Neuroscience Letters, 141 (1992) 123 126 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
123
NSL 08753
Differential effects of in vivo estrogen administration on hypothalamic growth hormone releasing hormone and somatostatin gene expression R . M . Sefiaris a, F. L a g o a, M . D . Lewis b, F. D o m i n g u e z a, M . F . S c a n l o n b a n d C. D i e g u e z a "Department of Physiology, Faculty of Medicine, University of Santiago, Santiago de Compostela (Spain) and bDepartment of Medicine, University of Wales College of Medicine, Cardiff ( UK)
(Received 26 November 1991; Revised version received 13 April 1992; Accepted 13 April 1992) Key words: Growth hormone releasing hormone, somatostatin, estrogens, rat hypothalamus
The aim of this study was to investigatethe effectof in vivo estrogen administration on hypothalamic growth hormone releasinghormone (GHRH) and somatostatin (SS) gene expression. We found that estrogen administration (estradiol valerate 250/tg/every 3 days, subcutaneously) to male rats induced a decrease in both hypothalamic GHRH mRNA levels and GHRH content, that was significant after 3 and 8 days of treatment. In contrast SS mRNA levelswere transiently elevated after 1 and 3 days of estrogen administration, returning to normal values after 8 days of treatment. These data suggest that the existence of sexual dimorphism in GH secretion in the rat could be mediated to some extent by gonadal hormones regulating somatostatin and GHRH gene expression in the hypothalamus.
Since G H secretion in the rat clearly shows sexual dimorphism, there is no doubt that gonadal hormones play a major role in the neuroregulation of G H secretion. The pituitary content of G H and the rate of G H synthesis are greater in male than in female rats [4]. Also, adult male rats exhibit regular and marked G H pulses that occur every 3-3.5 h, with almost undetectable troughs, while adult female rats have an irregular pattern of low amplitude G H pulses with higher troughs [7, 15, 18]. Although these differences are not completely understood, it is likely that they are due to the effect of gonadal hormones at both the hypothalamic and the pituitary levels [4]. The first possibility is supported by the existence of estrogen receptors in several hypothalamic regions, including the periventricular, arcuate and ventromedial nuclei, where there is an abundance of S S and G H R H - p r o d u c i n g neurons [11, 13, 17]. Furthermore, sex differences in hypothalamic SS m R N A levels have been shown, these being higher in male than in female rats [3]. Also, it was recently reported that a decrease in hypothalamic SS m R N A levels occurs after gonadectomy in both male and female rats, without significant changes in cerebral cortical SS m R N A [19].
Correspondence: C. Di6guez, Department of Physiology, Faculty of Medicine, University of Santiago de Compostela, c/S. Francisco s/n, 15705 Santiago de Compostela, Spain.
In the present study we have investigated the effect of estrogen administration on SS and G H R H m R N A levels and peptide content in the male rat hypothalamus in vivo. Adult male Sprague-Dawley rats (200-250 g) were used throughout. The animals were maintained in a constant l i g h t , l a r k cycle (lights on 08.00-20.00 h) with laboratory chow and tap water available ad libitum. Rats were treated with either vehicle (control group) or estrogen (estradiol valerate 250/,tg/every 3 days, subcutaneously) for 1, 3 and 8 days. Using a similar experimental approach, others have previously reported that estrogen administration induces a feminization of hepatic steroid metabolism and spontaneous G H secretion [12]. Rats were killed by decapitation and the hypothalamus quickly removed and frozen for later use. Individual hypothalami were homogenized in 2 ml 1 M HC1, and the homogenates heated at 100°C for 10 min. After centrifugation the supernatant was extracted using a C-18 silica SepPak cartridge (Waters Associates) using standard procedures (elution of the sample from the SepPak with 80% acetonitrile in 0.1% trifluoroacetic acid). The eluate was evaporated to dryness under vacuum prior to be assayed for G H R H and SS. The protein content of the cell extract pellet was assayed using the Lowry method [10]. G H R H content was measured by a two-site immunochemiluminometric assay [14], and SS content by conventional R I A as previously described [9].
124
G H R H and somatostatin m R N A levels were determined by Northern blot hybridization using antisense G H R H and SS cRNA probes respectively. Total RNA was extracted from the tissue as described by Chirgwin et al. [1], and size-separated by electrophoresis on MOPSformaldehyde-agarose (1.5%) gels. RNAs were then transferred to a Gene Screen membrane (Dupont). Following fixation by baking with ultraviolet, membranes were hybridized with [32p]cRNA probes complementary either to p r e p r o G H R H or preproSS mRNA. A 360 bp fragment of a rat p r e p r o G H R H cDNA was kindly provided by Dr. Mayo (Salk Institute, San Diego, CA) and it was subcloned into the transcription vector p G E M 4 (Promega Biotec). A plasmid pSP65 with a 450 bp fragment of a rat preproSS cDNA was kindly provided by Dr. Goodman (New England Medical Center, Boston, MA). Antisense cRNA probes were made in vitro by transcribing the insert sequence according to the recommendations of the supplier (Promega Biotec). Membranes were prehybridized for 6 h at 42°C in 50% formamide, 0.2% polyvinyl-pyrrolidone, 0.2% BSA, 0.2% Ficoll, 0.05 M Tris, 1 M NaC1, 0.1% sodium pyrophosphate, 0.1% SDS, 10% dextran sulphate and 0.1 mg/ ml denaturated salmon sperm DNA. Membranes were then hybridized for 24~48 h at 60-65°C under the same conditions with the addition of lxl06 cpm/ml of the SS probe, and 4x106 cpm/ml of the G H R H probe. After hybridization membranes were washed twice with each of the following solutions: firstly with 2xSSC (0.3 M sodium citrate, 3 M NaC1) at room temperature for 5 min, secondly with 2xSSC, 0.5% SDS at 65°C for 30 min, and finally with 0.2xSSC at 65°C for 30 min. Membranes were exposed to Hyperfilm (Amershan) at -80°C with intensifying screens. Northern blots were analyzed with a scanning densitometer (BioRad). Membranes were rehybridized with a cDNA probe of rat fl-actin as control probe. All data were expressed as mean + S.E.M. The results of m R N A levels were expressed as the ratio G H R H or SS mRNA/fl-actin mRNA, and were evaluated using the Mann-Whitney test. The results of peptide content were evaluated by analysis of variance (multiple range analysis, Sheffe). Estrogen administration to male rats in vivo significantly decreased the levels of G H R H mRNA/fl-actin m R N A after 3 and 8 days of treatment (% control = 58 _+ 25% after 1 day of treatment, 51 _+ 9% after 3 days (P < 0.01 vs. control), and 9.2 _+ 4.8% after 8 days, P < 0.01 vs. control, n = 3 different experiments) (Fig. 1). Also G H R H content was decreased after 3 and 8 days of estrogen administration (2.14 + 0.15 ng/mg protein in control animals, 1.74 + 0.18 ng/mg protein after 3 days of treatment (P < 0.05 vs. control), 1.19 _+ 0.25 ng/mg
8-ACTIN~
ss~
% Control < z
% Control
140
250-
120
"~ z 225-
z 100 i(_)
z 200~-
,~ & z "1-
**
60 40 20
**
0
&
150-
~: z
12s-
E
100-
T
*
75 E1
E3
E8
E1
E3
E8
Fig. 1. Hypothalamic p r e - p r o G H R H m R N A (left) and preproSS m R N A levels (right) after treatment with estrogens for ! day (El), 3 days (E3), 8 days (E8), or saline (control = C). Northern blot analysis. 50 ,ug (let1) and 20 ~ g (right) of total R N A from rat hypothalami were run in formaldehyde-agarose gels and blotted to a nylon membrane as described in the text. Blots were hybridized with the indicated 32p-labelled G H R H and SS c R N A probes. Only a band of approximately 750 bp was detected in the case of G H R H m R NA, and one of 650 bp in the case of SS m R N A . fl-actin m R N A hybridization is also shown. The results were corrected using the fl-actin as control probe. Data {mean + S . E M . ) were expressed as percentage change in relation to control values, n - 3 4 different experiments. *P < 0.05 vs. control, •*P < 0.01 vs. control.
protein after 8 days of treatment (P < 0.01 vs. control, n = 12-15 rats/group) (Fig. 2). In contrast, we have found increased levels of SS mRNA/fl-actin mRNA after 1 and 3 days of treatment (% control = 130 _+ 16% after 1 day (P < 0.05 vs. control), and 180 _+ 26% after 3 days (P < 0.01 vs. control), n - - 4 different experiments). Nevertheless, longer administrations of estrogens (8 days) returned SS m R N A content/fl-actin m R N A to control levels (% control -- 89 + 11%, n --4 different experiments) (Fig. 1). Somatostatin content did not show a significant increase until after 8 days of treatment (15.98 + 0.75 ng/mg protein in control rats and 19.42 _+ 1.35 ng/mg protein after 8 days of tretment, P < 0.05 vs. control, n -- 12-15 rats/ group) (Fig. 2). Our data show that both G H R H and SS gene expression in the rat hypothalamus are regulated by estrogens. Previous work regarding the effects of estrogens on hypothalamic G H R H gene expression has proved to be inconclusive. Although some authors have described an increase in hypothalamic G H R H content after 2 weeks
125
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22
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20
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18
O
2.25 *a
1.75~-r
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1.50 .*-,
(3. 16 l E:
2.00 I
~
1.2050 O
12
0.75
10
0.50
C
E1
E3
E
cn E
--...,,.
EB
Fig. 2. Hypothalamic SS (open bars) and GHRH (hatched bars) content after treatment with estrogens for 1 day (El), 3 days (E3), 8 days (E8), or saline (control -- C). Data were expressed as ng/mg protein (mean_+ S.E.M.). n-- 12 15 rats/group. *P < 0.05 vs. control, *'P < 0.01 vs. control, "P < 0.05 vs. E1 and E3, a~p < 0.01 vs. El and E3.
of estrogen treatment of gonadectomized male rats [5], others have found no alteration in either G H R H content or G H R H m R N A levels after gonadectomy [2]. In the present report we describe a parallel decrease in hypothalamic G H R H content and G H R H m R N A levels, thus suggesting that estrogens have an inhibitory effect on hypothalamic G H R H gene expression. It is unclear whether this effect reflects changes in transcription rate or m R N A stabilization. Nevertheless, inhibition of both G H R H m R N A levels and G H R H content by estrogens could explain the fact that despite lower somatostatinergic tone G H pulse a m p l i t u d e ~ h i c h is mainly dependent on G H R H surges--is decreased in female rats. Although some workers have failed to detect any change in hypothalamic somatostatin content in male rats following gonadectomy and estrogen administration [5], others have found a decrease [3, 19] in hypothalamic somatostatin m R N A after gonadectomy that was reversed [19] or unaffected [3] by estrogen administration. Since in none of these studies both parameters, SS content and SS m R N A , were reported it is difficult to assess whether these differences were related to the period of time after gonadectomy, to the dose of estrogen administered, or to the sex of the animal used. In any event our data regarding the effects of estrogens on SS m R N A levels show a transient increase that returns to control levels after 8 days of treatment. The different time-course of estrogen-induced increase in SS m R N A levels and SS content reported here could be explained because m R N A availability could be considered as an index of protein biosynthesis (although it cannot be assumed that all SS m R N A produced is trans-
lated into the mature peptide). In contrast the peptide content reflects the ratio of synthesis to release, and therefore our data suggest a different time-course effect of estrogens on somatostatin synthesis and secretion. Finally, from our data it is not possible to conclude whether estrogen administration increases the transcription rate of the SS gene or induces m R N A stabilization. Nevertheless, since according to the GenBank genetic sequence data, no estrogen receptor consensus binding sequence seems to be present in the SS gene [19], it is likely that their effects could be mediated through other neurotransmitters or neuropeptides. In any case, our findings support the view that in addition to their pituitary effects [2, 6, 8, 16], estrogens are involved in the neuroregulation of G H secretion by acting at the hypothalamic level. In summary, we have found that estrogen administration produced opposite effects on hypothalamic SS and G H R H gene expression in the hypothalamus, which may explain to some extent the existence of a sexual dimorphism of G H secretion in the rat. This work was supported by the D G I C Y T (87/0488 and 90/0771). 1 Chirwgwin, J.M., Przybyla, A.E., McDonald, R.J. and Runer, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299. 2 Chomczymsky, P., Downs, T. and Frohman, L.A., Feedback regulation of growth hormone releasing hormone gene expression by GH in rat hypothalamus, Mol. Endocrinol., 2 (1988) 23(~241. 3 Chowen-Breed, J.A., Steiner, R.A. and Clifton, D.K., Sexual dimorphism and testosterone-dependent regulation of somatostatin gene expression in the periventricular nucleus of the rat brain, Endocrinology, 125 (1989) 357 362. 4 Di6guez, C., Page, M.D. and Scanlon, M.F., Growth hormone neuroregulation and its alterations in disease states, Clin. Endocrinol., 28 (1988) 109-143. 5 Gabriel, S.M., Millard, W.I., Koenig, J.I., Badger, T.M., Russell, W.E., Maiter, D.M. and Martin, J.B., Sexual and developmental differences in peptides regulating growth hormone secretion in the rat, Neuroendocrinology, 50 (1989) 299 307. 6 Ho, K.Y., Leong, D.A., Sinha, Y.N., Johnson, M.L., Evans, W.S., Thorner, M.O., Sex related differences in GH secretion in rat using reverse haemolytic plaque assay, Am. J. Physiol., 250 (1986) E650654. 7 Jansson, J.-O., Edberg, S., lsaksson, O.P.G. and Eden, S., Influence of gonadal steroids on age- and sex-related secretory patterns of growth hormone in the rat, Endocrinology, 114 (1984) 1287 1294. 8 Krieg, R.J., Thorner, M.O. and Evans, W.S., Sex differences in beta-adrenergic stimulation of growth hormone secretion in vitro, Endocrinology, 119 (1986) 1339 1342. 9 Lewis, B.M., Dieguez, C., Lewis, M., Hall, R. and Scanlon, M.F., Hypothalamic D2 receptors mediate the preferential release of somatostatin-28 in response to dopaminergic stimulation, Endocrinology, 119 (1986) 1712-1717. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265 275.
126 11 McEwen, B.S., Binding and metabolism of sex steroids by the hypothalamic-pituitary unit: physiological implications, Annu. Rev. Physiol., 42 (1980) 97 110. 12 Mode, A., Gustafson, J.A., Jansson, J.O., Eden, S. and Isaksson O., Association between plasma levels of growth hormone and sex differentiation of hepatic steroid metabolism in the rat. Endocrinology 111 (1982) 1692-1697. 13 Morrell, J.I., Krieger, M.S. and Pfaff, D.W., Quantitative autoradiographic analysis of estradiol retention by cells in the preoptic area, hypothalamus and amygdala, Exp. Brain Res., 62 (1986) 343-354. 14 Page, M., Lewis, M., Lewis, B., Weeks, I. and Scanlon, M., Development and validation of a two-site inmunochemiluminometric assay for rat growth hormone releasing hormone, J. Neuroendocrinol., 1 (1989)433~136. 15 Saunders, A., Terry, L.C., Audet, J., Brazeau, P. and Martin, J.B., Dynamic studies of growth hormone and prolactin secretion in the female rat, Neuroendocrinology 21 (1976) 193-203.
16 Simard, J., Hubert, J.F., Hosseinzadeh, T. and Labrie, F., Stimulation of growth hormone release and synthesis by estrogens in rat anterior pituitary cells in culture, Endocrinology, 119 (1986) 20042011. 17 Sawchenko, P.E., Swanson, L.W., Rivier, J. and Vale, W.W., The distribution of growth hormone releasing factor inmunoreactivity in the central nervous system of the rat: an inmunohistochemical study using antisera directed against rat hypothalamic GRF, J. Comp. Neurol., 237 (1985):100-115. 18 Tannenbaum, G.S. and Martin, J.B., Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98 (1976) 562 570. 19 Werner, H., Koch, Y., Baldino, F. and Gozes, I., Steroid regulation of somatostatin mRNA in the rat hypothalamus, J. Biol. Chem., 263 (1988) 7666-7671.
123
NSL 08753
Differential effects of in vivo estrogen administration on hypothalamic growth hormone releasing hormone and somatostatin gene expression R . M . Sefiaris a, F. L a g o a, M . D . Lewis b, F. D o m i n g u e z a, M . F . S c a n l o n b a n d C. D i e g u e z a "Department of Physiology, Faculty of Medicine, University of Santiago, Santiago de Compostela (Spain) and bDepartment of Medicine, University of Wales College of Medicine, Cardiff ( UK)
(Received 26 November 1991; Revised version received 13 April 1992; Accepted 13 April 1992) Key words: Growth hormone releasing hormone, somatostatin, estrogens, rat hypothalamus
The aim of this study was to investigatethe effectof in vivo estrogen administration on hypothalamic growth hormone releasinghormone (GHRH) and somatostatin (SS) gene expression. We found that estrogen administration (estradiol valerate 250/tg/every 3 days, subcutaneously) to male rats induced a decrease in both hypothalamic GHRH mRNA levels and GHRH content, that was significant after 3 and 8 days of treatment. In contrast SS mRNA levelswere transiently elevated after 1 and 3 days of estrogen administration, returning to normal values after 8 days of treatment. These data suggest that the existence of sexual dimorphism in GH secretion in the rat could be mediated to some extent by gonadal hormones regulating somatostatin and GHRH gene expression in the hypothalamus.
Since G H secretion in the rat clearly shows sexual dimorphism, there is no doubt that gonadal hormones play a major role in the neuroregulation of G H secretion. The pituitary content of G H and the rate of G H synthesis are greater in male than in female rats [4]. Also, adult male rats exhibit regular and marked G H pulses that occur every 3-3.5 h, with almost undetectable troughs, while adult female rats have an irregular pattern of low amplitude G H pulses with higher troughs [7, 15, 18]. Although these differences are not completely understood, it is likely that they are due to the effect of gonadal hormones at both the hypothalamic and the pituitary levels [4]. The first possibility is supported by the existence of estrogen receptors in several hypothalamic regions, including the periventricular, arcuate and ventromedial nuclei, where there is an abundance of S S and G H R H - p r o d u c i n g neurons [11, 13, 17]. Furthermore, sex differences in hypothalamic SS m R N A levels have been shown, these being higher in male than in female rats [3]. Also, it was recently reported that a decrease in hypothalamic SS m R N A levels occurs after gonadectomy in both male and female rats, without significant changes in cerebral cortical SS m R N A [19].
Correspondence: C. Di6guez, Department of Physiology, Faculty of Medicine, University of Santiago de Compostela, c/S. Francisco s/n, 15705 Santiago de Compostela, Spain.
In the present study we have investigated the effect of estrogen administration on SS and G H R H m R N A levels and peptide content in the male rat hypothalamus in vivo. Adult male Sprague-Dawley rats (200-250 g) were used throughout. The animals were maintained in a constant l i g h t , l a r k cycle (lights on 08.00-20.00 h) with laboratory chow and tap water available ad libitum. Rats were treated with either vehicle (control group) or estrogen (estradiol valerate 250/,tg/every 3 days, subcutaneously) for 1, 3 and 8 days. Using a similar experimental approach, others have previously reported that estrogen administration induces a feminization of hepatic steroid metabolism and spontaneous G H secretion [12]. Rats were killed by decapitation and the hypothalamus quickly removed and frozen for later use. Individual hypothalami were homogenized in 2 ml 1 M HC1, and the homogenates heated at 100°C for 10 min. After centrifugation the supernatant was extracted using a C-18 silica SepPak cartridge (Waters Associates) using standard procedures (elution of the sample from the SepPak with 80% acetonitrile in 0.1% trifluoroacetic acid). The eluate was evaporated to dryness under vacuum prior to be assayed for G H R H and SS. The protein content of the cell extract pellet was assayed using the Lowry method [10]. G H R H content was measured by a two-site immunochemiluminometric assay [14], and SS content by conventional R I A as previously described [9].
124
G H R H and somatostatin m R N A levels were determined by Northern blot hybridization using antisense G H R H and SS cRNA probes respectively. Total RNA was extracted from the tissue as described by Chirgwin et al. [1], and size-separated by electrophoresis on MOPSformaldehyde-agarose (1.5%) gels. RNAs were then transferred to a Gene Screen membrane (Dupont). Following fixation by baking with ultraviolet, membranes were hybridized with [32p]cRNA probes complementary either to p r e p r o G H R H or preproSS mRNA. A 360 bp fragment of a rat p r e p r o G H R H cDNA was kindly provided by Dr. Mayo (Salk Institute, San Diego, CA) and it was subcloned into the transcription vector p G E M 4 (Promega Biotec). A plasmid pSP65 with a 450 bp fragment of a rat preproSS cDNA was kindly provided by Dr. Goodman (New England Medical Center, Boston, MA). Antisense cRNA probes were made in vitro by transcribing the insert sequence according to the recommendations of the supplier (Promega Biotec). Membranes were prehybridized for 6 h at 42°C in 50% formamide, 0.2% polyvinyl-pyrrolidone, 0.2% BSA, 0.2% Ficoll, 0.05 M Tris, 1 M NaC1, 0.1% sodium pyrophosphate, 0.1% SDS, 10% dextran sulphate and 0.1 mg/ ml denaturated salmon sperm DNA. Membranes were then hybridized for 24~48 h at 60-65°C under the same conditions with the addition of lxl06 cpm/ml of the SS probe, and 4x106 cpm/ml of the G H R H probe. After hybridization membranes were washed twice with each of the following solutions: firstly with 2xSSC (0.3 M sodium citrate, 3 M NaC1) at room temperature for 5 min, secondly with 2xSSC, 0.5% SDS at 65°C for 30 min, and finally with 0.2xSSC at 65°C for 30 min. Membranes were exposed to Hyperfilm (Amershan) at -80°C with intensifying screens. Northern blots were analyzed with a scanning densitometer (BioRad). Membranes were rehybridized with a cDNA probe of rat fl-actin as control probe. All data were expressed as mean + S.E.M. The results of m R N A levels were expressed as the ratio G H R H or SS mRNA/fl-actin mRNA, and were evaluated using the Mann-Whitney test. The results of peptide content were evaluated by analysis of variance (multiple range analysis, Sheffe). Estrogen administration to male rats in vivo significantly decreased the levels of G H R H mRNA/fl-actin m R N A after 3 and 8 days of treatment (% control = 58 _+ 25% after 1 day of treatment, 51 _+ 9% after 3 days (P < 0.01 vs. control), and 9.2 _+ 4.8% after 8 days, P < 0.01 vs. control, n = 3 different experiments) (Fig. 1). Also G H R H content was decreased after 3 and 8 days of estrogen administration (2.14 + 0.15 ng/mg protein in control animals, 1.74 + 0.18 ng/mg protein after 3 days of treatment (P < 0.05 vs. control), 1.19 _+ 0.25 ng/mg
8-ACTIN~
ss~
% Control < z
% Control
140
250-
120
"~ z 225-
z 100 i(_)
z 200~-
,~ & z "1-
**
60 40 20
**
0
&
150-
~: z
12s-
E
100-
T
*
75 E1
E3
E8
E1
E3
E8
Fig. 1. Hypothalamic p r e - p r o G H R H m R N A (left) and preproSS m R N A levels (right) after treatment with estrogens for ! day (El), 3 days (E3), 8 days (E8), or saline (control = C). Northern blot analysis. 50 ,ug (let1) and 20 ~ g (right) of total R N A from rat hypothalami were run in formaldehyde-agarose gels and blotted to a nylon membrane as described in the text. Blots were hybridized with the indicated 32p-labelled G H R H and SS c R N A probes. Only a band of approximately 750 bp was detected in the case of G H R H m R NA, and one of 650 bp in the case of SS m R N A . fl-actin m R N A hybridization is also shown. The results were corrected using the fl-actin as control probe. Data {mean + S . E M . ) were expressed as percentage change in relation to control values, n - 3 4 different experiments. *P < 0.05 vs. control, •*P < 0.01 vs. control.
protein after 8 days of treatment (P < 0.01 vs. control, n = 12-15 rats/group) (Fig. 2). In contrast, we have found increased levels of SS mRNA/fl-actin mRNA after 1 and 3 days of treatment (% control = 130 _+ 16% after 1 day (P < 0.05 vs. control), and 180 _+ 26% after 3 days (P < 0.01 vs. control), n - - 4 different experiments). Nevertheless, longer administrations of estrogens (8 days) returned SS m R N A content/fl-actin m R N A to control levels (% control -- 89 + 11%, n --4 different experiments) (Fig. 1). Somatostatin content did not show a significant increase until after 8 days of treatment (15.98 + 0.75 ng/mg protein in control rats and 19.42 _+ 1.35 ng/mg protein after 8 days of tretment, P < 0.05 vs. control, n -- 12-15 rats/ group) (Fig. 2). Our data show that both G H R H and SS gene expression in the rat hypothalamus are regulated by estrogens. Previous work regarding the effects of estrogens on hypothalamic G H R H gene expression has proved to be inconclusive. Although some authors have described an increase in hypothalamic G H R H content after 2 weeks
125
24 D
22
tn
20
'*"
18
O
2.25 *a
1.75~-r
T -*a,
1.50 .*-,
(3. 16 l E:
2.00 I
~
1.2050 O
12
0.75
10
0.50
C
E1
E3
E
cn E
--...,,.
EB
Fig. 2. Hypothalamic SS (open bars) and GHRH (hatched bars) content after treatment with estrogens for 1 day (El), 3 days (E3), 8 days (E8), or saline (control -- C). Data were expressed as ng/mg protein (mean_+ S.E.M.). n-- 12 15 rats/group. *P < 0.05 vs. control, *'P < 0.01 vs. control, "P < 0.05 vs. E1 and E3, a~p < 0.01 vs. El and E3.
of estrogen treatment of gonadectomized male rats [5], others have found no alteration in either G H R H content or G H R H m R N A levels after gonadectomy [2]. In the present report we describe a parallel decrease in hypothalamic G H R H content and G H R H m R N A levels, thus suggesting that estrogens have an inhibitory effect on hypothalamic G H R H gene expression. It is unclear whether this effect reflects changes in transcription rate or m R N A stabilization. Nevertheless, inhibition of both G H R H m R N A levels and G H R H content by estrogens could explain the fact that despite lower somatostatinergic tone G H pulse a m p l i t u d e ~ h i c h is mainly dependent on G H R H surges--is decreased in female rats. Although some workers have failed to detect any change in hypothalamic somatostatin content in male rats following gonadectomy and estrogen administration [5], others have found a decrease [3, 19] in hypothalamic somatostatin m R N A after gonadectomy that was reversed [19] or unaffected [3] by estrogen administration. Since in none of these studies both parameters, SS content and SS m R N A , were reported it is difficult to assess whether these differences were related to the period of time after gonadectomy, to the dose of estrogen administered, or to the sex of the animal used. In any event our data regarding the effects of estrogens on SS m R N A levels show a transient increase that returns to control levels after 8 days of treatment. The different time-course of estrogen-induced increase in SS m R N A levels and SS content reported here could be explained because m R N A availability could be considered as an index of protein biosynthesis (although it cannot be assumed that all SS m R N A produced is trans-
lated into the mature peptide). In contrast the peptide content reflects the ratio of synthesis to release, and therefore our data suggest a different time-course effect of estrogens on somatostatin synthesis and secretion. Finally, from our data it is not possible to conclude whether estrogen administration increases the transcription rate of the SS gene or induces m R N A stabilization. Nevertheless, since according to the GenBank genetic sequence data, no estrogen receptor consensus binding sequence seems to be present in the SS gene [19], it is likely that their effects could be mediated through other neurotransmitters or neuropeptides. In any case, our findings support the view that in addition to their pituitary effects [2, 6, 8, 16], estrogens are involved in the neuroregulation of G H secretion by acting at the hypothalamic level. In summary, we have found that estrogen administration produced opposite effects on hypothalamic SS and G H R H gene expression in the hypothalamus, which may explain to some extent the existence of a sexual dimorphism of G H secretion in the rat. This work was supported by the D G I C Y T (87/0488 and 90/0771). 1 Chirwgwin, J.M., Przybyla, A.E., McDonald, R.J. and Runer, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299. 2 Chomczymsky, P., Downs, T. and Frohman, L.A., Feedback regulation of growth hormone releasing hormone gene expression by GH in rat hypothalamus, Mol. Endocrinol., 2 (1988) 23(~241. 3 Chowen-Breed, J.A., Steiner, R.A. and Clifton, D.K., Sexual dimorphism and testosterone-dependent regulation of somatostatin gene expression in the periventricular nucleus of the rat brain, Endocrinology, 125 (1989) 357 362. 4 Di6guez, C., Page, M.D. and Scanlon, M.F., Growth hormone neuroregulation and its alterations in disease states, Clin. Endocrinol., 28 (1988) 109-143. 5 Gabriel, S.M., Millard, W.I., Koenig, J.I., Badger, T.M., Russell, W.E., Maiter, D.M. and Martin, J.B., Sexual and developmental differences in peptides regulating growth hormone secretion in the rat, Neuroendocrinology, 50 (1989) 299 307. 6 Ho, K.Y., Leong, D.A., Sinha, Y.N., Johnson, M.L., Evans, W.S., Thorner, M.O., Sex related differences in GH secretion in rat using reverse haemolytic plaque assay, Am. J. Physiol., 250 (1986) E650654. 7 Jansson, J.-O., Edberg, S., lsaksson, O.P.G. and Eden, S., Influence of gonadal steroids on age- and sex-related secretory patterns of growth hormone in the rat, Endocrinology, 114 (1984) 1287 1294. 8 Krieg, R.J., Thorner, M.O. and Evans, W.S., Sex differences in beta-adrenergic stimulation of growth hormone secretion in vitro, Endocrinology, 119 (1986) 1339 1342. 9 Lewis, B.M., Dieguez, C., Lewis, M., Hall, R. and Scanlon, M.F., Hypothalamic D2 receptors mediate the preferential release of somatostatin-28 in response to dopaminergic stimulation, Endocrinology, 119 (1986) 1712-1717. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265 275.
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16 Simard, J., Hubert, J.F., Hosseinzadeh, T. and Labrie, F., Stimulation of growth hormone release and synthesis by estrogens in rat anterior pituitary cells in culture, Endocrinology, 119 (1986) 20042011. 17 Sawchenko, P.E., Swanson, L.W., Rivier, J. and Vale, W.W., The distribution of growth hormone releasing factor inmunoreactivity in the central nervous system of the rat: an inmunohistochemical study using antisera directed against rat hypothalamic GRF, J. Comp. Neurol., 237 (1985):100-115. 18 Tannenbaum, G.S. and Martin, J.B., Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98 (1976) 562 570. 19 Werner, H., Koch, Y., Baldino, F. and Gozes, I., Steroid regulation of somatostatin mRNA in the rat hypothalamus, J. Biol. Chem., 263 (1988) 7666-7671.
