Thyroid status regulates particulate but not soluble TRH-degrading pyroglutamate aminopeptidase activity in the rat liver

Rat liver contains two topologically different TRH-degrading pyroglutamate aminopeptidases. The particulate pyroglutamate aminopeptidase, unlike the soluble one, was highly specific for TRH and shared many physico-chemical properties with serum thyroliberinase, which is controlled by thyroid hormones. Both enzymes convert pGlu His Pro NH2 into His Pro NH2; the latter may be cyclized to cyclo His Pro known to possess several biological activities and specific binding sites in liver. The aim of the present study was to determine the effects of thyroid status on the particulate and soluble enzymes activity and gain more insight into their biological role. The regulatory pathway for the particulate pyroglutamate aminopeptidase was found similar to that of serum thyroliberinase: their specific activities decrease in hypothyroid rats and increase in hyperthyroid rats, whereas that of soluble enzyme remains unchanged. We postulate that the particulate pyroglutamate aminopeptidase may be a determining factor in the concentrations of TRH and/or cyclo His Pro reaching liver cells and a possible source for serum thyroliberinase. Taken together, these data suggest that this "converting enzyme" acts as a physiological regulator.     

The effect of basal hypothalamic isolation on pituitary-thyroid activity and the response to propylthiouracil.

Basal hypothalamic deafferentation extending from the posterior border of the optic chiasm to the mid-mammillary bodies resulted in depression of plasma TSH, thyroxine (T4), and triiodothyronine (T3) concentration to 50% of normal controls within 7 days. Administration of 0.15% propylthiouracil (PTU) in the diet form postoperative day 26 caused a pronounced drop in the plasma T3 level and a rise in plasma TSH level within two days in the control animals, but had little effect during this interval in the deafferented animals. After 12 days of PTU, plasma T3 and T4 concentrations had dropped to undetectable concentrations in the control animals but both were still detectable in the deafferented animals. After 25 days of PTU, plasms T4 and T3 levels were undetectable and plasma TSH levels were significantly elevated above normal in all animals. Thyroid hypertrophy at that time was as great in the deafferented as in the control rats, although plasma TSH concentration was 50% lower in the former. Administration of 0.1 mug/100 g BW TRH iv on postoperative day 37, when plasma T4 and T3 were undetectable in the controls but still present in the deafferented animals, produced an equally high concentration of plasma TSH in all animals. We interpret these data to support the concepts that: 1) a major source of neural drive of that TRH which stimulates the secretion of TSH by the adenohypophysis lies outside the medial basal hypothalamus, 2) a decrease in TRH reaching the adenohypophysis causes a lower setting of the "thyrostat" sensitive to the concentration of circulating thyroid hormone, and 3) increased TSH secretion and resultant goitrogenesis is delayed in animals with impaired TRH secretion because of the slower rate of secretion of thyroid hormone than in intact controls and the longer time thus required to markedly reduce the concentration of circulating thyroid hormone.     

Increased sensitivity of the thyrotroph to thyrotropin-releasing hormones in rats with hypothalamic hypothyroidism

The status of TSH secretion in hypothalamic hypothyroidism was evaluated by using rats with anterior medial basal hypothalamic deafferentation as the experimental model of the disorder. In the deafferented rats, the basal serum thyroid hormone concentrations as well as that of TSH was significantly lower than normal and cold exposure failed to increase serum TSH, indicating they were in fact in a hypothalamic hypothyroid state. The minimum effective dose of TRH to elicit an increase in serum TSH was smaller in the deafferented rats than in the controls, whereas the response to the maximum dose of TRH was similar in both groups. Although the radioimmunoassayable TSH of the adenohypophysis was significantly decreased in the deafferented rats, it was qualitatively similar to that of the control rats, since the peak of TSH immunoreactivity was eluted at exactly the same position on the gel filtration column in the pituitaries from normal and deafferented rats. When the adenohypophysis was perifused in vitro with Krebs-Ringer solution buffered with Hepes, the minimum effective dose of TRH was similar in both cases. This finding suggests that the exposure to the perifusion medium completely devoid of thyroid and hypothalamic hormones erased the difference in sensitivity to TRH between the two groups as observed in vivo, although in vivo experiments on deafferented rats with normal thyroid hormone induced by exogenous thyroid hormone were not performed. Our results indicate that in hypothalamic hypothyroid rats: 1) the sensitivity but not the responsiveness of the thyrotroph to TRH is increased; and 2) the readily releasable fraction of pituitary TSH pool in response to exogenous TRH is increased. It is also suggested that the difference in the milieu between the pituitary of normal and deafferented rats in vivo is critically important for the latter to retain hypersensitivity to TRH.     

Thyroid status influences rat serum but not brain TRH pyroglutamyl aminopeptidase activities

Serum and brain cytosol contains pyroglutamyl aminopeptidase activity that converts TRH to His-ProNH2 (TRH PAPase). Whereas serum TRH PAPase has specificity for TRH, this is not the case for brain cytosol PAPase. Recent reports indicate that a brain membrane fraction contains TRH PAPase that is specific for TRH and has a remarkable similarity to serum TRH PAPase. In the present studies, a method for measuring serum TRH PAPase activity and the activities of the membrane and cytosol brain TRH PAPase enzymes are described. The effect of thyroid status on these different TRH PAPase activities was determined. In hypothyroid rats serum TRH PAPase activity was decreased, whereas in rats treated with supraphysiological doses of T4 it was increased. In contrast, the cytosolic and the membrane TRH PAPase enzymes in brain were not affected by thyroid status. It is concluded that the membrane-associated brain TRH PAPase differs from the serum TRH PAPase in terms of its response to thyroid hormone. In addition, the previously reported effects of thyroid status on rat serum TRH degrading activity are explained by the finding that thyroid hormone increases serum TRH PAPase activity.     

Regulation of thyroid hormone receptors and responses by thyrotropin-releasing hormone in GH4C1 cells

The present study was undertaken to test the effects of TRH on thyroid hormone receptors and responses in GH4C1 rat pituitary tumor cells. TRH caused a loss of up to 32% of specific nuclear thyroid hormone binding sites with an ED50 of approximately 1 nM, and this loss was additive to the receptor down-regulation caused by T3 itself. Scatchard analysis of nuclear T3 binding revealed that 10 nM TRH decreased the concentration of T3 receptors from Bmax (femtomoles per mg protein) of 110 to 50 while receptor affinity in serum-free medium changed from dissociation constant (Kd) 110 to 50 pM with TRH. TRH lowered the GH response to 0.5 nM T3 from 215% to 127% of control. The concentrations of TRH required to decrease T3 receptors and T3 responses were similar and indicated that these TRH effects are mediated by the TRH receptor. In the absence of added thyroid hormone TRH had little effect on the rate of GH synthesis. TRH did not affect the binding of 0.5 nM [125I]T3 to receptors during the first 8 h but reduced T3 receptor occupancy up to 25-50% in different experiments after 24 h. TRH blocked the induction of GH by T3 only after 48 h or longer. When cells were incubated for 2 weeks with or without 2 nM T3 and 10 nM TRH, the stimulation of cell growth by T3 was decreased by TRH (2- vs. 5-fold increase in cell number) as was stimulation of GH by T3 (5- vs. 13-fold). As expected, T3 blunted the PRL response to TRH from 19- to 3-fold. The effects of TRH on the density of thyroid hormone receptors could be mimicked by the calcium channel agonist BAY K8644 plus a protein kinase C-activating phorbol ester which together caused a 53% reduction in thyroid hormone binding. The dose-response and temporal relationships suggest a causal relationship between the TRH-mediated decrease in thyroid hormone receptors and the decrease in thyroid hormone responses in GH4C1 cells. It has previously been shown that thyroid hormones decrease the concentration of TRH receptors and TRH responsivity in pituitary cells. The results shown here for GH4C1 cells suggest that TRH regulation of T3 responses may also be important in feedback control at the pituitary level.     

Effects of thyroid state on ornithine decarboxylase activity of the adenohypophysis of the rat and chicken.

In White Leghorn chickens, 0.5 ng/kg of thyrotropin-releasing hormone (TRH) increased ornithine decarboxylase activity in the rostral lobe of the adenohypophysis without any alteration of enzyme activity in the caudal lobe. In rats, administration of TRH (250 ng/100 g body weight) increased thyroid-stimulating hormone (TSH) release but did not increase ornithine decarboxylase activity in the adenohypophysis. However, surgical thyroidectomy performed on rats resulted in declining levels of T₃ and T₄ in the plasma, an increase in the plasma level of TSH, and a twofold increase in ornithine decarboxylase activity in the adenohypophysis. In the chicken, administration of both methimazole and thyroxine caused elevated ornithine decarboxylase activity in the rostral and caudal lobes of the adenohypophysis. The hormone stimulation of ornithine decarboxylase activity, an early indicator of new biosynthesis, is suggested as a marker to study control mechanisms in the adenohypophysis.     

Modulation of pituitary thyrotropin releasing hormone receptor levels by estrogens and thyroid hormones.

The effect of estradiol and thyroid hormone treatment on pituitary TRH binding and TSH and PRL responses to the neurohormone was studied. A significant increase in the number of pituitary TRH binding sites was observed between 2 and 4 days after daily administration of estradiol benzoate with a plateau at 300% of control being reached at 7 days. Plasma PRL levels showed a similar early pattern of response. In animals rendered hypothyroid by a 2-month treatment with propylthiouracil or 1 month after surgical thyroidectomy, the level of pituitary TRH receptors was increased approximately 2-fold, this elevation being completely reversed by treatment with thyroid hormone. Estradiol-17beta administered with L-thyroxine partially reversed the inhibitory effect of thyroid hormone on TRH receptor levels in hypothyroid animals. The antagonism between estrogens and thyroid hormone is also apparent on the TSH response to TRH since estrogen administration can reverse the marked inhibition by thyroxine of the TSH response to TRH either partially or completely in intact and hypothyroid animals, respectively. The PRL response to TRH is 55 and 40% inhibited in hypothyroid and intact rats, respectively, by thyroid hormone when combined with estrogen treatment. The present data clearly show that estrogens and thyroid hormones can affect TSH and PRL secretion, the effect of estrogens being predominantly on PRL secretion while thyroid hormone affects mainly TSH. The close correlation observed between the level of TRH receptors and PRL and TSH responses to TRH suggests that estrogens and, to a lesser extent, thyroid hormones, exert their action by modulation of the level of receptors for the neurohormone in both thyrotrophs and mammotrophs.     

Regulation of adenohypophyseal pyroglutamyl aminopeptidase II activity by thyrotropin-releasing hormone and phorbol esters

Thyrotropin-releasing hormone (TRH) is inactivated by a narrow specificity ectopeptidase, pyroglutamyl aminopeptidase II (PPII), in the proximity of target cells. In adenohypophysis, PPII is present on lactotrophs. Its activity is regulated by thyroid hormones and 17β-estradiol. Studies with female rat adenohypophyseal cell cultures treated with 3,3′,5′-triiodo-l-thyronine (T₃) showed that hypothalamic/paracrine factors, including TRH, can also regulate PPII activity. Some of the transduction pathways involve protein kinase C (PKC) and cyclic adenosine monophosphate (cAMP). The purpose of this study was to determine whether T₃ levels or gender of animals used to propagate the culture determine the effects of TRH or PKC. PPII activity was lower in cultures from male rats. In cultures from both sexes, T₃ induced the activity. The percentages of decrease due to TRH or PKC were independent of T₃ or gender; the percentage of decrease due to cAMP may also be independent of gender. These results suggest that T₃ and hypothalamic/paracrine factors may independently control PPII activity in adenohypophysis, in either male or female animals.     

An immunocytochemical and electron microscopic study of the hyt mouse anterior pituitary gland

The hyt mutant mouse used in this study has a hypoplastic thyroid gland and is characterized by retarded somatic growth, very low to undetectable levels of plasma thyroxine (T4), and increased levels of plasma thyroid-stimulating hormone (TSH). This congenital hypothyroid mouse is therefore an ideal model for studying the effects of thyroid hypofunction on the adenohypophysis. The anterior pituitary of the hyt mouse appeared less granular than that of the normal control when viewed by light microscopy, owing to a decrease in the population of somatotrophs. Many cells, in various stages of transformation into 'thyroidectomy cells', were recognized by the appearance of the characteristic granules and dilated rough endoplasmic reticulum. In some cases, the enlarged rough endoplasmic reticulum also contained spherical electron-dense secretory granules. In addition there were many cells undergoing mitosis and these were identified as thyrotrophs by their characteristic granules. Administration of T4 during the first 40 days of life prevented the abnormal changes in the hyt anterior pituitary. A reduction in immunoreactive thyrotrophin-releasing hormone (TRH) levels was seen in the median eminence of the hyt mouse. Treatment with T4 restored this to normal, suggesting that the reduced TRH content of the hypothalamus of the mutant mouse may be due to T4 deprivation.     

Effects of hypo- and hyperthyroidism on pancreatic TRH-degrading activity and TRH concentrations in developing rat pancreas

High concentrations of thyrotrophin-releasing hormone (TRH) in the rat pancreas were detected during the first few days of life decreasing thereafter while pancreatic TRH-degrading activity (TRH-DA) absent at birth appeared on day 14 and increased to reach adult values by day 21. This period of life is also remarkable by the low level of circulating thyroid hormones. Since TRH-degrading activity may be thyroid hormone dependent it was of interest to study the effects of thyroid status fluctuations both on TRH-DA and TRH content during the neonatal period. In this study, hypo- and hyperthyroidism were induced by 6-n-propyl-2-thiouracil (PTU) and triiodothyronine (T3) respectively. Pancreatic TRH-DA and TRH concentrations were measured at different ages from birth until day 29, in treated animals and results compared to control age-matched rats. In hypothyroid rats, pancreatic TRH concentrations remained significantly higher after day 16 while TRH-DA was lower during the whole period studied. Following T3 treatment, pancreatic TRH concentrations decreased significantly from day 3 onwards. However, no significant changes were found for TRH-DA except a two-fold increase on day 28. These results suggest that two different mechanisms may account for thyroid hormones action: 1) a direct effect on pancreatic TRH 2) an inductive saturable effect on TRH-DA. Furthermore a fine tuner modulatory role of TRH-DA on TRH concentrations cannot be excluded.     

Response of hypothalamic peptide mRNAs to thyroidectomy.

Using in situ hybridization histochemistry, we have investigated the effect of thyroid hormone on the expression of several peptide mRNAs in the hypothalamic paraventricular nucleus (PVN) of adult male rats. Hypothyroidism was induced by surgical ablation of the thyroid gland. The animals (control sham-operated, thyroidectomized, thyroidectomized+T4 replaced rats) were studied 28 and 50 days after surgery. Sections of the PVN were hybridized using synthetic oligonucleotide probes complementary to mRNA for thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), galanin (GAL), enkephalin (ENK), neurotensin (NT), vasoactive intestinal polypeptide (VIP) and vasopressin (VP). GAL mRNA was also analyzed in the anterior paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus. At the PVN level, a feedback effect of thyroid hormone on TRH synthesis was demonstrated by the TRH mRNA increase in hypothyroidism and by its decrease in hyperthyroidism. Hypothyroidism caused a dramatic decrease in GAL mRNA in parvo- and magnocellular PVN neurons both 28 and 50 days after thyroid ablation, whereas no effect was seen in VP mRNA, the main peptide hormone coexisting with GAL. The T4 replacement prevented the GAL mRNA impairment. Hypothyroidism did not influence GAL mRNA in the anterior PVN, perifornical area or in the arcuate nucleus, whereas a decrease in GAL mRNA was observed in the dorsomedial nucleus. VIP mRNA, which is undetectable in the PVN of normal animals, was present in several PVN neurons after thyroidectomy. CRH mRNA was decreased after thyroidectomy, whereas the T4 restitution caused an upregulation. The levels of ENK or NT mRNA were not significantly affected by the thyroid status. The present results show that, in addition to TRH mRNA, other hypothalamic peptide mRNAs are affected by thyroid hormone levels.     

SPORADIC NON-TOXIC GOITRE: AN INVESTIGATION OF THE HYPOTHALAMIC–PITUITARY–THYROID AXIS

Thyrotrophin releasing hormone (TRH) tests have been carried out on sixty-two patients with sporadic non-toxic nodular goitre. 61% gave a subnormal thyroid stimulating hormone (TSH) response but had normal plasma thyroxine (T4) and triiodothyronine (T3) levels. T3 administration suppressed 131I uptake by the thyroid adequately in 74% of these and there was normal stimulation of thyroid uptake by exogenous TSH. Prolactin (PRL) rose normally after TRH in all the TRH non-responders. Normal TSH response to TRH was restored by partial thyroidectomy and in some cases by propyl thiouracil administration. Possible reasons for these findings are discussed. It is concluded that these cases were truly euthyroid.     

Lack of effect of thyroid hormones on the growth hormone response to thyrotropin-releasing hormone in acromegaly.

Serum growth hormone (GH) responses to thyrotropin-releasing hormone (TRH) were evaluated in 14 patients with acromegaly following treatment with thyroid hormones. After an initial TRH test, seven patients received L-triiodothyronine, 100 mug daily for seven days; the GH response to TRH was not significantly altered by this treatment. Similar findings were noted in two acromegalic subjects who were tested with TRH before and after longer periods of administration of L-thyroxine. Four of five additional subjects with acromegaly who had received replacement doses of thyroid hormones for an average of 6.6 yr demonstrated GH responses to TRH which were similar to those seen in subjects not receiving thyroid hormones. Acute or long-term administration of replacement doses of thyroid hormones seems to have minimal effect on the GH response to TRH in acromegaly.     

Hypothalamo-adenohypophyseal-thyroid interrelationships in the chick embryo. I TRH and TSH sensitivity.

The capabilities of the thyroid and adenohypophysis of the developing chick embryo to respond to exogenous thyrotropin (TSH) and thyrotropin-releasing hormone (TRH), respectively, were evaluated by means of radioimmunoassay measurements of plasma total thyroxine (T₄) levels. Chick embryos were treated with bovine TSH (2.5 mIU) on Days 6.5, 7.5, and 8.5 of incubation. The embryonic chick thyroid was shown to be sensitive to TSH as early as Day 6.5, as evidenced by an increase in plasma T₄ levels (P < 0.05). Additionally, the adenohypophyses of 6.5-day-old chick embryos were shown to respond to synthetic TRH (400 μg/100 g body weight) as indicated by a statistically significant rise in circulating thyroxine levels (P < 0.05). Possible underlying mechanisms to explain the increase in thyroidal activity that normally occurs during the Day 10.0–12.0 incubation interval in the chick embryo are discussed.     

Growth hormone response to thyrotropin-releasing hormone in the urethane-anesthetized rat: effect of thyroid status

To evaluate the role of thyroid hormones in regulation of the GH-stimulatory effects of TRH and human pancreatic GH-releasing factor (hpGRF-40), we studied the plasma GH responses to these secretagogues under conditions of thyroid hormone deprivation and replacement in the urethane-anesthetized rat. In euthyroid control rats, TRH (1 microgram/kg) elicited a small transient rise in plasma GH, which peaked at 2-5 min and returned to basal by 20 min. In chronically hypothyroid rats (10 weeks after thyroidectomy), intrapituitary GH was markedly depleted to less than 0.1% of normal, and TRH was completely ineffective in eliciting a plasma GH response to TRH. In chronically hypothyroid rats given T4 (20 micrograms/kg daily) for 4 days, intrapituitary GH was partially repleted, and the GH response to TRH was markedly enhanced compared to that in euthyroid rats. The extent to which the GH response to TRH was enhanced by chronic hypothyroidism, followed by short term T4 treatment, depended on the duration of T4 administration. The plasma GH response was greatest after 1-3 days of T4 treatment; treatment for 7 days, on the other hand, suppressed the GH response to below that of euthyroid rats. The minimal duration of hypothyroidism which, in combination with short term (2 days) T4 treatment, enhanced the plasma GH response to TRH, was 6 weeks, with 8 weeks or longer being optimal. The effect of TRH on plasma GH was dose dependent in thyroidectomized rats given T4 for 2 days; the lowest maximally stimulatory dose was 10 times less than that in the euthyroid rat (1 vs. 10 micrograms/kg). The GH-stimulatory effect of TRH in thyroidectomized rats given T4 for 2 days was abolished by the simultaneous administration of SRIF (40 micrograms/kg). That the failure of TRH to stimulate GH release in the chronically hypothyroid rat may have been the consequence of a depletion of intrapituitary GH available for release was suggested by the finding that in parallel studies, hpGRF-40, a more potent stimulator of GH release in the euthyroid rat, was also without effect. In contrast to the GH response to TRH, the GH response to hpGRF-40 was only partially restored by T4 treatment of chronically hypothyroid rats for 2 days. We conclude that chronic thyroid hormone deficiency selectively sensitizes the somatotroph to TRH. Short term thyroid hormone replacement is needed to replete intrapituitary GH and allow the expression of this enhanced GH secretory response to TRH. More prolonged treatment with T4, on the other hand, appears to desensitize the somatotroph to TRH.     

The effects of mammalian thyrotropin-releasing hormone on the pituitary-thyroid axis of teleost fish

The effect of synthetic mammalian thyrotropin-releasing hormone (TRH) on the pituitary-thyroid axis of poecilid fish is described. Interferometric measurement of thyroid gland activity was used to determine changes in thyrotropin [TSH] secretion, and histological examinations were also made of the pituitary and thyroid glands.The injection of TRH into intact male guppies significantly decreased thyroid activity and produced histological changes in the TSH cells which are classically associated with reduced activity.Experiments were also performed in which thyroid tissue was cultured in media in which there had been a prior pituitary incubation. The emanation from this pituitary produced an increase in activity of the thyroid tissue. This thyrotropic potential was significantly reduced when the pituitary had been cultured with TRH.Culture of thyroid tissue in media with TRH but without a pituitary preincubation showed no change in thyroid activity when compared with control incubations. Thus, the inhibition of thyroid activity by TRH in intact fish is probably not a direct action but is mediated by the pituitary gland.The present findings give support to the suggestion that the control of TSH in teleosts could be via an inhibitory hypothalamic factor (TRIH). Such a factor could well be similar to the tripeptide mammalian TRH which can obviously inhibit the pituitary-thyroid axis of fish.     

THYROID FUNCTION AND ANTIBODY STUDIES IN PERNICIOUS ANAEMIA

Thyroid antibodies were demonstrated in 57% of thirty pernicious anaemia patients without overt thyroid disease. Elevated basal thyroid stimulating hormone (TSH) levels and an enhaced TSH response to thyrotrophin releasing hormone (TRH) only occurred in thyroid antibody positive subjects; by contrast the thyroid antibody negative subjects in the older age group frequently and undetecable basal TSH levels and an impaired TRH response. Thyroid hormone concentrations provided no absolute evidence of hypothyroidism in any of the patients.     

Prostate-thyroid axis: prostatic TRH is one of the stimulators of thyroid hormone.

Ventral prostatectomy decreased serum thyroid hormones and histology of the thyroid gland indicate that hypothyroid condition. Co-culture of thyroid gland and ventral prostate stimulates thyroid hormone secretion. In the present study we report prostatic thyrotropin releasing hormone (TRH) is the stimulating factor of thyroid hormone secretion. Mature rat (90 days old) ventral prostate, anterior pituitary and thyroid glands were co-cultured in vitro with or without TRH antibody to assess the direct influence ofprostatic TRH on thyroid hormone secretion. Total thyroxine (T4) and triiodothyronine (T3) were increased significantly in the culture media of ventral prostate, anterior pituitary and thyroid gland when compared with thyroid gland plus anterior pituitary culture media. However, media T4 and T3 concentration decreased significantly in thyroid gland alone; also in thyroid gland plus ventral prostate, thyroid gland plus anterior pituitary and thyroid gland plus anterior pituitary plus ventral prostate were co-cultured with TRH antibody (Ab) in a dose dependent manner. The results suggest that ventral prostatic TRH is one ofthe stimulating factors of thyroid hormone secretion under these in vitro conditions.     

Prolactin secretion in mice with thyrotropin-releasing hormone deficiency

The physiological roles of TRH in pituitary lactotrophs, particularly during lactation, remain unclear. We studied the prolactin (PRL) status, including serum PRL and PRL mRNA levels in the pituitary, in nonlactating and lactating TRH-deficient (TRH(-/-)) mice with a rescue study with thyroid hormone and TRH. We found that, as reported previously for male TRH(-/-) mice, neither the morphology of the lactotrophs, PRL content in the pituitary, nor the serum PRL concentration was changed in nonlactating female TRH(-/-) mice. However, concurrent hypothyroidism induced a mild decrease in the PRL mRNA level. In contrast, during lactation, the serum PRL level in TRH(-/-) mice was significantly reduced to about 60% of the level in wild-type mice, and this was reversed by prolonged TRH administration, but not by thyroid hormone replacement. The PRL content and PRL mRNA level in the mutant pituitary during lactation were significantly lower than those in wild-type mice, and these reductions were reversed completely by TRH administration, but only partially by thyroid hormone replacement. Despite the low PRL levels, TRH(-/-) dams were fertile, and the nourished pups exhibited normal growth. Furthermore, the morphology of the pituitary was normal, and high performance gel filtration chromatography analysis of the PRL molecule revealed no apparent changes. We concluded that 1) TRH is not essential for pregnancy and lactation, but is required for full function of the lactotrophs, particularly during lactation; and 2) the PRL mRNA level in the pituitary is regulated by TRH, both directly and indirectly via thyroid hormone.