Masculinization of growth hormone (GH) secretory pattern by dihydrotestosterone is associated with augmentation of hypothalamic somatostatin and GH-releasing hormone mRNA levels in ovariectomized adult rats

Peptides.Vol. 13, pp. 475--481, 1992

0196o9781/92$5.00 + .00 Copyright© 1992PergamonPressLtd.

Printedin the USA.

Masculinization of Growth Hormone (GH) Secretory Pattem by Dihydrotestosterone Is Associated With Augmentation of Hypothalamic Somatostatin and GH-Releasing Hormone mRNA Levels in Ovariectomized Adult Rats O S A M U H A S E G A W A , H I T O S H I S U G I H A R A , S H I R O M I N A M I A N D ICHIJI W A K A B A Y A S H I 1

Department of Medicine, Nippon Medical School, Sendagi 1-1-5, Bunkyoku, Tokyo 113, Japan Received 6 N o v e m b e r 1991 HASEGAWA, O., H. SUGIHARA, S. MINAMI AND I. WAKABAYASHI.Masculinization ofgrowth hormone (GH) secretory pattern by dihydrotestosteroneis associated with augmentation of hypothalamic somatostatin and GH-releasinghormone mRNA levels in ovariectomizedadult rats. PEPTIDES 13(3) 475-481, 1992.--The role of androgen in the sexual dimorphism in hypothalamic growth hormone (GH)-releasinghormone (GHRH) and somatostatin (SS) gene expression was examined in rats. In the first study, the SS and GHRH mRNA levels were measured in both male and female rats at 4, 6, 8, and 10 weeks of age. A significantsex-related difference in the SS and GHRH mRNA levelswas observed after 8 weeks of age, when sexual maturation is fully attained. Male rats had higher SS and GHRH mRNA levelsthan the female rats. In the second study, adult ovariectornized rats received daily injection of dihydrotestosterone (DHT), nonaromatizable testosterone, at a dose of 2 rag/rat for 21 days. The DHT treatment maseulinizedthe GH secretorypattern, whichwas indistinguishablefrom that of intact male rats, and simultaneously augmented the SS and GHRH mRNA levels. The DHT treatment of ovariectomized rats after hypophyseetomy significantly raised the level of SS mRNA, but not that of GHRH mRNA compared to the control animals. These findings suggest that the activation of the SS gene expressionthrough androgen receptor plays an important role in the maintenance of sexual dimorphism in GH secretion in rats. Sexual dimorphism Gene expression

Secretorypattern

Growth hormone

IN rats, the growth hormone (GH) secretory pattern is related to age and sex (1,11,17,18,28). The adult GH secretory pattern is established with the onset of puberty. In adult male rats, GH is secreted in episodic bursts at 2.5-3-h intervals, while in female rats, GH is secreted with more frequent bursts. In addition, the GH levels between bursts are lower in male rats than in female rats. In prepuberal male rats, GH secretory bursts occur at irregular intervals and the baseline GH levels are between those of adult male and female rats. The principal control of GH secretion is accomplished by interactions between two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SS) (12,18,29). A significant sex-related difference is observed in the gene expression of hypothalamic GHRH and SS (2,3,7,24,38,42,43), and hypothalamic contents of SS and G H R H in rats (13,14,19,23). Several observations indicate that the sex-related difference in neuroen-

Requests for reprints should be addressed to Dr. Ichiji Wakabayashi.

475

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docrine control of GH secretion depends on gonadal steroids, particularly androgens. For instance, neonatal or prepubertal gonadectomy of male rats causes a reduction in GH pulse height to levels observed in normal female rats (17), and the administration of testosterone to ovariectomized adult rats masculinizes the GH secretory pattern (1,25). The GH secretory pattern of androgen-resistant (testieular feminized) rats resembled that of female rats (28). Testosterone or dihydrotestosterone (DHT) treatment of castrated rats augmented the SS mRNA level (2,3,7,43) or the GHRH mRNA level (3,42). Finally, the nuclear androgen receptors are present in both the arcuate and periventricular nuclei where GHRH and SS neurons reside, respectively (33,34), and male rats have significantlymore androgen receptors than female rats in the periventricular nucleus (34). We examined whether the administration of DHT, nonaromatizable testosterone, to female rats after ovariectomy mas-

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FIG. 1. The SS, GHRH, and GH mRNA levels of male and female rats at different weeks of age. mRNA levels were quantified by dot blot hybridization of total RNA and scanning densitometry of the autoradiogram as described in the Method section. Each column indicates the mean of 8 rats and the vertical bar represents the SEM. Open column: male rats, closed column: female rats. SS mRNA: F(7, 56) = 20.98; GHRH mRNA: F(7, 56) = 38.93; GH mRNA: F(7, 56) = 78.74. a: p < 0.05 as compared to the mean of female rats; b: p < 0.05 as compared to the mean of male rats at 4 weeks of age; c: p < 0.05 as compared to the mean of female rats at 4 weeks of age.

culinizes both the G H secretory pattern and the gene expression of SS and GHRH. Dihydrot~osterone was used because estrogen is shown to increase the androgen receptor in the medial preoptic area (15). We also made an observation after hypophysectomy in ovadectomized rats, because the expression of hypothalamic G H R H and SS is influenced by G H (6,10,21,31,32). METHOD

Animals Neonatal pups delivered by pregnant Wistar rats were housed together with their mothers. At 28 days of age, pups were weaned, sexed, and caged in groups. They were maintained in air-conditioned animal quarters with a lighting schedule of 0800-2000

h and fed food and water ad lib. They were killed by decapitation at 4, 6, 8, and 10 weeks of age. The brain and pituitary gland were removed. Hypothalamic fragments defined by the anterior margin of the optic chiasm, the anterior margin of mammillary body, the lateral hypothalamic sulci, and a depth of 3 mm from the attachment of the stalk were dissected. The pituitary gland was separated into the anterior and neurointermediate lobes. Only anterior pituitary tissue was used in the present study. Four-week-old female Wistar rats were either ovariectomized or sham operated under ether anesthesia. Two weeks after the operation, DHT (Sigma Chemical Co., St. Louis, MO, 2 mg/ rat/day, SC) or sesame oil was given for 21 days. We chose rather large dose of DHT, because in our previous study, daily administration of testosterone propionate to ovariectomized rats at a dose of 2 mg, but not 1 mg, altered the GH secretory pattern indistinguishable from that of intact male rats (1). All rats were provided with indwelling right atrial cannula under ether anesthesia for blood sampling. Four days after the cannulation, serial blood samples were drawn every 10 min for 9 h using an automatic blood microsampling system similar to that described by Clark et al. (9). This collection method avoided major blood loss and did not require transfusions of donor blood to maintain blood volume (9). Growth hormone concentrations were measured by double-antibody radioimmunoassay using materials supplied by NIDDK, NIH. All values are expressed as ng/ml in terms of the NIDDK reference preparation rat GH-RP-1. To identify the GH pulses and trough periods, data obtained from each rat were subjected to the Pulsar computer program developed and described by Merriam and Wachter (26). A group of rats was ovariectomized at 4 weeks of age and was hypophysectomized at 6 weeks of age. They were given daily SC injections of sodium 1-thyroxine (Sigma Chemical Co., St. Louis, MO, 2 #g/100 g b.wt.) and cortisone acetate (Sigma Chemical Co., St. Louis, MO, 50 izg/100 g b.wt.). They also received daily SC injections of DHT (2 mg) or sesame oil for 21 days. At the end of DHT treatment, rats were killed by decapitation. Hypothalamic and pituitary tissue were removed as described above.

Isolation of RNA Tissues were immediately frozen in liquid nitrogen and stored at - 8 0 ° C until RNA extraction. Total RNA from the anterior pituitary gland and hypothalamic tissue fragment was extracted by the single-step acid guanidinium thioeyanate-phenol-chloroform method of Chomczynski and Sacchi (5).

Riboprobe Synthesis and Labeling The rat GH, rat GHRH, and rat SS eDNA probes were kindly provided by Dr. R. M. Evans, Dr. K. E. Mayo, and Dr. M. R. Montminy, respectively. The rat GH eDNA probe was subcloned into pBluescript SK-II (+). The rat G H R H and rat SS eDNA probes were subcloned into pBluescript KS-II (+). These plasmids were used to produce respective antisense RNA probes. The plasmids that subclonod rat G H or rat G H R H eDNA were linearized with Eco RI. The plasmid subcloned rat SS eDNA was linearized with Hind III. The lineafizcd plasmids were extracted with phenol-chloroform, precipitated with ethanol, and solubilized at a concentration of 1 mg/ml in 1 × TE (I0 mM Tris-HC1, pH 7.4, and 1 mM EDTA). Radioactive ¢RNA copies were synthesized from the linearized plasmids with u p using Sp6/T7 transcription kit (Boehringer Mannbeim Yamanouchi Co. Ltd., Tokyo, Japan). The synthc~i_zed cRNA copies were precipitated with ethanol and solubilized in 1 × TE.

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FIG. 2. Effect of DHT on SS, GHRH, and GH mRNA levels among ovariectomized rats. Each column indicates the percent of the levels observed in sham-operated control animals and the vertical bar indicates SEM. Eight rats were used in each group. Open column: sham-operated female rats, dotted column: ovariectomized rats, dosed column: DHT-treated ovariectomized rats. SS mRNA: F(2, 21) = 23.18; GHRH mRNA: F(2, 21) = 7.83; GH mRNA: F(2, 21) = I 1.36. *p < 0.05.

Dot Blot Analysis The GHRH, SS, and GH m R N A levels were analyzed by dot blot hybridization. This was chosen since the mRNA of GHRH, SS, or GH was detected as a single band by Northern blot analysis under the same hybridizing and washing conditions. The RNA from hypothalamic or pituitary tissue was dissolved in 50% deionized formamide, 6% formaldehyde, l X MOPS (20 mM 3-[N-morpholino]-propane-sulfonic acid, 5 mM sodium acetate, pH 7.0, l mM EDTA), and incubated for 5 min at 65°C to denature RNA. The solution was chilled on ice and cold 20 × SSC was added so that the final concentration became 5 × SSC. Aliquots of hypothalamic RNA (1, 2, 4/~g) or pituitary RNA (0.125, 0.25, 0.5, 1 #g) in a volume of 50 #l were applied on the membrane (Hybond-N+, Amersham, Tokyo, Japan) with the SRC 96 Minifold I dot blotter (Schleicher & Schuell, Dassel, Germany). The RNA was fixed to the membrane with 50 mM NaOH and the membrane was rinsed briefly with 2 × SSC. The membrane was prehybridized at 65°C for 2 h in the solution containing 50% deionized formamide, 5 X SSC, l X PE (50 mM Tris-HC1, pH 7.5, 0.1% sodium pyrophosphate, 1% SDS, 0.2% polyvinyl pyrrolidone, 0.2% ficoll, 5 m M EDTA, 0.2% BSA), and 150 #g/ml denatured salmon sperm DNA. Hybridization was performed at the same temperature overnight in the same solution containing 32P-labeled GHRH, SS, or G H cRNA. The membrane was washed for 15 min twice in 2 × SSC, 0.1% SDS at 70°C and for 15 rain twice in 0.1 X SSC, 0.1% SDS at the same temperature. The membranes were exposed to Kodak XA R5 film at - 8 0 ° C for 10-30 min in G H m R N A analysis and 1-4 days in G H R H or SS m R N A analysis.

Data Analysis The results of the autoradiography were quantified by a scanning densitometer and expressed as arbitrary units or percent

of control. Each sample was subjected to dot blot hybridization at three or four doses. The blot corresponding to the same amount of total RNA was used when comparisons were made among groups. Statistical analysis was performed by analysis of variance followed by Duncan's multiple range test or by Student's t-test to compare means between two groups. RESULTS In male rats, hypothalamic SS mRNA levels at 8 and 10 weeks of age were significantly higher than those at 4 weeks of age, and in female rats, the SS mRNA levels at 8 weeks of age were significantly higher than those at 4 weeks of age (Fig. 1). The hypothalamic SS mRNA level at 4 and 6 weeks of age did not differ with sex, but was significantly higher in male rats than female animals at 8 and l0 weeks of age. In male rats, G H R H mRNA levels at 8 and l0 weeks of age were significantly higher than those at 4 weeks of age, and in female rats, the G H R H mRNA levels at 6, 8, and l0 weeks of age were significantly higher than those at 4 weeks of age (Fig. 1). Hypothalamic G H R H mRNA levels at 4 and 6 weeks of age did not differ with sex, while they were significantly higher in male rats than in female rats at 8 and 10 weeks of age. In male rats, pituitary G H mRNA levels were significantly higher at 6, 8, and 10 weeks of age than those at 4 weeks of age (Fig. 1). In female rats, GH mRNA levels were significantly higher at 8 weeks of age than at 4 weeks of age. The GH m R N A level did not differ with sex at 4 weeks of age, but was significantly higher in male rats than in female rats at 6, 8, and l0 weeks of age. Ovariectomy did not alter the SS and G H R H m R N A levels significantly, while the procedure significantly increased GH mRNA levels (Fig. 2). Daily administration of DHT significantly increased the SS and G H R H mRNA levels in ovariectomized rats, but the treatment did not alter the G H m R N A levels in

478

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ANDROGEN AND G H SECRETORY PATTERN

479

ovariectomized rats. Throughout this study, the levels of SS, GHRH, and G H m R N A in ovariectomized rats or ovariectomized DHT-treated rats were compared with those of random cycling sham-operated rats. Others showed that G H R H mRNA concentrations were not affected by the phase of estrous cycle in rats (24), and GH secretory pattern did not show significant alterations by the phase of estrous cycle in rats (8). The variabilities of SS and GH m R N A levels of random cycling intact female rats did not appear to differ from those of intact male rats as judged by values of SEM. Based on these data, we assumed that SS, GHRH, and G H mRNA levels would not be affected significantly by estrous cycle in rats. The baseline G H level of ovariectomized rats was significantly lower than that of shamoperated female rats, but pulsatility of G H secretion did not differ between the two groups of rats (Fig. 3, Table 1). In DHTtreated ovariectomized rats, the baseline G H levels and frequency of pulsatile GH secretion decreased significantly and the amplitude of GH secretion increased significantly as compared to those of sham-operated female rats. The pattern of pulsatile GH secretion was indistinguishable from that of intact male rats (Table 1). After hypophysectomy in ovariectomized rats, hypothalamic SS mRNA levels were significantly higher in DHTtreated rats as compared to control animals, while the treatment did not significantly alter the G H R H m R N A levels (Fig. 4). DISCUSSION We showed previously that testosterone administration to ovariectomized rats masculinized the G H secretory pattern in a dose-dependent manner (1). This phenomenon was accompanied by the increased inhibitory tone provided by SS and the association appeared to be casually related. The present study extends our previous study and shows that masculinization of the GH secretory pattern by DHT among ovariectomized rats accompanies significant augmentations of the hypothalamic mRNA levels of both SS and GHRH. The observed changes in the mRNA levels of G H R H and SS caused by DHT treatment could be secondary to the alteration of G H secretory pattern and/or the amount of G H secreted (6,10,21,31,32). To answer this question, we examined the effect of DHT on the hypothalamic SS and G H R H m R N A levels after hypophysectomy in ovariectomized rats. Our findings were consistent with the interpretation that DHT acts directly on the hypothalamus and increases the SS mRNA level through the activation of androgen receptors. This is also supported by the studies of others who observed the stimulatory effect of testosterone or DHT on SS gene expression in the presence of the pituitary (2,3,7,43). Although the gene expression of G H R H has also been suggested to be stimulated by testosterone through androgen receptors

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FIG. 4. Effect of DHT on SS and GHRH mRNA levels after hypophyscctomy in ovaricctomized rats. Each column indicates the percent of the levels observed in hypophysectomized-ovaricctomized control animals and the vertical bar indicates SEM. Eight rats were used in each group. Open column: hypophysectomized-ovadcctomized rats, closed column: DHT-treated hypophysectomized-ovadcctomized rats. SS mRNA: F(1, 14) = 93.71; GHRH mRNA: F(1, 14) = 2.98. *p < 0.05.

(3,42), our findings appear to suggest that the augmentation of the G H R H mRNA level caused by DHT is related to the altered GH secretory pattern. However, this interpretation requires precautions. The G H R H m R N A levels increase 3-7-fold following hypophysectomy (6,10,21). Under such conditions, the stimulatory effect of DHT on G H R H gene expression may be obscured. Therefore, we do not deny the possibility that DHT augments the G H R H mRNA level by acting directly on the hypothalamus. One wonders whether gender-related dimorphism in G H secretion is attained either by organizational or activational effect of androgen on SS and G H R H gene expression. Janssen et al. consider that testicular activity during early life is essential for the high GH pulses in adult male rats (17), while others report that neonatal testosterone treatment did not influence the secretory pattern of G H in female rats (27). The study of Argente et al. has shown that sexual dimorphism in the SS m R N A level

TABLE 1 COMPARISON OF GH SECRETORY PATTERN PARAMETERS USING PULSAR ANALYSIS

Group

Interpcak Interval(rain)

BaselineLevels(ng/ml)

Peak Amplitude(ng/ml)

Sham-operated female rats [8] Ovariectomized rats [8] DHT-trcated ovariectomized rats [8] Intact male rats [8]

77.1 _+6.0 72.8 _+4.7 155.0 _+8.4"t 161.3 _+7.2"t

56.8 _+2.4 19.7 _+ 1.4" 6.7 _+0.3*t 8.3 + 0.5*t

150.3 + 7.2 217.0 + 13.3" 312.2 _+ 18.9"t 304.5 _+22.6"t

Values arc mean _+SEM; numbers in brackets indicate the sample size. Interpeak interval: F(3, 134) = 50.39, baseline levels: F(3, 529) = 258.9, peak amplitude: F(3, 156) = 27.13. * p < 0.05, compared to sham-operated rats. t P < 0.05, compared to ovaricctomized rats.

480

HASEGAWA ET AL.

is evident as early as l0 days of age and persists throughout development, whereas the G H R H gene expression is clearly sexually dimorphic only in the late neonate and adult (3). It is expected that male rats are exposed to high levels of circulating testosterone at l0 days of age. They consider that in male rats, androgen plays both organizational and activational roles to influence SS gene expression, while the sex steroid exerts a predominantly activational effect on G H R H gene expression (3). By contrast, Maiter et al. report that neither gonadectomy of male rats or testosterone administration to ovariectomized rats alters the G H R H mRNA level (24). We observed that sexual dimorphism in SS and G H R H levels became manifest after 8 weeks of age, when sexual maturation of rats is fully attained. Coupled with the data, DHT augmented hypothalamic SS and G H R H mRNA levels in ovariectomized rats, and masculinized the G H secretory pattern. Therefore, we are inclined to believe that circulating testosterone plays important roles in the maintenance of sex-related differences in SS and G H R H gene expression. Growth hormone-releasing hormone is essential for the stimulation of GH secretion. Thus spontaneous G H secretion and GH stimulation by morphine are shown to require the presence of G H R H (39,41). Somatostatin may also play an important role in the dynamic regulation of GH secretion. For instance, G H R H secretion into hypophyseal portal vessels occurs only under the reduced SS secretion (30), and the removal of SS influence either by passive immunization or hypothalamic deafferentation abolishes the pulsatile GH release and morphineinduced G H release (12,20). Morphologically, SS-containing nerve terminals are present in GHRH-containing neuronal cells (22). In addition, a recent study suggested that SS exerts influences on G H R H gene expression in rats (21). Taken together, SS may exert important influences on G H R H secretion and synthesis, and thereby play a central role in the regulation of GH secretion. The effects of gonadal steroids on somatotroph function have been extensively studied. The apparent effect of gonadal steroids

on parameters of pituitary GH synthesis and release may be mediated indirectly through alterations in the pattern of hypothalamic G H R H and SS release to which the pituitaries have been exposed, because the evidence for a direct effect of gonadal steroids on the pituitary is limited and conflicting (35,37,40). Our finding that the pituitary GH mRNA level ofovariectomized rats is significantly higher than that of sham-operated female rats is consistent with the previous findings that GH synthesis (4), pituitary GH content (36), and G H responses to G H R H (37) are enhanced in female rats after ovariectomy, and that estradiot is inhibitory on the secretory function ofsomatotrophs (16). Estradiol reduces the proportion of somatotrophs (16). Therefore, the relative number of somatotrophs and GH mRNA level of individual somatotrophs are both significantly greater in male rats than female rats after the onset of puberty. This will explain, in part, the marked sex-related difference in GH mRNA levels, as well as the finding of a significant sex-related difference in the pituitary GH mRNA levels at 6 weeks of age, without accompanying significant sex-related alterations of hypothalamic G H R H and SS mRNA levels. In conclusion, the sex-related difference in hypothalamic G H R H and SS mRNA levels in rats occurs after 8 weeks of age, when the rats have reached full sexual maturation. Dihydrotestosterone treatment of ovariectomized rats increases hypothalamic SS and G H R H gene expression, and masculinizes the GH secretory pattern, which is indistinguishable from that of adult male rats. Therefore, we consider that the activation of gene expression of SS and G H R H by androgen plays a pertinent role in the gender-related difference in the growth of rats. ACKNOWLEDGEMENTS We thank Mrs. Sumiyo lzaki and Mrs. Masayo Ashizawa for their excellent technical assistance, and the NIDDK for the materials used to assay GH. We are grateful to Dr. Kelly E. Mayo, Dr. Marc R. Montminy, and Dr. Ronald M. Evans for their generous gifts of the GHRH eDNA, SS cDNA, and GH eDNA. This work was supported in part by a grantin-aide from the Japanese Ministry of Education, Science and Culture (02771818 to H. Sugihara).

REFERENCES

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8. Clark, R. G.; Carlsson, L. M. S.; Robinson, I. C. A. F. Growth hormone secretory profiles in conscious female rats. J. Endoerinol. 114:399-407; 1987. 9. Clark, R. G.; Chambers, G.; Lewin, J.; Robinson, I. C. A. F. Automated repetitive microsampiing of blood: ~ hormone profiles in conscious male rats. J. Endoerinol. I ! 1:27-35; 1986. 10. De Gennam Colonna, V.; Cattaneo, E.; Cocehi, D.; MUller, E. E.; Maggi, A. Growth hormone regulation of growth hormone.releasing hormone gene expression. Peptides 9:985-988; 1988. I 1. Eden, S. Age- and sex-relateddifferencesin episodic growth hormone secretion in the rat. Endocrinology 105:555-560; 1979. 12. Frohman, L. A.; Downs, T. R.; Katakami, H.; Jansson, J-O. The interaction of growth hormone-releasing hormone and somatostatin in the regulation of growth hormone secretion. In: Isaksson, O.; Binder, C.; Hall, K.; HSkfelt, B., ¢ds. Growth hormone: Basic and clinical aspects. Proceedings of the 1st Nordisk Insulin Symposium. Amsterdam: Exeerpta Medica; 1987:63-77. 13. Gabriel, S. M.; Millard, W. J.; Koeni8, J. I,; Badger, T. M.; Russell, W. E.; Maiter, D. M.; Martin, J. B. Sexual and developmental differences in peptides r ~ l a t i n g growth hormone secretion in the rat. Neuroendocrinology 50:299-307; 1989. 14. Gross, D. S. Role ofsomalostatin in the modulation of hypophysial growth hormone production by gonadal steroids. Am. J. Anat. 158: 507-519; 1980. 15. Handa, R. J.; Roselli, C. E.; Horton, L; Resko, J. A. The quantitative distribution of cytosolic androgen receptors in microdis,~'ted areas

A N D R O G E N A N D G H SECRETORY P A T T E R N

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25. 26. 27.

28. 29.

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