José Carretero

Aromatase, the enzyme responsible for estrogen biosynthesis, is expressed by human and rat glioblastomas

The biosynthesis of estradiol and related estrogens is catalyzed by the enzyme aromatase. Among other tissues, aromatase is expressed in the brain, where it is involved in the regulation of neuroendocrine events and reproduction. Under physiological conditions, the expression of aromatase in the mammalian brain is restricted to neurons. However, recent studies have shown that reactive astrocytes express aromatase after brain injury. This opens the possibility for the expression of the enzyme in other altered forms of glial cell, such as gliomas. In the present study, the expression of aromatase has been assessed, by RT-PCR and immunocytochemistry, in the rat glioblastoma C6 and in two human glioblastoma cell lines T98G and U373MG. The three cell lines expressed aromatase mRNA and showed a cytoplasmic pattern of aromatase immunoreactivity. In addition, the three cell lines express estrogen receptor alpha, suggesting that estradiol formed by aromatase may act as an autocrine or paracrine factor for glioblastoma cells. By analogy to the implication of aromatase into the growth of other forms of estrogen-sensitive tumors, such as some breast cancers, it is conceivable that the expression of aromatase may play a role in the growth of glioblastomas.

CaBP D-28k and NADPH-diaphorase coexistence in the magnocellular neurosecretory nuclei.

COEXISTENCE of the calcium binding protein calbindin D-28k and NADPH-diaphorase activity was studied in the magnocellular secretory nuclei of the rat hypothalamus using both immunocytochemical and histochemical techniques. Coexistence was found in all the nuclei considered (supraoptic, paraventricular, circularis and fornicals nuclei) with the exception of the hypothalamic area situated between the supraoptic and the paraventricular nuclei. Since both stainings are reliable markers, not based upon the physiological characteristics at a given moment, our study provides a further characterization of the neurons in the magnocellular neurosecretory nuclei.

Immunohistochemical evidence of the presence of aromatase P450 in the rat hypophysis

Abstract In order to analyze whether aromatase is present in the hypophysis of adult rats, we have performed an immunohistochemical study in young adult male and female rats. Our study has revealed that the hypophysis of adult rats contains aromatase, although marked differences are found between the sexes. The hypophyses of male rats have cells immunoreactive for the enzyme, 34.40% of these hypophyseal cells showing reaction. By contrast, cells from female rats show very little reaction, only 0.84% of them being reactive. No significant differences in the percentage of immunoreactive cells between one phase and another are observed during the estrous cycle. Our results point to the immunohistochemical expression of aromatase in the hypophysis of adult rats and at the same time suggest that its expression is sex-dependent. The enzyme may therefore be involved in the regulation of adenohypophyseal cytology by androgens.

Swim stress enhances the NADPH-diaphorase histochemical staining in the paraventricular nucleus of the hypothalamus.

Regulatory control of the hypothalamic-pituitary-adrenocortical axis (HPA) originates principally from the paraventricular nucleus (PVN), which contains an important population of nitric oxide synthesizing (NOS) neurons. In the present study, the effect of swim stress upon these neurons was investigated by means of the NADPH-diaphorase (ND) histochemical technique. A significant increase in the number of ND-neurons was observed following forced swim, especially after 30 min. These data confirm the involvement of NOS-neurons of the PVN in the response to different types of acute stressors.

Expression of Aromatase P450 is Increased in Spontaneous Prolactinomas of Aged Rats

We have recently reported the presence of aromatase P450 in the rat hypophysis. This enzyme is responsible for the aromatization of testosterone to estradiol. Since the induction of prolactinomas has been demonstrated in the rat following chronic treatment with estradiol, the aim of the present study was to analyze whether a relationship exists between the presence of pituitary aromatase and the appearance of spontaneous prolactinomas in aged rats. Of a series of 90 adenomas studied, 53% showed prolactin immunoreactive cells and were classified as prolactinomas; only 33% of the adenomas were pure prolactinomas and the other 20% were multi-hormonal protactinomas. Moreover, 60% of the adenomas were aromatase-positive tumors. Interestingly, 100% of the pure prolactinomas were aromatase-positive while only 60% of the multi-hormonal prolactinomas expressed the enzyme. Western blotting with anti-aromatase antibodies revealed a 3.8-fold increase in expression of aromatase in pituitary tumors as compared to normal rat pituitary gland. Double immunohistochemical labeling detected the coexistence of prolactin and aromatase P450 in prolactinoma cells. ACTH- and LH-positive adenomas were considered as controls; only multi-hormonal ACTH and LH tumors display aromatase-positive cells and all of these also contained prolactin-positive cells. Our results demonstrate for the first time that aromatase is expressed in pituitary adenomas and that it is related to the functional nature of the tumor, especially in the case of pure prolactinomas, suggesting the possibility that an abnormally high conversion of testosterone into estradiol in pituitary cells may contribute to the genesis of spontaneous prolactinomas in aged rats.

Expression of the μ-opioid receptor in the anterior pituitary gland is influenced by age and sex

Abstract To analyze whether opioids are able to modulate endocrine regulation by acting directly on rat pituitary cells, an immunohistochemical study of μ-opioid receptor expression in these cells was performed, with attention given to the analysis of potential age- and sex-related variations in receptor expression patterns. In both sexes, the μ-opioid receptor was detected in the pituitary pars distalis. However, significant age-related differences were observed. Both in male and female rats, the percentage of μ-opioid receptor-expressing cells decreased significantly from postnatal week one through the 24 months of our study. Interestingly, pituitary cells containing the μ-opioid receptor were significantly more numerous in male than in female, with exception of the pre-pubertal phase and old rats. According to two-way analysis of variance, the gender-related differences in μ-receptor expression were independent of age-related variations. Thus, without excluding hypothalamic actions, our results suggest that opioids may exert their endocrine function by acting directly on pituitary cells.

Calbindin D-28K- and parvalbumin-reacting neurons in the hypothalamic magnocellular neurosecretory nuclei of the rat

The distribution of calbindin D-28K- and parvalbumin-reacting neurons in the hypothalamic magnocellular neurosecretory nuclei of the rat was studied using the avidin-biotin-immunoperoxidase method and highly specific monoclonal antibodies. Incubation with anticalbindin D-28K-antiserum revealed immunoreactive neurons in the following nuclei: supraoptic, paraventricular (both in the magnocellular and parvicellular regions), circularis, fornicals and medial forebrain bundle. Incubation with parvalbumin antiserum displayed immunoreactive neurons only in the circularis nucleus. Additionally, it was possible to observe scattered calbindin and parvalbumin immunoreactive neurons (which do not form part of the nuclei considered) located in the hypothalamic area between the supraoptic and the paraventricular nuclei, especially for the calbindin D-28K antiserum.

Partial co-existence of NADPH-diaphorase and acetylcholinesterase in the hypothalamic magnocellular secretory nuclei of the rat

Co-localization of NADPH-diaphorase (ND) and acetylcholinesterase (AChE) activities were explored in the magnocellular secretory nuclei of the rat hypothalamus by means of a double histochemical staining of the same sections. Partial co-existence was found in all the nuclei studied (paraventricular, supraoptic, fornicals and circular nuclei). No particular location of the neurons expressing both markers was found, although in the paraventricular nucleus all of them (ND +, AChE + and neurons expressing both markers) were preferentially located in the magnocellular subdivisions whereas in the parvicellular ones only some neurons belonging to all three types were detected, mainly located in the periventricular and medial subdivisions. The lowest degree of co-existence was found at the level of the main magnocellular nuclei (supraoptic and paraventricular) when compared with the accessory magnocellular nuclei, especially the posterior fornical and the circular nuclei. These results extend previous data on the chemical nature of the neurons producing nitric oxide in the neurosecretory nuclei and the possible functional role of this atypical messenger in the hypothalamus.

Reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity in the paraventricular nucleus of the rat hypothalamus is modulated by estradiol

Based on previous studies demonstrating that reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (ND) activity is modulated by estradiol and the discovery of a new subtype of estrogen receptor highly expressed in the paraventricular nucleus of the hypothalamus (PVN), a possible estrogen influence on this activity was investigated in the neuronal populations (magno- and parvicellular) of this nucleus. Cryostat sections were cut and processed for the histochemical detection of the ND activity. Following ovariectomy (14 days), numerical data displayed a slight decrease in the number of ND-neurons, especially in the posterior magnocellular and the medial parvicellular subdivisions, which was reversed after daily treatment with estradiol benzoate. Administration of estradiol benzoate to male rats (14 days) induced a significant increase (P < 0.05) in the number of ND-neurons, mainly at the level of the posterior magnocellular subdivision. These data indicate that paraventricular ND-neurons are influenced by estradiol.

IN VITRO MORPHOMETRIC AND PROLIFERATIVE VARIATIONS IN VIP-IMMUNOREACTIVE PITUITARY CELLS INDUCED BY ESTRADIOL

Using double labelling for proliferating cell nuclear antigen (PCNA) and vasoactive intestinal peptide (VIP), a study was conducted to elucidate the repercussions of the addition of estradiol to monolayer pituitary cultures on the morphology, size, number, mitotic index and PCNA labelling index of VIP-immunoreactive cells, comparing the results with those obtained in control cultures. The addition of estradiol (100 nM) over 3 h of incubation led to a significant increase (p < 0.01) in the cellular, cytoplasmic and nuclear areas of VIP-immunoreactive cells and in the number of these cells. The same treatment elicited an even greater increase in the mitotic index (p < 0.01) and PCNA labelling index (p < 0.01) of VIP-immunoreactive cells. The results obtained suggest that estrogens induce proliferation and hyperplasia of in vitro VIP-immunoreactive pituitary cells.