Testosterone and GHRH gene expression in adult and old rats

Peptides,Vol. 12, pp. 309-312. ©Pergamon Press plc, 1991. Printed in the U.S.A.

0196-9781/91 $3.00 + .00

Testosterone and GHRH Gene Expression in Adult and Old Rats 1 VITO DE GENNARO COLONNA, DANIELA COCCHI, LUIS LIMA,t A D R I A N A M A G G I * A N D E U G E N I O E. M O L L E R 2

Department of Pharmacology, Chemotherapy and Toxicology, University of Milan, 20129 Milan, Italy *Institute of Pharmacological Sciences, University of Milan, 20133 Milan, Italy "/'Department of Physiology, School of Medicine, University of Santiago de Compostela Santiago de Compostela, Spain R e c e i v e d 9 M a y 1990

DE GENNARO COLONNA, V., D. COCCHI, L. LIMA, A. MAGGI AND E. E. MULLER. Testosteroneand GHRHgene expression in adult and old rats. PEPTIDES 12(2) 309-312, 1991.--The effect of castration and testosterone replacement on GHRH gene expression was evaluated in adult male rats. Castration for 21 days did not affect GHRH mRNA levels, and also ineffective in this context was acute or chronic administration of testosterone to castrated rats. Hypothalamic GHRH mRNA was significantly reduced in aged male rats, but restoration of plasma testosterone concentrations via implanted capsules did not modify the low levels of GHRH mRNA. All in all, these findings support the notion that in adult and aged male rats GHRH-producing structures are insensitive to androgens. Testosterone

Growth hormone-releasing factor

Gonadectomy

THE existence of a positive relationship between androgens and growth hormone (GH) secretion is suggested by a wealth of experimental evidence. In humans, an increased secretion of GH occurs at puberty (23) and in pubertal males chronic treatment with testosterone increases the 24-h mean concentrations of GH (17). In rats, the unique male pattern of GH secretion characterized by high-amplitude peaks that appear at regular intervals is abrogated by neonatal orchidectomy (14). A sex difference in the hypothalamic content of GH-releasing hormone (GHRH) has been described in the rat, with male rats having higher levels of the peptide than female rats (5,15). This sex difference, which can contribute to establish different modes of GH secretion, develops at puberty between days 25 and 35 of age. Hypothalamic levels of GHRH are instead moderately influenced by androgens in adulthood (15). Evaluation of absolute levels of a neurohormone barely reflects the functional activity of a given neuronal system (8). We decided, therefore, in this study to evaluate in adult animals the role of androgens on GH secretion, by studying the effect of their administration or withdrawal on the expression of hypothalamic GHRH. It is known that old male rats have a decreased GHRH-like immunoreactivity content and GHRH mRNA in the hypothalamus (7) and, in addition, have hypogonadism (21). We thought,

Aging

therefore, it would be of interest to evaluate in these rats a testosterone replacement therapy on hypothalamic GHRH mRNA. METHOD Adult (3 months) and old (20-24 months) male Sprague-Dawley rats (Charles River, Calco, Italy) were used in this study. They were housed under controlled conditions ( 2 2 - 2 ° C , 65% humidity and artificial light from 0600-2000 h) with standard pellet food and water available ad lib. Adult rats underwent bilateral orchidectomy or sham operation and starting from the day of operation some of them were implanted with testosterone-filled Silastic capsules (0.062 in i.d.; 0.125 in o.d., length 1.5 cm) capable of decreasing to normal the high plasma luteinizing hormone (LH) levels of gonadectomized animals (authors' unpublished results). In addition, a group of adult castrated and sham-operated (sham-op) rats underwent single subcutaneous injection of 150 Ixg testosterone propionate (TP), 18 h before sacrifice. Old rats were sham implanted or received the same testosterone capsules used for adult rats. Twenty-one days after orchidectomy or testosterone implantation, starting at 0900 and in random order to avoid diurnal variations, rats were killed by decapitation and the hypothalami were removed for GHRH mRNA determination. A 21-day period was chosen since it corresponds to the time of testosterone discharge

1Supported by the CNR Target Project on Biotechnology and Bioinstrumentation. 2Requests for reprints should be addressed to Prof. Eugenio E. MUller, Department of Pharmacology, University of Milan, Via Vanvitelli, 32-20129 Milan, Italy.

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from a single implanted capsule.

Sham-op

Cx

Sham-op T ac

Cx T ac

GHRH mRNA Immediately after brain removal, the hypothalami were carefully dissected as previously described (6). They were collected in pools of two samples (about 70 mg of tissue), immediately frozen on dry ice, and stored at - 7 0 ° C until used. Total RNA was isolated from pooled hypothalamic tissue by the guanidium thiocyanate/CsCl method (3,9). Poly(A) + RNA was then purified by chromatography on oligo (dT)-cellulose (Pharmacia type 7; Pharmacia, Uppsala, Sweden) (1). Ultraviolet absorbance analysis was utilized for RNA quantitation. Starting from 70 mg of frozen tissue, routinely 30-35 p~g of total RNA and 1-1.5 p,g of poly(A) + RNA were obtained. Poly(A) + RNA samples (1 ixg per sample) were spotted onto nitrocellulose sheets (BA 85, 0.45 p~m; Schleicher & Schnell, Dassel, West Germany) (prewetted with 5 x SSC) using a slot blot apparatus (Minifold II; Schleicher & Schnell). The filters were then baked in vacuum at 80°C for 2 h and hybridized with the 32p-labeled probe. To determine the range of linearity of the autoradiographical signal, known dilutions (8, 1.6, 0.32, 0.064, 0.012 ng) of pBr 322 were used and processed similarly as the RNA samples. Control of the amount of the RNA blotted was performed by reprobing the slot blots with oligo (dA), oligo (dT) 12-18 (Pharmacia) elongated via terminal transferase with a_32p dTTP (Amersham, Little Chalfont, UK). A plasmid containing the rat GHRH cDNA sequence prGRF 2 (18) was utilized as a probe. The prGRF 2 was labeled by multiprime DNA labeling system (Amersham, UK) with ot-32p dCTP to a specific activity of 2 x 108 dpm/txg cDNA. Hybridization conditions have been previously described (8). Representative autoradiographical signals were obtained using as little as 0.25 txg poly(A) ÷ RNA. As negative hybridization control, poly(A) ÷ RNA was also isolated from a brain area (cerebellum) lacking GHRH-producing neurons. Hybridization of slot blots from cerebellar poly(A) + RNA with the prGRF 2 produced, as expected, no autoradiographical signal. To test the size of the mRNA hybridized with the prGRF 2, a Northern analysis was performed, concomitantly to the slot blot hybridization. Poly(A) + RNA samples (1 Ixg per sample) isolated from hypothalamic tissue from each experimental group were electrophoresed on 1.2% formaldehyde-agarose gel, blotted onto a nitrocellulose sheet and probed with the 32p-labeled prGRF 2. A unique mRNA species of approximately 750 bases was detected after hybridization, a result consistent with the reported size of the mature GHRH mRNA (11,20). For quantitative measurements of slot blots, autoradiograms were subjected to densitometry with an LKB Ultroscan XL Laser Densitometer. The individual densitometric values were averaged for each experimental group and expressed as arbitrary densitometric units (ADU). Testosterone Assay At sacrifice blood was collected from the different experimental groups. After centrifugation plasma was separated and frozen until testosterone assay. Testosterone levels were determined by a solid phase radioimmunoassay using reagents provided by Zer (Jerusalem, Israel). The sensitivity of the assay is 0.2 ng/ml. The intraassay variability is less than 10%. In order to avoid interassay variability, all the samples were included in the same RIA. RESULTS

Hypothalamic GHRH mRNA was similar in sham-op and castrated adult rats, 21 days after surgery. In these rats, neither acute

Sham-op T chr

a

Q

Cx T chr

Old

t

g

Old T chr

FIG. 1. Slot blot analysis of hypothalamic GHRH mRNA levels in shamoperated (sham-op) or castrated (Cx) adult (3 months) male rats or in old (20-24 months) male rats, treated or not with testosterone acutely (T ac) or chronically (T chr). Each slot represents poly(A) + RNA (1 gg) from two pooled hypothalamic samples.

treatment with testosterone propionate nor chronic implantation of testosterone-filled capsules modified hypothalamic levels of GHRH mRNA. A significant reduction of GHRH mRNA was present in the hypothalamus of old rats. In these rats, a 21-day period of chronic implantation of testosterone did not modify hypothalamic levels of GHRH mRNA (Table 1, Fig. 11. Plasma levels of testosterone were undetectable in gonadectomized rats. Three-week implantation with testosterone restored plasma testosterone of castrated rats to levels not different from those of sham-op adult rats (Fig. 2). Acute administration of testosterone increased the levels of the hormone in plasma of adult castrated rats, although they were still significantly lower than those of sham-op rats. Either acute or chronic testosterone administration to sham-op adult rats did not induce any significant variation of plasma levels of the hormone (Fig. 2). Testosterone plasma levels were significantly lower in old than in adult rats but were restored to normal by three-week implantation of testosterone (Fig. 2). DISCUSSION

Many studies have clearly demonstrated a role for sex steroids in modulating GH secretion in humans and rats (see the Introduction). In the former, puberty is characterized by an increased se-

TABLE 1 HYPOTHALAMIC GHRH mRNA LEVELS EXPRESSED AS ARBITRARY DENSITOMETRIC UNITS IN SHAM-OPERATED (SHAM-OP) OR CASTRATED (Cxl ADULT (3 MONTHS) MALE RATS AND IN OLD (20-24 MONTHS) MALE RATS, TREATED OR NOT WITH TESTOSTERONE ACUTELY (T ac) OR CHRONICALLY (T chr)

Sham-op Cx Old

Control

T ac

T chr

4.36 -+ 0.30 4.32 -+ 0.18 2.20 ± 0.21"

4.18 + 0.18 4.24 ± 0.23 ND

4.24 ~ 0.19 4.20 -+ 0.28 2.24 ± 0.26*

Each point is the mean and SEM of 5 determinations. *p<0.01 vs, sham-op and Cx rats (treated or not with testosterone). ND = not determined.

TESTOSTERONE AND GHRH EXPRESSION

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Testosterone ng/rnl

3

i i i!i ! *

.....

Sharn-o

Cx

Cx T BC

4

Cx Sham-opSham-op Old T chr T ac T chr

01d T chr

FIG. 2. Plasma testosterone concentrations in sham-operated (sham-op) or castrated (Cx) adult (3 months) male rats or old (20-24 months) male rats, treated or not with testosterone acutely (T ac) or chronically (T chr). Each point is the mean and SEM of 9-10 determinations. *p<0.01 vs. adult sham-op rats. cretion of GH (23), and in peripubertal males chronic administration of testosterone induces a higher GH pulsatile release (17). In the rat, GH secretion is sexually dimorphic; both the total GH content (2) and the rate of GH synthesis (12) are greater in pituitaries from male than female rats. Moreover, GHRH-induced GH release is greater from cells of male than female rats both in vivo (24) and in vitro (19). The finding that the male rat hypothalamus contains more GHRH than the female one (5,15) suggests an action of gonadal steroids at this site in dictating a sexually-dimorphic pattern of GH secretion. Interestingly, the typical masculine pattern of GH secretion (high pulses and very low trough levels) is abrogated by neonatal orchidectomy (13,14). With the reservation in mind that androgens may affect GHRH not at the transcriptional but at translation or posttranslation level, as suggested for GH-GHRH (16), the present results bespeak the

ineffectiveness of gonadectomy and/or testosterone replacement to modify GHRH gene expression. It is evident, however, that gonadal steroids may modulate the secretion of GH also during adulthood. In fact, postpubertal administration of testosterone reverses the expression of the GH secretory pattern of female rats to the masculine pattern (13,14). Such effect, however, would be mediated by an action of gonadal steroids on other hypothalamic factors, i.e., somatostatin (SS). For instance, castration reduces the amount of immunoreactive SS-LI and of SS mRNA in the median eminence of male rats, effects that are counteracted by testosterone (4,10). Conversely, in female rats ovariectomy increases the amount of immunoreactive SS-LI in the median eminence and concomitantly reduces SS mRNA levels, and these effects are reversed by estradiol (25). In all, these findings indicate that testosterone is capable of augmenting SS activity in the hypothalamus, likely acting on androgen receptors located in this area (18). In view of androgen inability to affect hypothalamic GHRH function in postpubertal rats (this study), androgen modulation of somatotrophic function would likely occur in the hypothalamus via SS, whose role in maintaining pulsatile GH release is crucial (22). Though aged rats have a lower content of hypothalamic GHRH-LI and GHRH mRNA (7), our present data would rule out the possibility that the age-related hypogonadism may contribute to this alteration. In fact, replacement therapy with testosterone did not restore the low hypothalamic levels of GHRH mRNA in old rats to levels present in adult rats. All in all, our findings strongly support the view that in male rats GHRH-producing structures become less sensitive to androgens after puberty. ADDENDUM While our paper was in press, Zeitler et al. (Endocrinology 127:1362; 1990), using in situ hybridization, have reported reduced GHRH mRNA in the hypothalamus of castrated male rats and restoration of GHRH mRNA levels by testosterone replacement. In this study, however, the length of the castration period was considerably shorter than in ours.

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