Autolytic changes in the rat adenohypophysis

Exp. Path. 17, S. 185-195 (1979) Electron Microscopic Unit, Department of Pathology, St.lI1i ehael's Hospital, University of Toronto, and Ortho Pharmaceutical (Canada) Ltd., Toronto, Ontario, Canada

Autolytic changes in the rat adenohypophysis A hi sto lo gic, immunocytologic and electron microscopic st ud y By G. lLSE, K. KOVACS, N. RYAN, E. HORVATH and D. ILsE With 10 figures (Received December 20, 1978)

Address for correspondence: Dr. G. ILSE, R. T., Electron Microscopic Unit, Department of Pathology St. Michael's Hospital, 30 Bond Street, Toronto, Ontario M5B 1 W8, Canada . Key w 0 r ds: adenohypophysis; autolysis ; ultrastructure; adenohypophysis: cell types; illllllunocytology; electron microscopy; immune peroxidase technique; rat Summary

Thirty-nine adult fem ale Long Evans rats were decapitated and the heads stored at room temperature. The pituitaries were re moved at intervals from 30 minutes to seven days, fixed, embedded and studied by histology, immunocytology and electron microscopy. Histologically, changes were noticeable after two hours postmortem. lmmunoperoxidase staining showed positivity for growth hormone, prolactin, FSH, LH and TSH up to seven days after saerifice, appearing even stronger in the advanced stages of autolysis. Fine structural alterations were evident at 30 minutes and more conspicuous later. Changes included dilation, partial degranulation and whorl formation of RER, swelling of Golgi complexes and mitochondria, chromatin clumping, lysis, rhexis and pyknos is of nuclei, cytosegresome formation and disruption of cell membranes. Secretory granules remained well preserved throughout, although some exhibited fusion or reduced electron density. Dilation of capillaries with accumulation of erythrocytes, platelets and fibrin fibers were prominent findings. The severity of changes varied considerably from cell to cell indicating that the rate of autolysis is not the same among different cell types and is possibly affected by the actual funetional state of the cell. It appears that increased membrane permeability and disruption of plasma.lemma represent important steps in the autolyti c process.

Structural abnormalities whieh aecompany cell death, have a significant impaet on the evaluation of pathologic changes. The process of cell death in the living animal is often complicated by other abnormal events such as hemorrhage, inflammation and regeneration, whereas studies of morphologic alterations which occur during autolysis may yield information about the stability and fate of cellular constituents. Autolytic changes have been studied morphologically by many authors (DAWKINS et al. 1959; MAJNO et al. 1960 ; TRUMP et al. 1962; NOVIKOFF and SHIN 1964 ; TRUMP and ERICSSON 1965; VAN Nn.rWEGEN and SHELDON 1966; HERDSON et al. 1969; TRUMP and ARSTILA 1971; REIMER et al. 1972; KARASEK 1975; COLLAN and SALMENPERA 1976 ; PENTTILA and AHONEN 1976; NEVALAINEN and ANTTINEN 1977; MANN et al. 1978), in a variety of organs such as brain, kidney, liver, striated museIe , myocardium and pancreas, but to our knowledge the sequence of events of autolysis has not yet been investigated in the anterior pituitary. The purpose of the present communication is to define the progression of postmortem changes in the rat adenohypophysis by means of histologic, immunocytologie and electron microscopic observations. Jll aief'ials and methods Thirty-nine Long Eva.ns femaJe rats, fifteen months of age, were used. The animals were individIUtlly eaged in a c.limatically eontrolled room at 26 °C. They were fed Purina rat ehow and a.!lowed tap water ad libitum and were not handled for a period of one year other than during regul ar cage cleaning. The rats were decapita.ted with a gui llotine without anesthesia and the heads stored in a 13 Exp . Path. 17, H. 4

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Fig. 1. Adenohypophysis of a rat 12 hours following decapitation. The capillaries are dilated and contain many erythrocytes. Some adenohypophyseal cells are separated and have pyknotic nuclei. Hematoxylin-phloxine-saffron. X 250. well ventihtted fume hood at 20°C. The pituitaries were removed at zero time, 30 min., 1 hour, 90 min., 3, 4, 6, 8 and 12 hours, 1, 2, 3, 4 and 7 days postmortem and were fixed for histology, immunocytology and electron mi croscopy. For histology and immunocytology, tissues were fixed in buffered 4 % formaldehyde, and embedded in paraffin. Sections of 4- 6 fun thickness were stained with hematoxylin-phloxine-saffron (HPS) and the PAS technique. For immunocytological localization of growth hormone, prolactin, FSH, LH and TSH , the immunoperoxidase method was applied as described in detail elsewhere (MASON et al. 1969 ; STERNBERGER et al. 1970; KOVACS et al. 1976). The specific antibodies used were donated by Dr. ALBERT F. P ARLOW, National Institute of Arthritis, Metabolism and Digestive Diseases (NIAMDD), Rat Pituitary Hormo ne Distribution Program, Harbor General Hospital, Un iversity of California, Los Angeles, California, U.S.A. For electron microscopy, the pituitaries were cut into approximately 1 mm3 pieces, fixed in 2.5 % glutaraldehyde for 2 hours, rinsed in Sorensen's washing solution , post-fixed inl % osmium tetroxide, dehydrated in a graded ethanol series, infiltrated and embedded in Epon-Araldite. Semithin sections were stained with toluidine blue and examined by light microscopy in order to select areas suitable for the fine structural study. Ultrathin sections were stained with uranyl acetate and lead citrat e and investigated with a Philips 300 electron microscope.

Results L i ght Microscopic Findings : Changes were noticeable at 2 hours postmortem. Early alterations included dilation of capillaries and interstitial edema, with widening of the perivascular spaces and separation of individual adenohypophysiocytes. The vascular changes rapidly progressed and within 24 hours became very marked (fig. 1). The distended capillaries were densely packed with aggregated erythrocytes. Later, the vascular walls sho wed int erruptions and erythrocytes began to appear in the expanded extracellular space.

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., Fig. 2. Adenoh ypophysis of a control rat. Immunoreactive pro h~ ctin is evident in the cytophLsm of many cells. Immunoperoxidase t echnique for prolactin. X 250. Fig. 3. Adenohypophysis of a mt 4 days postmortem showing strong immunostaining for prola ctin in autolytic cells. Immunoperoxidase te chnique for prolactin. X 250.

The erythrocytes showed loss of hemoglobin and th e capillary lumina were filled with an acidophilic, pink mass. Changes of adenohypophysio cyt es were evident at 4-6 hours postmortem. First , the nuclear chromatin showed aggregation and chromatin clumps were attached to the internal aspects of the nuclea.r membrane. P yknosis, rhexis and lysis of nuclei soon became pronounce d. The nuclear membranes showed interruptions followed by complete disintegration of the nuclear content. Cytoplasmic changes occurred somewhat lat er than those observed in the nu clei. The cytoplasm had a vacuolated appearance while the cell borders became indistinct with subsequent rupture and disappearance of cell membranes. At 48 hours after decapitation, patchy areas of cell dcbris were apparent and by seven days the outlines of only a few cells were visible. PAS positivity in the cytoplasm was evident for up to seven days postm ortem. Immunoc y tologi c Finding s: The immunoperoxidase t echnique revealed the presence of growth hormone, prolactin , F SH, LH and TSH in the adenoh ypoph ysis for up to seven days postmortem. The intensity of immunostaining varied from cell to cell, not only in t he experimental animals but also in the control mat erial, making it difficult to establish any semi-quantitative comparison between the various groups. Nevertheless , the posit ivity app eared stronger with progression of time (fig. 2 and 3) and appeared to be maxim al by one to two days postmortem. At seven days disintegrating cells still contained brown deposits re presenting immunoreactive hormones. 13"

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Fig. 4. Electron micrograph of the adenohypophysis of a rat 1 hour after decapitation. Dilation of the capillary with accumulation of plasma fluid and endothelial blebbing is evident. Widening of perivascular space is noted. x 8,000.

Fig. 5. Electron micrograph of the adenohyp ophysis of a rat (j hours ,tfter sacrifice. So me cells are swollen and electron lucent while others are shrunken and electron dense. x 5,200.

Electron Microscopic Findings: Fine structural alterations were already apparent 30 minutes following decapitation. The extent and severity of the changes varied not only from one pituitary to another but also from cell to cell within the same adenohypophysis. Vascular changes were conspicuous from 30 minutes after decapitation (fig. 4), with capillaries appearing distended and filled with erythrocytes, platelets and fibrin fibers leading to obstruction of the capillary lumina. By six hours, plasma fluid had accumulated underneath the endothelial cells, resulting in widening of the perivascular spaces, development of interstitial edema, enlargement of intercellular recesses and separation of individual adenohypophyseal cells. The endothelial lining showed focal loss of fenestrae and in some places the endothelial cells became swollen, with vacuolization of the cytoplasm. In other places, the capillary lumina contained disintegrated cytoplasmic constituents. The endothelial nuclei showed pyknosis and rhexis and by seven days almost complete disappearance of nuclei was noted. From 24 hours the vascular walls exhibited disruptions and could only be recognized as individual membrane fragments. Erythrocytes became less electron dense and fragmented during the later phases of autolysis so that by seven days most were indistinguishable from nuclear remnants. The adenohypophyseal cells lost their structural integrity only gradually (fig. 5). Some of them became swollen and electron lucent while others were shrunken and electron dense. At 30 minutes postmortem the nuclei exhibited abnormalities including aggregation of nuclear chromatin with chromatin clumps attached to the internal aspects of the nuclear membrane. From three hours postmortem some nuclei became swollen with gradual disappearance of chromatin while in others the nuelear chromatin became condensed and fragmented (fig. 5) and the nuclear membranes showed disruptions. During the later phases of autolysis, the nuclei almost completely disappeared, with only a few fragmented nuclear membranes or very electron dense chromatin eIumps remaining recognizable. In some cells only nueIear outlines containing membranous structures were noticeable while in others the nuclei were relatively well preserved despite advanced cytoplasmic changes. In the cytoplasm, early changes included a striking decrease in the number of long RER cisternae and the preponderance of circular or elliptic profiles, many of which resembled nebenkerns. The circular membrane configurations enclosed various cytoplasmic constituents such as mitochondria and secretory granules. Golgi complexes were dilated at one hour (fig. 6) and immature granules within the sacculi had fused with one another. Clusters of large circular membranous profiles and lysosomal dense bodies were conspicuous around the Golgi complexes. The mitochondria were swollen with reduced electron density of the matrix. Alterations proceeded rapidly and by three hours postmortem the cytoplasm had become translucent in some cells. From 12 hours progressive fragmentation (fig. 7) of RER occurred with the appearance of indistinct profiles along the surfaces of many RER cisternae. Ribosomes had become detached and free ribosomes were randomly dispersed in the cytoplasm. Golgi complexes had collapsed, vesiculated or disintegrated with loss of their secretory granules. Mitochondria became irregular in shape and the mitochondrial envelope showed thickening with vesiculation of cristae and accumulation of small flocculent densities in the matrix or microcrystal formation in the inner compartments. Many mitochondria were still detectable at seven days postmortem but neither RER cisternae nor Golgi complexes were noticeable from 48 hours. Free ribosomes, however, were numerous. Cytosegresomes were noted at four hours postmortem and became conspicuous from eight hours. Numerous elongated agranular membranes appeared in the cytoplasm which surrounded various cytoplasmic constituents. Lys080mes were increased in size, number and electron density. The secretory granules in the cytoplasm were remarkably well preserved even in those cells which showed advanced autolysis (fig. 8). Initially, the only change was fusion of secretory granules but from 24 hours they became mottled in appearance or pleomorphic or less electron dense. The limiting membranes became more conspicuous and in some secretory granules a wide electron lucent halo appeared between the limiting membrane and the

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Fig. 6. Electron micrograph of the adenohypophysis of a rat 1 hour after decapitation. A lactotroph cell with markedly dilated Golgi complex (arrowhead) and whorl formation of RER can be note d. The Goigi saccul i are devoid of secretory granules. Mitochondria (M)show swelling and rarefaction with loss of cristae. X 7,400.

Fig. 8. Electron mierograph of the adenohypophysis of a rat 3 days after sacrifice. Secretory granules are generally well preserved and can be noted within the boundaries of their respective cells. x 7,000

electron dense core, while in others th e limiting membrane sho wed disruptions and disappeared. Many secretory granules were t aken up by lysosome8. Many seeretory granules however , appeared to be well preserved at seven days po stmortem and were notice d within the boundaries of their respective cells. From 90 minutes a prominent finding was the presenee of various vacuoles in the cytopl as m, principally in the swollen electron lu cent cells. Som e of the vacuoles were ass umed to correspond to lipid droplets, cystically dilated and degranulate d RER membranes, focal cytoplasmic degradations or remnants of mitochondria. :Many large vacuoles were filled with plasma fluid and seemed to represent invaginations of the plasma membranes. From about 12 h ours postmortem th e plasm a membranes became disrupted. This chan ge rapidly progressed an d various cytoplas mie constituents were seen in the edemato us, dist ende d, extracellular space. No intact plasma membranes were noted from about 48 hours postmortem. Since the rat adenohyp ophysis is co mpose d of five distinct cell typ es (som atotrophs, laetotrophs, go nadotrophs, thyrotrophs and corticotrophs) (COSTOFF 1973 ; Kmwsmn and F U J ITA 1975) , it seemed of importance t o determine whether antolysis affe cte d one parti cular cell type earlier an d 111 ore severel y t han others. This qu estio n re mained however , unanswered, since wit h advancin g a utolysis, cell identification became impossible (fig. 9). In th e early phases marked differ ences were apparent in the progression of autolysis among the various cell t ypes. In general, lactotrophs, a nd to a lesser extent somatotrophs un derwent autolysis more rapidl y than other cell types. The lact otrophs wer e especially Fig. 7. Electron micrograph of the adenohypophys is of a rat 24 ho urs after decapitation. The RER exhibits whorl format ion and vesiculation. F ocal Joss of cytoplas m, cytosegresome for mation (arrows) are prominent. X 7,400.

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Fig. 9. Electron micrograph of the adenohypophysis of a rat 7 days after decapitation. No cell membranes can be recognized and the subcellular details are lost. The remnants of a blood vessel is still seen. X 5,400.

prone to develop swelling (fig. 10) and to exhibit fusion of secretory granules as well as alterations of RER membranes and Golgi complexes. These changes were noted from 30 minutes postmortem. Thyrotrophs and corticotrophs also exhibited fusion of secretory granules while such a change was evident neither in the somatotrophs, nor in the gonadotrophs. Nuclear changes were prominent and more advanced in somatotrophs and corticotrophs. Karyolysis was a prominent finding in lactotrophs whereas pyknosis was more frequent in thyrotrophs and corticotrophs. Progression of mitochondrial, lysosomal and cell membrane changes were similar in all cell types except for the mitochondria of somatotrophs which showed a more rapid autolysis.

Discussion Present results clearly show that the rat adenohypophysis is prone to undergo rapid autolysis after cessation of its blood supply. The morphologic changes were similar to those seen following destruction of the pituitary stalk which is known to interrupt portal blood flow, and cause ischemia (KOVACS et al. 1977). With conelusive evidence that the adenohypophysis is sensitive to oxygen deficiency it is therefore imperative that for successful electron microscopical studies, the pituitary be fixed immediately upon removal. Any delay in fixation would permit rapid progression of autolytic alterations and would jeopardize effective assessment of the morphologic changes. It is also apparent that tissue obtained at autopsy is of very limited value if one wishes to reach meaningful conclusions from fine structural investigations of the pituitary gland. Studies dealing with postmortem autolysis always seek answers to the question which of the cellular constituents may be most susceptible to discontinuation of blood flow. Since the rate of autolysis varies considerably not only from one pituitary to another, but also from cell to cell even within the same adenohypophysis, it is not readily feasible to identify those cellular eonstituents which are more vulnerable than others to the arrest of circulation. Alterations were evident in the nuclei, RER, Golgi apparatus and mitochondria within 30 minutes after decapitation, consistent with the assumption that the autolytic process affected several cellular constituents simultaneously. The rapid autolysis of the Golgi apparatus was of particular importance. By 12 hours, the Golgi complexes were dilated or appeared collapsed while the formation and maturation of secretory granules seemed to have stopped inside the Golgi apparatus. The secretory granules were absent from the Golgi sacculi, indicating, that hormone synthesis must have been interrupted at an early phase of autolysis. Subendothelial, perivascular and interstitial edema was noted from one hour postmortem, and was followed by prominent abnormalities in the blood vessel walls. The vascular changes, parallel with the alterations disclosed in the parenchymal cells, advanced rapidly after decapitation and became conspicuous within three hours. Swelling or shrinkage of adenohypophyseal cells can be interpreted in functional terms as uptake of plasma fluid or loss of intracellular water due to damage of the cell membrane, and may suggest rapid alterations of membrane permeability during the conrse of autolysis. Rupture of cell membranes was detected by electron microscopy only at 12 hours postmortem. Although the rupture of the plasma membrane appeared to accelerate the disintegration of adenohypophyseal cells, more work is required to elucidate the significance of membrane changes in the pituitary during autolysis. Since the adenohypophysis is composed of 5 different cell populations (COST OFF 1973; KUROSUMI and FUJITA 1975), it seemed reasonable to suggest that the various cell types are not equally sensitive to oxygen deficiency. The present study lends support to this supposi-

Fig. 10. Electron micrograph of the adenohypophysis of a rat 3 hours postmortem. Lactotroph cells (L) exhibit severe damage. One corticotroph cell (C) and one gonadotroph (G) elm be identified. X 3,000.

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tion and clearly shows that the acidophil cell types (somatotrophs and laetotrophs) are more sensitive to arrest of blood circulation than the basophil cell types (gonadotrophs, thyrotrophs and corticotrophs). It appears that the lactotrophs which contain well-developed HER and large Golgi complexes are especially vulnerable to hypoxia. Lactotrophs with these fine structural features are regarded as representing actively secreting cells (HYMER et al. 1961). The question of whether the functional state of the cell may affect the rate of autolysis remains to be established. In the later phases of autolysis fine structural changes were very marked and cell identification became impossible, thus preventing further correlation of autolysis among the various cell types. The immunoperoxidase technique showed considerable quantities of immunoreactive hormones in the adenohypophysis for all postmortem intervals studied. Consistent with these findings it was evident from electron microscopic observations that the secretory granules remained well preserved up to seven days postmortem. These results indicate that despite advanced autolysis, the adenohypophyseal hormones had not decomposed and had retained their antigenicity. In some cells, the intensity of immnnostaining was more pronounced than in those of the control pituitaries fixed immediately after decapitation. Although the 2ignificance of this finding is not entirely clear, it seems conceivable that in autolyzing cells, antigenic sites may have become more accessible to the action of antibody. It remains to be determined as to whether the intense immul10staining could have been due to easier penetration of the antibody or to the unmasking of antigenic sites due to loss of protein binding, or to some unknown mechanisms. The immunoperoxidase technique is claimed to provide information only on hormone storage. However, variations in antigen accessibility may account for an enhanced or reduced immunopositivity without an actual change in cytoplasmic hormone content.

Acknowledgements This work was supp~rted in part by the Medical Research Council of Canada (Grant MA-6349). The authors wish to thank Mrs. Cynthia Edwards and Mrs. Judy Bruno for excellent technical assistance and Mrs. Linda Traeger for valuable secretarial help.

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