Propylthiouracyl-induced hypothyroidism results in reversible transdifferentiation of somatotrophs into thyroidectomy cells. A morphologic study of the rat pituitary including immunoelectron microscopy

Two-month-old female Fischer-344 rats were rendered hypothyroid by ingestion of propyl-thiouracyl (PTU) (0.1% in drinking water) and sacrificed 3, 7, 14, and 28 days after the start of PTU administration as well as 3, 7 and 14 days after interruption of a 14-day PTU treatment. Controls received no PTU. The pituitaries were studied by histology, immunohistochemistry, electron microscopy, and immunoelectron microscopy, using the immunogold double-labeling technique. In the course of hypothyroidism, pituitary thyrotrophs had undergone the well-known thyroidectomy change. In addition, a contingent of growth hormone (GH) cells lost their large secretory granules, enlarged, displayed progressive dilation of rough endoplasmic reticulum, thereby transforming into thyroidectomy cells. These bihormonal thyrosomatotrophs contained gH in their secretory granules and thyrotropin in the dilated rough endoplasmic reticulum as documented by the immunogold double-labeling method for GH and thyrotropin. After discontinuation of PTU treatment, a rapid increase in size, number and GH labeling of secretory granules and simultaneous involution of distended rough endoplasmic reticulum with reduction of thyrotropin labeling took place in thyrosomatotrophs. A practically complete restitution of normal pituitary structure was seen in 2 weeks. Results implicate that, contrary to previously accepted concepts, adenohypophysial cells may not be irreversibly committed to one morphologically recognizable cell line.     

Morphometric study of normal and adenomatous pituitary somatotrophs in humans

Somatotrophs from ten pituitary adenomas were evaluated morphometrically by light and electron microscopy using the following parameters: a) nuclear, cytoplasmic and cell volumes; b) volume density, total volume, surface density, total surface and surface/volume ratio of secretory granules, mitochondria, rough endoplasmic reticulum and Golgi apparatus, and c) the number of secretory granules and mitochondria per micron3 of cytoplasm and per cell. The results were compared (p less than 0.05 and p less than 0.10) with those obtained from somatotrophs identified in five normal pituitaries. The data obtained indicate that: a) in the adenomas, the number of secretory granules per cell cannot be accurately evaluated from their apparent number in sectioned cell profiles; b) there are two basic sub-types of adenomatous somatotrophs defined according to the mean secretory granule diameter; cells in which granule diameter is inferior to 180 nm exhibit distinct morphological features such as nuclear pleomorphism, the presence of gross bundles of intermediate sized filaments or fibrous bodies in the cytoplasm and a variable number of secretory granules. Adenomas constituted mainly by these cells were found in younger patients, suggesting the more aggressive nature of these tumours, thus warranting close clinical follow-up of such patients; and c) in both types of adenomatous cells, the organelles directly involved in the secretory process, i.e., the rough endoplasmic reticulum and Golgi apparatus, are larger than in the control cells; however, the ratio between the surfaces of these two compartments does not differ among the three groups studied.     

Mechanism of spontaneous and receptor-controlled electrical activity in pituitary somatotrophs: experiments and theory

Cultured pituitary somatotrophs release growth hormone in response to spontaneous Ca(2+) entry through voltage-gated calcium channels (VGCCs) that is governed by plateau-bursting electrical activity and is regulated by several neurohormones, including GH-releasing hormone (GHRH) and somatostatin. Here we combine experiments and theory to clarify the mechanisms underlying spontaneous and receptor-controlled electrical activity. Experiments support a role of a Na(+)-conducting and tetrodotoxin-insensitive channel in controlling spontaneous and GHRH-stimulated pacemaking, the latter in a cAMP-dependent manner; an opposing role of spontaneously active inwardly rectifying K(+) (K(ir)) channels and G-protein-regulated K(ir) channels in somatostatin-mediated inhibition of pacemaking; as well as a role of VGCCs in spiking and large conductance (BK-type) Ca(2+)-activated K(+) channels in plateau bursting. The mathematical model is compatible with a wide variety of experimental data involving pharmacology and extracellular ion substitution and supports the importance of constitutively active tetrodotoxin-insensitive Na(+) and K(ir) channels in maintaining spontaneous pacemaking in pituitary somatotrophs. The model also suggests that these channels are involved in the up- and downregulation of electrical activity by GHRH and somatostatin. In the model, the plateau bursting is controlled by two functional populations of BK channels, characterized by distance from the VGCCs. The rapid activation of the proximal BK channels is critical for the establishment of the plateau, whereas slow recruitment of the distal BK channels terminates the plateau.     

Retention of peptide hormones during partial secretion in pituitary somatotrophs and corticotrophs

The secretion of peptide hormones during exocytosis of an individual vesicle can result in either complete discharge of vesicle content or can occur in a partial manner in which some hormone is retained during transient fusion. In anterior pituitary lactotrophs, the retained hormone prolactin was internalized and recycled into a pool of vesicles that underwent preferential use during subsequent exocytic stimulations [Bauer et al., (2004) J Cell Sci. 117:2193–2202]. The aim of the present study was to determine whether retention and preferential recycling of retained hormones occurred in other anterior pituitary cells. Stimulation of somatotrophs with high K⁺ resulted in 50 discrete puncta per cell that were positive for growth hormone immunoreactivity. Identical stimulation of corticotrophs resulted in 150 puncta per cell that were anti-adrenocorticotrophic hormone (ACTH) positive. However, unlike what was observed for lactotrophs, the number of structures containing retained growth hormone and ACTH decreased to less than 10% of the initial value in 80 min in somatotrophs and in less than 10 min in corticotrophs. Our results indicate that functional recycling of retained hormones is not shared by all anterior pituitary cell types.     

Cellular and functional interactions between gonadotrophs and lactotrophs in pituitary cell cultures

Lactotroph secretory activity is regulated by hypothalamic stimulating and inhibiting factors as well as peripheral endocrine hormones. In addition to this important control domain, the pituitary gland displays an intrinsic regulatory ability through autocrine and paracrine signals. To evaluate the role of gonadotrophs in the regulation of prolactin (PRL) secretion, a comparative study was performed applying two regulatory agents that operate through different physiological mechanisms: gonadotropin-releasing hormone (GnRH), which releases regulatory factors co-localized in secretory granules of gonadotrophs, stimulating PRL secretion from lactotrophs; and angiotensin II (AII), with direct effects on lactotroph secretion through specific receptors. In these studies performed in regular and purified primary pituitary cell cultures from female rats, the lactotrophs comprised the largest population of cells (about 51%), whereas gonadotrophs represented only a small fraction (3%) of the total pituitary cell count. In regular cell cultures treatments with AII and GnRH showed a similar secretory behavior, increasing PRL output 73% and 63%, respectively. The stimulation with GnRH and AII of cell cultures with purified lactotrophs and gonadotrophs provided comparable results, but the response of lactotrophs was significantly higher (106% and 138%, respectively) than that recorded in regular cell cultures. Simultaneous AII treatment with an antipeptide antagonist to AII receptor (AII-antipep) completely blocked the PRL release induced by AII. The co-incubation of cells with GnRH and AII-antipep suppressed the peak of PRL release caused by GnRH, confirming that AII is a paracrine agent released by gonadotrophs stimulated with GnRH. The different secretory behavior of lactotrophs treated with AII and GnRH in both regular and purified cell cultures is indicative of the degree of functional interactions between different pituitary cell types. The present study supplies morphological and functional information on the cell-to-cell interactions, which plays an important role in the intrinsic regulatory control of PRL secretion.     

Reversible papillary hyperplasia of the rat urinary bladder.

A rapid reproducible method for the production of ulcers and reversible regenerative hyperplasia of the urinary bladder of rats is described. This method does not involve administration of a toxic chemical or retention of a foreign body. Ulceration was produced by means of applying a steel rod, 5 mm in diameter, frozen at -78 C, to the serosal surface of the bladder for 2 seconds, twice, with a 5-second interval between each application. Sequential histologic observations showed that ulcers were accompanied by necrosis of the entire thickness of the bladder at the site of freezing with extensive acute inflammation. Regenerative hyperplasia was evident at the edge of the ulcer by the second day, with greatest severity at 5 days when papillary and nodular hyperplasia were present. At 15 days the bladders were normal or had only minimal hyperplasia. The labeling index by autoradiography was highest in the mildly hyperplastic area near the ulcer at 2 days and remained relatively high through 5 days. The hyperplastic epithelium surrounding the ulcer observed by scanning electron microscopy had numerous degenerative cells on the surface, and small epithelial cells with numerous short, uniform microvilli on their luminal surface were also observed. The numerous short, uniform microvilli on their luminal surface were also observed. The microvilli had a symmetric luminal membrane, as observed on trasmission electron microscopy. More peripherally the cells had microridges only or microvilli and microridges on their luminal surface. Neither pleomorphic microvili nor a structured glycocalyx fuzz on microvilli was observed during the process of regenerative hyperplasia, distinguishing it from neoplastic bladder proliferations.     

Appearance and disappearance of tubular paracrystalline structures in somatotrophs and lactotrophs during the annual life cycle of the bat (Myotis lucifugus)

The fine structure of the endocrine (granulated) cells of the adenohypophysis of the bat was examined at different periods of the annual life cycle. During the active homeothermic period (early spring until late summer) the endocrine cells were heavily granulated, and evidence of granule release was commonly seen. At this time, but not at other times (hibernation and arousal), prominent paracrystalline structures were numerous and transiently appeared in the cytoplasm of acidophils (apparently both lactotrophs and somatotrophs) but not in the cytoplasm of basophils. These paracrystalline bodies were composed of densely packed, parallel arrays of tubules. The paracrystalline structures in lactotrophs differed in size, shape, and the pattern of arrangement of component tubules from those of somatotrophs. During hibernation no evidence of exocytosis was seen, and secretory granules accumulated in the cytoplasm. Prior to arousal, secretory granules were removed by autophagy.     

Proliferation and differentiation of pituitary somatotrophs and mammotrophs during late fetal and postnatal periods

Proliferation of somatotrophs and mammotrophs in the rat pituitary during late fetal and postnatal periods up to 4 weeks after birth was quantitatively studied with the double immunostaining of bromodeoxyuridine and the hormones produced by them. Somatotrophs were first detected in 18.5-day fetuses and rapidly increased in number throughout the periods studied. The cells labeled with both anti-BrdU and anti-GH were few in number until shortly before birth and then increased conspicuously during the first 10 days after birth. Mammotrophs were detected at gestational day 19.5 but they were few until the second week after birth, when their number began to increase rapidly. The percentage of the number of the cells double-labeled with both anti-BrdU and anti-GH to all somatotrophs was 8.3% at the most. This was about the same as that of corticotrophs during the late fetal period and that of thyrotrophs in the early postnatal period. In contrast, the percentage of double-labeled cells to all mammotrophs was 3.8% as a maximum, which is lower than the values for somatotrophs, corticotrophs, or thyrotrophs, indicating a smaller contribution of mitosis to mammotroph proliferation. It is possible that this smaller contribution is compensated for by transdifferentiation of cells committed to become the somatotroph lineage. However, coexistence of GH and PRL was not observed in the present material.     

Effects of pergolide on diethylstilbestrol-induced rat pituitary hyperplasia.

Hyperplastic anterior pituitary glands were produced in female rats by treatment with 10 mg of diethylstilbestrol in Silastic tubing. This led to increased numbers of immunoreactive prolactin cells and increased serum prolactin levels. After 6 weeks of diethylstilbestrol treatment, one group of rats was treated with daily injections of pergolide for 3 weeks. Pergolide produced a significant decrease in pituitary gland weight and in serum prolactin levels but did not change the percentage of prolactin cells significantly, compared with that of control rats. Ultrastructural studies showed a significant increase in the numbers of prolactin secretory granules and numerous large intracellular bodies with associated secretory granules in pituitaries from rats treated with pergolide. In one group of rats in which the diethylstilbestrol was discontinued for 3 weeks after 6 weeks of treatment there was a significant decrease in pituitary gland weight and serum prolactin and a significant decrease in the percentage of prolactin cells, compared with values in the rats treated with diethylstilbestrol for 9 weeks. These results indicate that pergolide causes decreased release of prolactin from secretory granules in anterior pituitary prolactin cells and an increase in the numbers of PRL secretory granules per cell but does not change the percentage of prolactin-producing pituitary cells after 3 weeks of treatment.     

Transdifferentiation of somatotrophs to thyrotrophs in the pituitary of patients with protracted primary hypothyroidism

In patients with protracted primary hypothyroidism, the pituitary is enlarged due to the lack of feedback inhibition by thyroid hormone. In the present work, adenohypophysial biopsies from three women with protracted primary hypothyroidism were investigated by routine histology, immunocytochemistry, double immunostaining, immunoelectron microscopy, and combined immunocytochemistry – in situ hybridization. These methods confirmed the presence of massive thyrotroph hyperplasia and the formation of ”thyroidectomy” or ”thyroid deficiency” cells. A number of thyroidectomy cells were found to be immunoreactive for growth hormone (GH). Double immunostaining and immunoelectron microscopy revealed the presence of bihormonal cells containing both GH and thyroid stimulating hormone (TSH). Immunostaining combined with in situ hybridization revealed GH immunoreactive cells expressing TSH mRNA as well as TSH immunopositive cells expressing GH mRNA. Our findings provide conclusive evidence that somatotrophs may transform to thyrotrophs. Thus, in addition to multiplication of thyrotrophs, transdifferentiation of GH cells to thyrotrophs contributes to the increase of TSH-producing cells. The presence of such bihormonal cells best termed ”thyrosomatotrophs” supports the concept that adenohypophysial cells are not irreversibly committed to the production of one single hormone and that their phenotype can change in response to functional demand. Received: 16 March 1999 / Accepted: 4 July 1999     

转染腺病毒载体对雌二醇促垂体催乳素细胞增殖的影响

目的探讨转染腺病毒载体对雌二醇促垂体催乳素细胞增殖的影响及可能的分子机制。方法应用BrdU标记法检测转染腺病毒载体后雌二醇促垂体细胞增殖作用的变化,实时荧光定量PCR方法检测腺病毒载体转染组雌激素反应基因ABCG2的mRNAs表达水平,Western Blot方法检测腺病毒载体对垂体前叶细胞磷酸化p38MAPK蛋白表达的影响。结果转染腺病毒载体显著下调了雌二醇的促催乳素细胞增殖作用(P0.01),腺病毒转染组磷酸化p38MAPK蛋白表达显著增加,ABCG2基因的表达显著下降(P0.01)。结论腺病毒载体转染下调雌二醇促垂体催乳素细胞的增殖作用,其机制可能与腺病毒载体改变p38MAPK和ABCG2的表达有关。     

Effect of intravenous infusion of growth hormone-releasing hormone on the morphology of rat pituitary somatotrophs

The effect of GRH infusion on rat adenohypophysial morphology was studied by light microscopy, immunocytochemistry, in situ hybridization, and electron microscopy. Synthetic rat GRH was intravenously administered by osmotic minipumps at 14.4, 72, 360 and 720 μg/ day/rat for 1 week. In one group treated for 1 week with a daily dose of 720 μg GRH, the rats were killed 7 days after withdrawal of GRH. Control rats in which GRH was replaced by excipient, or those that received no treatment, were included as well. GRH infusion with daily doses of 360 and 720 μg resulted in a significant increase in pituitary weight and weaker GH immunoreactivity compared with other groups. Ultrastructurally, the somatotrophs were increased in size and became sparsely granulated, and the organelles involved in hormone sythesis were very prominent. The intensity of the GH mRNA signal did not differ from control animals, suggesting the desensitization of somatotrophs to GRH. The highest GRH dose induced an increased number of nuclei immunoreactive for proliferation cell nuclear antigen (PCNA). One week after GRH withdrawal, shrinkage of cytoplasm, involution of RER and Golgi complex, and a decrease of cell attachment sites indicated the reversibility of changes induced by GRH. In conclusion, GRH infusion induced, within days, hypertrophy and proliferation of somatotrophs with ultrastructural features of highly stimulated, sparsely granulated cells. Morphological changes were reversible.Endocr Pathol 4:131–139, 1993.     

Estrogen-induced hyperplasia and neoplasia in the rat anterior pituitary gland. An immunohistochemical study

Diethylstilbestrol (DES) treatment of weanling F344 female rats resulted in enlarged pituitary glands and diffuse pituitary prolactin (PRL) cell hyperplasia in all animals after 9 and 12 weeks of treatment. Serum PRL was significantly greater than in control rats (P less than 0.001). Immunohistochemical studies showed that most of the pituitary gland cells consisted of PRL cells. Ultrastructural studies showed increased numbers of PRL cells with hyperplasia of the rough endoplasmic reticulum and decreased numbers of secretory granules. There was a decrease in the relative number of growth hormone (GH) and other cell types in the anterior pituitary. Pituitary tumors and normal pituitary glands were dissociated with trypsin and maintained in culture for 3 weeks. The numbers of PRL and GH cells decreased with time in both groups, and there was an increase in the number of fibroblasts. Staining of the culture cells with neuron-specific enolase showed that the anterior pituitary cells were positive for this enzyme, while the fibroblastic cells were negative. When dissociated pituitary cells were cultured in the presence of 10(-9) M DES for 7 days, there was a 42% increase in the number of immunoreactive PRL cells. These results indicate that DES-treated rats provide an excellent model for study of the in vivo and in vitro regulation of pituitary hyperplasia and neoplasia.     

Evidence for growth hormone (GH) autoregulation in pituitary somatotrophs in GH antagonist-transgenic mice and GH receptor-deficient mice

Growth hormone (GH) modulates the hypothalamic release of somatostatin and GH-releasing hormone; however, there has been no evidence of GH autoregulation on the pituitary somatotroph. To determine the effects of GH on its own regulation, we examined the pituitaries of giant transgenic mice expressing a GH agonist (E117L), dwarf transgenic mice expressing a GH antagonist (G119K), and dwarf mice devoid of the GH receptor/binding protein (GHR/BP). In the E117L transgenic mice, the number and distribution of pituitary GH-immunoreactive cells were unchanged from nontransgenic littermate controls; an ultrastructural examination revealed typical, densely granulated somatotrophs. In contrast, the pituitaries of the G119K mice contained both moderately granulated somatotrophs and a sparsely granulated (SG) population with well-developed synthetic organelles and a distinct juxtanuclear globular GH-staining pattern. GHR/BP-deficient mice exhibited a marked reduction in the intensity of cytoplasmic GH immunoreactivity; however, prominent GH staining in the juxtanuclear Golgi was seen. GH-immunoreactive cells were increased in number, and the reticulin network pattern was distorted; stains for proliferating cell nuclear antigen confirmed mild hyperplasia. Electron microscopy showed that the somatotrophs were hyperactive SG cells with prominent endoplasmic reticulum membranes, large Golgi complexes, and numerous mitochondria. These findings are consistent with synthetic and secretory hyperactivity in pituitary somatotrophs due to the reduced GH feedback regulation. The changes are most striking in animals that are devoid of GHR/BP and less marked in animals expressing a GH antagonist; both models had reduced insulin-like growth factor-I levels, but the more dramatic change in the GHR/BP animals can be explained by abrogated GH signaling. This represents the first evidence of direct GH feedback inhibition on pituitary somatotrophs, which may have implications for the use of GH analogs in different clinical settings.     

Secretory activity of lactotrophs and its regulation by hypothalamic hormones in primary pituitary cell cultures from rats of different ages

Laboratory of Biological Research into Hormonal Compounds, Institute of Experimental Endocrinology, All-Union Endocrinologic Research Center, Academy of Medical Sciences, Moscow. (Presented by Academician Yu. A. Pankov, Academy of Medical Sciences.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 4, pp. 402–404, April, 1992.     

Morphologic effects of bromocriptine on spontaneously occurring pituitary prolactin-cell hyperplasia in old Long-Evans rats.

The effect of bromocriptine (BEC), a dopaminergic agonist, on nontumorous pituitary prolactin (PRL) cells of aging female Long-Evans rats, was studied histologically, immunocytologically, electron-microscopically, and morphometrically. Rats were arbitrarily divided into two control groups, one with normal (less than 20 ng/ml) and one with elevated serum PRL concentrations, and into four BEC-treated groups, all of which had increased serum PRL levels prior to commencement of BEC administration. In hyperprolactinemic control rats, compared with normoprolactinemic control rats, pituitary weight and percentage of pituitary PRL cells were increased. The morphologic features of PRL cells in these two groups did not differ markedly, which suggested that hyperprolactinemia was due to increased PRL-cell number and not increased PRL-cell function. Compared with age-matched hyperprolactinemic control rats, hyperprolactinemic rats treated with BEC showed a reversible decrease in serum PRL levels, pituitary weight as well as percentage of pituitary PRL cells, and by ultrastructural morphometry an increase in the volume density of lysosomes. BEC caused no striking changes in nuclear and cytoplasmic areas, volume densities of RER, Golgi regions, mitochondria, lipid droplets, and size and volume densities of forming and storage granules. Since spontaneously hyperplastic PRL cells show less conspicuous morphologic changes following BEC treatment than PRL cells rendered hyperplastic by estrogen administration or pituitary transplantation, it is suggested that PRL cells with no increased endocrine function respond less markedly to dopaminergic suppression than endocrinologically hyperactive PRL cells. It can be concluded that BEC suppresses spontaneous proliferation of PRL cells which occurs with aging.     

The role of the pituitary hormones in hyperplasia of the mammary glands

Summary Combined administration of estrogens and prolactin did not induce any hyperplasia of the mammary glands of female suckling rats. However, combined administration of estrogens and gonadotropic hormones regularly caused a marked hyperplasia of the mammary glands. The extent of hyperplasia was connected with the amount of follicle-stimulating hormone administered, but did not depend upon the dose of the luteinizing hormone given; this points to the decisive role played by the follicle-stimulating hormone in the mammary gland hyperplasia occurring in female suckling rats.(Presented by Active Member AMN SSSR L. M. Shabad) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 55, No. 1, pp. 104–107, January 1963