Immunocytochemical Localization of Parathyroid Hormone in Bovine Parathyroid Glands and Human Parathyroid Adenomas Josephine M. Futrell, PhD, Sanford 1. Roth, MD, Sendy P. C. Su, BS, Joel F. Habener, MD, Gino V. Segre, MD, and John T. Potts, Jr., MD
Light and electron microscopic localization of parathyroid hormone (PTH) in human and bovine parathy roid tissue has been achiev ed using an indirect peroxidase labeled antibodv method. Granular deposition of the reaction product was found throughout the chief cell cytoplasm. There was no nuclear staining. At the ultrastructural lesvel, parathyroid hormone localized by this method appeared to be largely confined to the secretory granules in the cy toplasm of cells. MIitochondria and nuclei were free of reaction product. Aggregated sacs of granular endoplasmic reticulum were minimally reactive, and Golgi apparatuses did not show reaction product. (NM J Pathol 94:615622, 1979)
THE CHIEF CELLS of normal parathyroid glands of humans and other mammals have a continuous secretorv c-cle. consisting of an inactix-e resting phase. followed consecutix-elx- by synthesizing. packaging. secreting. and reco-erx phases.3 The parath! roid hormone is synthesized as preproparathy roid hormone presumably on the aggregated granular endoplasmic reticulum.45 The preprohormone is rapidly converted by proteo(l-tic cleavage to an intermediate precursor. ie. proparathyroid hormone. The prohormone is then transferred to an enlarging Golgi apparatus. w-here it is conv-erted bx- enzymic processes to parathyroid hormone which. in turn. is packaged into membrane-limited secretory granules.6 These granules containing parathyroid hormone, along X ith l-sosomelike granules,3 then move to the periphery of the cell m.-here their membranes fuse with the cell membrane and the contents are emptied into the extracellutlar space. The cells then involute to return to the resting or inactive phase characterized by a small Golgi apparatus. abundant glycogen. and fexx- secretory granules. Althotugh these small membrane-limited granules hav-e been inferred to be the site of storage of parathv-roid hormone in the gland. some authors F ron the Department o)f PatholzxI.Unix ersitx of XrkansaS for \Medical Sciences. Collegze of ledicinee I ittlt RwxAk. \rkansa.s and(l the Departmenit (f \ledicine. Har\ard \ledical Schx(d. and the \laa,achuistts General H 'spital. B1',tn.n \Massachuisetts Supported ir part b\ Grants C-X-IS616 and A\11794 from the Public Health Ser- ice Dr Hahener P, anl Iise"stiuratur at the Hox\ard Hughes \Medical Instittite Dr Segzre is the recipient of Research Career Dexelopment As-;ard .\100070 from NI\IDD Portuiouts of this stuidx "cere presented at the Federated Americ;anl Societx fo)r Experimental Bioluugx ' and at the Sixth International Parathv roid Conferencee. anricnixer. British Colnlmbia. 1977,2 Xddre,ss reprint requciests to Dr Sanford 1 Roth. Departmenrlt of Patholog-. College of Mledicine. Unnix\ersitx of rkana,as for \Medical Sciences. 4301 West \larkham. Little Rock. AR 72201. 0002-9440/79/0308-061 5$01 .00 615 (3 American Association of Pathologists
616
FUTRELL ET AL
American Journal of Pathology
have postulated that in at least some cases parathyroid hormone may bypass these granules.7 Attempts to prove that the granules are the secretory form of the hormone by isolation and analysis of granules obtained from subcellular fractions have met with only limited success.8'0 Studies with fluorescent labeled antibodies to parathyroid hormone have localized the hormone in the chief cells.1",2 This investigation was undertaken to confirm the granular nature of the storage form of the hormone and to determine if the pathway of hormone synthesis in the parathyroid is similar to that postulated for other endocrine glands. Materials and Methods Bovine parathyroid glands obtained from 3- to 4-year-old steers fresh from a local abattoir and human parathyroid adenomas were fixed for 4 to 6 hours at 4 C in Zamboni's 13 picric acid paraformaldehyde (PAF) or periodate-lysine-paraformaldehyde (PLP) fixative.' The tissues were washed overnight with 0.01 M phosphate-buffered saline with 10% sucrose and subsequently embedded in polyethylene glycol (average molecular weight, 950 to 1050) (Matheson Coleman & Bell, Norwood, Ohio). Five- to 10-u sections, floated on a 5% glycerol solution for 20 minutes, were transferred with a wire loop to an albumin-coated slide and dried in a 40 C oven for 30 to 60 minutes.15 The sections were covered for 1 hour with 1: 100 normal sheep serum to minimize nonspecific reactions.'" They were washed three times in 0.1 M phosphate-buffered (pH 7.4) normal saline (PBS) and then treated at 100% relative humidity for 24 hours with either diluted normal rabbit serum (Cappel Laboratories, Pa) or rabbit antiserum (R12) to bovine parathyroid hormone (PTH). After three washes in PBS, the sections were treated with 1:50 peroxidase-labeled goat antirabbit IgG (Cappel) in PBS. Following another 24-hour incubation and washing, the tissues were incubated for 30 minutes in a 3,3'-diaminobenzidine substrate solution without hydrogen peroxide." For the last 1 to 5 minutes, hydrogen peroxide was added to a final concentration of 0.005%. After two additional washings, glass coverslips were placed on the slides with a 90% glycerol solution for light microscopic viewing. For ultrastructural examination, the coverslip was removed and the tissue was washed in PBS. The section was then exposed to 2% osmium tetroxide in distilled water for 2 hours and dehydrated in graded ethanol. While still wet with 100% ethanol, a gelatin capsule filled with epon 17 was inverted over the tissue and the plastic was polymerized at 60 C for 72 hours. The hardened block was removed from the slide by gentle heating over a bunsen burner. The block was trimmed under a dissecting microscope. Thin (70-nm) sections were made on Porter-Blum MT2B ultramicrotomes, mounted on copper grids, and examined without additional staining in a Siemens Elmskop 101 or 102 at 5000 to 20,000 X initial magnification.
Results
Light microscopic studies of human bovine parathyroid glands revealed that the reaction product was deposited diffusely throughout the cytoplasm (Figures 1 and 2) with numerous more intensely staining granules (Figure 1). Nuclei, vascular endothelium, and interstitial tissue appeared free of reaction product. All control sections, including normal rabbit
Vol. 94, No. 3 March 1979
IMMUNE LOCAUZATION OF PARATHYROID HORMONE
617
serum controls (Figures 3 and 4), were virtually free of reaction product and showed no granular staining. Oxvphil cells seen in one adenoma were free of reaction product. At the ultrastructural level, deposition of the reaction product was limited largely to the smaller membrane-limited granules (Figures 5 through 7). No reaction product was seen in the nuclei. Mitochondria (Figure 6) and Golgi apparatuses were free of reaction product; however, the aggregated sacs of granular endoplasmic reticulum were minimallv stained. Not all granules showed staining, even in a cell with maximal deposition of reaction product. There was also considerable variabilitv in the amount of stained granules vs nonstained granules in chief cells from a single tissue section. Dispersed sacs of granular endoplasmic reticulum were free of reaction product. However, control cells (Figure 4) showed only minimal osmium staining and their granules were clearly lighter than experimental sections. There was, in addition, increased electron densitv of the plasma membrane in the experimental sections (Figure 7). The reaction product usually presented as multiple small granules of electrondense material (Figures 5 through 7). Of the two fixatives employed in these experiments, PLP was the most effective in preserving both antigenicity and tissue structure. Although there was some tissue swelling as a result of this technique, preservation was adequate, particularly at the electron microscopic level. Glutaraldehvde fixation, which provided better tissue preservation, destroyed the antigenicity of the parathyroid hormone in the tissue.
These studies demonstrate that the parathyroid hormone is largelv confined to the chief cell cytoplasm of the human and bovine parathyroid gland. The nuclei, vasculature, and intestitial tissue are free of sufficient parathyroid hormone to be identified by inununolocalization. The studies are in agreement of the observations made earlier , but further demonstrate the predominately granular nature of the hormone in the cvtoplasm. Oxyphil cells in a human adenoma did not contain inmmunoreactive parathyroid hormone, confirming the contention of Munger and Roth 6 that the parathyroid oxyphil cell does not generally synthesize parathyroid hormone. At the ultrastructural level, the hormone is largely confined to the small membrane-limited granules which have been postulated to be the sites of parathvroid hormone storage." These granules are smaller and more numerous than the granules which contain acid phosphatase.3 All of the small granules do not contain immunoreactive parathyroid hormone, and
618
FUTRELL ET AL
American Journal of Pathology
the possibility must be considered that they may contain substances other than parathyroid hormone, ie, parathyroid secretory protein,4 degraded hormone,5 other secretory proteins, or even catecholamines. In contrast to these observations, Ravazzola et al 18 found in similar immunocytochemical studies that all granules in chief cells reacted with antiserum to parathyroid hormone. One possible explanation for these differences in results is that Ravazzola used parathyroid glands obtained from newborn calves, whereas the glands used in the present studies were from adult bovines. The moderately increased background density of the experimental chief cells seen over the control chief cells is probably due to contamination of the cell cytoplasm with parathyroid hormone leeched from the secretory granules. Studies attempting to isolate parathyroid secretory granules have been complicated by lysis of the granules and contamination of the supernatants and other granular fractions with PTH.8-10 No parathyroid hormone is identifiable in the nuclei, mitochondria, or the Golgi complexes, and only small amounts are seen in the aggregates of granular endoplasmic reticulum. The lack of any detectable reaction product in the Golgi region may be a result of its absence in the resting phase Golgi (which comprise 60 to 80% of the cells), its low concentrations in the active Golgi, and/or the relatively high solubility of the hormone when it is in the Golgi region. The minimal reaction in the aggregated granular endoplasmic reticulum could also simply be a consequence of low concentrations of hormone in the region before condensation of the hormone occurs in the secretory granules. On the other hand, the antigenic sites on the hormone may not be as reactive when the hormone is in the form of the precursors preproparathyroid hormone and proparathyroid hormone, characteristic of the hormone during its transport in the membrane and cisternae of the granular endoplasmic reticulum. Further experiments under conditions of stimulation and suppression and with antiserums specific for the "pre-" and the "pro-" portions of the molecule will give additional information on the sites of hormone transport metabolism in the parathyroid gland. References 1. Roth SI, Su SP, Segre GV, Habener JF, Potts JT Jr: The immunocytochemical localization of parathyroid hormone (PTH) in the bovine. Fed Proc 33:241, 1974
(Abstr)
2. Futrell JM, Su SP, Roth SI, Habener JF, Segre GV, Potts JT Jr: Localization of parathyroid hormone in bovine and human parathyroid glands using a peroxidaselabelled antibody. Endocrinology of Calcium Metabolism. Proceedings of the Sixth
Vol. 94, No. 3 March 1979
3.
4.
5.
6. 7.
8.
9.
10. 11. 12. 13.
14.
15. 16. 17.
18.
IMMUNE LOCALIZATION OF PARATHYROID HORMONE
619
International Parathvroid Converence, Vancouver, BC. Edited by DH Copp, RV Talmage. Amsterdam, Excerpta Medica, 1978, p 353 Shannon WA Jr, Roth SI: An ultrastructural study of acid phosphatase activity in normal, adenomatous and hvperplastic (chief cell tvpe) human parathvroid glands. Am J Pathol 77:493-506, 1974 Habener JF: New concepts in the formation, regulation of release, and metabolism of parathyroid hormone. Polvpeptide Hormones: Molecular and Cellular Aspects. Ciba Foundation Svmposium 41 (new series). New York, Elsevier North-Holland, 1976, pp 197-224 Habener JF, Kemper B, Potts JT Jr: Calcium-dependent intracellular degradation of parathyroid hormone: A possible mechanism for the regulation of hormone stores. Endocrinology 97:431-441, 1975 Munger BL, Roth SI: The cytology of the normal parathvroid glands of man and Virginia deer: A light and electron microscopic study with morphologic evidence of secretion. J Cell Biol 17:379-400, 1963 Cohn DV, MacGregor RR, Chu LLH, Huang DWY, Anast CS, Hamilton JW: Biosynthesis of proparathyroid hormone and parathyroid hormone: Chemistry, physiology, and role of calcium in regulation. Am J Med 56:767-773, 1974 L'Heureux MV, Mellius P: Differential centrifugation of bovine parathvroid tissue. Biochim Biophvs Acta 20:447448, 1956 MacGregor RR, Chu LLH, Hamilton JW, Cohn DV: Studies on the subcellular localization of proparathvroid hormone and parathvroid hormone in the bovine parathvToid gland: Separation of newlv synthesized from mature forms. Endocrinology 93:1387-1397, 1973 Roth SI: Uinpublished data Hargis GK, Yakulis VJ, Williams GA, White AA: Cvtological detection of parathvroid hormone by imrnmunofluorescence. Proc Soc Exp Biol Med 117:836-839, 1964 Palmieri GA, Nordquist RE, Omenn GS: Immunochemical localization of parathvroid hormone in cancer tissue from patients with ectopic hvperparath,voidism. J Clin Invest 53:1726-1735, 1974 Stefanini NI, DeMartino C, Zamboni L: Fixation of ejaculated spermatozoa for electron microscopy, Nature [Lond] 216:173-174, 1967 McLean IW, Nakane PK: Periodate-lvsine-paraformaldehvde fixative: A new fixative for immunoelectron microscopy. J Histochem Cvtochem 22:1077-1083. 1974 Mazurkiewicz JR, Nakane PK: Light and electron microscopic localization of antigens in tissues embedded in polvethvlene glvcol with a peroxidase-labelled antibodv method. J Histochem Cvtochem 20:969-974, 1972 Graham RC Jr, Karnovskv MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidnev: Ultrastructural cvtochemistry by a new technique. J Histochem Cvtochem 14:291-302, 1966 Luft JH: Improvements in epoxy resin embedding methods. J Biophvs Biochem Cvtol 9:409-414, 1961 Ravazzola NM, Orci L, Habener JF, Potts JT Jr: Parathyroid secretory protein: ImmunocN-tochemical localization within cells that contain parathyroid hormone. Lancet 2:371-372, 1978
620
FUTRELL ET AL
American Journal of Pathology
[Illustrations follow]
001
*
-~~~~~~~~~~~~~~~~~~~~~~~~
Figure 1-Normal bovine parathyroid gland. The hormone is located in chief cell cytoplasm. A diff use background staining of the cytoplasm with superimposed more intensly stained granules are seen. Nuclei and capillary endothelium are free of reaction. (X 525) Figure 2-Human parathyroid adenoma. The hormone is localized in granules of chief cell cytoplasm. Nuclei, endothelium, and interstitium are free of reaction product. (X 120) Figure 3-Normal bovine parathyroid gland. Normal rabbit serum control with no reaction product in the chief cells. (X 250) Figure 4-N orm al bovine parathyroid. Secretory granules in chief cells of normal rabbit serum control showing osmium staining only. (x 47,000)
5
'~ ~ ~ ~ ~ ~~~~~.1 ~ ~1~
6
Figure 5-Normal bovine parathyroid. Chief cells with extensive hormone localization in the secretory granules. A granular deposition of the reaction product on an overall increase density of cell cytoplasm can be seen. (x Figure 6-Normal bovine parathyroid. The granular nature of the reaction product in secretory 44,800) Figure 7granules is clear. The mitochondrium and nucleus (N) are free of reaction product. (x 100,000) Normal bovine parathyroid. Peroxidase-positive secretory granules. The plasma membranes and the background cytoplasm show an increased density compared with the control glands. (x 71,000)
Light and electron microscopic localization of parathyroid hormone (PTH) in human and bovine parathy roid tissue has been achiev ed using an indirect peroxidase labeled antibodv method. Granular deposition of the reaction product was found throughout the chief cell cytoplasm. There was no nuclear staining. At the ultrastructural lesvel, parathyroid hormone localized by this method appeared to be largely confined to the secretory granules in the cy toplasm of cells. MIitochondria and nuclei were free of reaction product. Aggregated sacs of granular endoplasmic reticulum were minimally reactive, and Golgi apparatuses did not show reaction product. (NM J Pathol 94:615622, 1979)
THE CHIEF CELLS of normal parathyroid glands of humans and other mammals have a continuous secretorv c-cle. consisting of an inactix-e resting phase. followed consecutix-elx- by synthesizing. packaging. secreting. and reco-erx phases.3 The parath! roid hormone is synthesized as preproparathy roid hormone presumably on the aggregated granular endoplasmic reticulum.45 The preprohormone is rapidly converted by proteo(l-tic cleavage to an intermediate precursor. ie. proparathyroid hormone. The prohormone is then transferred to an enlarging Golgi apparatus. w-here it is conv-erted bx- enzymic processes to parathyroid hormone which. in turn. is packaged into membrane-limited secretory granules.6 These granules containing parathyroid hormone, along X ith l-sosomelike granules,3 then move to the periphery of the cell m.-here their membranes fuse with the cell membrane and the contents are emptied into the extracellutlar space. The cells then involute to return to the resting or inactive phase characterized by a small Golgi apparatus. abundant glycogen. and fexx- secretory granules. Althotugh these small membrane-limited granules hav-e been inferred to be the site of storage of parathv-roid hormone in the gland. some authors F ron the Department o)f PatholzxI.Unix ersitx of XrkansaS for \Medical Sciences. Collegze of ledicinee I ittlt RwxAk. \rkansa.s and(l the Departmenit (f \ledicine. Har\ard \ledical Schx(d. and the \laa,achuistts General H 'spital. B1',tn.n \Massachuisetts Supported ir part b\ Grants C-X-IS616 and A\11794 from the Public Health Ser- ice Dr Hahener P, anl Iise"stiuratur at the Hox\ard Hughes \Medical Instittite Dr Segzre is the recipient of Research Career Dexelopment As-;ard .\100070 from NI\IDD Portuiouts of this stuidx "cere presented at the Federated Americ;anl Societx fo)r Experimental Bioluugx ' and at the Sixth International Parathv roid Conferencee. anricnixer. British Colnlmbia. 1977,2 Xddre,ss reprint requciests to Dr Sanford 1 Roth. Departmenrlt of Patholog-. College of Mledicine. Unnix\ersitx of rkana,as for \Medical Sciences. 4301 West \larkham. Little Rock. AR 72201. 0002-9440/79/0308-061 5$01 .00 615 (3 American Association of Pathologists
616
FUTRELL ET AL
American Journal of Pathology
have postulated that in at least some cases parathyroid hormone may bypass these granules.7 Attempts to prove that the granules are the secretory form of the hormone by isolation and analysis of granules obtained from subcellular fractions have met with only limited success.8'0 Studies with fluorescent labeled antibodies to parathyroid hormone have localized the hormone in the chief cells.1",2 This investigation was undertaken to confirm the granular nature of the storage form of the hormone and to determine if the pathway of hormone synthesis in the parathyroid is similar to that postulated for other endocrine glands. Materials and Methods Bovine parathyroid glands obtained from 3- to 4-year-old steers fresh from a local abattoir and human parathyroid adenomas were fixed for 4 to 6 hours at 4 C in Zamboni's 13 picric acid paraformaldehyde (PAF) or periodate-lysine-paraformaldehyde (PLP) fixative.' The tissues were washed overnight with 0.01 M phosphate-buffered saline with 10% sucrose and subsequently embedded in polyethylene glycol (average molecular weight, 950 to 1050) (Matheson Coleman & Bell, Norwood, Ohio). Five- to 10-u sections, floated on a 5% glycerol solution for 20 minutes, were transferred with a wire loop to an albumin-coated slide and dried in a 40 C oven for 30 to 60 minutes.15 The sections were covered for 1 hour with 1: 100 normal sheep serum to minimize nonspecific reactions.'" They were washed three times in 0.1 M phosphate-buffered (pH 7.4) normal saline (PBS) and then treated at 100% relative humidity for 24 hours with either diluted normal rabbit serum (Cappel Laboratories, Pa) or rabbit antiserum (R12) to bovine parathyroid hormone (PTH). After three washes in PBS, the sections were treated with 1:50 peroxidase-labeled goat antirabbit IgG (Cappel) in PBS. Following another 24-hour incubation and washing, the tissues were incubated for 30 minutes in a 3,3'-diaminobenzidine substrate solution without hydrogen peroxide." For the last 1 to 5 minutes, hydrogen peroxide was added to a final concentration of 0.005%. After two additional washings, glass coverslips were placed on the slides with a 90% glycerol solution for light microscopic viewing. For ultrastructural examination, the coverslip was removed and the tissue was washed in PBS. The section was then exposed to 2% osmium tetroxide in distilled water for 2 hours and dehydrated in graded ethanol. While still wet with 100% ethanol, a gelatin capsule filled with epon 17 was inverted over the tissue and the plastic was polymerized at 60 C for 72 hours. The hardened block was removed from the slide by gentle heating over a bunsen burner. The block was trimmed under a dissecting microscope. Thin (70-nm) sections were made on Porter-Blum MT2B ultramicrotomes, mounted on copper grids, and examined without additional staining in a Siemens Elmskop 101 or 102 at 5000 to 20,000 X initial magnification.
Results
Light microscopic studies of human bovine parathyroid glands revealed that the reaction product was deposited diffusely throughout the cytoplasm (Figures 1 and 2) with numerous more intensely staining granules (Figure 1). Nuclei, vascular endothelium, and interstitial tissue appeared free of reaction product. All control sections, including normal rabbit
Vol. 94, No. 3 March 1979
IMMUNE LOCAUZATION OF PARATHYROID HORMONE
617
serum controls (Figures 3 and 4), were virtually free of reaction product and showed no granular staining. Oxvphil cells seen in one adenoma were free of reaction product. At the ultrastructural level, deposition of the reaction product was limited largely to the smaller membrane-limited granules (Figures 5 through 7). No reaction product was seen in the nuclei. Mitochondria (Figure 6) and Golgi apparatuses were free of reaction product; however, the aggregated sacs of granular endoplasmic reticulum were minimallv stained. Not all granules showed staining, even in a cell with maximal deposition of reaction product. There was also considerable variabilitv in the amount of stained granules vs nonstained granules in chief cells from a single tissue section. Dispersed sacs of granular endoplasmic reticulum were free of reaction product. However, control cells (Figure 4) showed only minimal osmium staining and their granules were clearly lighter than experimental sections. There was, in addition, increased electron densitv of the plasma membrane in the experimental sections (Figure 7). The reaction product usually presented as multiple small granules of electrondense material (Figures 5 through 7). Of the two fixatives employed in these experiments, PLP was the most effective in preserving both antigenicity and tissue structure. Although there was some tissue swelling as a result of this technique, preservation was adequate, particularly at the electron microscopic level. Glutaraldehvde fixation, which provided better tissue preservation, destroyed the antigenicity of the parathyroid hormone in the tissue.
These studies demonstrate that the parathyroid hormone is largelv confined to the chief cell cytoplasm of the human and bovine parathyroid gland. The nuclei, vasculature, and intestitial tissue are free of sufficient parathyroid hormone to be identified by inununolocalization. The studies are in agreement of the observations made earlier , but further demonstrate the predominately granular nature of the hormone in the cvtoplasm. Oxyphil cells in a human adenoma did not contain inmmunoreactive parathyroid hormone, confirming the contention of Munger and Roth 6 that the parathyroid oxyphil cell does not generally synthesize parathyroid hormone. At the ultrastructural level, the hormone is largely confined to the small membrane-limited granules which have been postulated to be the sites of parathvroid hormone storage." These granules are smaller and more numerous than the granules which contain acid phosphatase.3 All of the small granules do not contain immunoreactive parathyroid hormone, and
618
FUTRELL ET AL
American Journal of Pathology
the possibility must be considered that they may contain substances other than parathyroid hormone, ie, parathyroid secretory protein,4 degraded hormone,5 other secretory proteins, or even catecholamines. In contrast to these observations, Ravazzola et al 18 found in similar immunocytochemical studies that all granules in chief cells reacted with antiserum to parathyroid hormone. One possible explanation for these differences in results is that Ravazzola used parathyroid glands obtained from newborn calves, whereas the glands used in the present studies were from adult bovines. The moderately increased background density of the experimental chief cells seen over the control chief cells is probably due to contamination of the cell cytoplasm with parathyroid hormone leeched from the secretory granules. Studies attempting to isolate parathyroid secretory granules have been complicated by lysis of the granules and contamination of the supernatants and other granular fractions with PTH.8-10 No parathyroid hormone is identifiable in the nuclei, mitochondria, or the Golgi complexes, and only small amounts are seen in the aggregates of granular endoplasmic reticulum. The lack of any detectable reaction product in the Golgi region may be a result of its absence in the resting phase Golgi (which comprise 60 to 80% of the cells), its low concentrations in the active Golgi, and/or the relatively high solubility of the hormone when it is in the Golgi region. The minimal reaction in the aggregated granular endoplasmic reticulum could also simply be a consequence of low concentrations of hormone in the region before condensation of the hormone occurs in the secretory granules. On the other hand, the antigenic sites on the hormone may not be as reactive when the hormone is in the form of the precursors preproparathyroid hormone and proparathyroid hormone, characteristic of the hormone during its transport in the membrane and cisternae of the granular endoplasmic reticulum. Further experiments under conditions of stimulation and suppression and with antiserums specific for the "pre-" and the "pro-" portions of the molecule will give additional information on the sites of hormone transport metabolism in the parathyroid gland. References 1. Roth SI, Su SP, Segre GV, Habener JF, Potts JT Jr: The immunocytochemical localization of parathyroid hormone (PTH) in the bovine. Fed Proc 33:241, 1974
(Abstr)
2. Futrell JM, Su SP, Roth SI, Habener JF, Segre GV, Potts JT Jr: Localization of parathyroid hormone in bovine and human parathyroid glands using a peroxidaselabelled antibody. Endocrinology of Calcium Metabolism. Proceedings of the Sixth
Vol. 94, No. 3 March 1979
3.
4.
5.
6. 7.
8.
9.
10. 11. 12. 13.
14.
15. 16. 17.
18.
IMMUNE LOCALIZATION OF PARATHYROID HORMONE
619
International Parathvroid Converence, Vancouver, BC. Edited by DH Copp, RV Talmage. Amsterdam, Excerpta Medica, 1978, p 353 Shannon WA Jr, Roth SI: An ultrastructural study of acid phosphatase activity in normal, adenomatous and hvperplastic (chief cell tvpe) human parathvroid glands. Am J Pathol 77:493-506, 1974 Habener JF: New concepts in the formation, regulation of release, and metabolism of parathyroid hormone. Polvpeptide Hormones: Molecular and Cellular Aspects. Ciba Foundation Svmposium 41 (new series). New York, Elsevier North-Holland, 1976, pp 197-224 Habener JF, Kemper B, Potts JT Jr: Calcium-dependent intracellular degradation of parathyroid hormone: A possible mechanism for the regulation of hormone stores. Endocrinology 97:431-441, 1975 Munger BL, Roth SI: The cytology of the normal parathvroid glands of man and Virginia deer: A light and electron microscopic study with morphologic evidence of secretion. J Cell Biol 17:379-400, 1963 Cohn DV, MacGregor RR, Chu LLH, Huang DWY, Anast CS, Hamilton JW: Biosynthesis of proparathyroid hormone and parathyroid hormone: Chemistry, physiology, and role of calcium in regulation. Am J Med 56:767-773, 1974 L'Heureux MV, Mellius P: Differential centrifugation of bovine parathvroid tissue. Biochim Biophvs Acta 20:447448, 1956 MacGregor RR, Chu LLH, Hamilton JW, Cohn DV: Studies on the subcellular localization of proparathvroid hormone and parathvroid hormone in the bovine parathvToid gland: Separation of newlv synthesized from mature forms. Endocrinology 93:1387-1397, 1973 Roth SI: Uinpublished data Hargis GK, Yakulis VJ, Williams GA, White AA: Cvtological detection of parathvroid hormone by imrnmunofluorescence. Proc Soc Exp Biol Med 117:836-839, 1964 Palmieri GA, Nordquist RE, Omenn GS: Immunochemical localization of parathvroid hormone in cancer tissue from patients with ectopic hvperparath,voidism. J Clin Invest 53:1726-1735, 1974 Stefanini NI, DeMartino C, Zamboni L: Fixation of ejaculated spermatozoa for electron microscopy, Nature [Lond] 216:173-174, 1967 McLean IW, Nakane PK: Periodate-lvsine-paraformaldehvde fixative: A new fixative for immunoelectron microscopy. J Histochem Cvtochem 22:1077-1083. 1974 Mazurkiewicz JR, Nakane PK: Light and electron microscopic localization of antigens in tissues embedded in polvethvlene glvcol with a peroxidase-labelled antibodv method. J Histochem Cvtochem 20:969-974, 1972 Graham RC Jr, Karnovskv MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidnev: Ultrastructural cvtochemistry by a new technique. J Histochem Cvtochem 14:291-302, 1966 Luft JH: Improvements in epoxy resin embedding methods. J Biophvs Biochem Cvtol 9:409-414, 1961 Ravazzola NM, Orci L, Habener JF, Potts JT Jr: Parathyroid secretory protein: ImmunocN-tochemical localization within cells that contain parathyroid hormone. Lancet 2:371-372, 1978
620
FUTRELL ET AL
American Journal of Pathology
[Illustrations follow]
001
*
-~~~~~~~~~~~~~~~~~~~~~~~~
Figure 1-Normal bovine parathyroid gland. The hormone is located in chief cell cytoplasm. A diff use background staining of the cytoplasm with superimposed more intensly stained granules are seen. Nuclei and capillary endothelium are free of reaction. (X 525) Figure 2-Human parathyroid adenoma. The hormone is localized in granules of chief cell cytoplasm. Nuclei, endothelium, and interstitium are free of reaction product. (X 120) Figure 3-Normal bovine parathyroid gland. Normal rabbit serum control with no reaction product in the chief cells. (X 250) Figure 4-N orm al bovine parathyroid. Secretory granules in chief cells of normal rabbit serum control showing osmium staining only. (x 47,000)
5
'~ ~ ~ ~ ~ ~~~~~.1 ~ ~1~
6
Figure 5-Normal bovine parathyroid. Chief cells with extensive hormone localization in the secretory granules. A granular deposition of the reaction product on an overall increase density of cell cytoplasm can be seen. (x Figure 6-Normal bovine parathyroid. The granular nature of the reaction product in secretory 44,800) Figure 7granules is clear. The mitochondrium and nucleus (N) are free of reaction product. (x 100,000) Normal bovine parathyroid. Peroxidase-positive secretory granules. The plasma membranes and the background cytoplasm show an increased density compared with the control glands. (x 71,000)
