Postnatal differentiation of the immunohistochemical expression of aromatase P450 in the rat pituitary gland

At our laboratory, we have recently demonstrated the immunohistochemical expression of aromatase P450 in the pituitary glands of adult rats; this expression was seen to be sex-dependent. In order to determine whether the changes in the expression of the enzyme are related to changes in the gonadal sphere and whether the expression of the enzyme is related to the postnatal differentiation of hypophyseal cytology, in the present work we performed an immunohistochemical study in the rat pituitary gland from birth to old age. The immunohistochemical reaction to aromatase was evident and very generalized at 7 days after birth, with no large differences between the male and female animals. At 14 days the immunohistochemical reaction was decreased in the females, with no changes in the males. At 17 days, aromatase immunoreactivity in the pituitary glands of female rats was very weak whereas the males showed large numbers of reactive cells. These observations were further pronounced at 21 days and 2 months of life. At 24 months, the immunoreactivity found in the pituitary glands of the male rats had almost completely disappeared. Our results show that a postnatal differentiation in the immunohistochemical expression of aromatase occurs; this is tightly linked to sexual activity and is lost in old age. This suggests that hypophyseal aromatase would be related to the mechanisms of action of gonadal steroids on hypophyseal differentiation and secretion.     

Cellular localization of somatolactin in the pars intermedia of some teleost fishes

Summary We report here on the cellular localization in the fish pituitary of somatolactin (SL), a putative new pituitary hormone related to growth hormone and prolactin, which has been recently identified in the piscine pituitary gland. Immunocytochemical staining, using anti-cod SL serum, revealed that in the cod pituitary gland, SL is produced by cells in the intermediate lobe, bordering the neural tissue. These cells, staining weakly with periodic-acid-Schiff (PAS), are distinct from the melanocyte stimulating hormone (MSH) cells which, as in all teleosts, are PAS-negative. SL-immunoreactivity was observed in the same location in all other teleost species examined: flounder, rainbow trout, killifish, molly, catfish and eel. In most fish the SL-immunoreactive cells are either strongly or weakly PAS-positive but in rainbow trout are chromophobic, indicating that the SL protein can probably exist in glycosylated and non-glycosylated forms. Thus, in demonstrating the cellular localization of SL, this study provides the first identification of the enigmatic, second cell-type of the fish pars intermedia.     

Spontaneous pituitary abnormalities and mammary hyperplasia in FVB/NCr mice: implications for mouse modeling

The FVB/N mouse strain is widely used in the generation of transgenic mouse models. We have observed that mammary glands of wild-type virgin female FVB/NCr mice frequently have the morphologic and histologic appearance of a gland during pregnancy. By 13 months of age, the mammary glands of more than 40% of the mice examined had lobuloalveolar hyperplasia that was characterized by the presence of secretory alveoli and distended ducts apparently containing secretory material. The prevalence of this phenotype further increased with age. The mammary phenotype was highly correlated with the presence of proliferative, prolactin-secreting lesions in the pituitary gland. In mice aged 18 to 23 months, hyperplasia of the pars distalis was seen in 11 of 21 mice (52%), and a further 4 of 21 mice (19%) had pituitary adenomas. Pituitary hyperplasia was already evident in some mice as young as nine months. The pituitary phenotype was also associated with high prevalence (4/6 mice) of spontaneous mammary tumors in aged multiparous, but not virgin FVB/NCr mice. This high prevalence of pituitary abnormalities and their effects on the mammary gland have important consequences for the interpretation of new phenotypes generated in transgenic models using this mouse substrain.     

Effect of testosterone and progesterone on carboxypeptidase H activity in the hypothalamo-hypophyseal-gonadal axis and the adrenal gland in mice

Testosterone propionate decreased activity of carboxypeptidase H in the male mongrel mice pituitary gland, activity of the CP H in testicles, and increased these parameters in 4 hours after treatment. Progesterone decreased the CP H activity in pituitary gland and increased it in testicles. Progesterone increased the CP H activity in female pituitary gland and decreased it in adrenal gland and hypothalamus. The role of the CP H in the effects of the sex steroids on the function of peptidergic systems is discussed.     

Precocious differentation of mouse parotid glands and pancreas induced by hormones

Changes of α-amylase activity (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) in mouse parotid gland pancreas were investigated during embryonic and postnatal development. Amylase activity in the parotid gland increased from around day 12 and reached the adult level on day 30. On the other hand, the activity in the pancreas increased during the last stage of gestation, decreased after birth, and then gradually increased from around day 15, reaching the adult level on day 35. Precocious differentiation of the parotid gland was induceed by injections of hydrocortisone or thyroxine after birth, but these hormones did not have additive effects on the parotid gland. Injection of insulin had little effect when given alone, but suppressed the effects of the other two hormones on the gland. Only hydrocortisone increased the amylase activity in mouse pancreas during postnatal development, the other two hormones causing slight decrease in pancreatic amylase. Adrenalectomy and injection of hydrocortisone affected the parotid gland but not the pancreas of adult mice. These results suggest that hydrocortisone involved in cytodifferentiations of the parotid gland and pancreas, and in maintenance of the parotid gland. Thyroxine may also be important in differentiation of the parotid gland.     

Mex3c mutation affects lactation through impairing milk ejection in female mice

Mouse Mex3c encodes RNA-binding proteins of variant length through alternative splicing. Its mutation results in multiple defects including growth retardation, perturbed energy balance, and defective antiviral innate immunity. Here we report that Mex3c mutation affects mammary gland development and lactation in female mice. Pups of Mex3c mutant dams die of starvation soon after birth. Milk contents are present in the alveoli but deficient in the ducts of the mammary glands in mutant mice. Mutant mice do not show prolactin or oxytocin deficiency. They also develop myoepithelial cells in the mammary glands. Mex3c is expressed in the mammary gland epithelium. Our data suggest that functional defects in mammary gland epithelium or myoepithelial cells could cause lactation defects.     

Determination of free d-aspartic acid,d-serine and d-alanine in the brain of mutant mice lacking d-amino-acid oxidase activity

A simple and precise method for the simultaneous determination of free d-aspartic acid, d-serine and d-alanine in mouse brain tissues was established, using a reversed-phase HPLC system with widely used pre-column derivatizing reagents, o-phthaldialdehyde and N-t-butyloxycarbonyl-l-cysteine. With the present method, the contents of these three d-amino acids in hippocampus, hypothalamus, pituitary gland, pineal gland and medulla oblongata as well as cerebrum and cerebellum of mutant mice lacking d-amino-acid oxidase activity were determined and compared with those obtained for control mice. In both mice, extremely high contents of d-serine were observed in forebrain (100–400 nmol/g wet tissue), and the contents were small in pituitary and pineal glands. While, d-serine contents in cerebellum and medulla oblongata of mutant mice were about ten times higher than those in control mice. In contrast, d-alanine contents in mutant mice were higher than those in control mice in all brain regions and serum.     

Correlated changes in the structure of the anterior pituitary gland,testes and interrenal tissue during sexual maturation of male lizards

Summary The anterior pituitary gland, testes and interrenal glands of a series of young males of the teiid Cnemidophorus l. lemniscatus (L.) have been studied by light microscopy in order to correlate the changes occurring during sexual maturation. In the testes of the smallest animals, spermatogenesis does not progress beyond primary spermatocytes and there is no differentiated interstitial tissue. In medium-sized animals, spermatids and some interstitial cells appear, and in the largest lizards, spermatogenesis is completely established and Leydig cells abound. Simultaneously with the development of the testes, interrenal glands undergo great hypertrophy and hyperplasia, especially in the peripheral reactive zone. Starting in animals of intermediate size, the anterior hypophysis exhibits a considerable hypertrophy of two rostral cell types: the chromophobic corticotrophs and the acidophilic PAS-positive cells considered to be interstitiotrophs. These cells show large, vesicular nuclei and prominent nucleoli, signs of enhanced cellular activity. The hypertrophy begins in the dorso-rostral region of the gland close to the median eminence, at the site of entry of the portal vessels. This suggests a hypothalamic influence on the function of these pars distalis cells. The scattered basophilic gonadotrophs or folliculotrophs are scarce, small, and do not vary appreciably among the animals studied. The hyperactivity of corticotrophs may account for enlargement of the interrenal glands. Testicular development is apparently related to an increased activity of interstitiotrophs but to a stable level of activity in folliculotrophs.This research forms part of project No. 31.26.S1-0244 supported by the Consejo Nacional de Investigaciones Científicas y Tecnológicas.     

Requirement of thyrotropin-releasing hormone for the postnatal functions of pituitary thyrotrophs: ontogeny study of congenital tertiary hypothyroidism in mice

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

Testosterone metabolism in neuroendocrine organs in male rats under atrazine and deethylatrazine influence

The inhibitory influence of atrazine and deethylatrazine on testosterone metabolism in male rat anterior pituitary and hypothalamus were studied under in vivo and in vitro experimental conditions. In vivo strong influence of atrazine (12 mg/100 g by wt. daily during 7 days) on 5 alpha-R, 3 alpha- and 17 beta-HSD activities was detected in the anterior pituitary. This dose provokes a significant increase in the weight of the pituitary gland, with hyperemia and hypertrophy of chromophobic cells with vacuolar degeneration. In vivo treatment of male rats with the same dose of deethylatrazine markedly inhibited 5 alpha-R activity in the anterior pituitary. The rate of 5 alpha-R activity inhibition in the anterior pituitary was the same after in vivo treatment with atrazine (37.3%) as with deethylatrazine (33.9%). This could suggest that the mechanism of inhibition of deethylatrazine is similar to that of atrazine. In vitro atrazine or deethylatrazine addition into the incubation medium significantly (P less than 0.01) inhibited 5 alpha-R, 3 alpha- and 17 beta-HSD activities in the anterior pituitary. The inhibition of 5 alpha-R activity was marked more by atrazine than deethylatrazine, while 3 alpha- and 17 beta-HSD activities were inhibited at the same rate. In vivo treatment with the same dose of atrazine or deethylatrazine (12 mg/100 g by wt daily 7 days) significantly inhibited (P less than 0.01) 5 alpha-R and 17 beta-HSD at the male rat hypothalamic level. 3 alpha-HSD activity inhibition was not significant for either compound. The in vitro addition of deethylatrazine was much more effective (P less than 0.01) in inhibiting 5 alpha-R, 3 alpha- and 17 beta-HSD in male rat hypothalamus than atrazine. In spite of this, deethylatrazine seems to be less toxic in in vivo experiments due to its higher polarity and faster biodegradation.     

Ubiquitin C-Terminal Hydrolase L1 Is Expressed in Mouse Pituitary Gonadotropes In Vivo and Gonadotrope Cell Lines In Vitro

The ubiquitin-proteasome system (UPS) plays a fundamental role in regulating various biological activities. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme, belonging to the UPS. To date, it has been reported that UCH-L1 is highly and restrictedly expressed in neural and reproductive tissues and plays significant roles in these organs. Although the expression of UCH-L1 in the anterior pituitary gland has been reported, the detailed localization and the role of UCH-L1 remain obscure. In the present study, we detected UCH-L1 protein exclusively in hormone-producing cells, but not non-hormone producing folliculostellate cells in the anterior pituitary lobe. In addition, the cytoplasmic expression of UCH-L1 varied and was limited to gonadotropes and mammotropes. To investigate the role of UCH-L1 in anterior pituitary cells, we performed a comparative analysis using genetically UCH-L1-deficient gad mice. Significant decreases in the numbers of gonadotropes and mammotropes were observed in gad mice, suggesting a close involvement of UCH-L1 in these cells. Moreover, we also determined the expression of UCH-L1 in cultured gonadotropes. Taken together, this is the first report to definitely demonstrate the presence of UCH-L1 in mouse anterior pituitary gland, and our results might provide a novel insight for better understanding the role of UCH-L1 in the hypothalamic-pituitary-gonadal axis and in the reproduction.     

p19Ink4d Is a Tumor Suppressor and Controls Pituitary Anterior Lobe Cell Proliferation

Pituitary tumors develop in about one-quarter of the population, and most arise from the anterior lobe (AL). The pituitary gland is particularly sensitive to genetic alteration of genes involved in the cyclin-dependent kinase (CDK) inhibitor (CKI)–CDK-retinoblastoma protein (Rb) pathway. Mice heterozygous for the Rb mutation develop pituitary tumors, with about 20% arising from the AL. Perplexingly, none of the CKI-deficient mice reported thus far develop pituitary AL tumors. In this study, we show that deletion of p19^Ink4d (p19), a CKI gene, in mice results in spontaneous development of tumors in multiple organs and tissues. Specifically, more than one-half of the mutant mice developed pituitary hyperplasia or tumors predominantly in the AL. Tumor development is associated with increased cell proliferation and enhanced activity of Cdk4 and Cdk6 and phosphorylation of Rb protein. Though Cdk4 is indispensable for postnatal pituitary cell proliferation, it is not required for the hyperproliferative pituitary phenotype caused by p19 loss. Loss of p19 phosphorylates Rb in Cdk4^−/− pituitary AL cells and mouse embryonic fibroblasts (MEFs) and rescues their proliferation defects, at least partially, through the activation of Cdk6. These results provide the first genetic evidence that p19 is a tumor suppressor and the major CKI gene that controls pituitary AL cell proliferation.     

Proteolytic processing of TGFalpha redirects its mitogenic activity: the membrane-anchored form is autocrine, the secreted form is paracrine

Wild-type transforming growth factor alpha (TGFalpha) expression in lactotrope cells in the pituitary gland led to lactotrope-specific pituitary hyperplasia and adenomata. To indicate whether the EGF receptor is involved in this TGFalpha-mediated phenotype, we bred TGFalpha mice with mice expressing the cytoplasmic truncated-EGF receptor (EGFR-tr), which is dominant-negative in other models. These bitransgenic mice developed pituitary pathology despite expression of the dominant-negative receptor. To further characterize this observation, we generated two lineages of transgenic mice that overexpress mutant forms of TGFalpha: a processed soluble form (s TGFalpha) and a cytoplasmic-deleted form (TGFalphaDeltaC). While sTGFalpha expression in lactotrope cells failed to induce autocrine lactotrope hyperplasia, the pituitary became very enlarged due to proliferation of neighboring interstitial cells. In contrast, the TGFalphaDeltaC mice did not develop a phenotype, although the mRNA and protein were present in the pituitary and this form of TGFalpha was confirmed to be biologically active and targeted properly to the plasma membrane of cultured CHO cells. The results suggest that the cytoplasmic domain of TGFalpha is required for autocrine parenchymal tumor formation in the pituitary gland. This signal cannot be inhibited by the EGFR-tr. Conversely, the released form of TGFalpha appears to have primarily paracrine activity.     

Nestin-Cre Mice Are Affected by Hypopituitarism,Which Is Not Due to Significant Activity of the Transgene in the Pituitary Gland

Nestin-Cre mice express Cre recombinase under control of the rat nestin promoter and central nervous system (CNS) enhancer. While endogenous Nestin is expressed in some other tissues including the pituitary gland, Nestin-Cre mice induce recombination predominantly in the CNS. For this reason, they have been widely used to explore gene function or cell fate in the latter. Pituitary hormonal deficiencies, or hypopituitarism, are associated with a wide range of symptoms and with a significant morbidity. These can have a neural and/or a pituitary origin as the gland''s secretions are controlled by the hypothalamus. We report here that Nestin-Cre mice themselves are affected by mild hypopituitarism. Hence, physiological consequences are expected, especially in combination with defects resulting from Cre mediated deletion of any gene under investigation. To further investigate the origin of this phenotype, we re-examined the activity of the transgene. We compared it with expression of Nestin itself in the context of the hypothalamo-pituitary axis, especially in the light of a recent report showing pituitary Nestin-Cre activity, which contrasts with previous data. Our results disagree with those of this recent study and do not support the claim that Nestin positive cells are present in the pituitary anlagen, the Rathke''s pouch (RP). Moreover we did not observe any significant activity in the post-natal pituitary, in agreement with the initial report.