Sensitizing effect of treatment with estrogens on TSH response to TRH in male rats.

Daily administration of estradiol benzoate (10 microgram/100 g body wt) to intact male rats led to a twofold increase of the plasma TSH (thyroid-stimulating hormone) response to thyrotropin-releasing hormone (TRH) after 4 and 7 days of treatment whereas the basal plasma TSH level was not affected. The basal plasma PRL concentration and the PRL response to TRH were both markedly increased by estrogen treatment. The TSH pituitary content remained unchanged, whereas the PRL pituitary content increased in parallel with the effect on PRL secretion. Treatment with estrogens for 1 wk sensitized the TSH secretory response to low doses of TRH (10 ng), whereas no significant effect on the response was found at high doses of the neurohormone. The present data show that the stimulatory effect of estrogens on the TSH response to TRH is due to true sensitization of the thyrotrophs to the action of the neurohormone, whereas that on prolactin secretion can result partly from increased pituitary prolactin content.     

Effect of diphenylhydantoin on hypothalamic-pituitary-thyroid function in the rat

Chronic diphenylhydantoin (DPH) administration (5 mg x 100 g body wt-1 x day-1) to the normal rat is associated with a decrease in the serum thyroxine (T4) and triiodothyronine (T3) concentrations without an appropriate rise in the serum thyrotropin (TSH) concentration, suggesting a possible direct effect of DPH on TSH secretion. To further study this possibility, DPH was administered chronically to thyroidectomized, hypothyroid rats. In the hypothyroid rats treated chronically with DPH, serum TSH did not increase, pituitary TSH content was significantly decreased, and the serum TSH response to thyrotropin-releasing hormone (TRH) was decreased compared to that of diluent-treated, hypothyroid rats. Hypothalamic TRH content was similar in DPH and diluent-treated rats. These findings suggest that DPH suppresses pituitary TSH secretion, probably as a thyroid hormone agonist. The effect of a single large dose of DPH (20 mg/100 g body wt) administered to thyroidectomized rats also decreased serum tSH but, in contrast to the findings in chronically treated rats, hypothalamic TRH and pituitary TSH content and the serum TSH responses to TRH were increased. These differences may be due to the acute inhibitory effect of a large dose of DPH on hypothalamic TRH release. Furthermore, because the effect of thyroid hormone on regulating pituitary TSH synthesis and release is dose and time dependent, the effect of DPH as a thyroid hormone agonist on pituitary TSH dynamics may also be variable.     

Hypothyroidism increases prolactin secretion and decreases the intromission threshold for induction of pseudopregnancy in adult female rats

In order to understand the mechanism by which thyroid hormones alter prolactin (PRL) secretion, we investigated the role of tuberoinfundibular dopamine (TIDA) neurons and pituitary and hypothalamus vasoactive intestinal peptide (VIP) in thiouracil- (0. 03% in drinking water for 16 days) induced-hypothyroid adult female rats. The intromission threshold for induction of pseudopregnancy also was examined to evaluate the PRL response to coital stimulation in hypothyroid rats. Hypothyroidism in adult female rats did not affect TIDA neuronal activity as measured by tyrosine hydroxylase activity (DOPA accumulation 30 min after administration of m-hydroxybenzylhydrazine dihydrochloride, 100 mg/kg, i.p.) in the stalk-median eminence compared with that in euthyroid rats, whereas pituitary concentration of VIP was dramatically increased. Plasma concentration of PRL was higher at 1100 h of proestrus and estrus in hypothyroid rats as compared with that of euthyroid rats. The proportion of female rats exhibiting pseudopregnancy was higher in hypothyroid animals (100%) receiving seven intromissions than in euthyroid animals (43%). Administration of L-thyroxine in hypothyroid rats decreased the proportion of pseudopregnancy (40%) to the level of euthyroid animals. These results indicate that the increased level of pituitary VIP probably affects PRL secretion in a paracrine or autocrine manner and account for the hyperprolactinemia induced in hypothyroid female rats. No role for TIDA neurons in PRL elevation can be ascribed. A decrease in the intromission threshold for induction of pseudopregnancy might be due to increased levels of PRL in hypothyroid female rats.     

A Plurihormonal TSH-Secreting Pituitary Microadenoma: Report of a Case with an Atypical Clinical Presentation and Transient Response to Bromocriptine Therapy

Inappropriate secretion of thyrotropin (TSH) is a rare cause of hyperthyroidism, and it is caused by either a TSH-producing pituitary adenoma (usually a macroadenoma) or to selective pituitary resistance to thyroid hormone. The case of a 31-yr-old male who presented with clinical features of thyrotoxicosis, including episodes of thyrotoxic paralysis, and a thyroid profile characterized by free hyperthyroxinemia and hypertriiodothyronemia with a nonsuppressed, inadequately normal TSH is reported. Dynamic testing showed both, lack of TSH stimulation by thyroid-releasing hormone (TRH), and lack of suppression by T3, consistent with autonomous TSH secretion. Pituitary MRI revealed a microadenoma. Seventy five percent of the patient’s serum TSH immunoreactivity eluted as α-subunit in Sephadex G-100 chromatography. A diagnosis of TSH-secreting microadenoma was established, and the patient was treated successfully with bromocriptine, which resulted in both clinical and biochemical resolution of his hyperthyroidism. Two months later, he became hyperthyroid again during bromocriptine therapy. Octreotide was started with adequate control of his symptoms and normalization of his free T4 level. He eventually underwent transsphenoidal surgery with successful resection of a chromophobic microadenoma which immunostained for TSH, growth hormone (GH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH). One month postoperatively he is clinically and biochemically euthyroid on no medications.     

Effect of thyrotropin-releasing hormone on development of the pituitary–thyroid system in fetal rats in organ culture

Six groups of thyroid glands from 18-day fetal rats were explanted to organ culture for 2 days. In one group, thyroid was cultured alone and in the remaining five groups thyroid was cocultured with pituitaries from fetuses ranging in age from 17 to 21 days. In each of the groups, half of the cultures had thyrotropin-releasing hormone (TRH) added to the medium. Histometric parameters of the thyroid follicle such as diameter and cell height were used as indicators of development of the thyroid gland. When 18-day thyroid was cultured alone, addition of TRH did not accelerate development. When either one 18-day or two 17-day pituitaries were cocultured with thyroid, a significant increase in diameter and cell height was seen. Addition of TRH to the medium induced little or no further change. When the thyroid was cultured with 19- to 21-day pituitaries, a marked increase in thyroid development was observed; and the addition of TRH caused further acceleration in thyroid development. These results suggest that, in organ culture, 17- to 18-day pituitary glands can release some thyrotropin (TSH) with or without additional TRH. Older pituitaries (19- to 21-day) apparently can release an amount of TSH in the presence of TRH that is greater than their own spontaneous TSH secretion.     

Effect of thyrotropin-releasing hormone on development of the pituitary-thyroid system in fetal rats in organ culture

Six groups of thyroid glands from 18-day fetal rats were explanted to organ culture for 2 days. In one group, thyroid was cultured alone and in the remaining five groups thyroid was cocultured with pituitaries from fetuses ranging in age from 17 to 21 days. In each of the groups, half of the cultures had thyrotropin-releasing hormone (TRH) added to the medium. Histometric parameters of the thyroid follicle such as diameter and cell height were used as indicators of development of the thyroid gland. When 18-day thyroid was cultured alone, addition of TRH did not accelerate development. When either one 18-day or two 17-day pituitaries were cocultured with thyroid, a significant increase in diameter and cell height was seen. Addition of TRH to the medium induced little or no further change. When the thyroid was cultured with 19- to 21-day pituitaries, a marked increase in thyroid development was observed; and the addition of TRH caused further acceleration in thyroid development. These results suggest that, in organ culture, 17- to 18-day pituitary glands can release some thyrotropin (TSH) with or without additional TRH. Older pituitaries (19- to 21-day) apparently can release an amount of TSH in the presence of TRH that is greater than their own spontaneous TSH secretion.     

Growth hormone and thyrotropin secretory profiles and provocative testing in aged rats

The impact of aging on both basal and induced GH and TSH secretion in male and female rats was investigated. Analysis of the individual GH secretory profiles in young (3-4 month) and old (19-20 month) rats indicated that sex-dependent patterns of GH secretion was preserved in old animals. However, we observed a reduction of individual GH peak amplitudes of 66% in old males and 53% in old females when compared to their respective young animals. Further, the GH response to an intravenous bolus of GH-releasing factor (GRF), morphine and clonidine was dramatically blunted or absent in old male and female animals. In contrast to GH, basal TSH secretion was elevated, while the TSH response to thyrotropin-releasing hormone (TRH) was not significantly affected in old animals of either sex. The present data provide evidence that reduced pituitary sensitivity to GRF may be a possible cause for reduced GH secretion in old animals. Further, the elevation in plasma TSH observed in old animals is not the result of an increased pituitary sensitivity to TRH.     

Interaction between vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in stimulating the secretion of prolactin from rat anterior pituitary cells in vitro

Interaction between vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in regulating the secretion of prolactin (PRL) from the pituitary was investigated in the rat in vitro using two different methods: (1) short incubation of anterior pituitary cells and (2) superfusion of the pituitary cell column. PRL levels in the incubation medium were raised by addition of either VIP or PHI in concentrations of 10(-9) M to 10(-7) M in a dose-related manner. When both peptides were simultaneously added, additive stimulating effect on PRL release was obtained below the VIP concentration of 10(-7) M, in which no additive effect of PHI was revealed. PRL release from superfused rat pituitary cells was stimulated by 5-min pulses of VIP (10(-7) M), PHI (10(-7) M) and TRH (10(-8) M). Infusion of VIP for 200 min in the concentration of 0.3 x 10(-7) M resulted in an increase in basal release of PRL and blunted PRL release induced by not only VIP but also PHI stimulation, whereas PRL release induced by TRH was not affected. Infusion of PHI (0.3 x 10(-7) M, 0.7 x 10(-7) M and 10(-7) M) for 200 min also dose-relatedly suppressed PRL release induced by not only PHI but also VIP without any change in PRL release induced by TRH. These findings suggest that VIP and PHI may act through a common binding site on the pituitary lactotroph in the rat.     

Changes in pituitary and thyroid function with increasing age in young male rats.

As the age of young adult male rats increased from 30 to 150 days, the serum thyroxine (T4) decreased by 50% and the serum thyroid-stimulating hormone (TSH) increased by 250%. There was no change in the serum triiodothyronine (T3). The increment in serum TSH after injection of thyrotropin-releasing hormone (TRH) was not significantly different at any of the ages studied, but the old animals had significantly lower increments in serum T4 and T3 after subcutaneous administration of bovine TSH. Despite a higher basal serum TSH, the older rats had a lesser increase in serum TSH after thyroidectomy or propylthiouracil. Thus, 1) there is a progressive decline in intrinsic thyroid function between 30 and 150 days of age in male rats, and 2) pituitary TSH response to fall in serum concentration of thyroid hormones is also decreased with age.     

The ultrasensitive determination of TSH permits the prediction of the response to TSH in the TRH test

A new ultrasensitive TSH immunoradiometric assay (IRMA) using two monoclonal antibodies is now able to distinguish between euthyroid and hyperthyroid patients. The aim of this study was to compare data given by ultrasensitive basal TSH (IRMA) and by the response of TSH to TRH test considered until now as the more reliable test in case of mild or atypical hyperthyroidism. Basal plasma TSH levels were determined in euthyroid (n = 80), hyperthyroid (n = 30), hypothyroid (n = 14) and pituitary deficient patients (n = 8) before and 30 minutes after a TRH test (250 micrograms i.v.). A close linear correlation was found between basal and post-stimulative TSH levels. Normal TSH response ranged from 2 to 22 uU/ml. The sensibility and the specificity of these two parameters appeared comparable in the case of primary dysthyroidism; on the contrary basal TSH levels were not sufficient for the diagnosis of central hypothyroidism. In conclusion, excepted for pituitary deficiency, basal plasma TSH (IRMA) levels are accurate and sufficient in the evaluation of the thyroid function and make the TRH-test useless.     

Recombinant human thyroid-stimulating hormone: pharmacology,clinical applications and potential uses

The major functions of pituitary thyroid-stimulating hormone (TSH) are to maintain the biosynthesis and secretion of the thyroid hormones L-thyroxine (T4) and L-3,5,3'triidothyronine (T3). The TSH core contains two apoproteins, the alpha and beta subunits. The alpha subunit is identical to that of pituitary follitropin, pituitary lutropin and placental chorionic gonadotropin, whereas the beta subunit is unique. TSH is a glycoprotein; the glycoprotein components of the alpha and beta subunits account for more than 10% of their mass and are essential for normal thyrotropic action and intravascular kinetics. The hypothalamic tripeptide, TSH-releasing hormone (TRH) is required for optimum TSH biosynthesis, particularly as far as addition of the glycoprotein components is concerned. TRH deficiency is associated with secretion of TSH molecules that are appropriately measured in most assays but have reduced bioactivity. In previous years the TSH used in clinical practice was obtained and purified from bovine pituitaries. Bovine TSH was used to test thyroid function and to augment the uptake of radioiodine in patients with thyroid cancer. Bovine TSH has been largely abandoned as a clinical agent because of adverse immune reactions. A recombinant human TSH (rhTSH; Thyrogen), has been approved by the US FDA for diagnostic use in patients with thyroid cancer. The alpha and beta subunits of Thyrogen are identical to those of human pituitary TSH. Thyrogen has a specific activity of approximately 4 IU/mg and is a potent stimulator of T4, T3 and thyroglobulin (Tg) secretion in healthy volunteers. It also increases thyroid iodide uptake in patients with thyroid cancer or multinodular goitre and in volunteers, even those exposed to large amounts of stable iodide. Thyroid cancer patients who have been treated by thyroidectomy and radioiodine ablation but are at risk of harbouring residual thyroid cancer are candidates for Thyrogen administration to prepare them for whole body iodide scans and serum Tg measurements. In thyroidectomised thyroid cancer patients who are unable to secrete pituitary TSH upon thyroid hormone withdrawal, Thyrogen is the only acceptable method to prepare them for these procedures. Thyrogen has been used on a compassionate basis to prepare patients for radioiodine ablation. rhTSH, in addition to being useful in the management of patients with thyroid cancer, is potentially useful to test thyroid reserve and to aid in thyroid-related nuclear medicine procedures. In the future, TSH analogues that have superagonist or antagonist properties may become available as therapeutic agents.     

Thyroxine feed-back on the regulation of thyrotrophin secretion

1. The role of thyroid hormone as a feed-back at the hypothalamic level in the control of thyrotrophin (TSH) secretion and release has been investigated by estimating the plasma and pituitary TSH levels following intrahypophysial and intrahypothalamic thyroid autotransplants.2. Thyroidectomized rats bearing thyroid autotransplants in the pituitary had a significantly lower (P < 0.001) plasma TSH than that of controls at 26 degrees C but not at 4 degrees C.3. Thyroidectomized rats bearing thyroid autotransplants in the supraoptic area showed a significantly lower (P < 0.001) level of plasma TSH and higher pituitary TSH at 4 degrees C but not at 26 degrees C.4. Study with both unilaterally and bilaterally thyroidectomized rats bearing thyroid autotransplants either in the pituitary or in the hypothalamus revealed that thyroxine feed-back operates at pituitary level in normal situations (26 degrees C) and there exists a feed-back through higher centres, specifically the TSR secreting area of the hypothalamus, in situations demanding higher thyroid function, as in cold exposure.     

Transdifferentiation of pituitary thyrotrophs to lactothyrotrophs in primary hypothyroidism: case report

Primary hypothyroidism causes adenohypophysial hyperplasia via stimulation by hypothalamic thyrotropin-releasing hormone (TRH). The effect was long thought to simply result in thyroid-stimulating hormone (TSH) and prolactin (PRL) cell hyperplasia, an increase in TSH and PRL blood levels with resultant pituitary enlargement, often mimicking adenoma. Recently, it was shown that transformation of growth hormone (GH) cells into TSH cells takes place in both clinical and experimental primary hypothyroidism. Such shifts from one cell to another with a concomitant change in hormone production are termed "transdifferentiation" and involve the gradual acquisition of morphologic features of thyrotrophs ("somatothyrotrophs"). We recently encountered a unique case of pituitary hyperplasia in a 40-year-old female with primary hypothyroidism wherein increased TSH production was by way of PRL cell recruitment. The resultant "lactothyrotrophs" maintained TSH cell morphology (cellular elongation and prominence of PAS-positive lysosomes) but expressed immunoreactivity for both hormones. No co-expression of GH was noted nor was thyroidectomy cells seen. This form of transdifferentiation has not previously been described.     

Effects of propylthiouracil on growth hormone and prolactin messenger ribonucleic acids in the rat pituitary

The effects of hypothyroidism induced by propylthiouracil (PTU) treatment on growth hormone (GH) and prolactin (PRL) messenger ribonucleic acid (mRNA) levels were analyzed in adult female rat adenohypophyses by in situ hybridization histochemistry and Northern hybridization analyses. Twenty-eight days of PTU treatment produced a significant decrease in GH mRNA levels and a smaller decrease in PRL mRNA determined by both in situ hybridization histochemistry and Northern hybridization analyses. A combined procedure of in situ hybridization histochemistry followed by immunochemistry on the same sections revealed mammosomatotropic cells expressing GH mRNA and PRL protein in the same pituitary cells from all treatment groups. Cells expressing GH mRNA and thyroid-stimulating hormone protein were not detected by this method. Immunochemical staining revealed a decrease in GH cells and an increase in thyroid-stimulating hormone cells in hypothyroid rats. Cells expressing both GH and thyroid-stimulating hormone protein were not detected by immunostaining. These results indicate that hypothyroidism produces significant decreases in GH mRNA and also decreases PRL mRNA and that mammosomatotropic cells can be detected in pituitaries from normal and hypothyroid rats.     

Acute inhibition of PRL and TSH secretion after intraventricular injection of PRL in ovariectomized rats

Prolactin (PRL) is under short-loop inhibitory control via the hypothalamus. However, earlier studies evaluated the effects on PRL secretion of PRL levels elevated for periods of days. In this study we evaluated the acute effects of intraventricular and systemic injection of PRL on the release of a variety of pituitary hormones. Ovariectomized (OVX) rats, bearing implanted third ventricular and jugular cannulas were used. Blood was withdrawn in unanesthetized, freely moving animals before and after intraventricular injection of 0.9% NaCl or 1 or 3 micrograms of bovine (b) or ovine (o) PRL. Prolactin was also administered intravenously in doses of 3 or 6 micrograms. No effect on plasma levels of any of the pituitary hormones occurred after intraventricular or systemic injection of saline. Intraventricular injection of both doses of bPRL or oPRL significantly lowered plasma PRL within 15-30 min. In animals with elevated initial PRL values because of stress or estradiol (E) priming, greater lowering of PRL occurred. Inconsistent reductions in plasma PRL occurred after intravenous injection of oPRL but not bPRL, which elevated PRL values via cross-reaction in the immunoassay. In contrast, only small and inconsistent declines in luteinizing hormone (LH) were seen after intraventricular injection of PRL in either OVX or OVX E-primed rats. Plasma follicle-stimulating hormone (FSH) and growth hormone (GH) were not affected by PRL in any of the experiments; however, a significant lowering of thyrotropin (TSH) occurred in OVX or OVX E-primed rats within 30 min after intravenous injection of 3 micrograms of oPRL, but no change occurred after intravenous PRL. The data indicate that PRL can acutely inhibit PRL and TSH release via a hypothalamic action, whereas release of LH is only slightly inhibited and that of FSH and GH is unaltered.     

Immunocytochemical detection of effects of TRH and T4 on prolactin- and TSH-producing cells in the pituitary gland of Rana ridibunda

Effects of synthetic thyrotropin-releasing hormone (TRH) and various doses of thyroxin (T4) on prolactin (PRL)-producing cells and thyrotropic cells in the pituitary were investigated in adult male and female Rana ridibunda frogs. Animals were given 200 microg TRH once a week for 4 weeks and 0.2-0.5 mg T4 during 3 days per week for a period of 2 weeks by injections in the groin. PRL-producing cells and thyrotropic cells were identified with light microscopical and electron microscopical immunocytochemical methods, using rabbit anti-PRL and rabbit anti-thyroid stimulating hormone (TSH) as primary antibodies. TRH caused cytological changes in both cell types, which were consistent with increased synthesis and release of both PRL and TSH. Treatment with 0.5 mg T4 activated both cell types less than TRH treatment did, whereas 0.2 and 0.4 mg T4 caused inactivation of both cell types. In conclusion, mammalian TRH is effective on both types of frog pituitary cells. Our study suggests that T4 has a positive rather than a negative effect when concentrations above a certain threshold are given.     

Serum thyroid-stimulating hormone in cerebrovascular disease

A thyrotropin-releasing hormone (TRH) test with serum thyroid-stimulating hormone (TSH) assays was performed in 22 euthyroid stroke patients without thyroid disease and the results were compared with those in 17 age-matched euthyroid controls. Basal and maximum TSH levels after TRH injection were significantly lower in the stroke group without elevation of basal serum thyroid hormone levels. There was a tendency towards an inverse relationship between TSH levels and the degree of pareses of the extremities. The test was repeated in 7 stroke patients 3-4 months after the onset of stroke with essentially the same results. The abnormal TSH parameters in stroke patients seem to be the result of the brain lesion per se.     

Evidence that TRH stimulates secretion of TSH by two calcium-mediated mechanisms

Thyrotropin-released hormone (TRH) stimulation of thyrotropin (TSH) release from mouse thyrotropic tumor (TtT) cells is dependent on Ca2+. We demonstrate that TRH action in TtT cells does not require extracellular Ca2+ but that Ca2+ influx induced by TRH can augment TSH secretion. TRH caused a 46% increase in 45Ca2+ uptake by TtT cells in medium with 100 micro M Ca2+. The increment in 45Ca2+ uptake caused by TRH was dependent on the concentration of Ca2+ in the medium. In contrast to the effect of 50 mM K+, which also causes Ca2+ influx, TRH caused 45Ca2+ efflux and TSH release from TtT cells even when the concentration of Ca2+ in the medium was lowered below 100 micro M. TRH stimulated TSH release during perifusion in medium in which the free Ca2+ concentration was lowered to approximately 0.02 micro M, and reintroduction of Ca2+ into the medium simultaneously with TRH markedly increased TSH release. We suggest that TRH may affect Ca2+ metabolism in TtT cells by both extracellular Ca2+-independent and -dependent mechanisms and that this dual mechanism of action serves to augment further TSH secretion induced by TRH.     

The TSH secretion in the human pituitary adenomas

TSH secretion by a pituitary tumor is very rare (2%) and it is often associated with another hormone: GH or PRL essentially. We present here nine tumors in which the TSH secretion was proved by immunocytochemistry (ICC) and by RIA in the tumor extracts, in the serum and in the culture medium. Four tumors secreted TSH only. Five tumors secreted TSH and GH predominantly. In 3 of them traces of other hormones (PRL and FSH) were also detected. The "pure" TSH adenomas were monomorphous with typical ultrastructural and immunocytochemical features. Plurihormonal TSH adenomas were bimorphous with different cells secreting GH and TSH or monomorphous with one type of cell which secreted TSH or GH or both TSH and GH. In a majority of the cases, the tumoral TSH secretion induced hyperthyroidism but in 2 patients with TSH adenoma there was euthyroidism and in another with TSH-GH adenoma there was no sign of acromegaly and GH serum levels were normal.     

Effect of domperidone on serum TSH and growth hormone in thyroid patients

In euthyroid female patients, the release of TSH from the pituitary increases in response to domperidone, a dopaminergic-receptor blocking agent of peripheral action. The rate of increase varies with the functional state of the thyroid, as confirmed by results obtained in cases of euthyroidism, primary hypothyroidism, subclinical hypothyroidism and subclinical hyperthyroidism. Observations suggest that the more vigorous feedback-mechanism modulates the dopaminergic regulation of TSH-secretion at a high degree of sensitivity. Stimulation with 1-dopa intensifies the release of growth hormone from the pituitary which is, however, of lesser degree in hyperthyroid or hypothyroid than in euthyroid individuals. The GH-response to 1-dopa is enhanced by administration of propranolol, but the maximum serum GH levels in response to stimulation with 1-dopa are significantly lower in hyperthyroid than in euthyroid individuals. Administration of domperidone leaves the serum GH levels unaffected in euthyroid and hyperthyroid subjects, but causes a significant increase in a number of patients with primary hypothyroidism. The results suggest that the dopaminergic system plays a part in the regulation of TSH and GH secretion, asserting itself partly as a direct effect on the pituitary, and that the dopaminergic regulation may be affected by thyroid dysfunction.