riniker, B., P. Sieber, W. Rittel and H. Zuber 22: 209-227. 1972 Revised amino-acid sequences for por- Legait, E. 1963 Cytophysiologie du lobe inter- cine and human adrenocorticotrophic hormone. mediaire de l’hyyophyse des mammiferes. In: Nature New Biol., 235: 114-115. Cytologie de L’Adenohypophyse. J. Benoit and Rochefort, G. J., J. Rosenberger and M. Saffran C. Da Lage, eds. Sditions du Centre National 1959 Depletion of pituitary corticotrophin by de la Recherche Scientifique, Paris, pp. 215-227. various stresses and by neurohypophysial prep- Li, C. H. 1959 Proposed system of terminology arations. J. Physiol., 146: 105-116. for preparations of adrenocorticotropic hormone. Siperstein, E. R., and K. J. Miller 1970 Further Science, 129: 969-970. cytophysiologic evidence for the identity of the Mialhe-Voloss, C. 1958 Posthypophyse et activite cells that produce adrenocorticotrophic hor- corticotrope. Acta Endocr., Suppl. 35. mone. Endocrinology, 86: 451-486. Abbreviations N, pars nervosa 1, pars intermedia D, pars distalis

PLATE 1

EXPLANATION OF FIGURES

Figures 1-6 represent one-half of the hypophysis sectioned on a hori- zontal plane. x 38. Female control rat; staining with anti-pP*'-~g.corticotropin. The pars intermedia is well-stained and small corticotropic cells appear through- out the pars distalis. Female rat 14 days after adrenalectomy; staining with anti-p~"-~~- corticotropin. Corticotropic cells of the pars distalis are enlarged and probably increased in relative number. Female rat following treatment with cortisol; staining with anti-p~"-?~- corticotropin. As compared with the control (fig. 1 ), staining intensity of the pars intermedia is reduced and only a few corticotropic cells are visible jn the pars distalis (arrow). Female control rat; staining with anti-pr'-L4-corticotropin.The overall reaction of the pars intermedia and pars distalis is similar to that ob- tained with anti-pI.1~-3g-corticotropin(fig. 1 ).

402 ORIGIN OF CORTICOTROPIN AND MELANOTROPIN PLATE 1 Burton L. Baker and Sister Th&r&e Drummond

403 PLATE 2

EXPLANATION OF FIGURES

5 Female control rat; staining with anti-p-MSH. Staining is of moderate intensity in the pars nervosa and intense in the pars intermedia. x 38. 6 Female control rat; staining with anti-a-MSH. The partes nervosa and intermedia are unstained while some cells a9pear in the pars distalis that are most numerous along the cephalic and caudal borders of the lobe. x 38. 7 Female control rat; corticotropic cells (c) stained with anti-pp1i-39- corticotropin. x 1000. 8 Female rat 14 days after adrenalectomy; corticotropic cells staining with anti-pL.li-”-corticotropin. As compared with those in figure 7, corticotropic cells (c) are enlarged and vary in their staining capacity. In one instance, granules are visible peripherally (arrows) in the cell around an enlarged Golgi region. x 1000.

404 ORIGIN OF CORTICOTROPIN AND MELANOTROPIN PLATE 2 Burton L. Baker and Sister Therese Drummond

405 PLATE 3

EXPLANATION OF FIGURES

Figures 9-13 illustrate a region of the hypophysis extending from the pars distalis (D) above through the residual cleft (C), marginal zone of the pars intermedia (M), pars intermedia proper (I), and into the pars nervosa (N). X 400. 9 Control female rat; staining with anti-p,~1i-39-corticotropin.The mar- ginal zone of the pars intermedia is unstained. Most cells of the pars intermedia proper are stained but some (arrows) are not colored. 10 Female rat 14 days after adrenalectomy; staining with anti-pP”-””- corticotropin. Staining intensity of the pars intermedia proper is r6- duced slightly as compared with the control (fig. 9). There is no cellular enlargement in this specimen. 11 Female rat following treatment with cortisol; staining with ar~ti-p~”-~~- corticotropin. Staining intensity is reduced somewhat as compared with the control (fig. 9). 12 Female control rat; staining with anti-/3‘-21-corticotropin.In contrast to the lack of color in the marginal zone, the cells of the pars inter- media are rather uniformly stained. The darkest cells border the pars nervosa.

406 ORIGIN OF CORTICOTROPIN AND MELANOTROPIN PLATE 3 Burton L. Baker and Sister Therese Drummond

407 PLATE 4

EXPLANATION OF FIGURES

13 Female control rat; staining with anti-pmelanotropin. The marginal zone is uncolored whiie cells of the pars intermedia proper are stained with rather uniform intensity except for the apices of the cells border- ing the marginal zone and pars nervosa, which are much darker. Small areas of the pars nervosa are stained. 14 Pars nervosa of a female control rat; staining with anti-p-melanotropin. Numerous structures that appear to be nerve endings contain dark granules. These are most numerous around capillaries (cap.). i/ 400. 15 Pars intermedia of a female control rat; staining with anti-p131T~,J!'- corticotropin. Some cells (X) show little or no cytoplasmic staininq. In cthers granules (arrows) are evident, these being most numero'Js dongside the Sars nervosa. x 1000. 16 Pars intermedia of a female control rat; staining with anti-@'-"- corticorrqin. The general stainability of cells in the pars intermedia proper is iliustrated. Granulation (arrows) appears in the cytoplasm; the darkest cells border the pars nervosa and a connective tissue :,eptum. 1 1000.

408 ORIGIN OF CORTICOTROPIN AND MELANOTROPIN PLATE 4 Burton L. Baker and Sister Therese Drummond

409