Brain Research, 506 (1990) 307-310 Elsevier
307
BRES 23890
The cell population of somatostatin and growth hormone-releasing factor using quantitative immunohistochemistry in the isolated GH deficient dwarf rat Shuya Sakuma 1, Hiroshi Ishikawa 2 and Shinichi O k u m a 3 Department of 1Pediatrics and 2Anatomy, The Jikei University School of Medicine, Tokyo (Japan) and ~Research Laboratories. Morishita Pharmaceutical Co., Ltd., Shiga (Japan) (Accepted 19 September 1989)
Key words: Dwarf rat; Somatostatin; Growth hormone-releasing factor; Immunocytochemistry; Feedback regulation
We examined the cell population of somatostatin (SS) in the periventricular nucleus (PN) and growth hormone-releasing factor (GRF) in the arcuate nucleus (ARC) between spontaneous dwarf rats (SDRs; gene symbol: dr), which show isolated GH deficiency, and normal rats using avidin-biotin complex (ABC) immunohistochemistry. The total number of SS perikarya per brain weight in the PN of SDRs was 824.8 _+ 49.6 (mean _+ S.E.M., n = 4), whereas that of controls was 1108.5 .+--50.1 (n = 4). The GRF perikarya per brain weight in the ARC of SDRs numbered 1281.0 _+ 26.0 (n = 7), as compared to 685.4 + 64.6 (n = 7) in the controls. The SS perikarya in the PN of SDRs were significantly reduced (P < 0.05), while the GRF perikarya in the SDRs were significantly increased (P < 0.01). These results suggest that GH itself acts on SS to positively regulate its secretion and on GRF in a negative regulatory manner. T h e secretion of growth h o r m o n e ( G H ) is mainly r e g u l a t e d in an inhibitory fashion by somatostatin (SS) and is stimulated by growth hormone-releasing factor ( G R F ) 1. The f e e d b a c k mechanism on the secretion of SS and G R F by G H has been studied using hypophysectomized animals. H o w e v e r , in h y p o p h y s e c t o m i z e d animals, in addition to G H , o t h e r pituitary h o r m o n e s and their target h o r m o n e s are also deficient. T h e r e f o r e , it has been difficult to reveal the precise action of G H on the f e e d b a c k mechanism in the h y p o t h a l a m o - p i t u i t a r y axis. The best way to study the f e e d b a c k system is to use animals that are only G H deficient. T h o u g h a n u m b e r of dwarf animals, such as Snell (dw) 24, A m e s (df) 23 and little (lit) 7 mice, and rdw rats x4 are available, all these animals have deficiencies in thyroid-stimulating h o r m o n e (TSH) and/or prolactin as well as G H . The spontaneous dwarf rat ( S D R ; gene symbol: dr) is a new animal m o d e l for dwarfism. The S D R s were found in S p r a g u e - D a w l e y rats and exhibited an autosomal recessive mutation is. The S D R s are deficient only in G H and the etiology of the dwarfism in S D R s lies in the G H cells themselves 22'26. In an a t t e m p t to gain a greater understanding of the f e e d b a c k regulation system between SS or G R F and G H , this study was designed to quantitatively c o m p a r e the SS and G R F containing neurons in the brains of S D R s with n o r m a l S p r a g u e - D a w l e y rats using an a v i d i n - b i o t i n
complex ( A B C ) i m m u n o h i s t o c h e m i c a l m e t h o d . Ten- and 15-week-old male S D R s weighing 45-90 g and male S p r a g u e - D a w l e y rats of the same age, weighing 330-510 g, were housed under constant t e m p e r a t u r e (24 °C) and light (14 h ) - d a r k (10 h) cycles and provided with food pellets (CE-2, Clea Japan Inc., Tokyo, Japan) and water ad libitum. U n d e r p e n t o b a r b i t a l anesthesia (50 mg/kg b.wt., i.p.), all rats were placed into a stereotaxic a p p a r a t u s and colchicine (100 /~g dissolved in 5 or 10 ktl saline) was injected into the lateral ventricle. A f t e r 24 h all rats were anesthetized using p e n t o b a r b i t a l and perfused with 0.01 M p h o s p h a t e - b u f f e r e d saline (PBS), p H 7.2 followed by buffered Z a m b o n i ' s solution ( Z a m b o n i ' s solution in 0.1 M sodium p h o s p h a t e buffer, p H 7.2). The brains were then r e m o v e d , weighed, i m m e r s e d in the same fixative for 1 h at 4 °C, placed in PBS with 15% sucrose and frozen using dry-ice acetone. The frozen coronal sections from the face containing the o r g a n u m vasculosum of the lamina terminalis (OVLT) to the face containing mammillary body were serially sectioned at 30 j~m in thickness with a cryostat and stored in PBS at 4 °C. E v e r y set of 3 serial sections was e x a m i n e d as follows: the first section was processed i m m u n o c y t o c h e m i c a l l y for staining with an antibody against SS (1:1000), the second section was stained with the K l ~ v e r - B a r r e r a ' s technique
Correspondence: S. Sakuma, Department of Pediatrics, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
308 and the third section was processed immunocytochemically for staining with an antibody against rat GRF (1 : 1). The immunocytochemistry was performed on freefloating sections with the ABC method 9 using slight modifications as follows; the sections were placed into 0.3% Triton X-100 for 60 min followed by incubation for 72 h to 1 week at 4 °C with the primary antiserum. After rinsing in PBS the sections were incubated for 60 rain at room temperature with biotinylated anti-rabbit goat serum (1:100, Vector, Burlingame, CA), washed with PBS and incubated with an ABC with biotinylated peroxidase (1:200, Amersham, U.K.) under the same conditions. The immunoreaction was visualized in 100 ml of 0.05 M Tris/HC1 buffer, pH 7.6, containing 10 mg 3,3"-diaminobenzidine and 0.03% H20 2, for 8 rain at room temperature. The sections were placed into PBS and mounted on glass slides. They were then immersed in PBS and after dehydration through a graded ethanol series and cleaning in xylene, coverslipped. The anti-SS rabbit serum (Advance Lab., Tokyo, Japan) contained 0.3% Triton X-100, 0.1% bovine serum albumin (BSA), sodium azide and 50% liver extract. The anti-rat GRF rabbit serum was prepared from an antiserum against rat GRF for radioimmunoassay (RIA) (Peninsula Lab., Belmont, CA). The details and the specificities of the antisera are in a previous report 22. Briefly, the freeze-dried powder of anti-rat GRF rabbit serum was dissolved in 5 mi of distilled water and dialysed to eliminate the excess amount of salt. The specificities of the antisera were performed with preabsorption tests on the sections. Each antiserum was incubated with an excess of the following antigens: SS, secretin (Peptide Institute, Osaka, Japan), rat GRF (kindly gifted by Dr. Nicholas Ling, the Salk Institute, San Diego, CA, U.S.A.), fl-melanocyte-stimulating hormone (fl-MSH; Peninsula Lab., Belmont, CA, U.S.A.), and luteinizing hormone-releasing hormone (LHRH) (UCB Bioproducts S.A., Braine-lalleud, Belgium). All immunostaining was blocked by preincubation of the anti-SS serum with SS and was unchanged with the other antigens. The same results were observed in the preincubation tests of the anti-rat GRF serum with rat GRF and the other antigens. Immunoreactive perikarya were counted using a light microscope by a single individual. The total number of perikarya was calculated from the sum of the number of immunoreactive perikarya on each slide. SS-immunoreactive perikarya were counted in the periventricular nucleus (PN) and the GRF-immunoreactive perikarya were counted in the arcuate nucleus (ARC). The discriminating border of nucleus was determined by adjacent section staining with the Klfiver-Barrera's technique. Statistical analysis of the data was performed using the
I--
,500
1000
0
SS GRF Fig. 1. The total number of perikarya per brain weight in SDRs (hatched column), and controls (open column). Values given are the mean _+ S.E.M. Two bars on the left show the total number of somatostatin-immunoreactive perikarya per brain weight in the periventricular nucleus (n = 4) and the two bars on the right show the total number of GRF-immunoreactive perikarya per brain weight in the arcuate nucleus (n = 7). *P < 0.05. **P < 0.01.
Student's t-test. The PN of SDRs contained 989.8 + 59.6 (mean _+ S.E.M., n = 4) immunoreactive SS perikarya, whereas in the controls 1917.8 + 86.7 (n = 4) were observed representing a 52% (P < 0.01) difference between the two animal groups. In the A R C of the SDRs, the total number of G R F perikarya was 1588.4 + 33.0 (n = 7), while in the controls, 1309.1 + 123.4 (n = 7) were present. Apparently there was no significant difference between the SDRs and controls (P > 0.05) with respect to the immunostained GRF perikarya. However, the weight of the brain was 1.24 + 0.02 g in the SDRs (n = 5) and 1.91 _+ 0.05 g in the controls (n = 9), which was a significant reduction as a result of the dwarfism (P < 0.01). Therefore, we computed the total number of perikarya per brain weight in both the groups, which is shown in Fig. 1. The total number of SS perikarya per brain weight in the PN was 824.8 + 49.6 (n = 4) in the SDRs and 1108.5 + 50.1 (n = 4) in controls. Statistically the total number of SS perikarya in the PN per brain weight in the SDRs was significantly reduced compared to that in the controls (P < 0.05). The total number of GRF perikarya per brain weight in the A R C was 1.281.0 _ 26.6 (n = 7) in the SDRs and 685.4 + 64.6 (n = 7) in the controls. Thus, the total number of G R F perikarya per brain weight in the SDRs was significantly increased compared to that in the controls (P < 0.01). In our results the total number of SS perikarya in the PN of SDRs was significantly reduced compared to that of the controls (P < 0.01). It was reported that the content of SS in the hypothalamus and median eminence (ME) as determined by RIA 2s and immunocytochemistry2 decreased in the hypophysectomized rat, but was restored in the hypothalamus by GH treatment TM. The release of SS from the hypothalamus was reduced by. hypophysectomy and was increased by GH administration to normal rats. This effect was suggested to be a
309 direct action of G H itself 3. In the PN the content of SS d e c r e a s e d after h y p o p h y s e c t o m y 27. The administration of m o n o s o d i u m g l u t a m a t e , hypothalamic deafferentation 13, or double staining with r e t r o g r a d e transport methods using horseradish peroxidase ( H R P ) 1° revealed that SS n e u r o n s in the PN p r o j e c t e d to the M E . Recently the synthesis of 14-SS and 28-SS 29 and the content of p r e p r o S S - m R N A in the PN 2t decreased in the hypophysectomized rat and the content of p r e p r o S S - m R N A in the PN was elevated by the administration of rat G H . These studies have d e m o n s t r a t e d the following hypothesis: G H itself acts on the SS n e u r o n in the PN and feeds back on SS synthesis and is stimulatory to its release. The present study suggests that the reduction in the total n u m b e r of SS p e r i k a r y a in the PN of the S D R is due to a deficiency of the stimulatory action of G H . H o w e v e r , the brain weight of S D R s was smaller than that of the controls as was also the volume of the PN. Statistical c o m p u t a t i o n of the total n u m b e r of p e r i k a r y a p e r brain weight revealed that the total n u m b e r of SS p e r i k a r y a in the PN p e r brain weight in S D R s was significantly r e d u c e d c o m p a r e d to that in the controls ( P < 0.05). Therefore, this was considered to be an effect from the G H deficiency on the s h o r t - l o o p f e e d b a c k regulation b e t w e e n G H and SS. T h e total n u m b e r of G R F p e r i k a r y a in the A R C in S D R s was not significantly changed c o m p a r e d to that in controls ( P > 0.05). H o w e v e r , when expressed as a function of the brain weight, the total n u m b e r of G R F p e r i k a r y a was 1281.0 _ 26.6 in the S D R s and 685.4 _ 64.6 in the controls, which r e p r e s e n t e d a significant difference between the two animal groups ( P < 0.01). Thus, the absolute n u m b e r of G R F p e r i k a r y a of S D R s m a y be increased c o m p a r e d to that of controls. The effect of h y p o p h y s e c t o m y on the immunoreactive G R F fibers in the M E was to reduce their staining intensity 17'25. The content of r a d i o i m m u n o - a s s a y a b l e G R F in the hypothalamus was also r e d u c e d after h y p o p h y s e c t o m y 12. In contrast, h y p o t h a l a m i c G R F - m R N A levels were significantly increased after h y p o p h y s e c t o m y 5'16. Yokoe et al. r e p o r t e d that in spite of the reduction of hypothalamic G R F content, blood G R F concentration was increased after h y p o p h y s e c t o m y 3°. Usually, the i m m u n o c y t o c h e m -
ically stained h y p o t h a l a m i c G R F p e r i k a r y a are scarcely observed without colchicine t r e a t m e n t . Following disruption of the axon t r a n s p o r t with colchicine, many G R F p e r i k a r y a are observed. In the i m m u n o c y t o c h e m i c a l studies m e n t i o n e d above, Swachenko et al. 25 did not describe the use of colchicine in the h y p o p h y s e c t o m i z e d rat and M e r c h e n t h a l e r and A r i m u r a 17 did not inject colchicine since the animals they e m p l o y e d were dead. We p r e s u m e that the i m m u n o r e a c t i v e intensity of the M E is r e c o v e r e d using colchicine. The release of G R F in h y p o p h y s e c t o m i z e d rats d e c r e a s e d c o m p a r e d to that in the control animals but the fractional release rate was increased t2. These results may indicate that after hypophysectomy synthesis and release of G R F are increased but the G R F released is i m m e d i a t e l y t r a n s p o r t e d from the hypothalamus. We consider that an increase of G R F neurons in the A R C of S D R s is due to the deficiency of the inhibitory action of G H . The present study supports the premise that G H action on G R F neurons is inhibitory. SS content in the h y p o t h a l a m u s is influenced by thyroid-stimulating h o r m o n e (TSH) 2° and T34. A 24-h exposure to T S H increased SS release from hypothalamic primary cultures. SS content is r e d u c e d in long-term h y p o t h y r o i d rats and restored in h y p o t h y r o i d rats receiving T 3 treatment. G H secretion is influenced by thyroid h o r m o n e s aS, glucocorticoids ~9, and gonadal steroids 6. The data o b t a i n e d from h y p o p h y s e c t o m y include the effect of o t h e r h o r m o n e s beside G H . On the other hand, S D R s are isolated G H deficient rats. T h e r e f o r e , other h o r m o n e s are influenced much less in S D R s than in normal rats after h y p o p h y s e c t o m y . The present study d e m o n s t r a t e s that G H acts on both SS and G R F neurons and supports the hypothesis that its action on SS is stimulatory and on G R F is inhibitory.
1 Arimura, A. and Culler, M.D., Regulation of growth hormone secretion. In H. Imura (Ed.), Comprehensive Endocrinology, The pituitary Gland, Raven, New York, 1984, pp. 221-259. 2 Baker, B.L. and Yen, Y.Y., The influence of hypophysectomy on the stores of somatostatin in the hypothalamus and pituitary stem, Proc. Soc. Exp. Biol. Med., 151 (1976) 599-602. 3 Berelowitz, M., Firestone, S.L. and Frohman, L.A., Effects of growth hormone excess and deficiency of hypothalamic somatostatin content and release and on tissue somatostatin distribution, Endocrinology, 1(19 (1981) 714-719. 4 Berelowitz, M., Maeda, K., Harris, S. and Frohman, L.A., The effect of alternations in the pituitary-thyroid axis on hypothalamic content and in vitro release of somatostatin-like immuno-
reactivity, Endocrinology, 107 (1980) 24-29. 5 Chomczynski, P., Downs, T.R. and Frohman, L.A., Feedback regulation of growth hormone (GH)-releasing hormone (GRF) gene expression by GH in rat hypothalamus, 69th Annu. Meeting Endocr. Soc., 1987, p. 175 (abstr.). 6 Dave, J.R., Rubinstein, N. and Eskay, R.L., Evidence that fl-endorphin binds to specific receptors in rat peripheral tissue and stimulates the adenylate cyclase/cycle AMP system, Endocrinology, 117 (1985) 1389-1396. 7 Eicher, E.M. and Beamer, W.G., Inherited ateliotic dwarfism in mice, J. Herd, 67 (1976) 87-91. 8 Hoffman, D.L, and Baker, B.L., Effect of treatment with growth hormone on somatostatin in the median eminence of
This work was supported in part by a Grant-in-Aid for Scientific Research of Postgraduate Students 1988 from The Jikei University School of Medicine, We are grateful to Dr. Nicholas Ling for his gift of rat growth hormone-releasing factor and to Dr. D.C. Herbert (The University of Texas Health Science Center, San Antonio, TX, U.S.A.) and Prof. D.R. Naik (Utkal University, Bhubaneswar, India) for help in preparing the manuscript.
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hypophysectomized rats, Proc. Soc. Exp. Biol. Med., 156 (1977) 265-271. Hsu, S., Raine, L. and Franger, H., Use of avidin-biotinperoxidase complex (ABC) in immuno-peroxidase techniques, J. Histochem. Cytochem., 29 (1981) 577-580. Ishikawa, K., Taniguchi, Y., Kurosumi, K., Suzuki, M. and Shinoda, M., Immunohistochemical identification of somatostatin containing neurons projecting to the median eminence of the rat, Endocrinology, 121 (1987) 94-97. Kanatsuka, A., Makino, H., Matsushima, Y., Osegawa, M., Yamamoto, M. and Kumagai, A., Effect of hypophysectomy and growth hormone administration on somatostatin content in the rat hypothalamus, Neuroendocrinology, 29 (1979) 186-190. Katakami, H., Downs, T.R. and Frohman, L.A., Effect of hypophysectomy on hypothalamic growth hormone-releasing factor content and release in the rat, Endocrinology, 120 (1987) 1079-1082. Kawano, H., Daikoku, S. and Saito, S., Immunohistochemical studies of intrahypothalamic somatostatin-containing neurons in rat. Brain Research, 242 (1982) 227-232. Koto, M., Sato, T., Okamoto, M. and Adachi, J., rdw rats, a new hereditary dwarf model in the rat, Exp. Anita., 37 (1988) 21-30. Kunos, G., Thyroid hormone dependent interconvension of myocardial a and fl-adrenoceptors in the rat, Br. J, Pharmacol., 59 (1977) 177-189. Mayo, K.E., Structure and expression of hypothalamic GRF and CRF genes, 68th Ann. Meeting Endocr. Soc., 1986, p. 30 (abstr.). Merchenthaler, I., Thomas, C.R. and Arimura, A., Immunocytochemical localization of growth hormone releasing factor (GHRH)-containing structures in the rat brain using anti-rat G H R H serum, Peptides, 5 (1984) 1071-1075. Okuma, S., Study of growth hormone in spontaneous dwarf rat. Folia endocrinol., 60 (1984) 1005-1014. Preiksaitis, H.G. and Kunos, G., Adrenoceptor-mediated activation of liver glycogen phosphorylase: effects of thyroid state, Life Sci., 24 (1979) 35-41. Robbin, R.J., Leidy, J.W. and Landon, R.M., The effect of
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The cell population of somatostatin and growth hormone-releasing factor using quantitative immunohistochemistry in the isolated GH deficient dwarf rat Shuya Sakuma 1, Hiroshi Ishikawa 2 and Shinichi O k u m a 3 Department of 1Pediatrics and 2Anatomy, The Jikei University School of Medicine, Tokyo (Japan) and ~Research Laboratories. Morishita Pharmaceutical Co., Ltd., Shiga (Japan) (Accepted 19 September 1989)
Key words: Dwarf rat; Somatostatin; Growth hormone-releasing factor; Immunocytochemistry; Feedback regulation
We examined the cell population of somatostatin (SS) in the periventricular nucleus (PN) and growth hormone-releasing factor (GRF) in the arcuate nucleus (ARC) between spontaneous dwarf rats (SDRs; gene symbol: dr), which show isolated GH deficiency, and normal rats using avidin-biotin complex (ABC) immunohistochemistry. The total number of SS perikarya per brain weight in the PN of SDRs was 824.8 _+ 49.6 (mean _+ S.E.M., n = 4), whereas that of controls was 1108.5 .+--50.1 (n = 4). The GRF perikarya per brain weight in the ARC of SDRs numbered 1281.0 _+ 26.0 (n = 7), as compared to 685.4 + 64.6 (n = 7) in the controls. The SS perikarya in the PN of SDRs were significantly reduced (P < 0.05), while the GRF perikarya in the SDRs were significantly increased (P < 0.01). These results suggest that GH itself acts on SS to positively regulate its secretion and on GRF in a negative regulatory manner. T h e secretion of growth h o r m o n e ( G H ) is mainly r e g u l a t e d in an inhibitory fashion by somatostatin (SS) and is stimulated by growth hormone-releasing factor ( G R F ) 1. The f e e d b a c k mechanism on the secretion of SS and G R F by G H has been studied using hypophysectomized animals. H o w e v e r , in h y p o p h y s e c t o m i z e d animals, in addition to G H , o t h e r pituitary h o r m o n e s and their target h o r m o n e s are also deficient. T h e r e f o r e , it has been difficult to reveal the precise action of G H on the f e e d b a c k mechanism in the h y p o t h a l a m o - p i t u i t a r y axis. The best way to study the f e e d b a c k system is to use animals that are only G H deficient. T h o u g h a n u m b e r of dwarf animals, such as Snell (dw) 24, A m e s (df) 23 and little (lit) 7 mice, and rdw rats x4 are available, all these animals have deficiencies in thyroid-stimulating h o r m o n e (TSH) and/or prolactin as well as G H . The spontaneous dwarf rat ( S D R ; gene symbol: dr) is a new animal m o d e l for dwarfism. The S D R s were found in S p r a g u e - D a w l e y rats and exhibited an autosomal recessive mutation is. The S D R s are deficient only in G H and the etiology of the dwarfism in S D R s lies in the G H cells themselves 22'26. In an a t t e m p t to gain a greater understanding of the f e e d b a c k regulation system between SS or G R F and G H , this study was designed to quantitatively c o m p a r e the SS and G R F containing neurons in the brains of S D R s with n o r m a l S p r a g u e - D a w l e y rats using an a v i d i n - b i o t i n
complex ( A B C ) i m m u n o h i s t o c h e m i c a l m e t h o d . Ten- and 15-week-old male S D R s weighing 45-90 g and male S p r a g u e - D a w l e y rats of the same age, weighing 330-510 g, were housed under constant t e m p e r a t u r e (24 °C) and light (14 h ) - d a r k (10 h) cycles and provided with food pellets (CE-2, Clea Japan Inc., Tokyo, Japan) and water ad libitum. U n d e r p e n t o b a r b i t a l anesthesia (50 mg/kg b.wt., i.p.), all rats were placed into a stereotaxic a p p a r a t u s and colchicine (100 /~g dissolved in 5 or 10 ktl saline) was injected into the lateral ventricle. A f t e r 24 h all rats were anesthetized using p e n t o b a r b i t a l and perfused with 0.01 M p h o s p h a t e - b u f f e r e d saline (PBS), p H 7.2 followed by buffered Z a m b o n i ' s solution ( Z a m b o n i ' s solution in 0.1 M sodium p h o s p h a t e buffer, p H 7.2). The brains were then r e m o v e d , weighed, i m m e r s e d in the same fixative for 1 h at 4 °C, placed in PBS with 15% sucrose and frozen using dry-ice acetone. The frozen coronal sections from the face containing the o r g a n u m vasculosum of the lamina terminalis (OVLT) to the face containing mammillary body were serially sectioned at 30 j~m in thickness with a cryostat and stored in PBS at 4 °C. E v e r y set of 3 serial sections was e x a m i n e d as follows: the first section was processed i m m u n o c y t o c h e m i c a l l y for staining with an antibody against SS (1:1000), the second section was stained with the K l ~ v e r - B a r r e r a ' s technique
Correspondence: S. Sakuma, Department of Pediatrics, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
308 and the third section was processed immunocytochemically for staining with an antibody against rat GRF (1 : 1). The immunocytochemistry was performed on freefloating sections with the ABC method 9 using slight modifications as follows; the sections were placed into 0.3% Triton X-100 for 60 min followed by incubation for 72 h to 1 week at 4 °C with the primary antiserum. After rinsing in PBS the sections were incubated for 60 rain at room temperature with biotinylated anti-rabbit goat serum (1:100, Vector, Burlingame, CA), washed with PBS and incubated with an ABC with biotinylated peroxidase (1:200, Amersham, U.K.) under the same conditions. The immunoreaction was visualized in 100 ml of 0.05 M Tris/HC1 buffer, pH 7.6, containing 10 mg 3,3"-diaminobenzidine and 0.03% H20 2, for 8 rain at room temperature. The sections were placed into PBS and mounted on glass slides. They were then immersed in PBS and after dehydration through a graded ethanol series and cleaning in xylene, coverslipped. The anti-SS rabbit serum (Advance Lab., Tokyo, Japan) contained 0.3% Triton X-100, 0.1% bovine serum albumin (BSA), sodium azide and 50% liver extract. The anti-rat GRF rabbit serum was prepared from an antiserum against rat GRF for radioimmunoassay (RIA) (Peninsula Lab., Belmont, CA). The details and the specificities of the antisera are in a previous report 22. Briefly, the freeze-dried powder of anti-rat GRF rabbit serum was dissolved in 5 mi of distilled water and dialysed to eliminate the excess amount of salt. The specificities of the antisera were performed with preabsorption tests on the sections. Each antiserum was incubated with an excess of the following antigens: SS, secretin (Peptide Institute, Osaka, Japan), rat GRF (kindly gifted by Dr. Nicholas Ling, the Salk Institute, San Diego, CA, U.S.A.), fl-melanocyte-stimulating hormone (fl-MSH; Peninsula Lab., Belmont, CA, U.S.A.), and luteinizing hormone-releasing hormone (LHRH) (UCB Bioproducts S.A., Braine-lalleud, Belgium). All immunostaining was blocked by preincubation of the anti-SS serum with SS and was unchanged with the other antigens. The same results were observed in the preincubation tests of the anti-rat GRF serum with rat GRF and the other antigens. Immunoreactive perikarya were counted using a light microscope by a single individual. The total number of perikarya was calculated from the sum of the number of immunoreactive perikarya on each slide. SS-immunoreactive perikarya were counted in the periventricular nucleus (PN) and the GRF-immunoreactive perikarya were counted in the arcuate nucleus (ARC). The discriminating border of nucleus was determined by adjacent section staining with the Klfiver-Barrera's technique. Statistical analysis of the data was performed using the
I--
,500
1000
0
SS GRF Fig. 1. The total number of perikarya per brain weight in SDRs (hatched column), and controls (open column). Values given are the mean _+ S.E.M. Two bars on the left show the total number of somatostatin-immunoreactive perikarya per brain weight in the periventricular nucleus (n = 4) and the two bars on the right show the total number of GRF-immunoreactive perikarya per brain weight in the arcuate nucleus (n = 7). *P < 0.05. **P < 0.01.
Student's t-test. The PN of SDRs contained 989.8 + 59.6 (mean _+ S.E.M., n = 4) immunoreactive SS perikarya, whereas in the controls 1917.8 + 86.7 (n = 4) were observed representing a 52% (P < 0.01) difference between the two animal groups. In the A R C of the SDRs, the total number of G R F perikarya was 1588.4 + 33.0 (n = 7), while in the controls, 1309.1 + 123.4 (n = 7) were present. Apparently there was no significant difference between the SDRs and controls (P > 0.05) with respect to the immunostained GRF perikarya. However, the weight of the brain was 1.24 + 0.02 g in the SDRs (n = 5) and 1.91 _+ 0.05 g in the controls (n = 9), which was a significant reduction as a result of the dwarfism (P < 0.01). Therefore, we computed the total number of perikarya per brain weight in both the groups, which is shown in Fig. 1. The total number of SS perikarya per brain weight in the PN was 824.8 + 49.6 (n = 4) in the SDRs and 1108.5 + 50.1 (n = 4) in controls. Statistically the total number of SS perikarya in the PN per brain weight in the SDRs was significantly reduced compared to that in the controls (P < 0.05). The total number of GRF perikarya per brain weight in the A R C was 1.281.0 _ 26.6 (n = 7) in the SDRs and 685.4 + 64.6 (n = 7) in the controls. Thus, the total number of G R F perikarya per brain weight in the SDRs was significantly increased compared to that in the controls (P < 0.01). In our results the total number of SS perikarya in the PN of SDRs was significantly reduced compared to that of the controls (P < 0.01). It was reported that the content of SS in the hypothalamus and median eminence (ME) as determined by RIA 2s and immunocytochemistry2 decreased in the hypophysectomized rat, but was restored in the hypothalamus by GH treatment TM. The release of SS from the hypothalamus was reduced by. hypophysectomy and was increased by GH administration to normal rats. This effect was suggested to be a
309 direct action of G H itself 3. In the PN the content of SS d e c r e a s e d after h y p o p h y s e c t o m y 27. The administration of m o n o s o d i u m g l u t a m a t e , hypothalamic deafferentation 13, or double staining with r e t r o g r a d e transport methods using horseradish peroxidase ( H R P ) 1° revealed that SS n e u r o n s in the PN p r o j e c t e d to the M E . Recently the synthesis of 14-SS and 28-SS 29 and the content of p r e p r o S S - m R N A in the PN 2t decreased in the hypophysectomized rat and the content of p r e p r o S S - m R N A in the PN was elevated by the administration of rat G H . These studies have d e m o n s t r a t e d the following hypothesis: G H itself acts on the SS n e u r o n in the PN and feeds back on SS synthesis and is stimulatory to its release. The present study suggests that the reduction in the total n u m b e r of SS p e r i k a r y a in the PN of the S D R is due to a deficiency of the stimulatory action of G H . H o w e v e r , the brain weight of S D R s was smaller than that of the controls as was also the volume of the PN. Statistical c o m p u t a t i o n of the total n u m b e r of p e r i k a r y a p e r brain weight revealed that the total n u m b e r of SS p e r i k a r y a in the PN p e r brain weight in S D R s was significantly r e d u c e d c o m p a r e d to that in the controls ( P < 0.05). Therefore, this was considered to be an effect from the G H deficiency on the s h o r t - l o o p f e e d b a c k regulation b e t w e e n G H and SS. T h e total n u m b e r of G R F p e r i k a r y a in the A R C in S D R s was not significantly changed c o m p a r e d to that in controls ( P > 0.05). H o w e v e r , when expressed as a function of the brain weight, the total n u m b e r of G R F p e r i k a r y a was 1281.0 _ 26.6 in the S D R s and 685.4 _ 64.6 in the controls, which r e p r e s e n t e d a significant difference between the two animal groups ( P < 0.01). Thus, the absolute n u m b e r of G R F p e r i k a r y a of S D R s m a y be increased c o m p a r e d to that of controls. The effect of h y p o p h y s e c t o m y on the immunoreactive G R F fibers in the M E was to reduce their staining intensity 17'25. The content of r a d i o i m m u n o - a s s a y a b l e G R F in the hypothalamus was also r e d u c e d after h y p o p h y s e c t o m y 12. In contrast, h y p o t h a l a m i c G R F - m R N A levels were significantly increased after h y p o p h y s e c t o m y 5'16. Yokoe et al. r e p o r t e d that in spite of the reduction of hypothalamic G R F content, blood G R F concentration was increased after h y p o p h y s e c t o m y 3°. Usually, the i m m u n o c y t o c h e m -
ically stained h y p o t h a l a m i c G R F p e r i k a r y a are scarcely observed without colchicine t r e a t m e n t . Following disruption of the axon t r a n s p o r t with colchicine, many G R F p e r i k a r y a are observed. In the i m m u n o c y t o c h e m i c a l studies m e n t i o n e d above, Swachenko et al. 25 did not describe the use of colchicine in the h y p o p h y s e c t o m i z e d rat and M e r c h e n t h a l e r and A r i m u r a 17 did not inject colchicine since the animals they e m p l o y e d were dead. We p r e s u m e that the i m m u n o r e a c t i v e intensity of the M E is r e c o v e r e d using colchicine. The release of G R F in h y p o p h y s e c t o m i z e d rats d e c r e a s e d c o m p a r e d to that in the control animals but the fractional release rate was increased t2. These results may indicate that after hypophysectomy synthesis and release of G R F are increased but the G R F released is i m m e d i a t e l y t r a n s p o r t e d from the hypothalamus. We consider that an increase of G R F neurons in the A R C of S D R s is due to the deficiency of the inhibitory action of G H . The present study supports the premise that G H action on G R F neurons is inhibitory. SS content in the h y p o t h a l a m u s is influenced by thyroid-stimulating h o r m o n e (TSH) 2° and T34. A 24-h exposure to T S H increased SS release from hypothalamic primary cultures. SS content is r e d u c e d in long-term h y p o t h y r o i d rats and restored in h y p o t h y r o i d rats receiving T 3 treatment. G H secretion is influenced by thyroid h o r m o n e s aS, glucocorticoids ~9, and gonadal steroids 6. The data o b t a i n e d from h y p o p h y s e c t o m y include the effect of o t h e r h o r m o n e s beside G H . On the other hand, S D R s are isolated G H deficient rats. T h e r e f o r e , other h o r m o n e s are influenced much less in S D R s than in normal rats after h y p o p h y s e c t o m y . The present study d e m o n s t r a t e s that G H acts on both SS and G R F neurons and supports the hypothesis that its action on SS is stimulatory and on G R F is inhibitory.
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This work was supported in part by a Grant-in-Aid for Scientific Research of Postgraduate Students 1988 from The Jikei University School of Medicine, We are grateful to Dr. Nicholas Ling for his gift of rat growth hormone-releasing factor and to Dr. D.C. Herbert (The University of Texas Health Science Center, San Antonio, TX, U.S.A.) and Prof. D.R. Naik (Utkal University, Bhubaneswar, India) for help in preparing the manuscript.
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