0013-7227/91/1282-0937$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 2 Printed in U.S.A.
Growth Hormone Regulation in Primary Fetal and Neonatal Rat Pituitary Cell Cultures: The Role of Thyroid Hormone* SHEREEN EZZAT, DAN LAKS, JUDY OSTER, AND SHLOMO MELMED Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center- University of California School of Medicine, Los Angeles, California 90048
ABSTRACT. GH is first detectable in the fetal rat pituitary between gestational days 18 and 19. The reasons for the GH surge soon after birth and subsequent postnatal decline to adult levels remain unclear. We therefore determined whether GH gene regulation in the developing pituitary could be distinguished from adult rat somatotroph function. In primary cultures of fetal and neonatal rat pituitary cells, GH secretion was detected by the 20th gestational day. These cells were stimulated by GH-releasing hormone (GHRH), but not by T 3 or the morphogen retinoic acid. The stimulatory effect of T3 (0.25 HM) on GH secretion was detected only on the 2nd neonatal day and was similar to that seen in mature rat pituitary cell cultures. GHRH (10 nM) treatment for 24 h caused a 5-fold induction of GH secretion in pituitary cells derived from 2-, 5-, and 12-dayold neonatal rats. The presence or absence of T3 in the culture medium did not alter the response to GHRH. In contrast, only 2-fold induction of GH was observed in adult male pituitary cells
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during the same time course. Insulin-like growth factor-I (IGFI; 6.5 nM), the peripheral target hormone for GH, resulted in a modest (20%) attenuation of GH secretion from pituitary cells derived from 20-day-old fetuses. IGF-I, however, produced a 70% reduction in GH levels in adult male pituitary cells grown under similar conditions. The effects of IGF-I on adult pituitary cells grown in T3-depleted medium were blunted. Addition of T3 partially restored the responsiveness of these cells to IGF-I. The results suggest that the high circulating GH levels in the fetal and neonatal rat may be secondary to relative insensitivity of the immature somatotroph to the inhibitory actions of IGF-I in addition to enhanced responsiveness to GHRH compared with the adult rat pituitary. Relative thyroid hormone deficiency in the immature rat may be contributory to this early transient state of pituitary IGF-I resistance. (Endocrinology 128: 937943,1991)
stimulating SRIF release (8). Recently, the morphogen retinoic acid has also been identified as an inducer of GH secretion, probably due to its receptor DNA-binding similarity with other members of the steroid and T 3 receptor family (9). The objectives of this study were to compare the stimulatory and inhibitory signals on fetal and neonatal rat pituitary cells with those in adult rat pituitary cells. The results show that GH secretion in fetal rat pituitary cells is relatively resistant to T 3 induction. Furthermore, immature somatotrophs are shown here to be relatively insensitive to the inhibitory action of IGF-I. This in vitro difference appears to depend upon the presence or absence of T 3 in the medium.
ARKED elevation of circulating GH levels occurs in the mammalian fetus (1, 2). In rats, GH is first detectable in the pituitary between the 18th and 19th days of fetal development during a 21-day gestational period (3). GH levels surge soon after birth and subsequently decline to adult levels 2 weeks later (3). Pituitary GH secretion is under dual hypothalamic influence of GH-releasing hormone (GHRH) and somatostatin (SRIF). GHRH induces GH secretion and synthesis, while SRIF inhibits GH release (4). Peripheral hormones, such as T3, also modulate GH gene expression (5). In addition, the GH target growth factor insulin-like growth factor-I (IGF-I) has been shown to participate in a negative feedback loop to suppress both pituitary GH expression (6) and gene transcription (7). IGF-I suppression of GH also occurs at the hypothalamic level by
Materials and Methods
Received August 13, 1990. Address all correspondence and requests for reprints to: Shlomo Melmed, M.D., Division of Endocrinology and Metabolism, B-131 Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048. * This work was supported in part by NIH Grant DK-34824 and the Levien Family Foundation.
Animals Litters of timed Sprague-Dawley pregnant rats (date of plugging = 0; date of birth = G21) were purchased from Harlan Laboratories (Indianapolis, IN). Adult male rats were used as controls. Only male rats were used for the second neonatal day (N-2) and subsequent experiments. After decapitation, pitui-
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PITUITARY GH DEVELOPMENT
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tary glands were carefully identified under magnification and harvested under sterile conditions for primary monolayer cell cultures. These pituitaries were mechanically dispersed by repeated trituration through a narrow pipette tip after digestion in 0.35% collagenase and 0.1% hyaluronidase in 0.3% BSA. Approximately 8-10 litters were used for each experiment, and 4-6 fetal pituitary glands were required to yield sufficient cells for each culture well. Primary cultures Ham's F-10 medium was supplemented with L-glutamine, penicillin, streptomycin, and heat-inactivated fetal calf serum 10% (Hyclone Laboratories, Logan, UT). In some experiments thyronine-depleted serum-enriched medium was used where indicated. Depletion of T 3 and T4 from serum was achieved using an ion exchange resin (AG-lX-8) (10). The levels of T 3 and T4 in this serum were below 25 ng/dl and 1.5 Mg/dl, respectively. After dispersion, aliquots of cells were counted using an automated cell counter. Costar wells (9.5 cm2; Cambridge, MA) were plated equally at a density of 104-105 cells/ well in 3 ml 10% serum-enriched medium. After 24 h, medium was aspirated, and fresh medium of the same composition was replaced. At least four wells were selected randomly to serve as controls, while an equal number were treated with each of the different agents. At 24-h intervals thereafter, medium was collected for hormone RIA. At the end of some experiments, cells were harvested aseptically from plates with PBS (0.01 M) and EDTA (0.5 mM) for total RNA extraction. RIAs
Endo • 1991 Voll28-No2
Gel hybridization analysis At least 5 ng total RNA were loaded in each lane on a 1% agarose formaldehyde gel in 5 x gel running buffer [morpholino-propane sulfonic acid (MOPS; 0.2 M), Na acetate (50 mM), and EDTA (5 mM)]. Electrophoresis at 25 V was continued overnight. Transfer of RNA was performed by blotting the gel to Hybond filter for 16 h. This filter was dried and exposed under UV light for fixing and subsequent prehybridization. The blots were hybridized at 60 C overnight with 32P-labeled rat GH cDNA with a specific activity of about 2 X 108 cpm/^g DNA. The blots were then washed, dried, and exposed to x-ray film using an intensifying screen at -70 C for 24-72 h. For rehybridization, blots were washed again and exposed to confirm removal of the initial radiolabeled cDNA probe. Hybridization with the rat 7-actin-labeled cDNA was subsequently performed to normalize RNA loading in each lane. Materials Cell culture materials were purchased from Irvine Scientific Co. (Santa Ana, CA). T3, retinoic acid, and GHRH [human GRF-(l-44)] were purchased from Sigma (St. Louis, MO). Recombinant human IGF-I was a generous gift from Fujisawa Pharmaceutical Co. (Osaka, Japan). The rat GH cDNA was kindly provided by Drs. J. Baxter and N. Eberhardt, University of California, San Francisco. All reagents were freshly prepared and handled under subdued lighting when appropriate before their use in cultures. Statistics
Secreted GH in the culture medium was assayed by a double antibody method using specific rat GH reagents kindly provided by the National Hormone Distribution Program (Bethesda, MD). All samples were assayed in duplicate, with at least three different dilutions for optimal extrapolation from the standard curve. T4 was measured using a commercial kit (Ciba-Corning Diagnostics, Irvine, CA). The T 3 assay was measured by the Nichols Institute (San Juan Capistrano, CA). RNA extraction At the end of some experiments, cells from individual treatment groups were harvested by mechanical scraping and pelleted at 4 C for 15 min. These pellets were subjected to acid guanidinium thiocyanate-phenol-chloroform extraction, as previously described (11). Briefly, cell pellets were mixed sequentially with 1 ml guanidinium thiocyanate (83.3 g), diethylpyrocarbonate water (97.7 ml), Na citrate (pH 7; 5.87 ml), 10% Sarcosyl (8.8 ml), /3-mercaptoethanol (0.36 ml/50 ml), 0.1 ml 2 M Na acetate (pH 6), 1 ml phenol, and 0.4 ml chloroform-isoamyl alcohol (49:1). This mixture was subsequently cooled on ice for 15 min, followed by sedimentation at 7000 rpm for 20 min. The RNA-containing aqueous phase was transferred and mixed with 1 ml isopropranalol for 1 h at -20 C. RNA was then pelleted, resuspended, cooled to -20 C, washed and precipitated twice in 80% ethanol, and finally vaccum dried and resuspended in diethyl-pyrocarbonate water for measurements of optical density.
Values are depicted as the mean ± SEM, and differences were assessed by Student's t test.
Results Early detection and regulation of GH in fetal rat pituitary cells Gestational day 20 (G-20) was the earliest age from which adequate tissue could be harvested for primary cultures. These fetal rat cells secreted sufficient amounts of GH to be detected in vitro. Similarly, GH mRNA expression was detectable at this age (data not shown). The GH concentration in control wells from fetal, neonatal, and adult pituitary cells varied from 20-1000 ng/ ml depending on the sample size of the pituitary glands harvested. Due to the wide range of basal GH secretion rates, data are presented as a percentage of control secretion. Figure 1 depicts the effects of known GH inducers on GH secretion by G-20 pituitary cells. Neither T 3 (0.25 nM) nor retinoic acid (10 nM) altered GH secretion compared to that of control untreated cells. GHRH (10 nM), however, resulted in over 8-fold induction of GH secretion (P < 0.01) within 24 h. To examine the effect of inhibitory signals on the fetal rat pituitary, we tested the action of IGF-I on GH secretion. Figure 2
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PITUITARY GH DEVELOPMENT
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Effects of GHRH, T3, and IGF-I on neonatal rat pituitary cells
lOOOn
GHRH
24
48
72
HOURS
FIG. 1. Time course of T3) retinoic acid (RA), and GHRH effects on GH secretion by primary fetal rat pituitary cells. Gestational day 20 pituitary cells were incubated for 72 h in the presence of T3 (0.25 nM), RA (10 nM), or GHRH (10 nM). Each point represents mean ± SEM of four wells. *, P < 0.01 (vs. control). 100
To compare the effects of known inducers of GH on neonatal and mature somatotrophs, pituitary cells from N-2 and adult male rats were treated in vitro with T 3 and GHRH (Fig. 3). T 3 (0.25 nM) resulted in 1.9- and 2.0-fold induction of GH secretion above control in N-2 and adult cells, respectively. GHRH (10 nM), however, stimulated GH secretion by 491% (P < 0.01) in N-2 cells compared with 237% stimulation over control secretion in mature pituitary cells. To examine the role of T 3 on IGF-I action in immature pituitary cells, we determined the suppressive effect of IGF-I on GH secretion by neonatal pituitary cells in the presence and absence of thyroid hormone. Figure 4 depicts the GH response to IGF-I (6.5 nM) in T3-depleted medium. IGF-I attenuated GH secretion by only 29%. Cotreatment of cells with T 3 and IGF-I, however, resulted in 50% suppression of T3-induced GH secretion. The effect of T 3 depletion was also tested on IGF-I- or GHRH-mediated GH mRNA regulation in neonatal pituitary cells. Figure 5 depicts an autoradiograph of GH mRNA transcripts in N-5 rat pituitary cells treated in thyronine-depleted medium for 72 h and hybridized with rat GH cDNA. GHRH (10 nM) and T 3 (0.25 nM) both induced GH mRNA almost 2-fold after 72 h of treatment, as assessed by densitometric scanning of the blots.
75
500 r-
50
400 NEONATAL
25-
ADULT
300 * * * *
24
48
200 72 HRS.
FIG. 2. Time course of IGF-I effect on GH secretion from fetal or adult rat pituitary cells. Gestational day 20 pituitary cells or dispersed adult rat pituitary cells were incubated under the same conditions and treated with IGF-I (6.5 nM). GH secretion is expressed as a percentage of that in control wells not receiving IGF-I at all of the times indicated. Each point represents mean ± SEM of four wells. **, P < 0.03; ***, P < 0.005 (vs. adult).
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compares the effects of IGF-I (6.5 nM) on GH secretion by G-20 pituitary cells and adult male control cells. While IGF-I attenuated GH secretion by 25% in fetal cells, suppression of GH by 70-75% of control secretion was observed in adult pituitary cells treated with IGF-I.
• To
GHRH
To
GHRH
FIG. 3. Comparison of T3 and GHRH action on GH secretion from primary neonatal or adult pituitary cell cultures. N-2 or adult male rat pituitary cells were incubated for 24 h in the presence of thyroninedepleted medium and treated with T3 (0.25 nM) or GHRH (10 nM). Each bar represents the mean ± SEM of four wells. *, P < 0.01 (vs. neonatal control); **, P < 0.01 (vs. adult control).
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PITUITARY GH DEVELOPMENT
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200 i-
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150
100
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Endo • 1991 Voll28«No2
neonatal cells, we examined the effect of IGF-I on basal and GHRH-stimulated GH secretion in the presence and absence of T3. Figure 6 shows the time course of GH responses to GHRH and IGF-I in N-12 rat pituitary cells grown in T3-depleted medium. GHRH (10 nM) stimulated GH secretion by 500% (P < 0.01) after 24 h, declining to 261% of control values by 48 h and 213% at 72 h. Basal GH secretion in IGF-I (6.5 nM)-treated cells was suppressed maximally at 72 h by only 27% and was ineffective in suppressing GHRH-induced GH secretion throughout the 72-h time course. Similar experiments were repeated in the presence of T3-replete medium (Fig. 7). GHRH (10 nM) produced a similar degree of GH induction, but its effect was more sustained throughout the 72-h time course (363% of controls after 72 h). More significantly, IGF-I (6.5 nM) resulted in a greater degree of GH suppression to 48% (P < 0.03) of that in control cells.
IGF-1
FIG. 4. Effect of T3 on IGF-I-mediated GH attenuation in N-2 rat pituitary cells. Cells were incubated for 48 h in thyronine-depleted medium and treated with T3 (0.25 nM), IGF-I (6.5 nM), or both hormones. Each point represents the mean ± SEM of four wells. ***, P < 0.005 (vs. control); *, P < 0.01 (vs. T3 alone); NS, not significant (vs. control).
Discussion The developmental pattern of GH expression in several mammalian species has been well characterized in NS t.
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2o FIG. 5. Gel hybridization analysis of neonatal 5-day-old rat pituitary mRNA. After 72 h of incubation in thyronine-depleted medium, total RNA was electrophoresed and hybridized sequentially with 32P-labeled rat GH (lower panel) and 7-actin (upper panel) cDNAs. Treatments: C, control, GHRH, 10 nM; IGF-I, 6.5 nM; and T3, 0.25 nM. The OD values of rat GH transcripts are 1, 1.8, 0.8, and 1.8, respectively. The OD values of actin transcripts are 1, 1.2, 0.8, and 0.7, respectively. RNA MW markers (kb) are indicated by arrows.
GHRH was, therefore, effective in the absence of T 3 in the culture medium. The transcript signal derived from IGF-I (6.5 nM)-treated cells was, however, minimally attenuated compared to control mRNA levels. Hybridization with 7-actin cDNA confirmed equal RNA loading in each lane. To further elucidate the role of T 3 on IGF-I action in
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FIG. 6. Time course of GHRH and IGF-I effects on GH secretion by neonatal rat pituitary cells in the absence of T3. N-12 rat pituitary cells were grown in thyronine-depleted medium and treated with GHRH (10 nM), IGF-I (6.5 nM), or both as indicated. Each bar represents the mean ± SEM of three or four wells. *, P < 0.01 (vs. control); NS, not significant (vs. no added IGF-I).
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PITUITARY GH DEVELOPMENT
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FIG. 7. Time course of GHRH and IGF-I effects on GH secretion by neonatal rat pituitary cells in the presence of T3. N-12 rat pituitary cells were grown in thyronine-replete medium and treated with GHRH (10 nM) or IGF-I (6.5 nM). Each bar represents the mean ± SEM of four wells. ***, P < 0.005; **, P < 0.03 {vs. control).
vitro and in vivo (12-16). The ontogeny of GH in the fetal and neonatal rat pituitary, however, has so far mostly been studied using immunocytochemical techniques (17). Immunoreactive GH has been reported to be present in the fetal rat pituitary as early as gestational day 19 (18). GH mRNA has also been detected in intact pituitaries by in situ hybridization at this age (19). There is limited information, however, about GH synthesis and gene regulation during the perinatal period of rat development. The present studies confirm that fetal rat GH is sufficiently expressed by the 20th day of gestation to allow detection of GH secretion from primary cultures. Furthermore, the concordant temporal association of the onset of GH mRNA expression and GH secretion at this age suggests that translational regulation of GH is probably not a major determinant in the developing rat. This is in contrast with PRL gene expression, which has been reported to occur on the 4th day of neonatal life, at least 3 days before the onset of PRL secretion in newborn rats, as determined by the reverse hemolytic plaque assay (20), suggesting translational regulation in the onset of PRL gene expression and peptide secretion. Interestingly, T3, a known inducer of GH in the adult rat, failed to stimulate GH secretion in fetal pituitary
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cells as it did in neonatal (N-2) and adult pituitary cells. Retinoic acid, which may act similarly to T 3 in regulating the GH gene (9), also failed to induce fetal GH secretion. A similar nonresponsiveness of GH to T 3 has been shown in neonatal rats (21). Using an in vivo model of neonatal hypothyroidism, GH accumulation in rat pituitaries was shown to occur independently of thyroid hormone, and GH sensitivity to T 3 was acquired after the sixth day of postnatal life. The significance of these conclusions is less clear in view of previous work suggesting that the thyroid hormone receptor develops in brain, lung, liver, and heart at least 5 days before maturation of thyroid hormone secretion in the unborn rat (22). The elucidation of the ontogeny and tissue specificity of the various thyroid hormone receptor forms may explain the immature pituitary cell response to T3. Developing somatotrophs may also require a pituitary-specific factor necessary for T3-receptor-DNA binding interaction. The hyperresponsiveness of neonatal cells to GHRH compared with that of mature cells suggests a possible intrinsic difference between the immature somatotroph and its adult counterpart. Alternatively, somatotrophs may comprise a larger proportion of the pituitary cells in the immature rat, accounting for an exaggerated GH response of pituitary cells derived from fetal and neonatal rats to GHRH. These results are in agreement with previous findings, where an age-dependent pattern of GH responsiveness to GHRH, (Bu)2cAMP, and TRH has been documented (23, 24). Our findings also indicate that T 3 is neither necessary nor facilitatory to the action of GHRH on neonatal somatotrophs. This further supports the view that the relative deficiency of T 3 in the developing rat is not responsible for the sensitization of the immature somatotroph to such secretagogues as GHRH and TRH. The effect of hypersomatotropism on mammalian fetal and neonatal development remains uncertain. Fetal somatic growth appears to be independent of GH, as evidenced by the lack of growth retardation in the GHdeficient fetus (2). GH may play a role in fetal fat metabolism and corticosteroid production (3). IGF-I, a target growth factor for GH, regulates in vitro and in vivo GH secretion at the level of both the hypothalamus and pituitary (4, 8). Furthermore, as IGF-I participates in negative feedback regulation of rat pituitary GH gene transcription (7), it was used here to examine its role in regulating GH in the developing rat somatotroph. The dose selected in these experiments (6.5 nM) is within the physiological range for binding to the adult rat pituitary IGF-I receptor (25). The ontogeny of IGF-I gene expression has been documented in several fetal rat tissues (26). Our results, however, indicate that compared with the adult rat, the developing pituitary is relatively resistant to the in vitro inhibitory effects of
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PITUITARY GH DEVELOPMENT
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IGF-I. This finding is analogous to the relative insensitivity of the immature rat (27) and ovine (14) pituitary to SRIF suppression of GH. In the ovine fetal pituitary, relatively high doses of IGF-I were required to attenuate GHRH-induced GH secretion in euthyroid medium (14). We have previously shown that IGF-I is ineffective in suppressing GH in adult pituitary cells derived from hypothyroid rats (28). Replenishment of T 3 appears to restore the IGF-I responsiveness of the hypothyroid somatotroph (28). Pituitary IGF-I gene expression has been shown to be induced by T 3 (29), while the receptor itself may be regulated by T 3 (30). Thyroid hormone and GH have also been shown to interact in regulating hepatic IGF-I expression in hypophysectomized rats (31). Our present data on the in vitro effect of T 3 on IGF-I action in neonatal cells are consistent with these recent findings. As fetal rat tissues are known to be deficient in the outer ring monodeiodinase enzyme (32), they may also be relatively depleted of T3. These data, therefore, imply that fetal rat insensitivity to IGF-I may be attributable at least in part to the low circulating thyroid hormone state during fetal development. Hypersomatotropism in the immature rat may, therefore, be secondary to relative insensitivity to GH inhibitory signals, including SRIF and IGF-I, as a reflection of the developing endocrine environment in addition to the known intrinsic pituitary hyperresponsiveness to GHRH.
9.
10.
11.
12.
13.
14.
15.
17.
We wish to thank Fujisawa Pharmaceuticals (Osaka, Japan) for their generous contribution of recombinant human IGF-I. 18.
References 1. Gluckman PD 1983 The fetal neuroendocrine axis. Curr Top Exp Endocrinol 5:1-42 2. Fisher DA 1986 The unique endocrine milieu of the fetus. J Clin Invest 78:603-611 3. Jost A 1979 Fetal hormones and fetal growth. Contr Gynecol Obstet 5:1-20 4. Daughaday WH 1989 Growth hormone; normal synthesis, secretion, control and mechanisms of action. In: Degroot LJ (ed) Endocrinology Saunders, Philadelphia, pp 318-329 5. Halperin Y, Surks MI, Shapiro LE 1990 L-Triiodothyronine (T3) regulates cellular growth rate, growth hormone production, and levels of nuclear T3 receptors via distinct dose-response ranges in cultured GC cells. Endocrinology 126:2321-2326 6. Yamashita S, Melmed S 1986 Insulin-like growth factor I action on rat anterior pituitary cells: suppression of growth hormone secretion and messenger ribonucleic acid levels. Endocrinology 118:176-182 7. Yamashita S, Melmed S 1987 Insulin-like growth factor I regulation of growth hormone gene transcription in primary rat pituitary cells. J Clin Invest 79:449-452 8. Berelowitz M, Szabo M, Frohman LA, Firestone S, Chu L, Hintz RL 1981 Somatomedin-C mediates growth hormone negative feed-
back by effects on both the hypothalamus and the pituitary. Science 212:1279-1281 Forman BM, Yang CR, Au M, Casanova J, Ghysdael J, Samuels HH 1989 A domain containing leucine-zipper-like motifs mediate novel in vivo interactions between the thyroid hormone and retinoic acid receptors. Mol Endocrinol 3:1610-1626 Samuels HH, Stanley F, Casanova J 1979 Depletion of L-3,5,3'triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105:80-85 Chomczynski P, Sacchi N 1987 Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Ann Biochem 162:156-159 Blanchard MM, Goodyer CG, Charrier J, Barenton B 1988 In vitro regulation of growth hormone (GH) release from ovine pituitary cells during fetal and neonatal development: effects of GH-releasing factor, somatostatin, and insulin-like growth factor I. Endocrinology 122:2114-2120 De Zegher F, Styne DM, Daaboul J, Bettendorf M, Kaplan SL, Grumbach MM 1989 Hormone ontogeny in the ovine fetus and
16.
Acknowledgment
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neonatal lamb. XX. Effect of age, breading season, and twinning on the growth hormone (GH) response to GH-releasing factor: evidence for a homeostatic role of fetal GH. Endocrinology 124:124-128 Silverman BL, Bettendorf M, Kaplan SL, Grumbach MM, Miller WL 1989 Regulation of growth hormone (GH) secretion by GHreleasing factor, somatostatin and insulin-like growth factor I in ovine fetal and neonatal pituitary cells in vitro. Endocrinology 124:84-89 Slabaugh MB, Lieberman ME, Rutledge JJ, Gorski J 1982 Ontogeny of growth hormone and prolactin gene expression in mice. Endocrinology 110:1489-1497 Hall TR, Harvey S, Chadwick A 1985 Age-related changes in prolactin and growth hormone release from pituitary glands in vitro. Acta Endocrinol (Copenh) 108:479-484 Hemming FJ, Begeot M, Dubois MP, Dubois PM 1984 Fetal rat somatotropes in vitro: effects of insulin, cortisol, and growth hormone-releasing factor on their differentiation: a light and electron microscopic study. Endocrinology 114:2107-2113 Hemming FJ, Aubert ML, Dubois PM 1988 Differentiation of fetal rat somatotropes in vitro: effects of cortisol, 3,5,3'-triiodothyronine, and glucagon, a light microscopic and radioimmunologic study. Endocrinology 123:1230-1236 Nogami H, Suzuki K, Enomoto H, Ishikawa H 1989 Studies on the development of growth hormone and prolactin cells in the rat pituitary gland by in situ hybridization. Cell Tissue Res 255:23-28 Frawley LS, Miller HA 1989 Ontogeny of prolactin secretion in the neonatal rat is regulated posttranscriptionally. Endocrinology 124:3-6 Seo H, Wunderlich C, Vassart G, Refetoff S 1981 Growth hormone response to thyroid hormone in the neonatal rat. J Clin Invest 67:569-574 Perez-Castillo A, Bernal J, Ferreiro B, Pans T 1985 The early ontogenesis of thyroid hormone receptor in the rat fetus. Endocrinology 117:2457-2461 Szabo M, Cuttler L 1986 Differential responsiveness of the somatotroph to growth hormone-releasing factor during early neonatal development in the rat. Endocrinology 118:69-73 Welsh JB, Cuttler L, Szabo M 1986 Ontogeny of the in vitro growth hormone stimulatory effect of thyrotropin-releasing hormone in the rat. Endocrinology 119:2368-2375 Rosenfeld RG, Ceda G, Wilson DM, Dollar LA, Hoffman AR 1984 Characterization of high affinity receptors for insulin-like growth factor I and II on rat anterior pituitary cells. Endocrinology
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PITUITARY GH DEVELOPMENT 114:1571-1575 26. Lund PK, Moats-Staats BM, Hynes MA, Simmons JG, Jansen M, D'Ercole AJ, Van Wyk JJ 1986 Somatomedin-C/insulin-like growth factor I and insulin-like growth factor II mRNAs in rat fetal and adult tissues. J Biol Chem 261:14539-14544 27. Cuttler L, Welsh JB, Szabo M 1986 The effect of age on somatostatin suppression of basal, growth hormone-releasing factor-stimulated, and dibutyryl adenosine 3',5'-monophosphate-stimulated GH release from rat pituitary cells in monolayer culture. Endocrinology 119:152-158 28. Melmed S, Yamashita S 1986 Insulin-like growth factor I action on hypothyroid rat anterior pituitary cells: suppression of triiodothyronine-induced growth hormone secretion and messenger ribonucleic acid levels. Endocrinology 118:1483-1490
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29. Fagin JA, Fernandez-Mejia C, Melmed S 1989 Pituitary insulinlike growth factor-I gene expression: regulation by triiodothyronine and growth hormone. Endocrinology 125:2385-2391 30. Matsuo K, Yamashita S, Niwa M, Kurihara M, Harakawa S, Izumi M, Nagataki S, Melmed S 1990 Thyroid hormone regulates rat pituitary insulin-like growth factor-I receptors. Endocrinology 126:550-554 31. Wolf M, Ingbar SH, Moses AC 1989 Thyroid hormone and growth hormone interact to regulate insulin-like growth factor-I messenger ribonucleic acid and circulating levels in the rat. Endocrinology 125:2905-2914 32. Wu SY, Klein AH, Chopra IJ, Fisher DA 1978 Alterations in tissue thyroxine-5'-monodeiodinating activity in perinatal period. Endocrinology 103:235-239
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Vol. 128, No. 2 Printed in U.S.A.
Growth Hormone Regulation in Primary Fetal and Neonatal Rat Pituitary Cell Cultures: The Role of Thyroid Hormone* SHEREEN EZZAT, DAN LAKS, JUDY OSTER, AND SHLOMO MELMED Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center- University of California School of Medicine, Los Angeles, California 90048
ABSTRACT. GH is first detectable in the fetal rat pituitary between gestational days 18 and 19. The reasons for the GH surge soon after birth and subsequent postnatal decline to adult levels remain unclear. We therefore determined whether GH gene regulation in the developing pituitary could be distinguished from adult rat somatotroph function. In primary cultures of fetal and neonatal rat pituitary cells, GH secretion was detected by the 20th gestational day. These cells were stimulated by GH-releasing hormone (GHRH), but not by T 3 or the morphogen retinoic acid. The stimulatory effect of T3 (0.25 HM) on GH secretion was detected only on the 2nd neonatal day and was similar to that seen in mature rat pituitary cell cultures. GHRH (10 nM) treatment for 24 h caused a 5-fold induction of GH secretion in pituitary cells derived from 2-, 5-, and 12-dayold neonatal rats. The presence or absence of T3 in the culture medium did not alter the response to GHRH. In contrast, only 2-fold induction of GH was observed in adult male pituitary cells
M
during the same time course. Insulin-like growth factor-I (IGFI; 6.5 nM), the peripheral target hormone for GH, resulted in a modest (20%) attenuation of GH secretion from pituitary cells derived from 20-day-old fetuses. IGF-I, however, produced a 70% reduction in GH levels in adult male pituitary cells grown under similar conditions. The effects of IGF-I on adult pituitary cells grown in T3-depleted medium were blunted. Addition of T3 partially restored the responsiveness of these cells to IGF-I. The results suggest that the high circulating GH levels in the fetal and neonatal rat may be secondary to relative insensitivity of the immature somatotroph to the inhibitory actions of IGF-I in addition to enhanced responsiveness to GHRH compared with the adult rat pituitary. Relative thyroid hormone deficiency in the immature rat may be contributory to this early transient state of pituitary IGF-I resistance. (Endocrinology 128: 937943,1991)
stimulating SRIF release (8). Recently, the morphogen retinoic acid has also been identified as an inducer of GH secretion, probably due to its receptor DNA-binding similarity with other members of the steroid and T 3 receptor family (9). The objectives of this study were to compare the stimulatory and inhibitory signals on fetal and neonatal rat pituitary cells with those in adult rat pituitary cells. The results show that GH secretion in fetal rat pituitary cells is relatively resistant to T 3 induction. Furthermore, immature somatotrophs are shown here to be relatively insensitive to the inhibitory action of IGF-I. This in vitro difference appears to depend upon the presence or absence of T 3 in the medium.
ARKED elevation of circulating GH levels occurs in the mammalian fetus (1, 2). In rats, GH is first detectable in the pituitary between the 18th and 19th days of fetal development during a 21-day gestational period (3). GH levels surge soon after birth and subsequently decline to adult levels 2 weeks later (3). Pituitary GH secretion is under dual hypothalamic influence of GH-releasing hormone (GHRH) and somatostatin (SRIF). GHRH induces GH secretion and synthesis, while SRIF inhibits GH release (4). Peripheral hormones, such as T3, also modulate GH gene expression (5). In addition, the GH target growth factor insulin-like growth factor-I (IGF-I) has been shown to participate in a negative feedback loop to suppress both pituitary GH expression (6) and gene transcription (7). IGF-I suppression of GH also occurs at the hypothalamic level by
Materials and Methods
Received August 13, 1990. Address all correspondence and requests for reprints to: Shlomo Melmed, M.D., Division of Endocrinology and Metabolism, B-131 Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048. * This work was supported in part by NIH Grant DK-34824 and the Levien Family Foundation.
Animals Litters of timed Sprague-Dawley pregnant rats (date of plugging = 0; date of birth = G21) were purchased from Harlan Laboratories (Indianapolis, IN). Adult male rats were used as controls. Only male rats were used for the second neonatal day (N-2) and subsequent experiments. After decapitation, pitui-
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PITUITARY GH DEVELOPMENT
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tary glands were carefully identified under magnification and harvested under sterile conditions for primary monolayer cell cultures. These pituitaries were mechanically dispersed by repeated trituration through a narrow pipette tip after digestion in 0.35% collagenase and 0.1% hyaluronidase in 0.3% BSA. Approximately 8-10 litters were used for each experiment, and 4-6 fetal pituitary glands were required to yield sufficient cells for each culture well. Primary cultures Ham's F-10 medium was supplemented with L-glutamine, penicillin, streptomycin, and heat-inactivated fetal calf serum 10% (Hyclone Laboratories, Logan, UT). In some experiments thyronine-depleted serum-enriched medium was used where indicated. Depletion of T 3 and T4 from serum was achieved using an ion exchange resin (AG-lX-8) (10). The levels of T 3 and T4 in this serum were below 25 ng/dl and 1.5 Mg/dl, respectively. After dispersion, aliquots of cells were counted using an automated cell counter. Costar wells (9.5 cm2; Cambridge, MA) were plated equally at a density of 104-105 cells/ well in 3 ml 10% serum-enriched medium. After 24 h, medium was aspirated, and fresh medium of the same composition was replaced. At least four wells were selected randomly to serve as controls, while an equal number were treated with each of the different agents. At 24-h intervals thereafter, medium was collected for hormone RIA. At the end of some experiments, cells were harvested aseptically from plates with PBS (0.01 M) and EDTA (0.5 mM) for total RNA extraction. RIAs
Endo • 1991 Voll28-No2
Gel hybridization analysis At least 5 ng total RNA were loaded in each lane on a 1% agarose formaldehyde gel in 5 x gel running buffer [morpholino-propane sulfonic acid (MOPS; 0.2 M), Na acetate (50 mM), and EDTA (5 mM)]. Electrophoresis at 25 V was continued overnight. Transfer of RNA was performed by blotting the gel to Hybond filter for 16 h. This filter was dried and exposed under UV light for fixing and subsequent prehybridization. The blots were hybridized at 60 C overnight with 32P-labeled rat GH cDNA with a specific activity of about 2 X 108 cpm/^g DNA. The blots were then washed, dried, and exposed to x-ray film using an intensifying screen at -70 C for 24-72 h. For rehybridization, blots were washed again and exposed to confirm removal of the initial radiolabeled cDNA probe. Hybridization with the rat 7-actin-labeled cDNA was subsequently performed to normalize RNA loading in each lane. Materials Cell culture materials were purchased from Irvine Scientific Co. (Santa Ana, CA). T3, retinoic acid, and GHRH [human GRF-(l-44)] were purchased from Sigma (St. Louis, MO). Recombinant human IGF-I was a generous gift from Fujisawa Pharmaceutical Co. (Osaka, Japan). The rat GH cDNA was kindly provided by Drs. J. Baxter and N. Eberhardt, University of California, San Francisco. All reagents were freshly prepared and handled under subdued lighting when appropriate before their use in cultures. Statistics
Secreted GH in the culture medium was assayed by a double antibody method using specific rat GH reagents kindly provided by the National Hormone Distribution Program (Bethesda, MD). All samples were assayed in duplicate, with at least three different dilutions for optimal extrapolation from the standard curve. T4 was measured using a commercial kit (Ciba-Corning Diagnostics, Irvine, CA). The T 3 assay was measured by the Nichols Institute (San Juan Capistrano, CA). RNA extraction At the end of some experiments, cells from individual treatment groups were harvested by mechanical scraping and pelleted at 4 C for 15 min. These pellets were subjected to acid guanidinium thiocyanate-phenol-chloroform extraction, as previously described (11). Briefly, cell pellets were mixed sequentially with 1 ml guanidinium thiocyanate (83.3 g), diethylpyrocarbonate water (97.7 ml), Na citrate (pH 7; 5.87 ml), 10% Sarcosyl (8.8 ml), /3-mercaptoethanol (0.36 ml/50 ml), 0.1 ml 2 M Na acetate (pH 6), 1 ml phenol, and 0.4 ml chloroform-isoamyl alcohol (49:1). This mixture was subsequently cooled on ice for 15 min, followed by sedimentation at 7000 rpm for 20 min. The RNA-containing aqueous phase was transferred and mixed with 1 ml isopropranalol for 1 h at -20 C. RNA was then pelleted, resuspended, cooled to -20 C, washed and precipitated twice in 80% ethanol, and finally vaccum dried and resuspended in diethyl-pyrocarbonate water for measurements of optical density.
Values are depicted as the mean ± SEM, and differences were assessed by Student's t test.
Results Early detection and regulation of GH in fetal rat pituitary cells Gestational day 20 (G-20) was the earliest age from which adequate tissue could be harvested for primary cultures. These fetal rat cells secreted sufficient amounts of GH to be detected in vitro. Similarly, GH mRNA expression was detectable at this age (data not shown). The GH concentration in control wells from fetal, neonatal, and adult pituitary cells varied from 20-1000 ng/ ml depending on the sample size of the pituitary glands harvested. Due to the wide range of basal GH secretion rates, data are presented as a percentage of control secretion. Figure 1 depicts the effects of known GH inducers on GH secretion by G-20 pituitary cells. Neither T 3 (0.25 nM) nor retinoic acid (10 nM) altered GH secretion compared to that of control untreated cells. GHRH (10 nM), however, resulted in over 8-fold induction of GH secretion (P < 0.01) within 24 h. To examine the effect of inhibitory signals on the fetal rat pituitary, we tested the action of IGF-I on GH secretion. Figure 2
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PITUITARY GH DEVELOPMENT
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Effects of GHRH, T3, and IGF-I on neonatal rat pituitary cells
lOOOn
GHRH
24
48
72
HOURS
FIG. 1. Time course of T3) retinoic acid (RA), and GHRH effects on GH secretion by primary fetal rat pituitary cells. Gestational day 20 pituitary cells were incubated for 72 h in the presence of T3 (0.25 nM), RA (10 nM), or GHRH (10 nM). Each point represents mean ± SEM of four wells. *, P < 0.01 (vs. control). 100
To compare the effects of known inducers of GH on neonatal and mature somatotrophs, pituitary cells from N-2 and adult male rats were treated in vitro with T 3 and GHRH (Fig. 3). T 3 (0.25 nM) resulted in 1.9- and 2.0-fold induction of GH secretion above control in N-2 and adult cells, respectively. GHRH (10 nM), however, stimulated GH secretion by 491% (P < 0.01) in N-2 cells compared with 237% stimulation over control secretion in mature pituitary cells. To examine the role of T 3 on IGF-I action in immature pituitary cells, we determined the suppressive effect of IGF-I on GH secretion by neonatal pituitary cells in the presence and absence of thyroid hormone. Figure 4 depicts the GH response to IGF-I (6.5 nM) in T3-depleted medium. IGF-I attenuated GH secretion by only 29%. Cotreatment of cells with T 3 and IGF-I, however, resulted in 50% suppression of T3-induced GH secretion. The effect of T 3 depletion was also tested on IGF-I- or GHRH-mediated GH mRNA regulation in neonatal pituitary cells. Figure 5 depicts an autoradiograph of GH mRNA transcripts in N-5 rat pituitary cells treated in thyronine-depleted medium for 72 h and hybridized with rat GH cDNA. GHRH (10 nM) and T 3 (0.25 nM) both induced GH mRNA almost 2-fold after 72 h of treatment, as assessed by densitometric scanning of the blots.
75
500 r-
50
400 NEONATAL
25-
ADULT
300 * * * *
24
48
200 72 HRS.
FIG. 2. Time course of IGF-I effect on GH secretion from fetal or adult rat pituitary cells. Gestational day 20 pituitary cells or dispersed adult rat pituitary cells were incubated under the same conditions and treated with IGF-I (6.5 nM). GH secretion is expressed as a percentage of that in control wells not receiving IGF-I at all of the times indicated. Each point represents mean ± SEM of four wells. **, P < 0.03; ***, P < 0.005 (vs. adult).
I C
compares the effects of IGF-I (6.5 nM) on GH secretion by G-20 pituitary cells and adult male control cells. While IGF-I attenuated GH secretion by 25% in fetal cells, suppression of GH by 70-75% of control secretion was observed in adult pituitary cells treated with IGF-I.
• To
GHRH
To
GHRH
FIG. 3. Comparison of T3 and GHRH action on GH secretion from primary neonatal or adult pituitary cell cultures. N-2 or adult male rat pituitary cells were incubated for 24 h in the presence of thyroninedepleted medium and treated with T3 (0.25 nM) or GHRH (10 nM). Each bar represents the mean ± SEM of four wells. *, P < 0.01 (vs. neonatal control); **, P < 0.01 (vs. adult control).
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PITUITARY GH DEVELOPMENT
940 ***
200 i-
i
150
100
I
1 NS
T 50
IGF-1
T3
Endo • 1991 Voll28«No2
neonatal cells, we examined the effect of IGF-I on basal and GHRH-stimulated GH secretion in the presence and absence of T3. Figure 6 shows the time course of GH responses to GHRH and IGF-I in N-12 rat pituitary cells grown in T3-depleted medium. GHRH (10 nM) stimulated GH secretion by 500% (P < 0.01) after 24 h, declining to 261% of control values by 48 h and 213% at 72 h. Basal GH secretion in IGF-I (6.5 nM)-treated cells was suppressed maximally at 72 h by only 27% and was ineffective in suppressing GHRH-induced GH secretion throughout the 72-h time course. Similar experiments were repeated in the presence of T3-replete medium (Fig. 7). GHRH (10 nM) produced a similar degree of GH induction, but its effect was more sustained throughout the 72-h time course (363% of controls after 72 h). More significantly, IGF-I (6.5 nM) resulted in a greater degree of GH suppression to 48% (P < 0.03) of that in control cells.
IGF-1
FIG. 4. Effect of T3 on IGF-I-mediated GH attenuation in N-2 rat pituitary cells. Cells were incubated for 48 h in thyronine-depleted medium and treated with T3 (0.25 nM), IGF-I (6.5 nM), or both hormones. Each point represents the mean ± SEM of four wells. ***, P < 0.005 (vs. control); *, P < 0.01 (vs. T3 alone); NS, not significant (vs. control).
Discussion The developmental pattern of GH expression in several mammalian species has been well characterized in NS t.
500
48 hours
2.2
+•
400 NS
1 . 3 5 »•
o I-
300
o o
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2o FIG. 5. Gel hybridization analysis of neonatal 5-day-old rat pituitary mRNA. After 72 h of incubation in thyronine-depleted medium, total RNA was electrophoresed and hybridized sequentially with 32P-labeled rat GH (lower panel) and 7-actin (upper panel) cDNAs. Treatments: C, control, GHRH, 10 nM; IGF-I, 6.5 nM; and T3, 0.25 nM. The OD values of rat GH transcripts are 1, 1.8, 0.8, and 1.8, respectively. The OD values of actin transcripts are 1, 1.2, 0.8, and 0.7, respectively. RNA MW markers (kb) are indicated by arrows.
GHRH was, therefore, effective in the absence of T 3 in the culture medium. The transcript signal derived from IGF-I (6.5 nM)-treated cells was, however, minimally attenuated compared to control mRNA levels. Hybridization with 7-actin cDNA confirmed equal RNA loading in each lane. To further elucidate the role of T 3 on IGF-I action in
JL
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NS
100 NS
C
GHRH IGF-1 GHRH
+
IGF-1
ii
NS
4
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r3! 5,.;..,
C GHRH IGF-1 GHRH
C GHRH IGF-1 GHRH
IGF-1
IGF-1
FIG. 6. Time course of GHRH and IGF-I effects on GH secretion by neonatal rat pituitary cells in the absence of T3. N-12 rat pituitary cells were grown in thyronine-depleted medium and treated with GHRH (10 nM), IGF-I (6.5 nM), or both as indicated. Each bar represents the mean ± SEM of three or four wells. *, P < 0.01 (vs. control); NS, not significant (vs. no added IGF-I).
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PITUITARY GH DEVELOPMENT
***
***
500
1
|
I 24 hours
fr^| 48 hours QT] 72 hours
* * *
400
I
g 300 t-
z o o
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o X 200
o
100
X
**
C
n
GHRHIGF-1
**
* *
C
GHRH IGF-1
C
GHRHIGF-1
FIG. 7. Time course of GHRH and IGF-I effects on GH secretion by neonatal rat pituitary cells in the presence of T3. N-12 rat pituitary cells were grown in thyronine-replete medium and treated with GHRH (10 nM) or IGF-I (6.5 nM). Each bar represents the mean ± SEM of four wells. ***, P < 0.005; **, P < 0.03 {vs. control).
vitro and in vivo (12-16). The ontogeny of GH in the fetal and neonatal rat pituitary, however, has so far mostly been studied using immunocytochemical techniques (17). Immunoreactive GH has been reported to be present in the fetal rat pituitary as early as gestational day 19 (18). GH mRNA has also been detected in intact pituitaries by in situ hybridization at this age (19). There is limited information, however, about GH synthesis and gene regulation during the perinatal period of rat development. The present studies confirm that fetal rat GH is sufficiently expressed by the 20th day of gestation to allow detection of GH secretion from primary cultures. Furthermore, the concordant temporal association of the onset of GH mRNA expression and GH secretion at this age suggests that translational regulation of GH is probably not a major determinant in the developing rat. This is in contrast with PRL gene expression, which has been reported to occur on the 4th day of neonatal life, at least 3 days before the onset of PRL secretion in newborn rats, as determined by the reverse hemolytic plaque assay (20), suggesting translational regulation in the onset of PRL gene expression and peptide secretion. Interestingly, T3, a known inducer of GH in the adult rat, failed to stimulate GH secretion in fetal pituitary
941
cells as it did in neonatal (N-2) and adult pituitary cells. Retinoic acid, which may act similarly to T 3 in regulating the GH gene (9), also failed to induce fetal GH secretion. A similar nonresponsiveness of GH to T 3 has been shown in neonatal rats (21). Using an in vivo model of neonatal hypothyroidism, GH accumulation in rat pituitaries was shown to occur independently of thyroid hormone, and GH sensitivity to T 3 was acquired after the sixth day of postnatal life. The significance of these conclusions is less clear in view of previous work suggesting that the thyroid hormone receptor develops in brain, lung, liver, and heart at least 5 days before maturation of thyroid hormone secretion in the unborn rat (22). The elucidation of the ontogeny and tissue specificity of the various thyroid hormone receptor forms may explain the immature pituitary cell response to T3. Developing somatotrophs may also require a pituitary-specific factor necessary for T3-receptor-DNA binding interaction. The hyperresponsiveness of neonatal cells to GHRH compared with that of mature cells suggests a possible intrinsic difference between the immature somatotroph and its adult counterpart. Alternatively, somatotrophs may comprise a larger proportion of the pituitary cells in the immature rat, accounting for an exaggerated GH response of pituitary cells derived from fetal and neonatal rats to GHRH. These results are in agreement with previous findings, where an age-dependent pattern of GH responsiveness to GHRH, (Bu)2cAMP, and TRH has been documented (23, 24). Our findings also indicate that T 3 is neither necessary nor facilitatory to the action of GHRH on neonatal somatotrophs. This further supports the view that the relative deficiency of T 3 in the developing rat is not responsible for the sensitization of the immature somatotroph to such secretagogues as GHRH and TRH. The effect of hypersomatotropism on mammalian fetal and neonatal development remains uncertain. Fetal somatic growth appears to be independent of GH, as evidenced by the lack of growth retardation in the GHdeficient fetus (2). GH may play a role in fetal fat metabolism and corticosteroid production (3). IGF-I, a target growth factor for GH, regulates in vitro and in vivo GH secretion at the level of both the hypothalamus and pituitary (4, 8). Furthermore, as IGF-I participates in negative feedback regulation of rat pituitary GH gene transcription (7), it was used here to examine its role in regulating GH in the developing rat somatotroph. The dose selected in these experiments (6.5 nM) is within the physiological range for binding to the adult rat pituitary IGF-I receptor (25). The ontogeny of IGF-I gene expression has been documented in several fetal rat tissues (26). Our results, however, indicate that compared with the adult rat, the developing pituitary is relatively resistant to the in vitro inhibitory effects of
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PITUITARY GH DEVELOPMENT
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IGF-I. This finding is analogous to the relative insensitivity of the immature rat (27) and ovine (14) pituitary to SRIF suppression of GH. In the ovine fetal pituitary, relatively high doses of IGF-I were required to attenuate GHRH-induced GH secretion in euthyroid medium (14). We have previously shown that IGF-I is ineffective in suppressing GH in adult pituitary cells derived from hypothyroid rats (28). Replenishment of T 3 appears to restore the IGF-I responsiveness of the hypothyroid somatotroph (28). Pituitary IGF-I gene expression has been shown to be induced by T 3 (29), while the receptor itself may be regulated by T 3 (30). Thyroid hormone and GH have also been shown to interact in regulating hepatic IGF-I expression in hypophysectomized rats (31). Our present data on the in vitro effect of T 3 on IGF-I action in neonatal cells are consistent with these recent findings. As fetal rat tissues are known to be deficient in the outer ring monodeiodinase enzyme (32), they may also be relatively depleted of T3. These data, therefore, imply that fetal rat insensitivity to IGF-I may be attributable at least in part to the low circulating thyroid hormone state during fetal development. Hypersomatotropism in the immature rat may, therefore, be secondary to relative insensitivity to GH inhibitory signals, including SRIF and IGF-I, as a reflection of the developing endocrine environment in addition to the known intrinsic pituitary hyperresponsiveness to GHRH.
9.
10.
11.
12.
13.
14.
15.
17.
We wish to thank Fujisawa Pharmaceuticals (Osaka, Japan) for their generous contribution of recombinant human IGF-I. 18.
References 1. Gluckman PD 1983 The fetal neuroendocrine axis. Curr Top Exp Endocrinol 5:1-42 2. Fisher DA 1986 The unique endocrine milieu of the fetus. J Clin Invest 78:603-611 3. Jost A 1979 Fetal hormones and fetal growth. Contr Gynecol Obstet 5:1-20 4. Daughaday WH 1989 Growth hormone; normal synthesis, secretion, control and mechanisms of action. In: Degroot LJ (ed) Endocrinology Saunders, Philadelphia, pp 318-329 5. Halperin Y, Surks MI, Shapiro LE 1990 L-Triiodothyronine (T3) regulates cellular growth rate, growth hormone production, and levels of nuclear T3 receptors via distinct dose-response ranges in cultured GC cells. Endocrinology 126:2321-2326 6. Yamashita S, Melmed S 1986 Insulin-like growth factor I action on rat anterior pituitary cells: suppression of growth hormone secretion and messenger ribonucleic acid levels. Endocrinology 118:176-182 7. Yamashita S, Melmed S 1987 Insulin-like growth factor I regulation of growth hormone gene transcription in primary rat pituitary cells. J Clin Invest 79:449-452 8. Berelowitz M, Szabo M, Frohman LA, Firestone S, Chu L, Hintz RL 1981 Somatomedin-C mediates growth hormone negative feed-
back by effects on both the hypothalamus and the pituitary. Science 212:1279-1281 Forman BM, Yang CR, Au M, Casanova J, Ghysdael J, Samuels HH 1989 A domain containing leucine-zipper-like motifs mediate novel in vivo interactions between the thyroid hormone and retinoic acid receptors. Mol Endocrinol 3:1610-1626 Samuels HH, Stanley F, Casanova J 1979 Depletion of L-3,5,3'triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105:80-85 Chomczynski P, Sacchi N 1987 Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Ann Biochem 162:156-159 Blanchard MM, Goodyer CG, Charrier J, Barenton B 1988 In vitro regulation of growth hormone (GH) release from ovine pituitary cells during fetal and neonatal development: effects of GH-releasing factor, somatostatin, and insulin-like growth factor I. Endocrinology 122:2114-2120 De Zegher F, Styne DM, Daaboul J, Bettendorf M, Kaplan SL, Grumbach MM 1989 Hormone ontogeny in the ovine fetus and
16.
Acknowledgment
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19.
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25.
neonatal lamb. XX. Effect of age, breading season, and twinning on the growth hormone (GH) response to GH-releasing factor: evidence for a homeostatic role of fetal GH. Endocrinology 124:124-128 Silverman BL, Bettendorf M, Kaplan SL, Grumbach MM, Miller WL 1989 Regulation of growth hormone (GH) secretion by GHreleasing factor, somatostatin and insulin-like growth factor I in ovine fetal and neonatal pituitary cells in vitro. Endocrinology 124:84-89 Slabaugh MB, Lieberman ME, Rutledge JJ, Gorski J 1982 Ontogeny of growth hormone and prolactin gene expression in mice. Endocrinology 110:1489-1497 Hall TR, Harvey S, Chadwick A 1985 Age-related changes in prolactin and growth hormone release from pituitary glands in vitro. Acta Endocrinol (Copenh) 108:479-484 Hemming FJ, Begeot M, Dubois MP, Dubois PM 1984 Fetal rat somatotropes in vitro: effects of insulin, cortisol, and growth hormone-releasing factor on their differentiation: a light and electron microscopic study. Endocrinology 114:2107-2113 Hemming FJ, Aubert ML, Dubois PM 1988 Differentiation of fetal rat somatotropes in vitro: effects of cortisol, 3,5,3'-triiodothyronine, and glucagon, a light microscopic and radioimmunologic study. Endocrinology 123:1230-1236 Nogami H, Suzuki K, Enomoto H, Ishikawa H 1989 Studies on the development of growth hormone and prolactin cells in the rat pituitary gland by in situ hybridization. Cell Tissue Res 255:23-28 Frawley LS, Miller HA 1989 Ontogeny of prolactin secretion in the neonatal rat is regulated posttranscriptionally. Endocrinology 124:3-6 Seo H, Wunderlich C, Vassart G, Refetoff S 1981 Growth hormone response to thyroid hormone in the neonatal rat. J Clin Invest 67:569-574 Perez-Castillo A, Bernal J, Ferreiro B, Pans T 1985 The early ontogenesis of thyroid hormone receptor in the rat fetus. Endocrinology 117:2457-2461 Szabo M, Cuttler L 1986 Differential responsiveness of the somatotroph to growth hormone-releasing factor during early neonatal development in the rat. Endocrinology 118:69-73 Welsh JB, Cuttler L, Szabo M 1986 Ontogeny of the in vitro growth hormone stimulatory effect of thyrotropin-releasing hormone in the rat. Endocrinology 119:2368-2375 Rosenfeld RG, Ceda G, Wilson DM, Dollar LA, Hoffman AR 1984 Characterization of high affinity receptors for insulin-like growth factor I and II on rat anterior pituitary cells. Endocrinology
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PITUITARY GH DEVELOPMENT 114:1571-1575 26. Lund PK, Moats-Staats BM, Hynes MA, Simmons JG, Jansen M, D'Ercole AJ, Van Wyk JJ 1986 Somatomedin-C/insulin-like growth factor I and insulin-like growth factor II mRNAs in rat fetal and adult tissues. J Biol Chem 261:14539-14544 27. Cuttler L, Welsh JB, Szabo M 1986 The effect of age on somatostatin suppression of basal, growth hormone-releasing factor-stimulated, and dibutyryl adenosine 3',5'-monophosphate-stimulated GH release from rat pituitary cells in monolayer culture. Endocrinology 119:152-158 28. Melmed S, Yamashita S 1986 Insulin-like growth factor I action on hypothyroid rat anterior pituitary cells: suppression of triiodothyronine-induced growth hormone secretion and messenger ribonucleic acid levels. Endocrinology 118:1483-1490
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29. Fagin JA, Fernandez-Mejia C, Melmed S 1989 Pituitary insulinlike growth factor-I gene expression: regulation by triiodothyronine and growth hormone. Endocrinology 125:2385-2391 30. Matsuo K, Yamashita S, Niwa M, Kurihara M, Harakawa S, Izumi M, Nagataki S, Melmed S 1990 Thyroid hormone regulates rat pituitary insulin-like growth factor-I receptors. Endocrinology 126:550-554 31. Wolf M, Ingbar SH, Moses AC 1989 Thyroid hormone and growth hormone interact to regulate insulin-like growth factor-I messenger ribonucleic acid and circulating levels in the rat. Endocrinology 125:2905-2914 32. Wu SY, Klein AH, Chopra IJ, Fisher DA 1978 Alterations in tissue thyroxine-5'-monodeiodinating activity in perinatal period. Endocrinology 103:235-239
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