245 Effect of neonatal thyroidectomy on secretion in the rat

S Kitauchi, H Yamanouchi, N Hirano, S Tone´ 1 and M Shiino Department of Anatomy, Wakayama Medical College, Wakayama 640-8155, Japan and 1Department of Radiation Research, Tokyo Metropolitan Institute of Medical Science, Tokyo 133-0021, Japan (Requests for offprints should be addressed to S Kitauchi, Department of Anatomy, Wakayama Medical College, 27-kyubancho, Wakayama-shi, 640, Japan)

Abstract The influence of neonatal thyroidectomy (Tx) on GH pituitary GH cells were significantly decreased in number production was investigated by means of Northern blot 15 and 20 days after Tx. These data suggest that GH analysis. Tx resulted in a significant decrease in pituitary mRNA is transcribed, independent of hormone, GH mRNA levels after 10, 15 and 20 days. The changes in the rat anterior during early neonatal life. of pituitary GH mRNA were soon reflected in pituitary In addition, the present study ascertained that GH depen- GH content. There was, however, no significant differ- dence on thyroid hormone is acquired between the 5th ence in pituitary GH mRNA levels and GH content and 10th day of neonatal life. between Tx and sham-operated rats at 5 days old. The Journal of (1998) 157, 245–250

Introduction Materials and Methods

It is known that thyroid hormone is an important regulator Animals of growth hormone (GH) production in the rat somato- Sprague–Dawley derived rats were bred in our animal troph (Peake et al. 1973). Recent work suggests that quarters. Female and male neonatal rats were used in this thyroid hormone directly induces the transcription of the experiment. Tx was performed under anesthesia induced GH gene (Evans et al. 1982, Spindler et al. 1982, Casanova by cold temperature within 24 h of birth. After the et al. 1985, Brent et al. 1991). On the other hand, operation the animals were warmed under a tungsten lamp previous studies demonstrated that GH dependence on until they began to move actively, and then returned to thyroid hormone was not clear during the early neonatal their mothers. Controls were sham operated by only period (Coulombe et al. 1980, Seo et al. 1981). However, cutting the skin and exposing the gland. The mothers and the quantitative aspects of GH gene expression their pups were housed in individual cages under con- during neonatal hypothyroidism have not been well trolled illumination (ratio of 14 h light:10 h darkness) and investigated. In the rat, serum thyroid hormone concen- temperature (26 C). They were fed Purina Laboratory tration is low at birth and increases to adult values in ) chow with water freely available. On days 5, 10, 15 and the weaning stage (Dussault & Labrie 1975). The 20, the pups were weighed and killed by decapitation. thyroid hormone system is still immature in the neonatal Immediately after exsanguination pituitary glands were period. Is thyroid hormone really essential for GH produc- removed, and stored at 80 C until RIA and Northern tion in the neonatal rat, as in older animals? To answer " ) blot analysis. For immunocytochemistry and electron this question, we investigated the influence of neonatal microscopy, pituitary glands were fixed immediately. The thyroidectomy (Tx) on GH gene expression in the rat blood was centrifuged, and the serum was collected and by using Northern blot analysis. We also evaluated stored at 20 C. In all experiments, rats were killed the amount of pituitary GH content to compare with " ) between 1400 and 1600 h. The absence of the thyroid GH mRNA levels. Additionally, we performed a gland in Tx rats was verified at autopsy. morphometrical analysis of pituitary GH cells in parallel with the biochemical examinations using immuno- cytochemistry. Finally, we observed immunoelectron RNA extraction and Northern blot analysis microscopic profiles of GH cells at all experimental stages in order to confirm the effects of Tx on GH cells Total RNA was prepared by homogenization of pituitary morphologically. glands in guanidinium isothiocyanate followed by acid

Journal of Endocrinology (1998) 157, 245–250  1998 Society for Endocrinology Printed in Great Britain 0022–0795/98/0157–0245 $08.00/0

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phenol–chloroform extraction (Chomczynski & Sacchi were rinsed in PBS and stained by the streptavidin–biotin 1987). RNA was ethanol precipitated, redissolved in method using a HISTOFINE SABPO Kit (Nichirei Corp. sterile water and quantified by absorbance at 260 nm. Tokyo, Japan) with diaminobenzidine as a chromogen. Two µg of total RNA from one pituitary were separated Rabbit antibody against rat GH was provided by Dr K on a l% agarose gel containing formalin and trans- Wakabayashi of the Institute for Molecular and Cellular ferred onto a nylon membrane (Hybond N, Amersham Regulation, Gunma University. International plc, Amersham, Bucks, UK) by capillary The sections were visualized with a microscope action (Sambrook et al. 1989). The blot was hybridized equipped with a CCD TV camera. Ten images were with 32P-labeled rat GH cDNA. The rat GH cDNA insert captured at random for each pituitary and analyzed with a (pRGH-1, 0·8 kb (Seeburg et al. 1977)) was excised from Macintosh computer using Image 1·5 (NIH, USA; public the pBR322/Hind III site and radiolabeled with 32Pby domain). Background density points were removed by nick translation (Rigby et al. 1977) to about 108 c.p.m./µg thresholding the image. The number of the cells that DNA using a commercial kit (Takara Shuzo Co. Ltd, reacted with anti-GH antibody was counted. Kyoto, Japan). The GH cDNA probe was removed, and For immunoelectron microscopic examination, pitu- the filters were rehybridized with human ribosomal DNA itary tissues were cut into small pieces and fixed in a (5* portion, pHr21Ab (Safrany et al. 1989)) to hybridize solution consisting of 3% paraformaldehyde and 3% with 18S rRNA for the comparison of the amount of glutaraldehyde in 0·1 M cacodylate buffer (pH 7·4) for 2 h RNA in samples loaded on the gel between experimental at room temperature and then post-fixed in 0·1% osmium groups. The human ribosomal DNA was excised from tetroxide dissolved in 0·1 M cacodylate buffer for 1 h at the pUC13/EcoRI site and radiolabeled as GH cDNA. 0 )C. After routine dehydration, the tissues were em- Radioactivity on the filters was quantified using a Bio bedded in Epon-Araldyde mixture. The thin sections were Imaging Analyzer BAS 2000 (Fujix, Tokyo, Japan). The reacted by the protein A-gold method described pre- ratio of the intensity of the GH mRNA band to that of the viously (Bendayan & Zollinger 1983). Control immuno- 18S rRNA band was used to express the relative amounts cytochemical tests were carried out by substituting normal of mRNA present. Rat GH cDNA was provided by Dr rabbit serum or PBS for specific antiserum. Electron J D Baxter of the University of California, USA. Human microscopic observations were made with a Hitachi 7000 ribosomal DNA was provided by Health Science electron microscope. Research Resources Bank (Osaka, Japan). Data analysis RIA For Northern blot analysis and RIA, experiments were The pituitary glands were homogenized in 0·05 M reproduced six times. For immunocytochemistry and NaHCO3–Na2CO3 buffer (pH 10·0) and diluted with immunoelectron microscopic examination, experiments BSA-PBS. Pituitary GH content was measured by the were reproduced four times. Data are expressed as double-antibody RIA using reagents provided by the mean&.. Statistical analyses were performed by the National Hormone and Pituitary Program, Rockville, Mann–Whitney U test. We considered differences signifi- MD, USA (rat GH RIA kit, reference standard NIDDK- cant at P<0·05. rGH-RP2) and Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan (second antisera, HAC-MKA2–02GTP88). Results The intraassay and interassay coefficients of variation were 3·15 and 3·80% respectively. Serum thyroxine (T4)con- Serum T concentrations centration was measured using a commercial kit (Eiken 4 Co., Tokyo, Japan). Serum T4 concentrations were significantly decreased: 0·35&0·11 vs 1·75&0·78, 0·44&0·15 vs 4·29&1·99, 0·98&0·49 vs 8·24&2·98, 1·58&0·92 vs 5·72& Immunocytochemistry and morphometry 1·24 µg/dl (n=6, P<0·005) at 5, 10, 15 and 20 days after Pituitaries were fixed in Bouin’s fluid for 24 h at 4 )C, Tx respectively. dehydrated and embedded in paraffin. Serial frontal sec- tions (2 µm) were mounted on glass slides and deparaffin- GH mRNA levels of Tx and sham-operated rats in the ated. They were incubated in ethanol with 3% H O to 2 2 neonatal period inactivate endogenous peroxidase activity, and immersed in 10% normal goat serum in 0·9% NaCl–0·01 M phos- In the early neonatal period, pituitary GH mRNA content phate buffer, pH 7·5, at room temperature for 30 min. was not affected by Tx. As shown in Fig. 1, no significant Then, rabbit antibody against rat GH was applied to the differences were found between Tx and sham-operated sections. After overnight incubation at 37 )C, the sections pups in the amount of pituitary GH mRNA at the 5th day.

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Figure 1 Effect of Tx on the expression of pituitary GH mRNA. Pituitary GH mRNA and 18S rRNA in Tx and sham-operated (S) neonatal rats on days 5, 10, 15 and 20 were detected by Northern blot analysis as described in Materials and Methods. Figure 2 Effect of Tx on the amount of pituitary GH mRNA and (A) Representative autoradiograms for GH mRNA and 18S rRNA. GH content in rats at ages 5–20 days. (A) The amount of GH Each lane contained 2 µg total RNA prepared from a single mRNA per pituitary (relative value). The amount of GH mRNA was pituitary. The positions of the 18S and 28S rRNA subunits and the calculated by multiplying the GH mRNA level (data from Fig. 1B) size of the GH transcripts (1 kb) are indicated on the right. by the amount of total RNA per pituitary (data not shown). The (B) Relative expression of GH mRNA. Extent of hybridization with 5-day sham-operated value corresponds to 1·0. Each bar the 32P-labeled cDNA probe for GH mRNA and ribosomal DNA represents the mean&S.D. of six independent analyses. *P<0·05, probe was quantified by scanning with a densitometer. The levels **P<0·01, ***P<0·005 compared with sham-operated values of GH mRNA were expressed as signal intensity of GH mRNA (Mann–Whitney U test). (B) Effect on pituitary GH. Pituitary GH normalized for that of 18S rRNA (GH mRNA/18S rRNA ratio). content was measured by double-antibody RIA. Each bar Data are expressed as percent of the 5-day sham-operated value. represents the mean&S.D. of six independent analyses. **P<0·01 Each bar represents the mean&S.D. of six independent analyses. compared with sham-operated values (Mann–Whitney U test). **P<0·01 compared with sham-operated values (Mann–Whitney U test).

compared with pituitary GH mRNA content (Fig. 2A A significant decrease was first observed at the 10th day. and B). At the 5th day, no significant differences were Tx resulted in a significant decrease of pituitary GH found between Tx and sham-operated pups in the amount mRNA levels at the 10, 15 and 20th days. The amount of of pituitary GH mRNA and GH content. However, pituitary GH mRNA in the Tx rats decreased to levels significant decreases were observed 10, 15 and 20 days approximately 19, 18 and 16% of the control at the 10, 15 after Tx in the amount of both contents. The amount of and 20th days respectively. pituitary GH mRNA contents in Tx rats decreased to levels approximately 21, 18 and 13% of the control at the 10, 15 and 20th days respectively, and the amount of Pituitary GH mRNA and GH content pituitary GH content in Tx rats decreased to levels Differences of pituitary GH content between Tx and approximately 24, 17 and 28% of the control at the same sham-operated rats changed in the same manner when days respectively.

Journal of Endocrinology (1998) 157, 245–250

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Figure 3 Changes of body weights in rats at ages 5–20 days after Figure 5 An example of a GH cell 15 days after sham operation Tx. Data are expressed as mean&S.D. of six independent analyses. in the rat. The tissue was immunocytochemically reacted with *P<0·05 compared with sham-operated values (Mann–Whitney antiserum to rat GH. The inset is a higher magnification of the U test). area indicated by the arrow. One can see gold particles, indicating GH distribution, on the secretory granules.

Figure 4 Effect of Tx on the number of pituitary GH cells in rats at ages 5–20 days. Sections were visualized with a microscope fitted with a CCD TV camera. Ten images were captured at random for Figure 6 An example of a GH cell 15 days after Tx in the rat. The each pituitary and analyzed with a Macintosh computer using inset is a higher magnification of the area indicated by the arrow. Image 1·5 (NIH; public domain). Data are expressed as One can see smaller and lesser numbers of secretory granules mean&S.D. of four independent analyses. *P<0·05 compared with than was seen in sham-operated animals (Fig. 5). sham-operated values (Mann–Whitney U test).

Body weight observed on the 15th day. The number of pituitary GH cells in the Tx rats decreased to levels approximately The body weights of Tx and sham-operated rats are 60 and 61% of the control at the 15 and 20th days illustrated in Fig. 3. A significant difference was first respectively. observed at the 20th day between Tx and sham-operated rats. The body weight of Tx rats decreased to a level approximately 71% of the control at the 20th day. Electron microscopy The ultrastructural profiles of GH cells after Tx showed Morphometry rather depressed structures. These features of GH cells Figure 4 shows the number of pituitary GH cells in became clear on the 15th day after Tx; that is the size of pituitary glands. At the 5th and 10th day, no significant GH cells and the size and number of their secretory differences were found between Tx and sham-operated granules were reduced. The organelles of GH cells were pups in the number of pituitary GH cells. A significant also morphologically inactive as compared with those of decrease in the number of pituitary GH cells was first sham-operated rats (Figs 5 and 6).

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Discussion were slight rises in serum T4 concentrations at the 15th and 20th days. Although we verified the absence of The dependence of GH production on thyroid hormone remaining thyroid glands at autopsy in Tx rats, it may be has been previously demonstrated in the adult rat and in possible that residual minute thyroid tissue occasionally experiments using rat somatotropic cell lines (Peake et al. remaining unexpectedly after the operation regenerated 1973, Hervas et al. 1975). More recent works stated that and produced thyroid hormone at the 15th and 20th days. thyroid hormone acted directly on the thyroid hormone The first 3 weeks of life in the rat are characterized by response element of the GH gene and increased its the maturation of the thyroid hormone system (Walker transcriptional activity (Nyborg et al. 1984, Yaffe& et al. 1977). Serum thyroid hormone concentrations are Samuels 1984). It is generally accepted that, in the adult low at birth and increase rapidly to weaning, when they rat, thyroid hormone plays an important role in the reach adult values (Dussault & Labrie 1975). In this study regulation of GH gene expression. However, the effect of thyroid hormone replacement controls were not included. thyroid hormone on GH in the neonatal period is contro- Hypothyroidism during development has a number of versial. Coulombe et al. (1980) and Seo et al. (1981) physiological effects including the failure to thrive. In reported that in early neonatal life thyroid hormone turn, this may have an effect on the GH axis. This deficiency did not affect the accumulation of GH when possibility cannot be entirely excluded. Consequently the measured by RIA. Those studies suggested that the effects of the thyroid hormone on GH production were regulation of the GH gene by thyroid hormone is not not established in the early neonatal period, such as in the established in the early neonatal period. On the other 5 day old. hand, Rodriguez et al. (1995a) recently showed significant decreases of GH mRNA and GH in pituitaries of hypo- thyroid rats at the 19th fetal day and concluded that Acknowledgements thyroid hormone regulates GH gene at an early stage of pituitary development. We express our thanks to Dr John D Baxter for supplying In the present study, we have demonstrated that thyroid GH cDNA. We also wish to express our thanks to Dr hormone deficiency did not affect the amount of pituitary Edward G Rennels, Professor Emeritus, the University of GH mRNA and pituitary GH content in early neonatal Texas Health Science Center, for his kind advice in life, such as in the 5 day old. GH dependence on thyroid preparing the manuscript. hormone became obvious at the 10th day. Thyroid hor- mone deficiency was associated with a diminution in the amount of the pituitary GH mRNA and GH content. References There may be other factors that affect GH mRNA concentrations in this study. Rodriguez et al. (1995b) BendayanM&Zollinger M 1983 Ultrastructural localization of showed reduction in GH mRNA in food-restricted, antigenic sites on osmium-fixed tissues applying the protein A-gold fasting and diabetic rats, as in thyroidectomized rats. In our technique. Journal of Histochemistry and Cytochemistry 31 101–109. Brent GA, Williams GR, Harney JW, Forman BM, Samuels HH, study, all rats had food freely available and the changes of Moore DD & Larsen PR 1991 Effects of varying the position of body weight were not obvious in Tx rats. 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Hervas F, Morreale de EscobarG&EscobardelReyF1975 Rapid Sambrook J, Fritsch EF & Maniatis T 1989 Extraction, purification, effects of single small doses of -thyroxine and triiodo--thyronine and analysis of messenger RNA from eukaryotic cells. In Molecular on growth hormone, as studied in the rat by radioimmunoassay. Cloning, edn 2, pp 7·37–7·52. Ed C Nolan. Cold Spring Harbor: Endocrinology 97 91–101. Cold Spring Harbor Laboratory Press. Nyborg JK, Nguyen AP & Spindler SR 1984 Relationship between Seeburg PH, Shine J, Martial JA, Baxter JD & Goodman HM 1977 thyroid and glucocorticoid hormone receptor occupancy, growth Nucleotide sequence and amplification in bacteria of structural gene hormone gene transcription, and mRNA accumulation. Journal of for rat growth hormone. Nature 270 486–494. Biological Chemistry 259 12377–12381. Seo H, Wunderlich C, VassartG&Refetoff S1981Growthhormone Peake GT, Birge CA & Daughaday WH 1973 Alterations of responses to thyroid hormone in the neonatal rat: resistance and radioimmunoassayable growth hormone and prolactin during anamnestic response. Journal of Clinical Investigation 67 569–574. hypothyroidism. Endocrinology 92 487–493. Spindler SR, Mellon SH & Baxter JD 1982 Growth hormone gene Rigby PWJ, Dieckmann M, RhodesC&BergP1977 Labeling transcription is regulated by thyroid and glucocorticoid hormones in deoxyribonucleic acid to high specific activity in vitro by nick cultured rat pituitary tumor cells. Journal of Biological Chemistry 257 translation with DNA polymerase I. Journal of Molecular Biology 113 11627–11632. 237–251. Yaffe BM & Samuels HH 1984 Hormonal regulation of the growth hormone gene. Relationship of the rate of transcription to the level Rodriguez-Garcia M, Jolin T, SantosA&Perez-Castillo A 1995a of nuclear thyroid hormone–receptor complexes. Journal of Biological Effect of perinatal hypothyroidism on the developmental regulation Chemistry 259 6284–6291. of rat pituitary growth hormone and thyrotropin genes. Walker P, Dussault JH, Alvarado UG & Dupont A 1977 The Endocrinology 136 4336–4350. development of the hypothalamo–pituitary axis in the neonatal rat: Rodriguez M, Rodriguez F, Jolin T & Santisteban P 1995b hypothalamic and pituitary and serum growth hormone Comparative effects of food restriction, fasting, and concentrations. Endocrinology 101 782–787. thyroidectomy on growth hormone and thyrotropin gene expression in the rat pituitary. European Journal of Endocrinology 133 110–116. Safrany G, Kominami R, Muramatsu M & Hidvegi EJ 1989 Received 28 April 1997 Transcription of human ribosomal DNA may terminate at multiple Revised manuscript received 14 October 1997 sites. Gene 79 299–307. Accepted 25 November 1997

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