Nutr. Meta bol. 20: 67-75 (1976)
Effect of Starvation on Pituitary Growth Hormone Cells and Blood Growth Hormone and Prolactin Levels in the Rat A.M.T. Sirek, E. Horvath, C. Ezrin and K. Kovacs Department of Pathology, University of Toronto, Banting Institute, Toronto, Ont., and Departments of Medicine and Pathology, University of Toronto, St. Michael’s Hospital, Toronto, Ont.
Key Words. Starvation ■Pituitary • Pituitary morphology • Growth hormone • Prolactin Abstract. Growth hormone (GH) cells of rats were studied on days 2, 4 and 7 of starvation. Immunoperoxidase staining for light microscopy confirmed the presence of GH in the pituitaries of all groups of animals. Election microscopy revealed crinophagy in the cytoplasm of GH cells on days 4 and 7. By ultrastructural morphometry, volume density and the diameter of secretory granules in the cytoplasm of GH cells remained unchanged. Blood GH determinations showed a significant decrease on day 4 of the starvation period. On day 7 most of the values were in the range of the controls. Blood prolactin levels fell significantly on day 7. It appears that the pituitary is capable of secreting GH even in rats completely deprived of exogenous nutrients.
Starvation has profound effects on growth and on endocrine function, in cluding tire secretory activity of the pituitary gland. Many investigators have sought to help elucidate this controversial problem. In humans, fasting and hypoglycemia are associated with elevated plasma growth hormone (GH) levels ( 2,
12).
Received: December 8, 1975; accepted: February 16, 1976.
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
In rats deprived of food, but with free access to water, Mulinos and Pomerantz (10) documented a decrease in the weights of the male accessory sex organs and the pituitary. Because similar observations were noted in hypophysectomized animals, they concluded that inanition produced a pseudohypophysectomized state. Srebnik and Nelson (13) reported reduced pituitary content of GH and diminished size of pituitary acidophils in rats after 5 weeks on a protein deficient diet. Friedman and Reichlin (4) showed decreased pituitary GH content in rats after 1, 2 and 3 days of starvation. Dickerman etal. (3) observed
Sirek /Horvath /Ezrin/Kovacs
68
a fall in GH levels in the plasma and pituitaries of rats after 7 days of starvation. Dickerman et al. (3) and Meites and Fiel (9) have suggested that GH-releasing factors is reduced in the hypothalami of starved rats. All of tire quoted studies in rats were undertaken using the tibia bioassay technique to measure GH. Trenkle (16), in contrast to earlier authors, showed that in rats, the fall in pituitary GH concentration on the seventh day of starvation was preceded by a rise on tire second and fifth days. He also found a fall in plasma GH using a radioimmunoassay with guinea pig antiserum to porcine GH. Akikusa (1) found decreased pituitary GH levels in rats after 1, 3 and 5 days of starvation. Hormone assay in this case was done by incubation with 14Cleucine followed by polyacrylamide disc electrophoresis and radioactivity counting. No reports on rat pituitary ultrastructure during starvation were encountered in the literature available to us. The present study reports our observations on the morphology of the rat pituitary GH cell during starvation and the corresponding serum GH levels. The study was extended to assess serum prolactin (PRL) levels as well.
Materials and Methods
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
45 male Wistar rats with a mean initial body weight of 240 g, were starved, but given drinking water ad libitum and, with 15 fed control animals were sacrificed on the second, fourth, and seventh day of starvation. The fasting animals were housed in separate cages with wire-net floors which ensured against coprophagy. Animals were exposed to naturally occurring daylight and darkness by means of large windows in a room with the lights off. They were decapitated between 9 and 10 a.m. on the appropriate day. The testes, seminal vesicles, prostate, spleen and thymus glands were weighed using an analytical balance to the precision of 0.1 mg. Body weight before removal of food and on the morning of sacrificing were also recorded. Pituitaries for light microscopy were fixed in 10% neutral formol saline, and embedded in paraffin. Sections of 6 jam thickness were stained with haematoxylin and eosin and with the immunoperoxidase stain for GH. The technique was that of Mason et al. (6 ), with the following modifications: (1) The section was exposed to anti-GH serum for 15 min (antirat GH serum was obtained from the National Institute of Arthritis and Metabolic Diseases, NIH, Rat Hormone Distributing Programme, Bethesda. Md.). (2) In place of horse radish peroxidase followed by antiperoxidase, the horseradish-pcroxidase-antiperoxida.se complex of Sternberger et al. (14) was used. (The peroxidase-antiperoxidase complex was a gift of Dr. L.A. Sternberger. Edgewood Arsenal, Md.) The specificity of the technique was confirmed by lack of staining when anti-GH serum, preincubated with excess GH, was applied to the section. Exposing the section to rabbit 7 -globulin instead of anti-GH serum, also resulted in negative staining. Pituitaries for electron microscopy were dissected immediately, minced into small pieces with a razor blade and fixed in veronal acetate buffered 0 s 0 4. The pieces were dehydrated in graded ethanol and embedded in Epon-Araldite. Ultra-thin sections were cut, and double stained with uranyl acetate and lead hydroxide. The grids were studied by conventional transmission electron microscopy using a RCA EMU-3F electron microscope
Effect of Starvation on Growth Hormone and Prolactin
69
and with ultrastructural morphometry. Electron micrographs were made at X 4,100 magnifi cation; the developing of prints introduced an additional magnification factor of X 6. The diameter of approximately 2,000 secretory granules in the cytoplasm of GH cells was measured in each group of animals. Frequencies per 1,000 granules were graphed. Volume density counts were done by placing a transparent grid with evenly spaced, broken lines over the photograph. The end points of the line segments formed a network of points. The number of points superimposed over granules compared to the total number of points over the field yielded the volume density (17). Points over the nucleus were not counted. Seven cells per animal and five animals per group were evaluated in this way. Blood for hormone determination was collected from the trunk after decapitation. The serum was separated and frozen until the time of assay (the maximum time in deep freeze storage was 4 months). GH and PRL radioimmunoassays were done using a double antibody technique, according to the instructions accompanying the antisera from the National Insti tute of Arthritis and Metabolic Diseases. N1H. Rat GH labelled with 1311, and monkey antirat GH serum with rabbit antimonkey >-globulin serum as the second antibody, were the materials used for the GH determinations. Rat PRL, rabbit antirat PRL serum, and rhesus antirabbit 7 -globulin scrum were used in the PRL assay. The Student’s t test was used for the statistical evaluation of results.
Results Body and organ weights. Decrease in body weights occurred as expected in starvation (fig. 1). The testicular weights of starved animals did not differ signifi cantly from those of the controls. The prostate and seminal vesicles, however, showed progressive atrophy as the starvation period was prolonged (fig. 2). Spleen and thymus weights also decreased (fig. 2). Light microscopy. Haematoxylin and eosin staining showed no difference between the acidophils of starved as compared to control animals. Immunostaining. GH immunostaining showed dark brown deposits of equal intensity and distribution in all groups of animals (fig. 3). Electron microscopy. Electron micrographs of GH cells showed the charac teristic large, round, secretory granules and prominent, rough-surfaced endoplas-
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
Fig. I. Percentage changes in body weight during starvation.
Sirek /Horvath /Ezrin /Ko vacs
70
Fig. 2. Changes in organ weights during starvation.
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
/V ; Fig. 3. Light microscopic features of a pituitary immunostaincd for GH after 4 days of starvation. Dark deposits (arrowheads) represent immunoreactive GH in the cytoplasm of the cells, x 500.
Effect of Starvation on Growth Hormone and Prolactin
71
mic reticulum in both the control and the starved animals. Increased size and number of Iysosomes were present in the GH cells of starved animals, especially on the fourth and seventh days of starvation. Crinophagy was evident; Iysosomes were engulfing secretory granules (fig. 4, 5). EM morphometry. The frequency distribution of the GH-granule diameter showed no objective difference in the size of granules in starved as compared to control animals. The mode in each group occurred at 312 nm. There was no statistically significant skewing or kurtosis in any of the groups (fig. 6). The volume density of GH granules per cell profile indicated that there was no objective increase or decrease in granulation of the cells. The mean volume density of each group of starved animals was compared to the mean for the control animals using the Student’s t test. No significant change occurred (fig- 7). Serum GH and PRL. The serum GH values showed a large spread. There was a statistically significant fall on day 4 of starvation and a rise on day 7 (fig. 8). However, the biological significance of these changes is questionable. Serum PRL determinations also showed considerable variation. The decrease in serum PRL on the seventh day of starvation was significant (fig. 9).
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
Fig. 4. Electron micrograph of a GH-celi from a control animal. Note the large, round, secretory granules (arrowheads) and the well-developed, rough-surfaced endoplasmic reticu lum (RER). X 12,300.
Sirek /Horvath /Ezrin ¡Kovacs
72
Fig. 5. Electron micrograph of a GH cell after 4 (lays of starvation. Crinophagy is evident (arrowheads). X 12,300.
DAYS OF STARVATION
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
Fig. 6. Frequency distribution of secretory granule diameter in GH cells. Fig. 7. Volume density of secretory granules of GH cells per cell profile.
73
Effect of Starvation on Growth Hormone and Prolactin
.
100 * 90 300 80 70 -
•
60 -
200 -
50 40 100 80 40 -J
1
S'
20 -
* —
!
•
__
,
T
60 E
30 -
-
•
; ►
.
v
s
r
►
0
2
4
7
20 E
S
Days of starvation
10 -
: :
;
•
-f-
---1--------- 1--------- 1--------- 1 0 2 4 7 Days of starvation
Fig. 8. Serum GH values during starvation. Fig. 9. Scrum PRL values during starvation.
The weights of the accessory sex organs during starvation showed a pro nounced decrease while the testes showed only minimal change. These observa tions are in agreement with those of Negro-Vilar et al. (11). The decrease in thymus weight confirms the original observations of Jackson (5) and the more recent work of McAnulty and Dickerson (7). The decreased spleen weight is consistent with the reduction in size of tonsils and peripheral lymphatic tissues observed in malnutrition (7). To our knowledge, the morphology of the rat GH cell during starvation has not been previously reported. The presence of GH immunostaining of equal intensity in both fed and starved animals is complemented by the electron microscopic findings. No change in the size of GH granules nor in the granula tion of the GH cells was observed. The significance of the crinophagy observed in the GH cells of starved animals remains to be established. The wide range in serum GH values that we obtained confirms the findings of Takahashi et al. (15). This very large variation almost precludes biological
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
Discussion
Sirek/Horvath /Ezrin /Kovacs
74
significance despite statistical significance. Samples drawn at such relatively long intervals as 48 or 72 h may not reflect invariably the minute-to-minute changes in circulating GH levels. It would appear that the pituitary gland of the rat is still capable of synthesizing and secreting GH even when it is completely deprived of exogenous nutrients. There is very little literature on the behaviour of PRL in the rat during starvation. Akikusa (1) showed no change in PRL content of the rat pituitary gland after 1, 3 and 5 days of starvation. McAttie and Trenkle (8) reported decreased serum PRL by radioimmunoassay in heifers after 2 days of starvation. Our results in rats showed a fall in serum PRL only after a relatively prolonged period of starvation. The decrease in blood levels of PRL raises many speculative questions, and its significance remains to be elucidated.
A cknowledgements The authors are indebted to Mrs. Erika Johansson (Department of Neuroendocrinol ogy, Clarke Institute, University of Toronto) for the GH and PRL radioimmunoassays; to Mr. Gerhard Penz and Mr. Ernest Whitter for technical assistance and to Miss Elizabeth Warner for typing the manuscript.
1 2
3
4 5 6
7 8 9
Akikusa, Y.: Effect of starvation on synthesis and release of growth hormone and prolactin in the rat anterior pituitary. Endocr. jap. 18: 411-416 (1971). Cahill, G.F., jr.; Herrera, M.G.; Morgan, A.P.; Soeldner, J.S.; Steinke. J.; Levy, P.L.; Reichard, G.A., />., and Kipnis, D.M.: Hormone-fuel interrelationships during fasting. J. clin. Invest. 45: 1751-1769 (1966). Dickerman, E.; Negro-Vilar, A., and Meites. J.: Effects of starvation on plasma GH activity, pituitary GH and GH-RE levels in the rat. Endocrinology 84: 814-819 (1969). Friedman, R.C. and Reichlin, S.: Growth hormone content of the pituitary gland of starved rats. Endocrinology 76: 787 -788 (1965). Jackson, C.M.: The effects of inanition and malnutrition upon growth and structure (Blakiston, Philadelphia 1925). Mason, T.E.; Phifer. R.F.; Spicer, S.S.; Swallow, R.A., and Dreskin, R.B.: An immuno globulin-enzyme bridge method for localizing tissue antigens. J. Histochem. Cytochem. 17: 563 569(1969). McAnulty, P.A. and Dickerson, J.W.T.: The cellular response of the weanling rat thymus gland to undernutrition and rehabilitation. Pediat. Res. 9: 778-785 (1973). McAttee, J.W. and Trenkle, A.: Effects of feeding, fasting, glucose or arginine on plasma prolactin levels in the bovine. Endocrinology 89: 730 734 (1971). Meites, J. and Fiel, N.J.: Effect of starvation on hypothalamic content of ‘somato tropin releasing factor’ and pituitary growth hormone content. Endocrinology 77: 455 460(1965).
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
References
Effect of Starvation on Growth Hormone and Prolactin
75
10 Mulinos, M.G. and Pomerantz, Pseudo-hypophysectomy: a condition resembling hypophysectomy produced by malnutrition. J. Nutr. 19: 493-504 (1940). 11 Negro-Vilar. A.; Dickerman, E., and Meites, J.: Effects of starvation on hypothalamic FSH-RE and pituitary FSH in male rats. Endocrinology 88: 1246- 1249 (1971). 12 Roth. J.: Click, S.M.; Yalow, R.S., and Berson, S.A.: Hypoglycemia: a potent stimulus to secretion of growth hormone. Science 140: 987-988 (1963). 13 Srebnik. H.H. and Nelson, M.M.: Anterior pituitary function in male rats deprived of dietary protein. Endocrinology 70: 723- 730 (1962). 14 Sternberger. L.A.; Hardy, P.H., jr.; Cuculis, J.J., and Meyer, H.G.: The unlabelled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen antibody complex (horseradish peroxidase- antihorseradish peroxi dase) and its use in identification of spirochetes. J. Histochcm. Cytochem. 18: 315 333 (1970). 15 Takahashi, K.: Daughaday, W.H., and Kipnis, D.M.: Regulation of immunoreactive growth hormone secretion in male rats. Endocrinology 88: 909-917 (1971). 16 Trenkle, A.: Effect of starvation on plasma growth hormone in rats. Proc. Soc. exp. Biol. Med. 135: 77-80(1970). 17 Weibel, E.R.: Sterological principles for morphometry in electron microscopic cytol ogy. lnt. Rev. Cytol. 26: 235 302 (1969).
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
A.M.T. Sirek, MD, Department of Medicine, St. Michael’s Hospital, 30 Bond Street, Toronto, Ont. (Canada)
Effect of Starvation on Pituitary Growth Hormone Cells and Blood Growth Hormone and Prolactin Levels in the Rat A.M.T. Sirek, E. Horvath, C. Ezrin and K. Kovacs Department of Pathology, University of Toronto, Banting Institute, Toronto, Ont., and Departments of Medicine and Pathology, University of Toronto, St. Michael’s Hospital, Toronto, Ont.
Key Words. Starvation ■Pituitary • Pituitary morphology • Growth hormone • Prolactin Abstract. Growth hormone (GH) cells of rats were studied on days 2, 4 and 7 of starvation. Immunoperoxidase staining for light microscopy confirmed the presence of GH in the pituitaries of all groups of animals. Election microscopy revealed crinophagy in the cytoplasm of GH cells on days 4 and 7. By ultrastructural morphometry, volume density and the diameter of secretory granules in the cytoplasm of GH cells remained unchanged. Blood GH determinations showed a significant decrease on day 4 of the starvation period. On day 7 most of the values were in the range of the controls. Blood prolactin levels fell significantly on day 7. It appears that the pituitary is capable of secreting GH even in rats completely deprived of exogenous nutrients.
Starvation has profound effects on growth and on endocrine function, in cluding tire secretory activity of the pituitary gland. Many investigators have sought to help elucidate this controversial problem. In humans, fasting and hypoglycemia are associated with elevated plasma growth hormone (GH) levels ( 2,
12).
Received: December 8, 1975; accepted: February 16, 1976.
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
In rats deprived of food, but with free access to water, Mulinos and Pomerantz (10) documented a decrease in the weights of the male accessory sex organs and the pituitary. Because similar observations were noted in hypophysectomized animals, they concluded that inanition produced a pseudohypophysectomized state. Srebnik and Nelson (13) reported reduced pituitary content of GH and diminished size of pituitary acidophils in rats after 5 weeks on a protein deficient diet. Friedman and Reichlin (4) showed decreased pituitary GH content in rats after 1, 2 and 3 days of starvation. Dickerman etal. (3) observed
Sirek /Horvath /Ezrin/Kovacs
68
a fall in GH levels in the plasma and pituitaries of rats after 7 days of starvation. Dickerman et al. (3) and Meites and Fiel (9) have suggested that GH-releasing factors is reduced in the hypothalami of starved rats. All of tire quoted studies in rats were undertaken using the tibia bioassay technique to measure GH. Trenkle (16), in contrast to earlier authors, showed that in rats, the fall in pituitary GH concentration on the seventh day of starvation was preceded by a rise on tire second and fifth days. He also found a fall in plasma GH using a radioimmunoassay with guinea pig antiserum to porcine GH. Akikusa (1) found decreased pituitary GH levels in rats after 1, 3 and 5 days of starvation. Hormone assay in this case was done by incubation with 14Cleucine followed by polyacrylamide disc electrophoresis and radioactivity counting. No reports on rat pituitary ultrastructure during starvation were encountered in the literature available to us. The present study reports our observations on the morphology of the rat pituitary GH cell during starvation and the corresponding serum GH levels. The study was extended to assess serum prolactin (PRL) levels as well.
Materials and Methods
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
45 male Wistar rats with a mean initial body weight of 240 g, were starved, but given drinking water ad libitum and, with 15 fed control animals were sacrificed on the second, fourth, and seventh day of starvation. The fasting animals were housed in separate cages with wire-net floors which ensured against coprophagy. Animals were exposed to naturally occurring daylight and darkness by means of large windows in a room with the lights off. They were decapitated between 9 and 10 a.m. on the appropriate day. The testes, seminal vesicles, prostate, spleen and thymus glands were weighed using an analytical balance to the precision of 0.1 mg. Body weight before removal of food and on the morning of sacrificing were also recorded. Pituitaries for light microscopy were fixed in 10% neutral formol saline, and embedded in paraffin. Sections of 6 jam thickness were stained with haematoxylin and eosin and with the immunoperoxidase stain for GH. The technique was that of Mason et al. (6 ), with the following modifications: (1) The section was exposed to anti-GH serum for 15 min (antirat GH serum was obtained from the National Institute of Arthritis and Metabolic Diseases, NIH, Rat Hormone Distributing Programme, Bethesda. Md.). (2) In place of horse radish peroxidase followed by antiperoxidase, the horseradish-pcroxidase-antiperoxida.se complex of Sternberger et al. (14) was used. (The peroxidase-antiperoxidase complex was a gift of Dr. L.A. Sternberger. Edgewood Arsenal, Md.) The specificity of the technique was confirmed by lack of staining when anti-GH serum, preincubated with excess GH, was applied to the section. Exposing the section to rabbit 7 -globulin instead of anti-GH serum, also resulted in negative staining. Pituitaries for electron microscopy were dissected immediately, minced into small pieces with a razor blade and fixed in veronal acetate buffered 0 s 0 4. The pieces were dehydrated in graded ethanol and embedded in Epon-Araldite. Ultra-thin sections were cut, and double stained with uranyl acetate and lead hydroxide. The grids were studied by conventional transmission electron microscopy using a RCA EMU-3F electron microscope
Effect of Starvation on Growth Hormone and Prolactin
69
and with ultrastructural morphometry. Electron micrographs were made at X 4,100 magnifi cation; the developing of prints introduced an additional magnification factor of X 6. The diameter of approximately 2,000 secretory granules in the cytoplasm of GH cells was measured in each group of animals. Frequencies per 1,000 granules were graphed. Volume density counts were done by placing a transparent grid with evenly spaced, broken lines over the photograph. The end points of the line segments formed a network of points. The number of points superimposed over granules compared to the total number of points over the field yielded the volume density (17). Points over the nucleus were not counted. Seven cells per animal and five animals per group were evaluated in this way. Blood for hormone determination was collected from the trunk after decapitation. The serum was separated and frozen until the time of assay (the maximum time in deep freeze storage was 4 months). GH and PRL radioimmunoassays were done using a double antibody technique, according to the instructions accompanying the antisera from the National Insti tute of Arthritis and Metabolic Diseases. N1H. Rat GH labelled with 1311, and monkey antirat GH serum with rabbit antimonkey >-globulin serum as the second antibody, were the materials used for the GH determinations. Rat PRL, rabbit antirat PRL serum, and rhesus antirabbit 7 -globulin scrum were used in the PRL assay. The Student’s t test was used for the statistical evaluation of results.
Results Body and organ weights. Decrease in body weights occurred as expected in starvation (fig. 1). The testicular weights of starved animals did not differ signifi cantly from those of the controls. The prostate and seminal vesicles, however, showed progressive atrophy as the starvation period was prolonged (fig. 2). Spleen and thymus weights also decreased (fig. 2). Light microscopy. Haematoxylin and eosin staining showed no difference between the acidophils of starved as compared to control animals. Immunostaining. GH immunostaining showed dark brown deposits of equal intensity and distribution in all groups of animals (fig. 3). Electron microscopy. Electron micrographs of GH cells showed the charac teristic large, round, secretory granules and prominent, rough-surfaced endoplas-
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
Fig. I. Percentage changes in body weight during starvation.
Sirek /Horvath /Ezrin /Ko vacs
70
Fig. 2. Changes in organ weights during starvation.
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
/V ; Fig. 3. Light microscopic features of a pituitary immunostaincd for GH after 4 days of starvation. Dark deposits (arrowheads) represent immunoreactive GH in the cytoplasm of the cells, x 500.
Effect of Starvation on Growth Hormone and Prolactin
71
mic reticulum in both the control and the starved animals. Increased size and number of Iysosomes were present in the GH cells of starved animals, especially on the fourth and seventh days of starvation. Crinophagy was evident; Iysosomes were engulfing secretory granules (fig. 4, 5). EM morphometry. The frequency distribution of the GH-granule diameter showed no objective difference in the size of granules in starved as compared to control animals. The mode in each group occurred at 312 nm. There was no statistically significant skewing or kurtosis in any of the groups (fig. 6). The volume density of GH granules per cell profile indicated that there was no objective increase or decrease in granulation of the cells. The mean volume density of each group of starved animals was compared to the mean for the control animals using the Student’s t test. No significant change occurred (fig- 7). Serum GH and PRL. The serum GH values showed a large spread. There was a statistically significant fall on day 4 of starvation and a rise on day 7 (fig. 8). However, the biological significance of these changes is questionable. Serum PRL determinations also showed considerable variation. The decrease in serum PRL on the seventh day of starvation was significant (fig. 9).
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
Fig. 4. Electron micrograph of a GH-celi from a control animal. Note the large, round, secretory granules (arrowheads) and the well-developed, rough-surfaced endoplasmic reticu lum (RER). X 12,300.
Sirek /Horvath /Ezrin ¡Kovacs
72
Fig. 5. Electron micrograph of a GH cell after 4 (lays of starvation. Crinophagy is evident (arrowheads). X 12,300.
DAYS OF STARVATION
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
Fig. 6. Frequency distribution of secretory granule diameter in GH cells. Fig. 7. Volume density of secretory granules of GH cells per cell profile.
73
Effect of Starvation on Growth Hormone and Prolactin
.
100 * 90 300 80 70 -
•
60 -
200 -
50 40 100 80 40 -J
1
S'
20 -
* —
!
•
__
,
T
60 E
30 -
-
•
; ►
.
v
s
r
►
0
2
4
7
20 E
S
Days of starvation
10 -
: :
;
•
-f-
---1--------- 1--------- 1--------- 1 0 2 4 7 Days of starvation
Fig. 8. Serum GH values during starvation. Fig. 9. Scrum PRL values during starvation.
The weights of the accessory sex organs during starvation showed a pro nounced decrease while the testes showed only minimal change. These observa tions are in agreement with those of Negro-Vilar et al. (11). The decrease in thymus weight confirms the original observations of Jackson (5) and the more recent work of McAnulty and Dickerson (7). The decreased spleen weight is consistent with the reduction in size of tonsils and peripheral lymphatic tissues observed in malnutrition (7). To our knowledge, the morphology of the rat GH cell during starvation has not been previously reported. The presence of GH immunostaining of equal intensity in both fed and starved animals is complemented by the electron microscopic findings. No change in the size of GH granules nor in the granula tion of the GH cells was observed. The significance of the crinophagy observed in the GH cells of starved animals remains to be established. The wide range in serum GH values that we obtained confirms the findings of Takahashi et al. (15). This very large variation almost precludes biological
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
Discussion
Sirek/Horvath /Ezrin /Kovacs
74
significance despite statistical significance. Samples drawn at such relatively long intervals as 48 or 72 h may not reflect invariably the minute-to-minute changes in circulating GH levels. It would appear that the pituitary gland of the rat is still capable of synthesizing and secreting GH even when it is completely deprived of exogenous nutrients. There is very little literature on the behaviour of PRL in the rat during starvation. Akikusa (1) showed no change in PRL content of the rat pituitary gland after 1, 3 and 5 days of starvation. McAttie and Trenkle (8) reported decreased serum PRL by radioimmunoassay in heifers after 2 days of starvation. Our results in rats showed a fall in serum PRL only after a relatively prolonged period of starvation. The decrease in blood levels of PRL raises many speculative questions, and its significance remains to be elucidated.
A cknowledgements The authors are indebted to Mrs. Erika Johansson (Department of Neuroendocrinol ogy, Clarke Institute, University of Toronto) for the GH and PRL radioimmunoassays; to Mr. Gerhard Penz and Mr. Ernest Whitter for technical assistance and to Miss Elizabeth Warner for typing the manuscript.
1 2
3
4 5 6
7 8 9
Akikusa, Y.: Effect of starvation on synthesis and release of growth hormone and prolactin in the rat anterior pituitary. Endocr. jap. 18: 411-416 (1971). Cahill, G.F., jr.; Herrera, M.G.; Morgan, A.P.; Soeldner, J.S.; Steinke. J.; Levy, P.L.; Reichard, G.A., />., and Kipnis, D.M.: Hormone-fuel interrelationships during fasting. J. clin. Invest. 45: 1751-1769 (1966). Dickerman, E.; Negro-Vilar, A., and Meites. J.: Effects of starvation on plasma GH activity, pituitary GH and GH-RE levels in the rat. Endocrinology 84: 814-819 (1969). Friedman, R.C. and Reichlin, S.: Growth hormone content of the pituitary gland of starved rats. Endocrinology 76: 787 -788 (1965). Jackson, C.M.: The effects of inanition and malnutrition upon growth and structure (Blakiston, Philadelphia 1925). Mason, T.E.; Phifer. R.F.; Spicer, S.S.; Swallow, R.A., and Dreskin, R.B.: An immuno globulin-enzyme bridge method for localizing tissue antigens. J. Histochem. Cytochem. 17: 563 569(1969). McAnulty, P.A. and Dickerson, J.W.T.: The cellular response of the weanling rat thymus gland to undernutrition and rehabilitation. Pediat. Res. 9: 778-785 (1973). McAttee, J.W. and Trenkle, A.: Effects of feeding, fasting, glucose or arginine on plasma prolactin levels in the bovine. Endocrinology 89: 730 734 (1971). Meites, J. and Fiel, N.J.: Effect of starvation on hypothalamic content of ‘somato tropin releasing factor’ and pituitary growth hormone content. Endocrinology 77: 455 460(1965).
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 6:27:30 AM
References
Effect of Starvation on Growth Hormone and Prolactin
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10 Mulinos, M.G. and Pomerantz, Pseudo-hypophysectomy: a condition resembling hypophysectomy produced by malnutrition. J. Nutr. 19: 493-504 (1940). 11 Negro-Vilar. A.; Dickerman, E., and Meites, J.: Effects of starvation on hypothalamic FSH-RE and pituitary FSH in male rats. Endocrinology 88: 1246- 1249 (1971). 12 Roth. J.: Click, S.M.; Yalow, R.S., and Berson, S.A.: Hypoglycemia: a potent stimulus to secretion of growth hormone. Science 140: 987-988 (1963). 13 Srebnik. H.H. and Nelson, M.M.: Anterior pituitary function in male rats deprived of dietary protein. Endocrinology 70: 723- 730 (1962). 14 Sternberger. L.A.; Hardy, P.H., jr.; Cuculis, J.J., and Meyer, H.G.: The unlabelled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen antibody complex (horseradish peroxidase- antihorseradish peroxi dase) and its use in identification of spirochetes. J. Histochcm. Cytochem. 18: 315 333 (1970). 15 Takahashi, K.: Daughaday, W.H., and Kipnis, D.M.: Regulation of immunoreactive growth hormone secretion in male rats. Endocrinology 88: 909-917 (1971). 16 Trenkle, A.: Effect of starvation on plasma growth hormone in rats. Proc. Soc. exp. Biol. Med. 135: 77-80(1970). 17 Weibel, E.R.: Sterological principles for morphometry in electron microscopic cytol ogy. lnt. Rev. Cytol. 26: 235 302 (1969).
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A.M.T. Sirek, MD, Department of Medicine, St. Michael’s Hospital, 30 Bond Street, Toronto, Ont. (Canada)
