- Email: [email protected]
Regulation of hypothalamic somatostatin and growth hormone releasing hormone mRNA levels by inhibin
Molecular Brain Research 66 Ž1999. 191–194
Short communication
Regulation of hypothalamic somatostatin and growth hormone releasing hormone mRNA levels by inhibin E. Carro a , R.M. Senarıs ˜ ´ a
a,)
, F. Mallo b, C. Dieguez ´
a
Department of Physiology, Faculty of Medicine, UniÕersity of Santiago de Compostela, 15700 Santiago de Compostela, Spain b Department of Fundamental Biology, Faculty of Sciences, UniÕersity of Vigo, Spain Accepted 29 December 1998
Abstract Although it is well established that inhibin plays a major role in the regulation of the hypothalamic-pituitary-gonadal axis, its influence in the regulation of other neuroendocrine functions is still poorly understood. Recent results indicate that inhibin suppresses plasma GH levels, but its site of action is yet unknown. Therefore, in the present work we investigated the effects of inhibin on somatostatin and growth hormone releasing hormone ŽGHRH. mRNA levels in the hypothalamus by ‘in situ’ hybridization. We found that inhibin administration Ž4, 12 and 24 h, i.c.v.. led to an increase in somatostatin mRNA levels in the periventricular nucleus, and to a decrease in GHRH mRNA content in the arcuate nucleus of the hypothalamus. These findings indicate that inhibin regulates the hypothalamic levels of somatostatin and GHRH mRNA. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Inhibin; GH; Somatostatin; Growth hormone-releasing hormone; Hypothalamus
Inhibins are heterodimers composed of an a-subunit linked by disulfide bonds to one of two related b-subunits ŽbA or bB.. Although originally isolated as gonadal protein hormones which inhibit FSH secretion by the anterior pituitary gland, they are extensively distributed anatomically w11x, which suggests a variety of paracrine and autocrine roles in addition to their originally described endocrine functions w20x. Expression of a and b subunits has been found in the pituitary, and also in the brain. In particular, dense groups of stained fibers positive for the bA and bB subunits, and cells containing the mRNA encoding the a-subunit were found in the hypothalamus w14x. This suggests that this family of peptides are likely involved in neuroendocrine regulation at the hypothalamic level. Recent results obtained by our group have shown an inhibitory effect of inhibin on plasma GH levels w3x, which indicate that this protein is also involved in the regulation of the GH axis. Nevertheless it is still unclear whether inhibin is acting at the hypothalmic andror pituitary level.
)
Corresponding author. Fax: q34-981-574145; E-mail: [email protected]
The aim of this study was to determine the possible regulatory function of inhibin on GHRH and SRIF mRNA levels in the arcuate and periventricular neurons of the hypothalamus, respectively, since these two subpopulations of cells give rise to the neurosecretory projections to the hypophysial portal blood system controlling GH release by the pituitary. Adult male Sprague–Dawley rats Ž250–300 g. were used in this study. They were housed under constant light conditions Ž12 h light, 12 h darkness. in a temperature and humidity-controlled room. Chronic intra-cerebro-ventricular Ži.c.v.. canullae were implanted under sodium pentobarbital Ž50 mgrkg, i.p.. anaesthesia, as previously described w10x. After surgery, the animals were placed directly in isolation test chambers for 5 days and were given free access to regular Purina rat chow and tap water. They were treated through the i.c.v. route with vehicle Žsaline solution. or inhibin Ž20 mgrkg, Sigma.. The experiments started at 12 a.m. and finished at 12 p.m. of the following day. All rats were treated, simultaneously, with vehicle or inhibin, depending on the experimental group, and they were all killed at the same time at the end of the 24 h experiment. Rats were divided into 4 experimental groups: Ž1. Rats receiving inhibin during 24 h. These animals
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 0 2 5 - X
192
E. Carro et al.r Molecular Brain Research 66 (1999) 191–194
received 3 doses of inhibin Ž20 mgrkg.: at 12 a.m., 12 p.m. and 8 a.m. of the following day. Ž2. Rats receiving inhibin during 12 h. These animals received 1 dose of vehicle at 12 a.m., and 2 doses of inhibin Ž20 mgrkg.: at 12 p.m., and at 8 a.m. of the following day. Ž3. Rats receiving inhibin during 4 h. These animals received 2 doses of vehicle: at 12 a.m., and at 12 p.m., and 1 dose of inhibin Ž20 mgrkg. at 8 a.m. of the following day. Ž4. Control group: rats receiving 3 doses of vehicle: at 12 a.m., at 12 p.m. and at 8 a.m. of the following day. All rats were killed simultaneously at 12 a.m. Hypothalamic SRIF and GHRH mRNA levels were determined by in situ hybridization as previously shown w17x. Antisense oligodeoxynucleotides were used to detect
SRIF and GHRH mRNA levels. SRIF oligoprobe Ž30 mer. recognized nucleotides 310–339 of the preproSRIF cDNA. GHRH oligoprobe Ž35 mer. was complementary to nucleotides 51–85 of the GHRH cDNA. We have already demonstrated the specificity of the hybridization signal obtained with both probes w17x. The oligoprobes were 3X-end labeled with 35 S-dATP using terminal deoxynucleotydyl transferase. Fifteen-mm hypothalamic coronal sections were cut on a cryostat, and immediately stored at y808C until hybridization. Sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4. at room temperature for 30 min, dehydrated through 70, 80, 90, 95%, and absolute ethanol Ž5 min each., and dried. Hybridization was carried out overnight at 378C in a moist
Fig. 1. Brightfield photomicrographs from emulsion autoradiograms. Figures A and B correspond to cells in the periventricular nucleus of the hypothalamus that underwent in situ hybridization with a 35 S-oligoprobe complementary to pre-pro-SRIF mRNA. Magnification 40 = . Figures C and D correspond to cells in the arcuate nucleus of the hypothalamus that underwent in situ hybridization with a 35 S-oligoprobe complementary to pre-pro-GHRH mRNA. Magnification 40 = . A and C, rats that received saline solution Žcontrol rats.. B and D, rats treated with inhibin during a period of 24 h. Cells have been counterstained with methylene blue.
E. Carro et al.r Molecular Brain Research 66 (1999) 191–194
chamber. Hybridization solution was applied onto each slide and contained 4 = SSC, 50% deionized formamide, 1 = Denhardt’s solution, 500 mgrml sheared salmon sperm DNA, 10% dextran sulfate, 0.3% b-mercaptoethanol, and 5 = 10 5 dpm per slide of the labeled probe. After hybridization sections were rinsed in 1 = SSC with 0.001% b-mercaptoethanol at room temperature, and then sequentially washed in 1 = SSC containing 0.001% bmercaptoethanol at 558C Ž30 minrwash, three washes in total.; 1 = SSC with 0.001% b-mercaptoethanol at room temperature for 1 h, and then rinsed in 70% ethanol with 300 mM ammonium acetate. Finally, sections were airdried and exposed to Hyperfilm b-Max ŽAmersham, UK. at room temperature for 2 days. After the films were developed, sections were dipped in Ilford autoradiography emulsion ŽIlford, Nottingham, UK., and exposed for 1 week at 48C. Slides were then developed in Kodak D-19 developer ŽEastman Kodak, NY., fixed ŽKodak fixer., and counterstained with methylene blue. To compare anatomically similar regions, slides were matched according to the rat brain atlas of Paxinos and Watson w12x. Sections from at least 3 animals per experimental group were processed together. The entire experimental design was repeated 3 times. Image analysis. The number of silver grains per positively labeled cell was determined under darkfield optics using an automated image-processing system. This image analysis system has been described previously in detail w6x, and kindly supplied by Dr. D. Clifton and Dr. J. Chowen. In each tissue section, the number of silver grains over every identifiable cell was determined. Cells were identified by the presence of a cluster of silver grains over methylene blue counterstained cells. The number of grains per cell was referred to here as mRNA signal level. Data Žmean " S.E.M.. are presented as percentage change in relation to control values. Statistical analysis
193
was carried out using analysis of variance. N s number of repetitions of independent experiments. Inhibin administration Ži.c.v.. led to a significant increase on SRIF mRNA levels in the periventricular nucleus of the hypothalamus at all time-points studied: 4, 12 and 24 h Ž% controls 165 " 13%, p - 0.05, 173 " 8%, p - 0.05, and 188 " 10%, p - 0.01, respectively. N s 3 independent experiments. ŽFigs. 1 and 2.. In contrast, GHRH mRNA levels in the arcuate nucleus decreased 4 h after the treatment Ž% controls 69 " 13%, p - 0.05., and remained suppressed thereafter Ž% control s 58 " 10, p - 0.05%, and 55 " 8, p - 0.01%, after 12 and 24 h, respectively. N s 3 independent experiments. ŽFigs. 1 and 2.. Previous findings demonstrated an inhibitory effect of inhibin on plasma GH levels, and a decrease of in vivo GH responses to GHRH. w3x. To further clarify its place of action we determined hypothalamic SRIF and GHRH mRNA levels in order to evaluate whether inhibin was acting in the hypothalamus. In keeping with this possibility we found that inhibin increased SRIF mRNA levels in the periventricular nucleus of the hypothalamus, while it decreased GHRH mRNA levels in the arcuate nucleus. Taking into account that the genesis of spontaneous GH surges in the male rat appears to be dependent on an increase in hypothalamic GHRH-gene expression and secretion w18,22x, inhibin-induced decrease in GHRH mRNA levels could explain the reduction in plasma GH levels observed after administration of this protein. On the contrary, an increase in the hypothalamic somatostatinergic tone could explain the decrease on in vivo GH responses to GHRH as well as contribute to reduce spontaneous GH secretion. Although our data clearly show that inhibin suppresses in vivo GH secretion by acting at the hypothalamic level, a direct pituitary action cannot be ruled out. Furthermore, it is still unclear whether in the physiological setting this
Fig. 2. Pre-pro-SRIF mRNA levels in the periventricular nucleus, and pre-pro-GHRH mRNA content in the arcuate nucleus of the hypothalamus, determined by in situ hybridization Žsee material and methods., corresponding to rats treated with saline solution i.c.v. Žcontrol rats, C., and 4, 12 and 24 h after the administration of inhibin. Data were shown as percentage change in relation to control animals. ) p - 0.05 vs. control, )) p - 0.01 vs. control. N s 3 independent experiments.
194
E. Carro et al.r Molecular Brain Research 66 (1999) 191–194
effect could be mediated by inhibin produced in the gonads or inhibin synthesized in the central nervous system. The fact that within the hypothalamus, the paraventricular, periventricular and arcuate nuclei, areas where SRIF and GHRH-producing neurons are located, are rich in stained fibers positive for the inhibin bA and bB subunits, and in cells containing the mRNA encoding for the a-subunit w14x, supports this latter possibility. Nevertheless, that inhibin produced by the Sertoli cells of the testes might influence GH secretion cannot be discarded. Thus, although there is clear evidence that the sexual dimorphism observed in GHRH and SRIF gene expression and GH secretion is, at least in part, mediated by gonadal steroids w2,5,8,15,21x, recent indirect evidence also suggested the possible involvement of non-androgenic testicular factors. Therefore, it has been shown that in vivo GH responses to GHRH were reduced in pentobarbital anaesthetized gonadectomized male rats but remained normal after testosterone suppression by administration of a specific toxin for Leydig cells Žethylene dimethane sulphonate, EDS. w1x. Using a similar experimental model it was also shown that non-androgenic testicular factors regulate hypothalamic GHRH and SRIF mRNA levels as well as pituitary mRNA content of somatostatin receptor subtypes w9,16x. These findings suggest that testicular factors other than testosterone, secreted by cell types different of Leydig cells, may well be involved in the regulation of the GHRHSRIF-GH axis as they are in the LHRH-gonadotropins axis w4,7,13,19x. In conclusion, our data show that inhibin exerts a marked regulatory effect on somatostatin and GHRH mRNA levels in the hypothalamus. Therefore, the actions of inhibin on the neuroregulation of pituitary function appear to be more widespread than previously thought. Acknowledgements This work was supported by grants from Fondo de Investigaciones Sanitarias, Spanish Ministry of Health and the Xunta de Galicia. We are grateful to Ms. Luz Casas for her expert technical assistance. References w1x E. Aguilar, L. Pinilla, M. Tena-Sempere, Growth hormone-releasing hormone-induced growth hormone secretion in adult rats orchidectomized or injected with ethylene dimethane sulphonate, Neuroendocrinology 57 Ž1993. 132–134. w2x J. Argente, J. Chowen Breed, R.A. Steiner, D.K. Clifton, Somatostatin messenger RNA in hypothalamic neurons is increased by testosterone through activation of androgen receptors and not by aromatization to estradiol, Neuroendocrinology 52 Ž1990. 342–349. w3x E. Carro, R.M. Senarıs, Inhibin suppresses in ˜ ´ F. Mallo, C. Dieguez, ´ vivo GH secretion, Neuroendocrinology 68 Ž1998. 293–296. w4x R.S. Carroll, P.M. Kowash, J.A. Lofgren, R.H. Schwall, W.W. Chin, In vivo regulation of FSH synthesis by inhibin, Endocrinology 129 Ž1991. 3299–3304.
w5x J.A. Chowen-Breed, R.A. Steiner, D.K. Clifton, Sexual dimorphism and testosterone-dependent regulation of somatostatin gene expression in the periventricular nucleus of the rat brain, Endocrinology 125 Ž1989. 357–362. w6x J.A. Chowen-Breed, R.A. Steiner, D.K. Clifton, Semiquantitative analysis of cellular somatostatin mRNA levels by in situ hybridization histochemistry, Methods Neurosci. 5 Ž1991. 137–158. w7x M.D. Culler, A. Negro-Vilar, Destruction of testicular Leydig cells reveal a role of endogenous inhibin in regulating follicle-stimulating hormone secretion in the adult male rat, Mol. Cell Endocrinol 58 Ž1990. 263–273. w8x C. Dieguez, M.D. Page, M.F. Scanlon, Growth hormone neuroregu´ lation and its alterations in disease states, Clin. Endocrinol. 28 Ž1988. 109–143. w9x F. Lago, R.M. Senarıs, C. Dieguez, ˜ ´ P.C. Emson, F. Domınguez, ´ ´ Evidence for the involvement of non-androgenic testicular factors in the regulation of hypothalamic somatostatin and GHRH mRNA levels, Mol. Brain Res. 35 Ž1996. 220–226. w10x F. Mallo, J.A. Lamas, F.F. Casanueva, C. Dieguez, Effect of retinoic acid deficiency on in vivo and in vitro GH responses to GHRH in male rats, Neuroendocrinology 55 Ž1992. 642–647. w11x H. Meunier, C. Rivier, R.M. Evans, W. Vale, Gonadal and extragonadal expression of inhibin a , bA and bB subunits in various tissues predicts diverse functions, Proc. Natl. Acad. Sci. USA 85 Ž1988. 247–251. w12x G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, 2nd edn., Sydney, 1986. w13x C. Rivier, A. Corrigan, W. Vale, Effect of recombinant human inhibin on gonadotrophin secretion by the male rat, Endocrinology 129 Ž1991. 2155–2159. w14x V.J. Roberts, S.L. Barth, H. Meunier, W. Vale, Hybridization histochemical and immunohistochemical localization of inhibinractivin subunits and messenger ribonucleic acids in the rat brain, J. Comp. Neurol. 364 Ž1996. 473–493. w15x R.M. Senarıs, M.F. Scanlon, ˜ ´ F. Lago, M.D. Lewis, F. Domınguez, ´ C. Dieguez, Differential effects of in vivo estrogen administration on ´ hypothalamic growth hormone and somatostatin gene expression, Neurosci. Lett. 141 Ž1992. 123–126. w16x R.M. Senarıs, ˜ ´ F. Lago, C. Dieguez, Gonadal regulation of somatostatin receptor 1, 2 and 3 mRNA levels in the rat anterior pituitary, Mol. Brain Res. 38 Ž1996. 171–175. w17x R.M. Senarıs, ˜ ´ F. Lago, R. Coya, J. Pineda, C. Dieguez, Regulation of hypothalamic somatostatin, growth hormone-releasing hormone, and growth hormone receptor messenger ribonucleic acid by glucocorticoids, Endocrinology 137 Ž1996. 5236–5241. w18x G.S. Tannenbaum, N. Ling, The interrelationship of growth hormone ŽGH.-releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion, Endocrinology 115 Ž1984. 1952– 1957. w19x M. Tena-Sempere, L. Pinilla, E. Aguilar, Follicle-stimulating hormone and luteinizing hormone secretion in male rats orchidectomized or injected with ethylene dimethane sulphonate, Endocrinology 133 Ž1993. 1173–1181. w20x W. Vale, C. Rivier, A. Hsueh, C. Campen, H. Meunier, T. Bicsak, J. Vaughan, A. Corrigan, W. Bardin, P. Sawchenko, F. Petraglia, J. Yu, P. Plotsky, J. Spiess, J. Rivier, Chemical and Biological characterization of the inhibin family of protein hormones, Recent Prog. Horm. Res. 44 Ž1988. 1–34. w21x P. Zeitler, J. Argente, J.A. Chowen-Breed, D.K. Clifton, R.A. Steiner, Growth hormone releasing hormone messenger ribonucleic acid in the hypothalamus of the adult male rat is increased by testosterone, Endocrinology 127 Ž1990. 1362–1368. w22x P. Zeitler, G.S. Tannenbaum, D. Clifton, R.A. Steiner, Ultradian oscillations in somatostatin and growth hormone-relasing hormone mRNAs in the brains of adult male rats, Proc. Natl. Acad. Sci. USA 88 Ž1991. 8920–8924.
Short communication
Regulation of hypothalamic somatostatin and growth hormone releasing hormone mRNA levels by inhibin E. Carro a , R.M. Senarıs ˜ ´ a
a,)
, F. Mallo b, C. Dieguez ´
a
Department of Physiology, Faculty of Medicine, UniÕersity of Santiago de Compostela, 15700 Santiago de Compostela, Spain b Department of Fundamental Biology, Faculty of Sciences, UniÕersity of Vigo, Spain Accepted 29 December 1998
Abstract Although it is well established that inhibin plays a major role in the regulation of the hypothalamic-pituitary-gonadal axis, its influence in the regulation of other neuroendocrine functions is still poorly understood. Recent results indicate that inhibin suppresses plasma GH levels, but its site of action is yet unknown. Therefore, in the present work we investigated the effects of inhibin on somatostatin and growth hormone releasing hormone ŽGHRH. mRNA levels in the hypothalamus by ‘in situ’ hybridization. We found that inhibin administration Ž4, 12 and 24 h, i.c.v.. led to an increase in somatostatin mRNA levels in the periventricular nucleus, and to a decrease in GHRH mRNA content in the arcuate nucleus of the hypothalamus. These findings indicate that inhibin regulates the hypothalamic levels of somatostatin and GHRH mRNA. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Inhibin; GH; Somatostatin; Growth hormone-releasing hormone; Hypothalamus
Inhibins are heterodimers composed of an a-subunit linked by disulfide bonds to one of two related b-subunits ŽbA or bB.. Although originally isolated as gonadal protein hormones which inhibit FSH secretion by the anterior pituitary gland, they are extensively distributed anatomically w11x, which suggests a variety of paracrine and autocrine roles in addition to their originally described endocrine functions w20x. Expression of a and b subunits has been found in the pituitary, and also in the brain. In particular, dense groups of stained fibers positive for the bA and bB subunits, and cells containing the mRNA encoding the a-subunit were found in the hypothalamus w14x. This suggests that this family of peptides are likely involved in neuroendocrine regulation at the hypothalamic level. Recent results obtained by our group have shown an inhibitory effect of inhibin on plasma GH levels w3x, which indicate that this protein is also involved in the regulation of the GH axis. Nevertheless it is still unclear whether inhibin is acting at the hypothalmic andror pituitary level.
)
Corresponding author. Fax: q34-981-574145; E-mail: [email protected]
The aim of this study was to determine the possible regulatory function of inhibin on GHRH and SRIF mRNA levels in the arcuate and periventricular neurons of the hypothalamus, respectively, since these two subpopulations of cells give rise to the neurosecretory projections to the hypophysial portal blood system controlling GH release by the pituitary. Adult male Sprague–Dawley rats Ž250–300 g. were used in this study. They were housed under constant light conditions Ž12 h light, 12 h darkness. in a temperature and humidity-controlled room. Chronic intra-cerebro-ventricular Ži.c.v.. canullae were implanted under sodium pentobarbital Ž50 mgrkg, i.p.. anaesthesia, as previously described w10x. After surgery, the animals were placed directly in isolation test chambers for 5 days and were given free access to regular Purina rat chow and tap water. They were treated through the i.c.v. route with vehicle Žsaline solution. or inhibin Ž20 mgrkg, Sigma.. The experiments started at 12 a.m. and finished at 12 p.m. of the following day. All rats were treated, simultaneously, with vehicle or inhibin, depending on the experimental group, and they were all killed at the same time at the end of the 24 h experiment. Rats were divided into 4 experimental groups: Ž1. Rats receiving inhibin during 24 h. These animals
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 0 2 5 - X
192
E. Carro et al.r Molecular Brain Research 66 (1999) 191–194
received 3 doses of inhibin Ž20 mgrkg.: at 12 a.m., 12 p.m. and 8 a.m. of the following day. Ž2. Rats receiving inhibin during 12 h. These animals received 1 dose of vehicle at 12 a.m., and 2 doses of inhibin Ž20 mgrkg.: at 12 p.m., and at 8 a.m. of the following day. Ž3. Rats receiving inhibin during 4 h. These animals received 2 doses of vehicle: at 12 a.m., and at 12 p.m., and 1 dose of inhibin Ž20 mgrkg. at 8 a.m. of the following day. Ž4. Control group: rats receiving 3 doses of vehicle: at 12 a.m., at 12 p.m. and at 8 a.m. of the following day. All rats were killed simultaneously at 12 a.m. Hypothalamic SRIF and GHRH mRNA levels were determined by in situ hybridization as previously shown w17x. Antisense oligodeoxynucleotides were used to detect
SRIF and GHRH mRNA levels. SRIF oligoprobe Ž30 mer. recognized nucleotides 310–339 of the preproSRIF cDNA. GHRH oligoprobe Ž35 mer. was complementary to nucleotides 51–85 of the GHRH cDNA. We have already demonstrated the specificity of the hybridization signal obtained with both probes w17x. The oligoprobes were 3X-end labeled with 35 S-dATP using terminal deoxynucleotydyl transferase. Fifteen-mm hypothalamic coronal sections were cut on a cryostat, and immediately stored at y808C until hybridization. Sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4. at room temperature for 30 min, dehydrated through 70, 80, 90, 95%, and absolute ethanol Ž5 min each., and dried. Hybridization was carried out overnight at 378C in a moist
Fig. 1. Brightfield photomicrographs from emulsion autoradiograms. Figures A and B correspond to cells in the periventricular nucleus of the hypothalamus that underwent in situ hybridization with a 35 S-oligoprobe complementary to pre-pro-SRIF mRNA. Magnification 40 = . Figures C and D correspond to cells in the arcuate nucleus of the hypothalamus that underwent in situ hybridization with a 35 S-oligoprobe complementary to pre-pro-GHRH mRNA. Magnification 40 = . A and C, rats that received saline solution Žcontrol rats.. B and D, rats treated with inhibin during a period of 24 h. Cells have been counterstained with methylene blue.
E. Carro et al.r Molecular Brain Research 66 (1999) 191–194
chamber. Hybridization solution was applied onto each slide and contained 4 = SSC, 50% deionized formamide, 1 = Denhardt’s solution, 500 mgrml sheared salmon sperm DNA, 10% dextran sulfate, 0.3% b-mercaptoethanol, and 5 = 10 5 dpm per slide of the labeled probe. After hybridization sections were rinsed in 1 = SSC with 0.001% b-mercaptoethanol at room temperature, and then sequentially washed in 1 = SSC containing 0.001% bmercaptoethanol at 558C Ž30 minrwash, three washes in total.; 1 = SSC with 0.001% b-mercaptoethanol at room temperature for 1 h, and then rinsed in 70% ethanol with 300 mM ammonium acetate. Finally, sections were airdried and exposed to Hyperfilm b-Max ŽAmersham, UK. at room temperature for 2 days. After the films were developed, sections were dipped in Ilford autoradiography emulsion ŽIlford, Nottingham, UK., and exposed for 1 week at 48C. Slides were then developed in Kodak D-19 developer ŽEastman Kodak, NY., fixed ŽKodak fixer., and counterstained with methylene blue. To compare anatomically similar regions, slides were matched according to the rat brain atlas of Paxinos and Watson w12x. Sections from at least 3 animals per experimental group were processed together. The entire experimental design was repeated 3 times. Image analysis. The number of silver grains per positively labeled cell was determined under darkfield optics using an automated image-processing system. This image analysis system has been described previously in detail w6x, and kindly supplied by Dr. D. Clifton and Dr. J. Chowen. In each tissue section, the number of silver grains over every identifiable cell was determined. Cells were identified by the presence of a cluster of silver grains over methylene blue counterstained cells. The number of grains per cell was referred to here as mRNA signal level. Data Žmean " S.E.M.. are presented as percentage change in relation to control values. Statistical analysis
193
was carried out using analysis of variance. N s number of repetitions of independent experiments. Inhibin administration Ži.c.v.. led to a significant increase on SRIF mRNA levels in the periventricular nucleus of the hypothalamus at all time-points studied: 4, 12 and 24 h Ž% controls 165 " 13%, p - 0.05, 173 " 8%, p - 0.05, and 188 " 10%, p - 0.01, respectively. N s 3 independent experiments. ŽFigs. 1 and 2.. In contrast, GHRH mRNA levels in the arcuate nucleus decreased 4 h after the treatment Ž% controls 69 " 13%, p - 0.05., and remained suppressed thereafter Ž% control s 58 " 10, p - 0.05%, and 55 " 8, p - 0.01%, after 12 and 24 h, respectively. N s 3 independent experiments. ŽFigs. 1 and 2.. Previous findings demonstrated an inhibitory effect of inhibin on plasma GH levels, and a decrease of in vivo GH responses to GHRH. w3x. To further clarify its place of action we determined hypothalamic SRIF and GHRH mRNA levels in order to evaluate whether inhibin was acting in the hypothalamus. In keeping with this possibility we found that inhibin increased SRIF mRNA levels in the periventricular nucleus of the hypothalamus, while it decreased GHRH mRNA levels in the arcuate nucleus. Taking into account that the genesis of spontaneous GH surges in the male rat appears to be dependent on an increase in hypothalamic GHRH-gene expression and secretion w18,22x, inhibin-induced decrease in GHRH mRNA levels could explain the reduction in plasma GH levels observed after administration of this protein. On the contrary, an increase in the hypothalamic somatostatinergic tone could explain the decrease on in vivo GH responses to GHRH as well as contribute to reduce spontaneous GH secretion. Although our data clearly show that inhibin suppresses in vivo GH secretion by acting at the hypothalamic level, a direct pituitary action cannot be ruled out. Furthermore, it is still unclear whether in the physiological setting this
Fig. 2. Pre-pro-SRIF mRNA levels in the periventricular nucleus, and pre-pro-GHRH mRNA content in the arcuate nucleus of the hypothalamus, determined by in situ hybridization Žsee material and methods., corresponding to rats treated with saline solution i.c.v. Žcontrol rats, C., and 4, 12 and 24 h after the administration of inhibin. Data were shown as percentage change in relation to control animals. ) p - 0.05 vs. control, )) p - 0.01 vs. control. N s 3 independent experiments.
194
E. Carro et al.r Molecular Brain Research 66 (1999) 191–194
effect could be mediated by inhibin produced in the gonads or inhibin synthesized in the central nervous system. The fact that within the hypothalamus, the paraventricular, periventricular and arcuate nuclei, areas where SRIF and GHRH-producing neurons are located, are rich in stained fibers positive for the inhibin bA and bB subunits, and in cells containing the mRNA encoding for the a-subunit w14x, supports this latter possibility. Nevertheless, that inhibin produced by the Sertoli cells of the testes might influence GH secretion cannot be discarded. Thus, although there is clear evidence that the sexual dimorphism observed in GHRH and SRIF gene expression and GH secretion is, at least in part, mediated by gonadal steroids w2,5,8,15,21x, recent indirect evidence also suggested the possible involvement of non-androgenic testicular factors. Therefore, it has been shown that in vivo GH responses to GHRH were reduced in pentobarbital anaesthetized gonadectomized male rats but remained normal after testosterone suppression by administration of a specific toxin for Leydig cells Žethylene dimethane sulphonate, EDS. w1x. Using a similar experimental model it was also shown that non-androgenic testicular factors regulate hypothalamic GHRH and SRIF mRNA levels as well as pituitary mRNA content of somatostatin receptor subtypes w9,16x. These findings suggest that testicular factors other than testosterone, secreted by cell types different of Leydig cells, may well be involved in the regulation of the GHRHSRIF-GH axis as they are in the LHRH-gonadotropins axis w4,7,13,19x. In conclusion, our data show that inhibin exerts a marked regulatory effect on somatostatin and GHRH mRNA levels in the hypothalamus. Therefore, the actions of inhibin on the neuroregulation of pituitary function appear to be more widespread than previously thought. Acknowledgements This work was supported by grants from Fondo de Investigaciones Sanitarias, Spanish Ministry of Health and the Xunta de Galicia. We are grateful to Ms. Luz Casas for her expert technical assistance. References w1x E. Aguilar, L. Pinilla, M. Tena-Sempere, Growth hormone-releasing hormone-induced growth hormone secretion in adult rats orchidectomized or injected with ethylene dimethane sulphonate, Neuroendocrinology 57 Ž1993. 132–134. w2x J. Argente, J. Chowen Breed, R.A. Steiner, D.K. Clifton, Somatostatin messenger RNA in hypothalamic neurons is increased by testosterone through activation of androgen receptors and not by aromatization to estradiol, Neuroendocrinology 52 Ž1990. 342–349. w3x E. Carro, R.M. Senarıs, Inhibin suppresses in ˜ ´ F. Mallo, C. Dieguez, ´ vivo GH secretion, Neuroendocrinology 68 Ž1998. 293–296. w4x R.S. Carroll, P.M. Kowash, J.A. Lofgren, R.H. Schwall, W.W. Chin, In vivo regulation of FSH synthesis by inhibin, Endocrinology 129 Ž1991. 3299–3304.
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