Requirement of Thyrotropin-Releasing Hormone for the Postnatal Functions of Pituitary Thyrotrophs: Ontogeny Study of Congenital Tertiary Hypothyroidism in Mice

Nobuyuki Shibusawa, Masanobu Yamada, Junko Hirato Tuyoshi Monden, Teturou Satoh and Masatomo Mori

First Department of Internal Medicine (N.S., M.Y., T.M., T.S., M.M.) First Department of Pathology (J.H.) Gunma University School of Medicine Maebashi 371, Japan


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We recently reported that TRH-deficient mice showed characteristic tertiary hypothyroidism. In the present study, we investigated how this tertiary hypothyroidism occurred particularly in pre- and postnatal stages. Immunohistochemical analysis revealed a number of TSH-immunopositive cells in the TRH-/- pituitary on embryonic day 17.5 and at birth. The mutant pituitary at birth in pups born from TRH-deficient dams also showed no apparent morphological changes, indicating no requirement of either maternal or embryonic TRH for the development of pituitary thyrotrophs. In contrast, apparent decreases in number and level of staining of TSH-immunopositive cells were observed after postnatal day 10 in mutant pituitary. Similar decreases were observed in the 8-week-old mutant pituitary, while no apparent changes were observed in other pituitary hormone-producing cells, and prolonged TRH administration completely reversed this effect. Consistent with these morphological results, TRH-/- mice showed normal thyroid hormone levels at birth, but the subsequent postnatal increase was depressed, resulting in hypothyroidism. As expected, TSH content in the TRH-/- pituitary showed a marked reduction to only 40% of that in the wild type. Despite hypothyroidism in the mutant mice, both the pituitary TSHß and {alpha} mRNA levels were lower than those of the wild-type pituitary. These phenotypic changes were specific to the pituitary thyrotrophs. These findings indicated that 1) TRH is essential only for the postnatal maintenance of the normal function of pituitary thyrotrophs, including the normal feedback regulation of the TSH gene by thyroid hormone; 2) neither maternal nor embryonic TRH is required for normal development of the fetal pituitary thyrotroph; and 3) TRH-deficient mice do not exhibit hypothyroidism at birth. Moreover, reflecting its name, TRH has more critical effects on the pituitary thyrotrophs than on other pituitary hormone-producing cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TRH is a hypothalamic tripeptide, which is known to be a major stimulator of synthesis and secretion of TSH in the anterior pituitary gland (1 ). However, limited information is available regarding its physiological and developmental roles in the pituitary thyrotrophs. In rodents, the administration of TRH to the dam stimulates fetal pituitary and thyroid function (2 3 ). Employing primary cultures of embryonic pituitaries, a similar conclusion was reached by Heritier et al. (4 ) who suggested that TRH may regulate pituitary differentiation at the early embryonic stage. However, fetal encephalectomy has been found not to affect the pituitary-thyroid function (5 ). The anencephalic human fetus also showed normal serum TSH values and preserved secretory activity of pituitary thyrotrophs, suggesting that embryonic development of pituitary thyrotrophs is independent of hypothalamic TRH (6 7 ). In a subsequent study, however, the same authors observed that TRH values in the cerebrospinal fluid were similar to those of normal subjects. Thus, TRH of extrahypothalamic origin might be capable of reaching the pituitary by an alternate route (8 ). Moreover, we reported expression of the prepro-TRH gene in the human placenta in which TRH and TRH-related peptides are synthesized (9 ). As the placenta is permeable to TRH, maternal TRH may affect fetal development of pituitary thyrotrophs (10 11 ). As a result of these problems and lack of an appropriate animal model for laboratory studies, it has not been possible to determine conclusively and directly the contribution of TRH to embryonic development of pituitary thyrotrophs.

Tertiary (hypothalamic) and secondary (pituitary) hypothyroidism are categorized together into central hypothyroidism because of the difficulty of differential diagnosis between these two disorders (12 ). Although some patients with tertiary hypothyroidism have been reported, most of these cases were associated with other hormone deficiencies or brain injury, and no patients with isolated TRH deficiency have yet been reported (13 14 15 16 ). Therefore, the natural course of congenital TRH deficiency is unclear. Recently, a patient with compound heterozygosity for TRH receptor gene mutations was reported, and this patient did not have functional TRH receptors resulting in hypothyroidism (17 ). However, he was not diagnosed as having hypothyroidism by neonatal screening because of his normal TSH levels (15 mU/liter). His sole manifestation was a short stature at 8.9 yr of age. It is, therefore, expected that patients with isolated TRH deficiency may show similar manifestations to a patient with TRH receptor mutations. In fact, Niimi et al. (14 ) reported a 4.3-yr-old girl diagnosed with congenital tertiary hypothyroidism by several clinical tests whose only manifestation was short stature, and her hypothyroidism was not detected by neonatal screening for TSH.

We recently cloned the human and mouse prepro-TRH genes and generated TRH-deficient mice using homologous recombination in embryonic stem cells (18 19 20 21 ). These TRH-deficient mice were viable, developed normally, and were fertile. However, adult TRH-/- mice showed characteristic phenotypes including apparent hyperglycemia and tertiary hypothyroidism with mild elevation of serum TSH level. The availability of these TRH-/- mice will be useful for determining the role of TRH in the regulation of pituitary thyrotroph proliferation and to investigate the developmental course and molecular pathophysiology of congenital TRH deficiency and tertiary hypothyroidism, which could not be carried out in humans.

In the present study, we studied ontogeny of thyrotroph abnormalities and corresponding serum thyroid hormone levels and found that isolated TRH deficiency did not cause hypothyroidism at birth, but mild hypothyroidism developed within a few days.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Requirement of TRH for Postnatal Maintenance of Thyrotrophs but Not for Their Normal Embryonic Development
Figure 1Go shows the results of ontogeny study of TSH-producing cells in the wild-type and TRH-/- pituitary. TSHß-immunopositive cells were observed throughout the anterior lobe at embryonic day 17.5 (E17.5) in both the wild-type and TRH-/- pituitary (38.0 ± 6.1, 34.7 ± 3.9, means ± SE, numbers of thyrotrophs per section of the wild-type and TRH-/- pituitary; n = 4) (Fig. 1Go, A and B). Thyrotrophs increased in number and staining intensity until birth, and a significant number of thyrotrophs was observed at birth [postnatal day 0.5 (P0.5)] in the pituitary of the wild-type mice (70.0 ± 9.2, n = 9; 77.2 ± 9.9, n = 6) (panels C and D). Despite the lack of TRH, the staining and number of TSH-immunopositive cells in the TRH-/- pituitary were indistinguishable from those of wild-type controls, indicating the preservation of normal proliferation of pituitary thyrotrophs. However, marked decreases were seen in staining and number of TSH-immunopositive cells at P10 in the TRH-/- pituitary as compared with the wild-type controls (44.2 ± 4.9, n = 5; 18.5 ± 4.6, n = 6; numbers of thyrotrophs/320 x 210 µm field of the wild-type and TRH-/- pituitary section, P < 0.005)(E and F). Similar decreases were observed at 21 days (67.1 ± 5.7, n = 6; 44.0 ± 7.2, n = 5; P < 0.05)(G and H) and 8 weeks (69.9 ± 3.6, n = 5; 43.4 ± 5.4, n = 5; P < 0.05) (I and J) after birth, and this change in the 8-week pituitary was completely reversed by prolonged TRH administration (61.7 ± 9.8, n = 5)(K). These findings clearly demonstrated that there is no requirement of embryonic TRH for thyrotroph development, but this hormone is essential for the postnatal maintenance of normal thyrotroph function.



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Figure 1. Ontogeny of the Pituitary TSH-Immunopositive Cells in the Wild-Type and TRH-/- Mice

A number of TSH-immunopositive cells were observed in both the wild-type and TRH-/- pituitary on E17.5 and P 0.5 (A, B, and C, D). Marked decreases were observed in staining and number of TSH-immunopositive cells in TRH-/- pituitary after P10 compared with the wild-type pituitary (P10, E and F; P21, G and H; and 8-week, I and J). Prolonged administration of TRH using an osmotic pump reversed this change (K). +/+ Represents the wild-type, and -/- represents the homozygous pituitary. Magnification, x200 except for panels C and D (x100).

 
Maternal TRH Is Also Not Required for Embryonic Thyrotroph Development
As no morphological changes were seen in the pituitary thyrotrophs in TRH-/- embryos, maternal TRH may be involved in embryonic thyrotroph development. Therefore, we examined the TRH-/- pituitary in pups born from TRH-/- dams. As shown in Fig. 2Go, the number and level of staining of TSH-immunopositive cells were similar to those of the wild-type pituitary (70.6 ± 8.4, n = 5), indicating that neither maternal nor embryonic TRH is required for normal development of pituitary thyrotrophs.



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Figure 2. Maternal TRH Did Not Affect Embryonic Thyrotrophs (P0.5).

Thyrotrophs were intact on P0.5 in the TRH-/- mice born from TRH-/- dams. Magnification, x200.

 
Hypoplasia Occurred Specifically in the TRH-Deficient Thyrotrophs but Not in Other Pituitary Hormone-Producing Cells
To evaluate morphological changes of other pituitary hormone-producing cells in TRH-deficient mice, immunocytochemical analysis was performed on pituitary PRL-, GH-, LH-, FSH-, and ACTH-producing cells using specific antibodies. In contrast to the hypoplasia observed in the mutant pituitary thyrotrophs, a similar staining pattern of all other pituitary hormone-producing cells examined was observed in the 8-week-old TRH-deficient pituitary as compared with those in the wild-type (Fig. 3Go).



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Figure 3. Immunohistochemical Analysis of PRL-, GH-, LH-, FSH-, and ACTH-Producing Cells

No apparent changes were observed in the lactotrophs of the 8-week wild-type or TRH-/- pituitary (A and B), GH (C and D), LH (E and F), FSH (G and H), and ACTH (I and J)-producing cells.

 
Ontogeny of Thyroid Hormone Levels in TRH-/- Mice
We previously reported mild hypothyroidism in the 8-week-old TRH-/- mice showing approximately 50~60% of the normal thyroid hormone level. To assess thyroid hormone level at early fetal stage, which was expected to be lower than that in adults, we first examined whether the filter method could be used for measurement of thyroid hormone level. A preliminary experiment showed a good correlation between serum free T4, total T4 and free T4 levels determined by the filter method (data not shown). As shown in Table 1Go, thyroid hormone levels at birth (P0.5) in TRH-/- mice could be determined by the filter method and were equivalent to those of the wild-type controls. Furthermore, the thyroid hormone level in mice born from TRH-/- dams was also similar to that in controls (0.41 ± 0.05 ng/dl, n = 5), indicating no effect of maternal TRH on fetal thyroid hormone level. The thyroid hormone level then increased markedly by postnatal day 10 (~6-fold) in the wild-type mice. However, TRH-/- mice showed a lesser increase (~3-fold) at 5 days old, reaching a level of 68.8% of the normal thyroid hormone level. A similar decrease was also observed in 10-day-old TRH-/- mice, and the level was 28.3% of that in controls at 21 days old just before weaning. At 8 weeks old, the thyroid hormone level in TRH-/- mice was 50.3% of that in controls, and this decrease was completely reversed by prolonged TRH administration using an osmotic pump.


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Table 1. Thyroid Hormone Levels in 0.5, 5, 10, and 21 Days and 8 Weeks after Birth in Wild-Type and Homozygous Mice

 
Decrease in TSH Content, and TSHß and -{alpha} mRNA Levels in the TRH-/- Pituitary
To understand the mechanism of postnatal hypoplasia of pituitary thyrotrophs, we measured TSH content by specific RIA and the levels of RNAs for the {alpha}- and ß-chains of TSH by Northern blot analysis. As shown in Fig. 4AGo, unexpectedly the protein content of the mutant pituitary was higher than that in the wild-type (wild-type, 167.4 ± 10.2 µg/pituitary, n = 7; homozygotes, 210.8 ± 10.2, n = 11; P < 0.01). Although the mechanism of this increase is unclear at present, a significant decrease in TSH content was observed in the TRH-/- pituitary (wild-type, 61.7 ± 7.8 µg/pituitary, n = 7; homozygotes, 21.9 ± 2.4, n = 11; P < 0.01) (Fig. 4BGo).



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Figure 4. Protein and TSH Content in the Wild-Type and TRH-/- Pituitary

The protein content in the TRH-/- pituitary was higher than that of the wild-type, but TSH content was significantly reduced to approximately 40% of the control (P < 0.01).

 
Northern blot analyses showed single TSH {alpha} and ß mRNA bands of approximately 0.9 kb and 0.75 kb, respectively, in both the wild-type and TRH-/- pituitaries (Figs. 5Go and 6Go). The experimental hypothyroidism in the wild-type mice induced by MMI treatment increased TSHß mRNA levels by approximately 3-fold (Fig. 5AGo). In contrast, in the simple TRH-/- mice with the same thyroid hormone level as the experimental hypothyroid mice the TSH ß mRNA level was reduced to 79.1 ± 0.7% of that in the wild-type pituitary (Fig. 5BGo). Similarly, TSH{alpha} mRNA level in the TRH-/- mice was reduced to 71.8 ± 5.0% of that in the euthyroid mice, while the experimental hypothyroid wild-type mice showed a 2-fold increase (Fig. 6Go, A and B).



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Figure 5. Northern Blot Analysis of TSHß mRNA Level in the 8-Week Wild-Type and TRH-/- Pituitary

A, Experimental hypothyroidism induced by treatment with MMI induced an approximately 3-fold increase in TSHß mRNA level as compared with that of the control. B, TSHß mRNA level in the TRH-/- pituitary was reduced to approximately 79% of that in the wild-type controls. C, Ethidium bromide staining of rRNA confirmed equal application of each total RNA.

 


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Figure 6. Northern Blot Analysis of TSH{alpha} mRNA Levels in the 8-Week Wild-Type and TRH-/- Pituitary

A, Experimental hypothyroidism induced an approximately 2-fold increase as compared with the control. B, TSH{alpha} mRNA level was reduced to approximately 72% of that in the control wild-type pituitary. We stripped the probe from the filter which hybridized with the TSHß probe and then used the same filter for detection of TSH{alpha} mRNA signals.

 
Specific Effect of TRH Deficiency on the Pituitary TSH mRNA Levels
To evaluate the specificity of the effect of TRH deficiency on the pituitary thyrotrophs, we examined mRNA levels of other pituitary hormones including LHß, FSHß, POMC, PRL, and GH in the 8-week mutant pituitary. Northern blot analysis showed a single hybridization signal of each of LHß, FSHß, POMC, PRL, and GH mRNA at approximately 0.7, 1.7, 1.2, 1.0 ,and 1.0 kb, respectively, in both the wild-type and mutant pituitary (data not shown). As shown in Fig. 7Go, no apparent changes were observed in the LHß, FSHß, or POMC mRNA levels of the mutant pituitaries as compared with those in the wild-type controls (105.1 ± 1.6%, 100.8 ± 2.4%, 113.3 ± 3.4% of the controls; n = 6). In contrast, significant decreases of PRL and GH mRNA levels (63.4 ± 7.7% and 73.0 ± 1.1% of the controls, n = 6; P < 0.01) were observed in the mutant pituitary. However, these decreases were reversed to the normal levels by thyroid hormone replacement (115.9 ± 12.0%, n = 5 and 98.3 ± 3.7% of the controls, n = 10), indicating that the decreases in PRL and GH mRNA levels in the mutant mice were due to hypothyroidism rather than lack of TRH.



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Figure 7. mRNA Levels of LHß, FSHß, POMC, PRL, and GH in the 8-Week Wild-Type and TRH-/- Pituitary

The data are indicated as percentages of mRNA level of the mutant pituitary compared with that in the wild-type mice as 100%. +T4 indicates that TRH-/- mice were supplemented by thyroid hormone injection as described in Materials and Methods. There were no significant changes in LHß, FSHß, or POMC mRNA level between the wild-type and mutant pituitaries. Although PRL and GH mRNA levels of the mutant pituitary were decreased as compared with those in the wild-type pituitary, these decreases were reversed by thyroid hormone replacement.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The TRH-/- mice provided a good model in which to investigate the contribution of TRH to pituitary thyrotroph development. The demonstration of the presence of intact thyrotrophs in the TRH-/- mice at birth provided direct evidence that TRH is not physiologically required for the proliferation or differentiation of embryonic pituitary thyrotrophs. We further confirmed this result by observing thyrotrophs in the TRH-/- pups born from TRH-/- dams. These findings excluded the possibility of maternal contribution of TRH to the development of embryonic thyrotrophs. However, after birth marked decreases in staining and numbers of TSH-immunopositive cells were apparent. These findings clearly demonstrated the importance of TRH in maintenance of normal postnatal functions of the pituitary thyrotrophs.

It is of interest whether other hypothalamic hormones including CRF, GnRH, and GH releasing hormone (GRH) are involved in regulation of proliferation and differentiation of different pituitary cells. Mutant and gene knockout mice bearing specific deletions in the genes for these peptides or corresponding receptors have been reported. Adult CRF and CRF receptor-1 (CRFR1) knockout mice showed no abnormalities in the pituitary ACTH-producing cells, indicating no requirement of CRF for embryonic or postnatal function of ACTH-producing cells in the pituitary (22 23 ). In contrast, adult GRH receptor-deficient mice (litt/lit dwarf mice) showed hypoplasia of somatotrophs (24 ). Lack of functional GRH receptors disturbed the postnatal generation of caudomedial complement of proliferating somatotrophs. In GnRH-deficient mice (hypogonadal, hpg), hypoplasia of the pituitary gonadotrophs and hypogonadism occurred, and transplantation of the brain graft containing GnRH neurons reversed these changes (25 ). Similarly to the results observed in TRH-/- mice, these abnormalities were produced after birth. Therefore, none of these hypothalamic peptides appeared to be involved in embryonic pituitary proliferation or development.

Defects in TRH function resulted in approximately 60% reduction of the TSH content at 8 weeks of age, reflecting the postnatal decrease in the staining of TSH-immunopositive cells. Northern blot analysis showed that despite their hypothyroidism, both TSHß and -{alpha} mRNA levels in the TRH-/- mice were lower than those in the wild-type controls. Considering the mild elevation of the serum TSH level in TRH-/- mice, we speculated that the decreased TSH content in the pituitary may be due, at least in part, to an insufficient increase in TSH synthesis to compensate for the augmented release of TSH from the pituitary. These findings also suggested that TRH is essential for the normal regulation of TSHß and -{alpha} gene expression by thyroid hormone in vivo, although thyroid hormone is considered to be the most potent regulator of the TSH gene. Wondisford et al. (26 ) demonstrated cross-talk of TRH and thyroid hormone through the AP-1 binding site and the nearby negative thyroid hormone response element on the TSHß gene. They reported that TRH is functionally required for complete regulation of TSHß gene expression by thyroid hormone (26 ). This result and our observations suggested that TRH may alter the set point or sensitivity of thyroid hormone-mediated negative feedback of the TSHß and -{alpha} genes at the transcriptional level.

As mentioned above, TRH-/- mice were euthyroid at birth but developed hypothyroidism within a few days. During the second postnatal week, circulating thyroxine concentration has been shown to rise significantly to the adult level (27 28 29 ). Indeed, in the present study this increase in thyroid hormone level was observed in the wild-type mice. In contrast, TRH-/- mice showed an insufficient postnatal increase in the serum thyroid hormone level, resulting in hypothyroidism. Rodents are immature at birth and undergo major developmental changes at the end of the suckling period, and thyroxine is an important coordinator of this maturational event in many organ systems (30 ). We previously reported that TRH-/- mice showed growth retardation particularly after weaning, and thyroid hormone replacement almost completely reversed this effect. Furthermore, treatment of suckling rats with T4 has been reported to facilitate early weaning, and hypothyroidism apparently delays weaning (31 32 ). Therefore, the hypothyroidism in the TRH--/- mice may induce impairment of the suckling-weaning transition causing growth retardation. Further studies are required to determine the precise mechanism responsible for the growth retardation in TRH-/- mice. However, these findings indicated that TRH is an important factor in the postnatal increase in thyroid hormone level.

TRH is also known to be a potential stimulator of PRL secretion from the pituitary in human and other species (33 34 ). Furthermore, in rodents, TRH also stimulates GH secretion from the pituitary gland, and we recently reported that TRH receptor subtype 1 mRNA was expressed in both rat lactotrophs and somatotrophs (35 36 ). However, although there were decreases in PRL and GH mRNA levels in TRH-/- pituitary, these were corrected by thyroid hormone replacement, indicating that these decreases were not a direct effect of TRH. In addition, in our morphological and Northern blot analysis of other pituitary hormones, no significant effect induced by lack of TRH was observed on pituitary hormone-producing cells other than thyrotrophs in the TRH-deficient mice. These results clearly demonstrated the specificity of TRH action on the pituitary thyrotrophs.

In conclusion, we demonstrated here specific postnatal roles of TRH in the regulation of the pituitary thyrotroph functions driving a significant rise in serum thyroid hormone level during the early neonatal period.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals and Genotyping
The procedures used for animal care and use in this study were approved by the Review Committee on Animal Use at Gunma University, Maebashi, Japan. Animals were maintained under a 12-h light and 12-h dark schedule (light on at 0600 h) and fed laboratory chow and tap water ad libitum. All experiments were performed between 0900 and 1100 h. Animals bearing the targeted prepro-TRH allele were interbred, and the F2 offspring were analyzed by PCR and Southern blot analysis as described previously (20 ). Embryos were obtained from dated pregnancies. The day on which a vaginal plug was detected was considered E0.5. The TRH-/- mice were given TRH at 1.0 µg/kg/day (kindly provided by Tanabe Pharmaceutical Co., Osaka, Japan) using a continuous miniosmotic pump (Alzet Model 2002, Alza Corp., Palo Alto, CA) over a period of 14 days. Experimental hypothyroid mice were generated by providing drinking water containing 0.02% 2-mercapto-1-methyl-imidazole, methimazole (Sigma, St. Louis, MO) for 25 days. The mice with T4 replacement received 1.2 µg T4/100 g body weight subcutaneously for 10 days before the experiment.

Immunohistochemistry
The whole head at E17.5 and P0.5, and the pituitary removed from P10, P21, and at 8 weeks were fixed in 10% (wt/vol) phosphate-buffered formalin at room temperature overnight and embedded in paraffin wax. Sagittal sections through the pituitary were cut serially at a thickness of 10 µm. The sections were pretreated with 3% hydrogen peroxide for 15 min at room temperature, rinsed in PBS and incubated at 4 C overnight with rabbit antirat TSHß-IC-1 antibody (AFP-1274789, NIDDK), rabbit antimouse PRL (AFP-181078 a kind gift from Dr. Parlow, Harbor-UCLA Medical Center, Torrance CA), monkey antirat GH (AFP411S, NIDDK), rabbit antimouse LH (A582/R4H, Biogenesis), rabbit antimouse FSH (A581/R4H, Biogenesis, Poole, UK) or rabbit antihuman ACTH (DAKO Corp., Kyoto, Japan). Immunohistochemical staining was then visualized by the biotin-streptavidin method as described previously (20 ). The experiment were repeated at least three times using individual pituitaries. To compare the number of TSH-immunopositive cells between each pituitary, the numbers of positive cells were counted in a section of the pituitary or in the field of 320 x 210 µm. To control the variable distribution of cell types in the pituitary, the sections with the largest number of positive cells were selected among those from the middle of sagittal sections of the anterior pituitary.

Serum T4 Level Estimation
Serum T4 levels were determined by double antibody RIA (Dainabot Co., Ltd., Tokyo, Japan). For estimation of thyroid hormone levels on P0.5 and P5.0, the blood from the body after decapitation was applied to filter paper (Toyo filter paper no.1) and allowed to dry. To perform the assay, 3-mm dots were punched out from the filters and eluted with 0.2 ml of 0.05 M phosphate buffer (pH 8.0) containing 0.01 mg of 8-anilinonaphthalene sulfonic acid in 10 x 70 mm plastic tubes. The recovery of free T4 after elution was found to be more than 95%. All assays were performed in duplicate and the assay sensitivity was 0.10 ng/dl. The assay was performed as recommended by Eiken Immunochemical Laboratory (Tokyo, Japan). A previous study indicated that a single 3-mm dot contained approximately 5 µl of serum (37 ).

RIA of TSH in the Pituitary
After cervical dislocation, the removed pituitary was extracted with 1 ml of PBS using a Teflon homogenizer. After centrifugation, the supernatant was diluted 100-fold, and TSH was measured by specific mouse TSH RIA with mouse TSH/LH reference (AFP51718MP), mouse TSH antiserum (AFP98991), and rat TSH antigen (NIDDK-rTSH-I-9), all of which were obtained from Dr. A. F. Parlow, Harbor-UCLA Medical Center (Torrance CA). The detection limit of the assay was 15 ng TSH/ml. The intraassay variation was less than 6%, and all samples were measured in one series to prevent interassay variation. Protein content was measured by the Bradford method.

Northern Blot Analysis of TSHß, TSH{alpha}, LHß, FSHß, POMC, PRL, and GH mRNA in the Pituitary
Total RNA (20 µg) was extracted from the pituitaries by a modified acid-phenol method, resolved through a 1.2% formaldehyde agarose gel and transferred onto nylon membranes as reported previously (38 ). The membranes were hybridized under high stringency with the indicated probe. After overnight hybridization, the membrane was washed twice in 2x SSC and 1% SDS at room temperature for 5 min and twice in 0.1x SSC and 1% SDS at 68 C for 15 min, and then exposed to XAR-5 x-ray film (Kodak, Rochester, NY) for 16 h. The hybridization bands were quantitatively measured using Adobe Photoshop 4.0 (Adobe Systems Corp., San Jose, CA) and NIH Image (Scion Corp., Frederick, MD). Mouse TSHß, TSH{alpha}, LH, FSH, POMC, PRL, and GH cDNAs were generated by PCR with the following sets of primers and mouse pituitary cDNA as the template. TSHß sense primer 5'-gctgggtattgtatgacacgggata-3' and antisense primer 5'-ttacacttgccacacttgcagctta-3'; TSH{alpha} sense primer 5'-tgtccatgttcctgca tattcttca-3' and antisense primer, 5'-gtggcctccgaggtaatattctttg-3'; LH sense primer 5'-tcaccaccagcatc-tgtgcc-3' and antisense primer 5'-aggg ctacaggaaaggagac-tatgg-3'; FSH sense primer 5'-gctgtttacttcccagaccatgatg-3' and antisense primer 5'-gtattgatgcttatgcagaaacggc-3'; POMC sense primer 5'-gctcctactccatggagcacttcc-3' and antisense primer 5'-ctcttgaactctaggggaaaggcct-3'; PRL sense primer 5'-ggctacacctgaaga caaggaacaa-3' and antisense primer 5'-tgttcctcaatctctttggctcttg-3'; GH sense primer 5'-gattttcaccaacagcctgatgttc-3' and antisense primer 5'-ttggcgtcaaacttgtcataggttt-3'. These cDNAs encompassed the region between nucleotides 142 and 326 of TSHß cDNA from the translational start site; i.e. 44–257 of TSH{alpha}, 146- 317 of LHß, 52–201 of FSHß, 271–394 of POMC, 276–452 of PRL, and 360–518 of GH cDNA, respectively. All of these PCR products were subcloned into the pGEMT vector (Promega Corp., Madison, WI) and used to generate cRNA probes with appropriate digestion by endonuclease and RNA polymerase (SP6 or T7). A sample from an individual pituitary was used for each hybridization, and the experiment was repeated at least twice.

Statistical Analysis
All data were analyzed by ANOVA and Student’s t tests or Duncan’s multiple range test.


    ACKNOWLEDGMENTS
 
We thank Dr. K. Wakabayashi and H. Kobayashi for assistance with RIA and M. Yokota for assistance with immunocytochemistry.


    FOOTNOTES
 
Address requests for reprints to: Masanobu Yamada, M.D., Ph.D., First Department of Internal Medicine, Gunma University School of Medicine, 3–39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan.

This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (to M.Y. and M.M.).

Received for publication February 18, 1999. Revision received September 20, 1999. Accepted for publication September 21, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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