Neuroendocrinology 1990;51:241-248
© 1990 S. Karger AG, Basel 0028-3835/90/0513-0241 $ 2.75/0
Human Pituitary Corticotroph Adenomas in vitro: Morphologic and Functional Responses to Corticotropin-Releasing Hormone and Cortisol Susanna E. Horvatha, Sylvia L. Asaa. Kalman Kovacsa, Laura A. Adams*, William Singerb, Harley S. Smytfc Departments of aPathology and bMedicine, St. Michael's Hospital, and d e p a rtm e n t of Neurosurgery, th e Wellesley Hospital, University of Toronto, Canada
Key Words. Corticotroph adenoma • Cortisol • Corticotropin-releasing hormone • Tissue culture • Electron microscopy • Morphometry • Radioimmunoassay
Human pituitary corticotrophs show a spectrum of mor phologic changes which are thought to reflect their functional state. Stimulated corticotrophs in patients with adrenal cortical insufficiency undergo enlargement [1] and studies of adrenalectomized rats have shown that these adrenalectomy cells contain abundant rough endoplasmic reticulum and large Golgi appa ratus [2, 3]. In experimental animals, chronic continuous infu sion of corticotropin-releasing hormone (CRH) results in hy perplasia of corticotrophs with mild increase in number and size of secretory granules but no development of adrenalectomy cells [4], while in humans with CRH-producing neoplasms, hy perplasia of corticotrophs has been documented [5-7]; no par ticular ultrastructural appearance has been described to charac terize the stimulated human corticotroph [8]. Suppressed cor ticotrophs in patients with glucocorticoid excess have a paucity of rough endoplasmic reticulum and involuted Golgi regions; their cytoplasm is almost completely occupied by accumula tions of filaments recognized as Crooke’s hyalinization [9-12] Received: February 17, 1989 Accepted after revision: June 28, 1989
and now known to belong to the cytokeratin class of intermedi ate filaments [13]. Several morphologic abnormalities have been described in the pituitaries of patients with Cushing’s disease. Some have a solitary adenoma and Crooke’s hyaline change of nontumorous corticotrophs, suggesting that cortisol suppression selectively affects only the nonneoplastic corticotrophs whereas the neo plastic cells are autonomous, and not responsive to the negative feedback effect of cortisol [14], Others have corticotroph hyper plasia either alone or associated with adenoma; the hyperplastic cells may have variable numbers of stored secretory granules and show a spectrum of ACTH immunoreactivity, and some have a degree of Crooke’s hyalinization [ 14], indicating the pos sibility of underlying stimulation and an interaction between the stimulus and the suppressive effects of elevated circulating cor ticosteroid levels. To characterize structure-function correlations of human adenomatous corticotrophs, this study was designed to quantify and correlate the changes in the morphology and functional ac tivity of corticotroph adenoma cells in vitro when treated with two regulatory hormones, CRH and cortisol.
Downloaded by: King's College London 137.73.144.138 - 1/30/2019 4:44:52 AM
Abstract. We examined the direct effects of corticotropin-releasing hormone (CRH) and cortisol on the morphology of cells from 6 functioning human pituitary corticotroph adenomas in culture using both light and electron microscopic morphometry and correlated the structural changes with alterations in adrenocorticotropin (ACTH) release in each case. During incubations lasting 2 or 24 h, ACTH release was increased by CRH and reduced by cortisol. After incubations lasting from 2 to 72 h, light microscopic morphometric analysis showed no significant differences in cell size, nuclear area, cytoplasmic area or nuclear/cytoplasmic ratio between treated and control adenoma cells. Ultrastructural morphometry documented increased cytoplasmic volume density (CVD) of rough endoplasmic reticulum and/or Golgi apparatus and reduced CVD of secretory granules in cells incubated with CRH. There was no consistent change in CVD of endoplasmic reticulum, Golgi apparatus or secretory granules in adenoma cells incubated with cortisol, but in all tumors there were marked filament accumulations indicating a direct effect of cortisol on adeno matous corticotrophs. The changes were similar after 2- and 72-hour exposures. These results indicate that (1) some adenomatous corticotrophs can respond to CRH and cortisol; (2) the morphologic changes observed in cells treated with CRH correlate with increased ACTH release, and (3) accumulation of filaments is the direct effect of cortisol and is associated with reduced ACTH release.
Horvath/Asa/Kovacs/Adams/Singer/Smyth
242
Materials and Methods
O f 140 adenoma specimens obtained at selective pituitary microsurgical dissection, 6 were classified as well-differentiated, functioning corticotroph adenomas [14]. O f these 6, 4 were obtained from patients with clinically diagnosed Cushing's disease and 2 were from patients with Nelson’s syndrome. Table I summarizes the clinical data of each patient. At the time o f transsphenoidal surgery, each specimen was divided into 3 parts; one was fixed for light microscopic study, one for electron microscopic analysis, and one was used for tissue culture study. Morphologic Methods Tissue for light microscopy was fixed in 10% buffered formalin, de hydrated in graded ethanols and embedded in paraffin. Sections 4-6 pm thick were stained with hematoxylin and eosin and the periodic acid-Schiff technique, lmmunohistochemistry was performed on the paraffin-embedded tissue using the avidin-biotin-peroxidase complex technique as described previously [15]. For electron microscopy, adenoma tissues were fixed in 2.5% glutaraldehyde in Sorensen's buffer, postfixed in 1% OsO-i in Millonig's buf fer, dehydrated in graded ethanols and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and ex amined using a Philips 410-LS electron microscope.
Tissue Culture Methods Tissue for culture was placed aseptically in 10-mi culture medium (CMRL-1969, Connaught, Willowdale, Ont., Canada) containing peni cillin-streptomycin (10,000 pg/m l of each). As described previously [15, 16], cells were dispersed in collagenase solution for 35-45 min at 37 °C with periodic trituration, harvested, resuspended in CMRL-1969 medium with 10% fetal calf serum and then plated onto collagen-coated plastic multiwell culture dishes at a concentration of 2 x I04 cells/ ml/well. Cells were allowed to attach for 24-72 h, after which media were collected every 24 h for radioimmunoassay. Experimental incubations were begun on day 7-11 of culture. Media were removed, the cells were placed in 1 ml fresh medium and were incubated for 24 h. This was followed by a 2-hour incubation in medium without test substances. For each case studied, the cells were divided into three groups: one test group was incubated in CRH at a concentration of 2 x 10~7 M (courtesy of Dr. W. Vale, The Salk Insti tute, LaJolla, Calif.). The second test group was incubated in cortisol at a concentration o f 2.7 x 10"7 M (British Drug House, Toronto, Ont., Canada). Control cells were maintained in vehicle alone. Incubations in these substances were carried out on 4 tumors for 3 consecutive 2- and 22-hour periods and on 2 tumors (tumors 1 and 6) for a single 2-hour period. Upon termination of the experiment, monolayers were trypsinized, harvested by centrifugation into pellets, and fixed and embedded for electron microscopy as described above.
Table 1. Clinical information o f patients with corticotroph adenomas studied in vitro Age
Sex
Clinical presentation
Radiological findings
ACTH levels pmol/1
Cortisol levels nmol/1 a.m.
p.m.
Urinaiy free Cortisol nmol/day
Urinaiy 17 0H C S pm ol/day
1
34
F
Cushing’s disease
suprasellar extension, compression and invasion of sphenoid sinus
62.5
704
775
1,910
ND
2
26
F
Cushing’s disease
supradiaphragmatic extension
40
587
397
469
92
3
30
M
Cushing’s disease
sellar enlargement, cystic necrosis
220
ND
ND
46,000
400
4
26
F
Cushing’s disease
pituitary expanded on right 50% in height
25
880
680
3,000
ND
5
30
F
Nelson’s syndrome
enlarged right side, upward convexity of diaphragmatic sella, stalk deviation
505
ND
ND
ND
ND
6
22
F
Nelson’s syndrome
asymmetic gland enlargement and stalk deviation
104.2
ND
ND
ND
ND
© 1990 S. Karger AG, Basel 0028-3835/90/0513-0241 $ 2.75/0
Human Pituitary Corticotroph Adenomas in vitro: Morphologic and Functional Responses to Corticotropin-Releasing Hormone and Cortisol Susanna E. Horvatha, Sylvia L. Asaa. Kalman Kovacsa, Laura A. Adams*, William Singerb, Harley S. Smytfc Departments of aPathology and bMedicine, St. Michael's Hospital, and d e p a rtm e n t of Neurosurgery, th e Wellesley Hospital, University of Toronto, Canada
Key Words. Corticotroph adenoma • Cortisol • Corticotropin-releasing hormone • Tissue culture • Electron microscopy • Morphometry • Radioimmunoassay
Human pituitary corticotrophs show a spectrum of mor phologic changes which are thought to reflect their functional state. Stimulated corticotrophs in patients with adrenal cortical insufficiency undergo enlargement [1] and studies of adrenalectomized rats have shown that these adrenalectomy cells contain abundant rough endoplasmic reticulum and large Golgi appa ratus [2, 3]. In experimental animals, chronic continuous infu sion of corticotropin-releasing hormone (CRH) results in hy perplasia of corticotrophs with mild increase in number and size of secretory granules but no development of adrenalectomy cells [4], while in humans with CRH-producing neoplasms, hy perplasia of corticotrophs has been documented [5-7]; no par ticular ultrastructural appearance has been described to charac terize the stimulated human corticotroph [8]. Suppressed cor ticotrophs in patients with glucocorticoid excess have a paucity of rough endoplasmic reticulum and involuted Golgi regions; their cytoplasm is almost completely occupied by accumula tions of filaments recognized as Crooke’s hyalinization [9-12] Received: February 17, 1989 Accepted after revision: June 28, 1989
and now known to belong to the cytokeratin class of intermedi ate filaments [13]. Several morphologic abnormalities have been described in the pituitaries of patients with Cushing’s disease. Some have a solitary adenoma and Crooke’s hyaline change of nontumorous corticotrophs, suggesting that cortisol suppression selectively affects only the nonneoplastic corticotrophs whereas the neo plastic cells are autonomous, and not responsive to the negative feedback effect of cortisol [14], Others have corticotroph hyper plasia either alone or associated with adenoma; the hyperplastic cells may have variable numbers of stored secretory granules and show a spectrum of ACTH immunoreactivity, and some have a degree of Crooke’s hyalinization [ 14], indicating the pos sibility of underlying stimulation and an interaction between the stimulus and the suppressive effects of elevated circulating cor ticosteroid levels. To characterize structure-function correlations of human adenomatous corticotrophs, this study was designed to quantify and correlate the changes in the morphology and functional ac tivity of corticotroph adenoma cells in vitro when treated with two regulatory hormones, CRH and cortisol.
Downloaded by: King's College London 137.73.144.138 - 1/30/2019 4:44:52 AM
Abstract. We examined the direct effects of corticotropin-releasing hormone (CRH) and cortisol on the morphology of cells from 6 functioning human pituitary corticotroph adenomas in culture using both light and electron microscopic morphometry and correlated the structural changes with alterations in adrenocorticotropin (ACTH) release in each case. During incubations lasting 2 or 24 h, ACTH release was increased by CRH and reduced by cortisol. After incubations lasting from 2 to 72 h, light microscopic morphometric analysis showed no significant differences in cell size, nuclear area, cytoplasmic area or nuclear/cytoplasmic ratio between treated and control adenoma cells. Ultrastructural morphometry documented increased cytoplasmic volume density (CVD) of rough endoplasmic reticulum and/or Golgi apparatus and reduced CVD of secretory granules in cells incubated with CRH. There was no consistent change in CVD of endoplasmic reticulum, Golgi apparatus or secretory granules in adenoma cells incubated with cortisol, but in all tumors there were marked filament accumulations indicating a direct effect of cortisol on adeno matous corticotrophs. The changes were similar after 2- and 72-hour exposures. These results indicate that (1) some adenomatous corticotrophs can respond to CRH and cortisol; (2) the morphologic changes observed in cells treated with CRH correlate with increased ACTH release, and (3) accumulation of filaments is the direct effect of cortisol and is associated with reduced ACTH release.
Horvath/Asa/Kovacs/Adams/Singer/Smyth
242
Materials and Methods
O f 140 adenoma specimens obtained at selective pituitary microsurgical dissection, 6 were classified as well-differentiated, functioning corticotroph adenomas [14]. O f these 6, 4 were obtained from patients with clinically diagnosed Cushing's disease and 2 were from patients with Nelson’s syndrome. Table I summarizes the clinical data of each patient. At the time o f transsphenoidal surgery, each specimen was divided into 3 parts; one was fixed for light microscopic study, one for electron microscopic analysis, and one was used for tissue culture study. Morphologic Methods Tissue for light microscopy was fixed in 10% buffered formalin, de hydrated in graded ethanols and embedded in paraffin. Sections 4-6 pm thick were stained with hematoxylin and eosin and the periodic acid-Schiff technique, lmmunohistochemistry was performed on the paraffin-embedded tissue using the avidin-biotin-peroxidase complex technique as described previously [15]. For electron microscopy, adenoma tissues were fixed in 2.5% glutaraldehyde in Sorensen's buffer, postfixed in 1% OsO-i in Millonig's buf fer, dehydrated in graded ethanols and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and ex amined using a Philips 410-LS electron microscope.
Tissue Culture Methods Tissue for culture was placed aseptically in 10-mi culture medium (CMRL-1969, Connaught, Willowdale, Ont., Canada) containing peni cillin-streptomycin (10,000 pg/m l of each). As described previously [15, 16], cells were dispersed in collagenase solution for 35-45 min at 37 °C with periodic trituration, harvested, resuspended in CMRL-1969 medium with 10% fetal calf serum and then plated onto collagen-coated plastic multiwell culture dishes at a concentration of 2 x I04 cells/ ml/well. Cells were allowed to attach for 24-72 h, after which media were collected every 24 h for radioimmunoassay. Experimental incubations were begun on day 7-11 of culture. Media were removed, the cells were placed in 1 ml fresh medium and were incubated for 24 h. This was followed by a 2-hour incubation in medium without test substances. For each case studied, the cells were divided into three groups: one test group was incubated in CRH at a concentration of 2 x 10~7 M (courtesy of Dr. W. Vale, The Salk Insti tute, LaJolla, Calif.). The second test group was incubated in cortisol at a concentration o f 2.7 x 10"7 M (British Drug House, Toronto, Ont., Canada). Control cells were maintained in vehicle alone. Incubations in these substances were carried out on 4 tumors for 3 consecutive 2- and 22-hour periods and on 2 tumors (tumors 1 and 6) for a single 2-hour period. Upon termination of the experiment, monolayers were trypsinized, harvested by centrifugation into pellets, and fixed and embedded for electron microscopy as described above.
Table 1. Clinical information o f patients with corticotroph adenomas studied in vitro Age
Sex
Clinical presentation
Radiological findings
ACTH levels pmol/1
Cortisol levels nmol/1 a.m.
p.m.
Urinaiy free Cortisol nmol/day
Urinaiy 17 0H C S pm ol/day
1
34
F
Cushing’s disease
suprasellar extension, compression and invasion of sphenoid sinus
62.5
704
775
1,910
ND
2
26
F
Cushing’s disease
supradiaphragmatic extension
40
587
397
469
92
3
30
M
Cushing’s disease
sellar enlargement, cystic necrosis
220
ND
ND
46,000
400
4
26
F
Cushing’s disease
pituitary expanded on right 50% in height
25
880
680
3,000
ND
5
30
F
Nelson’s syndrome
enlarged right side, upward convexity of diaphragmatic sella, stalk deviation
505
ND
ND
ND
ND
6
22
F
Nelson’s syndrome
asymmetic gland enlargement and stalk deviation
104.2
ND
ND
ND
ND
