Effects of thyroidectomy, propylthiouracil, and thyroxine on pituitary content and immunocytochemical staining of thyrotropin (TSH) and thyrotropin releasing hormone (TRH)

Previous studies have demonstrated immunocytochemical staining for beta chains of thyroid stimulating hormone (TSH-beta) in rough endoplasmic reticulum of pituitary cells hypertrophied after thyroidectomy ("thyroidectomy cells") (Moriarty CG(1976): J Histochem Cytochem (24:846; Moriarty GC, Tobin RB (1976): J Histochem Cytochem 24:1140). Here we report the localization of thyrotropin releasing hormone (TRH) in serial sections of the same pituitaries to determine if it could be found at similar sites. No staining for TRH was found in hypertrophied TSH cells formed 42 days after the surgery, or after 14, 34, and 70 days of propylthiouracil (PTU) treatment. The loss in immunostaining in the PTU-treated rats was correlated with radioimmunoassay (RIA) measurements that showed a 65% reduction in anterior pituitary TRH content after 34, 70, and 98 days of PTU treatment (from 22.9--7.8 pg/mg wet wt) and a 50% reduction in TSH content after 34 days of treatment. When thyroxine was administered to hypothyroid rats for 3 days before death, our previous studies had demonstrated intense staining for TSH in granules inside the rough endoplasmic reticulum. In this study, the radioimmunoassay showed that TSH content rose dramatically in the hypothyroid animals treated with PTU for 77 days and thyroxine for 2 days before death (from 8.5--64.1 mU/mg wet wt); however, the rise in TRH content was minimal (5.8--9.8 pg/mg wet wt). The immunocytochemical stain for TRH correlated well with the RIA showing a weak reaction mainly on small granules in the cytoplasm. No reaction for TRH was found in rough endoplasmic reticulum. These results suggest that TRH and TSH storage sites are dissimilar in the hypothyroid rat. The presence of stain for TRH in granules in the cytoplasm suggests that it might play a role in the storage or packaging of TSH. Its absence in profiles of rough endoplasmic reticulum staining intensely for TSH suggests that it is not synthesized at this site. No definite conclusions about its origin can be drawn at this time.     

Smooth endoplasmic reticulum proliferation in mouse enterocytes induced by fructose feeding

Summary Adult male mice receiving a fructose diet show, after three days, a large proliferation of the smooth endoplasmic reticulum in the jejunal absorptive cells. This extension is a secondary stage of the trememdous increase of the glucose-6-phosphatase activity and seems to result from a crypt regulation. Glucose feeding does not provoke the same morphologic changes. This work was supported by a research grant from the Medical Research Council of Canada. Dr. D. Maestracci Ph. D. is a recipient of a Centennial Fellowship of the Medical Research Council of Canada. Mr. D. Ménard is a recipient of a studentship of the Medical Reserach Council of Canada. The authors are greatly indebted to Mrs. S. Sinave for her skillful technical assistance.     

Alteration of stimulated TSH and prolactin response in children treated with growth hormone.

The plasma TSH and prolactin responses to thyrotropin releasing hormone (TRH) were measured in 5 children with isolated growth hormone deficiency prior to, during and after the administration of human growth hormone (hGH). TSH and prolactin secretory patterns were not uniformly concordant. TSH responses to TRH infusion were suppressed in 4 subjects after 5 days or 1 month of hGH administration despite normal serum thyroxin concentrations. Prolactin responses were suppressed in all 5 subjects after 5 days of hGH administration. After 8 months of hGH therapy both TSH and prolactin responses returned toward pre-hGH values. Our finding that suppression of the TRH-induced TSH and prolactin secretory responses are reversible during hGH administration supports the concept of altered neuroregulation in this form of hypothalamic disorder.     

Ultrastructural localization of acid phosphatase in the posterior pituitary of the dehydrated rat

Summary Acid phosphatase (AcPase) was localized by means of electron-microscopic histochemistry and estimated biochemically in the posterior pituitary of rats deprived of water, given 2% NaCl ad libitum, or given tap water ad libitum over 6 days. Autophagic vacuoles, some of which gave a positive AcPase reaction, often contained neurosecretory granules (NSG) in nerve endings of control animals on tap water. Nerve endings of water-deprived or salt-treated rats were depleted of NSG, but frequently contained dense membranous residual bodies, some of which appeared to enclose microvesicles. Smooth endoplasmic reticulum located in axons and terminals appears to be a source of hydrolytic enzymes for neurohypophysial lysosomes. The total amount of AcPase per posterior lobe increased progressively to 40% above control levels after 6 days of water deprivation or salt administration, and this increase may reflect accelerated production of neuronal components in neurohypophysial cells whose secretory rate has been stimulated by elevated body osmolarity.Supported by the Medical Research Council of Canada.Medical Research Associate of the Medical Research Council of Canada.     

The endocrine cells of the upper gut mucosa in dogs with transplantation of the pyloric antrum to the colon

Summary A previous examination, done by others, of the endocrine cells of the canine pyloric antrum following its transplantation to the colon evidenced an increase in their number; the cell types involved, however, were not individualized. The present study further characterizes the changes occurring in the endocrine cells of the pyloric antrum of dogs following transplantation to the colon. The main change observed consists of hyperplasia of the enterochromaffin cells, with minor qualitative modifications in the antral gastrin-producing cells being noted only by electron microscopy. The response of the enterochromaffin cells cannot be readily explained but might be related to similar enterochromaffin cell hyperplasias found in human stomachs with peptic ulcers. Antrum transplantation to the colon in dogs appears to provide a promising experimental model for the study of the relationship between gastrin and other humoral factors (probably biogenic amines) in the mechanism of gastric secretion under normal as well as abnormal circumstances.Supported by a grant from the Medical Research Council of Canada. While performing this research, Dr. J. Lechago was a recipient of a fellowship from the Medical Research Council of Canada.     

Fine structure of the fetal rat thyroid and parathyroid glands at term and during prolonged gestation

Summary The fine structure of the fetal rat thyroid and parathyroid glands was studied at term and during prolonged gestation, which was induced by subcutaneous injections of progesterone to the mothers from gestational days 20 through 24. At term, the follicular and parafollicular cells of the thyroid as well as cells of the parathyroid exhibited well developed cytoplasmic organelles. Morphological changes were not detected in either of the endocrine glands during prolonged gestation. The results are discussed in relationship to 1) thyroid follicular cell activity during stress and 2) the function of thyroid parafollicular and parathyroid cells in calcium homeostasis.Supported by the Medical Research Council of Canada Grant No. MA4740.     

The ultrastructure of peripheral arteries during the development of DOCA hypertension in the rat

Summary The ultrastructure of the unconstricted superficial epigastric and femoral arteries is described in normal rats and in animals with hypertension induced by unilateral nephrectomy, by subcutaneous injections of desoxycorticosterone acetate and drinking of 1% NaCl. The femoral artery showed by far the greater response to the DOCA-saline treatment. In both vessels, the smooth-muscle cells changed from the normal spindle shape to a blunt ended outline with numerous pinocytotic vesicles and prolific collagen production. With long term hypertension, particularly in the femoral artery, the smooth-muscle cell profiles became very irregular. Hypertrophy of the organelles of the smooth-muscle cells was associated with an increase in the intercellular material which gradually changed from a mainly collagenous character to mainly vesicular. Lysosomal activity indicated cell disintegration. White blood cells adhere to the endothelium in hypertensive rats and there was an increase in subendothelial material. The number of intimal smooth-muscle cells increased noticeably in the femoral artery. In both arteries, the adventitial fibroblasts hypertrophied in hypertensive rats. In animals with an elevated blood pressure the morphological response was observed as early as 4 to 7 days after initiation of treatment.This work was supported by grants from the British Columbia Heart Foundation and the Medical Research Council of Canada.The authors are indebted to Miyoshi Nakashima for her invaluable assistance with these experiments.     

Influence of pimozide on a TRH induced TSH secretion in the rat during food deprivation.

Adult Wistar rats food deprived for 3 days had lower basal levels of TSH compared to normal fed animals. An increase of these lower levels to normal values was obtained following a prolonged (injections during 3 consecutive days) or acute treatment (single injection) with pimozide (1 mg/injection). Blood samples obtained after the last or an only injection of pimozide contained profound increased prolactin levels. Prolactin increase was more than 100-fold in fed and more than 30-fold in starved rats following prolonged pimozide treatment and more than 25-fold and 10-fold following a single injection of pimozide. An injection of 250 ng of TRH increased plasma concentrations of TSH in all groups, but this increase was more pronounced in fasted rats injected with pimozide during 3 consecutive days. It is concluded that fasting results in a dopaminergic inhibition of the sensitivity of the thyrotrophs to a TRH challenge.     

Cytological changes of the pituitary basophils in rats slowly infused with thyrotropin-releasing hormone (TRH).

Young male rats were slowly infused with synthetic TRH, 1 microgram/hr, for 1, 3, 24, 48 and 72 hr, respectively. In the control rats, the basophils of the pituitaries can be divided, in their cytological properties, into the II- (classical thyrotrophs), II/III-,III, (classical LH-cell), and III/IV-type cell. The typical IV-type cells (classical FSH-cell), however, are scarcely found in the young rats. Following 1-hr infusion of TRH, the II-type cells decrease in number with the advancement of granular release, but morphological changes are not yet concrete on the other types of basophils. The II-type cells are quickly invisible following a 3-hr infusion, while the III- and III/IV-type cells remain without any significant changes. The III- and III/IV-type cells are progressively degranulated after a 24-hr infusion. The diameter of secretory granules is reduced to 100--150 nm. The smallest ones below 50 nm in diameter, are disintegrated to disperse into the ground matrix. After degranutlaion, the III/IV-type cells appear to revert to the polygonal or stellate cells with the identical fine structure with the II-type cells. There is evidence that the thyroidectomy cells may develop from the III/IV-type cells only after a 48-hr infusion. After 72 hr, most basophils are provided with the uniform structure of "reversionary II-type cells". In reference to the high serum TSH concentration and no significant change of pituitary TSH concentration under the same experimental condition (Soji, 1978), the present author conclusively postulates that the degranulation of the III/IV-type cells may mainly reflect the conspicuous elevation of serum TSH concentration. The above morphological results are contradictory a plausible view that TRH acts only upon the thyrotrophs to release TSH. The fact that all the basophils ultimately take an appearance of "reversionary II-type cells" in the gland by the prolonged infusion of TRH may not only suggest the share of responsiveness of all the basophils to TRH, but also support the hypothesis of secretory cycle of the basophils.     

Theoretical and experimental derivation of the function relating the size distribution of spherical particles in a volume element to that obtained from images of sections taken through it

Summary The mathematical formulation of three classes of theories concerning the problem outlined in the title of this study was critically assessed and experimentally evaluated. It was found that only one class of the theories describes the relation between the distribution of particles in a volume element to that observed in sections through this volume in a satisfactory way. The errors in the remaining two classes could be traced and the corrections made after a mathematically rigorous analysis brought these theories to comply with a model which, as we could prove experimentally, deserves a high level of confidence.Supported by a grant from the Medical Research Council of Canada.In partial fulfillment of the requirements for obtention of the degree of a Master of Science. Supported by a Studentship from the Medical Research Council of Canada.Scholar of the Medical Research Council of Canada.     

Thyroid stimulating hormone and prolactin secretion: reduced sensitivity to TRH-stimulated prolactin release after midpregnancy in rats

Prolactin (PRL) and thyroid stimulating hormone (TSH) plasma concentrations were measured during the latter part of the dark period in early and mid-late pregnancy in the rat. On Days 4-5 and 7-8 of pregnancy, plasma PRL concentrations surged between 22:00 and 06:00 hr and TSH values increased between 22:00 and 02:00 hr. While the TSH pattern was maintained during the second-half of pregnancy, surges in PRL release ceased and PRL levels remained at less than 10 ng/ml. The effects of thyrotropin releasing hormone (TRH) administration on PRL and TSH secretion were then measured to determine whether the second-half of pregnancy is associated with a decrease in sensitivity to an agent that can stimulate PRL release. Injection (iv) of cannulated pregnant rats with a low dosage (20 ng) of TRH stimulated a twofold increase in plasma TSH during both early (Days 5-9) and later (Days 14-18) pregnancy but did not change plasma PRL levels. Treatment with a high dosage (2 micrograms) of TRH induced a sixfold rise in plasma TSH during both phases of gestation. The higher dose of TRH also stimulated elevations in plasma PRL during early and mid-late pregnancy; however, both the absolute increase in the amount of PRL in plasma and the percentage increase over baseline levels were greater from Days 5-9 than from Days 14-16 of gestation. These data indicate that the neuroendocrine sensitivity to factors that stimulate PRL secretion changes as pregnancy progresses, and suggest that nocturnal secretion of PRL and TSH during pregnancy may be regulated, in part, by a common trophic factor.     

Effect of prolonged intermittent thyrotropin-releasing hormone administration to fetal and neonatal rats.

Fetal and neonatal rats received daily subcutaneous injections of 10 microgram thyrotropin-releasing hormone (TRH) until 7 or 14 days postnatally. At 70 days the pups were challenged with 1 microgram TRH intravenously via an indwelling jugular cannula. Basal serum thyroxine, triiodothyronine, and thyroid-stimulating hormone (TSH) concentrations did not differ among the three groups. The mean TSH responses as determined by the mean peak TSH concentration and the total TSH response as determined by planimetry were not significantly different, and there was no significant difference in pituitary TSH content following the TRH challenge among the three groups. This study suggests that the integrity of the hypothalamo--pituitary axis in adult rats cannot be affected by the repeated administration of pharmacologic doses of TRH during the perinatal period.     

Estrogen-induced transformation of somatotrophs into mammotrophs in the rat

Summary In the normal male rat pituitary tritiated thymidine labeled mainly STH cells (somatotrophs), no labeled prolactin cell was found. Following estradiol treatment for 21 days tritiated thymidine labeled mainly prolactin cells (mammotrophs). To determine the origin of these mammotrophs tritiated thymidine was given before the estradiol treatment started, thus labeling many somatotrophs. After 21 days of estradiol, out of 42 labeled cells, 14 were mammotrophs and 13 were somatotrophs; these results suggest that there might be a true transformation of somatotrophs into mammotrophs under the influence of estradiol or that there exist two types of somatotrophs: 1) a committed somatotroph which is not transformed by estrogen treatment, and 2) an uncommitted mammosomatotroph, which under normal conditions bears the features of a somatotroph, but which transforms into a mammotroph under the influence of estradiol.This work was supported by grants MA-552 and MT-2701 from the Medical Research Council of Canada. The authors wish to thank Dr. G. M. Brown, Clarke Institute of Psychiatry, University of Toronto, for the radioimmunoassays of growth hormone and prolactin. Reagents for both radioimmunoassays were kindly provided by the National Institute of Arthritis and Metabolic Diseases, through the Rat Pituitary Hormone Distribution Program. — We are also thankful to Dr. L. Endrenyi, Department of Pharmacology, University of Toronto, for the statistical analysis of our data.Fellow of the Medical Research Council of Canada.     

Thyroxine accelerates the differentiation of granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse

Summary The effects of the administration of thyroxine (T4) on the postnatal cytodifferentiation of granular convoluted tubule (GCT) cells of the submandibular gland (SMG) of Lewiss-Webster mice were studied by light and electron microscopy. From birth, mice of both sexes were injected daily with T4 (sc 0.4 g/g BW) and were sacrificed 24 h after the last injection at 7, 9, 11, 14 and 21 days of age. Control mice received vehicle only. In control mice, granulated striated duct (SD) cells were first detected at 9 days and 7 days of age by light- and electron microscopy, respectively. Furthermore, a few scattered granulated SD cells were observed by light microscopy as early as day 7 in T4-treated mice of both sexes. At 21 days of age, in mice given T4, GCT cells were larger and more numerous and the Golgi apparatus, rough endoplasmic reticulum, and secretion granules were more abundant. In control mice, immunocytochemical staining for epidermal growth factor-(EGF) was first detectable at day 21 at the light- and electron-microscopic levels. However, positively stained cells were first observed in T4-treated mice by light- and electron-microscopic immunocytochemistry at 14 and 11 days of age, respectively. Moreover, in the 21-day-old T4-treated mice, the number of immunoreactive GCT cells, as well as the intensity of the staining per cell, was markedly increased as compared to controls. EGF immunostaining was restricted to GCT cells, and by immuno-electron-microscopy was only seen in apical secretory granules in granulated SD cells and GCT cells. There were no sex differences in the differentiation of the duct system under any conditions. It is concluded that T4 stimulates the biosynthesis of EGF by an acceleration of the differentiation of the GCT precursor cells to mature cells.Supported in part by grant no. MT-5730 from the Medical Research Council of CanadaHolder of a fellowship from the Medical Research Council of CanadaScholar of the Fonds de la Recherche en Santé du Québec     

Morphological and cytochemical modifications induced by colchicine treatment in the duodenal epithelium of mouse

Summary S.W.R. adult male mice received one injection of colchicine and were killed at various time-intervals from 4 to 24 hours. Ultrastructural morphology and cytochemical activity of acid phosphatase, arylsulfatase and thiamine pyrophosphatase were recorded in the duodenal epithelium. Extensive damage was observed in the undifferentiated cells with formation of large heterophagosomes and in the absorptive cells with apparition of cytolysomes. The activity of the acid phosphatase and thiamine pyrophosphatase decreased after 6 hours of treatment. Moreover, the migration of the acid phosphatase content of the Golgi vesicles was restraint. These observations were related to the disruptive action of the alcaloid towards the microtubules.This work was supported by a grant from the Medical Research Council of Canada.Mr. Raymackers was a recipient of a studentship of the Medical Research Council of Canada. The authors are greatly indebted to Mr. Michel Couture and Pierre Magny for their skilful technical aid.     

Changes of cell morphology and prolactin secretion induced by 2-Br- alpha-ergocryptine, estradiol, and thyrotropin-releasing hormone in rat anterior pituitary cells in culture

The secretion of prolactin in cultured pituitary cells was studied in correlation with the cellular changes induced by stimulatory or inhibitory agents. The techniques used in this study were: radioimmunoassay, immunocytochemistry, scanning (SEM) as well as transmission (TEM) electron microscopy. Prolactin secretion was stimulated by 17 beta-estradiol (10 nM) as well as thyrotropin- releasing hormone (TRH) (3 nM) and inhibited by 2-Br-alpha-ergocryptine (CB-154) (1 muM). The total prolactin (release and cell content) increased between 2 and 8 d of estradiol treatment, indicating an increase of both synthesis and release of prolactin. This finding was in agreement with TEM observations because, in estradiol-treated prolactin cells, the Golgi saccules were distended and Golgi elements were increased, thus indicating increased synthetic activity of these cells. The addition of TRH over a 4-h period resulted in a significant degranulation of prolactin cells. In contrast, prolactin secretory granules became accumulated in the cells after CB-154 treatment for a period ranging from 4 to 24 h. In agreement, light microscope immunocytochemistry showed an increased reaction for prolactin after short-term (< 24 h) incubation with CB-154. Because prolactin cells represent approximately 70% of the glandular cell population as revealed by immunocytochemistry, it was then possible to observe the changes of cell surface by SEM. In most cells, estradiol and TRH led to an increase in the number and prominence of microvilli and blebs, whereas CB-154 treatment resulted in a slightly decreased number of microvilli and an increased occurrence of membrane foldings. This report thus provides morphological evidence for the stimulatory effects of estradiol and TRH, and the inhibitory effects of CB-154 on prolactin secretion in pituitary cells in primary culture. These data, moreover, show that acute changes in secretory activity of prolactin-secreting cells are accompanied by marked changes of their morphological characteristics.     

Thyrotropin-releasing hormone stimulates the activity of the rat thyrotropin beta-subunit gene promoter transfected into pituitary cells

In order to investigate the molecular mechanism(s) by which TRH regulates the biosynthesis of TSH, we are studying the effects of TRH on the expression of the TSH subunit genes (alpha and TSH beta). To study the structure-function relation of TRH stimulation of the activity of the single rat TSH beta gene, chimaeric plasmids were constructed. The 5'-flanking region of the rat TSH beta gene including exon 1 (5'-untranslated region) was inserted into a promoterless, modified pBR, chloramphenicol acetyltransferase (CAT) expression vector. After transfection, specific TSH beta promoter activity was evident in both TRH-responsive pituitary-derived GH3 and primary pituitary cell cultures. To determine potential regulation of TSH beta promoter-directed activity in these cells by TRH, cells were incubated with media containing TRH (10(-7) to 10(-11) M) for 1 to 48 h. TRH stimulated a 1.5- to 3-fold increase in TSH beta promoter activity. Concomitant with an increase in CAT activity was an anticipated increase in PRL synthesis in the GH3 cells in response to TRH. The TRH effect on the TSH beta gene was specific; no increase in CAT activity was detected for TKCAT (thymidine kinase of herpes simplex virus promoter), pBRCAT (no promoter), or TSH beta CAT (3'-5'-orientation). Similar results were obtained using primary pituitary cell cultures. Deletion mutation analysis indicated that TRH sensitivity was detected in a 1.1 kilobase, but not in a 0.38 kilobase TSH beta gene fragment suggesting that the TRH responsive element(s) resides at least in part within the 700 base pairs of the 5'-flanking sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological studies of the stromal changes after artificial induction of the decidual reaction in rats

Summary Stromal changes during early oil-induced decidualization were studied. Rats previously sensitized with estradiol after an experimental delay were used. Among the initial stromal characteristics that developed after induction, the following sequence was found: ultrastructural alterations of stromal cell nucleoli, extracellular edema, and Pontamine Blue Reaction (PBR). The results suggest that stromal cell differentiation into decidual cells is initiated before the PBR becomes visible. This is in accordance with biochemical and morphological findings at blastocyst implantation. The appearance of an edema before the PBR furthermore suggests that also the increase in vascular permeability is initiated early and takes place slowly. The functional relationships between the vascular and stromal cell changes are, however, not determinable with the method used.This work was supported by grants from the Medical Faculty at the University of Uppsala and the Swedish Medical Research Council (Project No 12X-70 to O. Nilsson)Technical assistance: Mrs Marianne Ljungkvist and Miss Kerstin Rystedt     

The goldfish pituitary

Summary In the goldfish, Carassius auratus, morphological and functional aspects of the pituitary gland were studied at the ultrastructural level and six cell types could be distinguished in the pars distalis. Acidophilic cells of the rostral pars distalis were identified as prolactin cells, the chromophobic cells of the rostral pars distalis as ACTH cells, the non-globular basophilic cells of the rostral and the proximal pars distalis as TSH cells, the globular basophils of the proximal pars distalis as gonadotropic cells and the acidophils of the proximal pars distalis as somatotrophs.Besides some of the well established criteria of morphological and functional identification of different cell types, two new approaches have been used in the present study. One was to express the electron density of secretory granules objectively by means of a photometric method. It was found that both types of acidophilic cells which produce the proteohormones prolactin and somatotropin respectively, had granules with the highest electron densities. The basophilic cells producing the glycoproteins gonadotropin and TSH respectively, possessed granules of intermediate electron density whereas the chromophobic cells storing the peptide hormone ACTH had granules of lowest densities. The second new approach was the administration of the synthetic mammalian releasing hormones LH-RP and TRF, which helped in identifying gonadotropic and thyrotropic cells respectively. In the goldfish there is evidence for the presence of only one type of gonadotropic cell.Supported by a grant of the Science Research Council of Great Britain to Professor Sir Francis Knowles, F.R.S. The electron microscope used was provided by the Medical Research Council of Great Britain. The integrating photometer IPM2 was kindly on loan from Messrs. Carl Zeiss, Oberkochen, Germany. For technical advice we are greatly indebted to Mr. P. K. Kaul, B. E., M.I. Struct. E., C. Eng.     

Cellular defense in an Arthropod in response to infection with aSalmonella typhimurium strain

Larvae ofGalleria mellonella L. when injected with cells ofSalmonella typhimurium strain LT 2 responded by cellular defense: their hemocytes gathered and formed a pseudo-tissue by which the bacteria were encapsulated. In contrast,S. typhimurium strain 7 Suc LL caused lysis of about 64% of the hemocytes and cellular defense against this strain was lacking.This explains the difference in the mortality rate of the larvae which was 10% after intracoelomic injection with 2×10⁴ cells of strain LT 2 and 48% with the same number of cells of strain 7 Suc LL.After injection of strain 7 Suc LL, lysis of hemocytes preceded proliferation of bacteria; moreover such lysis also occurred after injection of cell-free culture filtrate of this strain. This suggests that lysis is due to either a toxic substance or a proteolytic enzyme produced by strain 7 Suc LL.These investigations were supported by the Medical Research Council of Canada Grant MA-2385 and the National Research Council of Canada Grant A-3746.