Local transformations of androgens into estradiol by aromatase P450 is involved in the regulation of prolactin and the proliferation of pituitary prolactin-positive cells.

In previous studies we demonstrated the immunohistochemical expression of aromatase in pituitary cells. In order to determine whether pituitary aromatase is involved in the paracrine regulation of prolactin-producing pituitary cells and the physiological relevance of pituitary aromatase in the control of these cells, an in vivo and in vitro immunocytochemical and morphometric study of prolactin-positive pituitary cells was carried out on the pituitary glands of adult male rats treated with the aromatase antagonist fadrozole. Moreover, we analyzed the expression of mRNA for the enzyme in pituitary cells of male adult rats by in situ hybridization. The aromatase-mRNA was seen to be located in the cytoplasm of 41% of pituitary cells and was well correlated with the immunocytochemical staining. After in vivo treatment with fadrozole, the size (cellular and nuclear areas) of prolactin cells, as well as the percentage of prolactin-positive cells and the percentage of proliferating-prolactin cells, was significantly decreased. Moreover, fadrozole decreased serum prolactin levels. In vitro, treatment with fadrozole plus testosterone induced similar effects on prolactin-positive cells, inhibiting their cellular proliferation. Our results suggest that under physiological conditions aromatase P450 exerts a relevant control over male pituitary prolactin-cells, probably transforming testosterone to estradiol in the pituitary gland.

The activity and proliferation of pituitary prolactin-positive cells and pituitary VIP-positive cells are regulated by interleukin 6.

Interleukins are proteins involved in the immune system and have been related to the endocrine regulation of the hypothalamic-pituitary-adrenal axis as well as to the secretion of ACTH, prolactin (PRL), GH and, possibly, LH. Like interleukin-6 (IL-6), vasoactive intestinal peptide (VIP) is synthesized in the pituitary gland and stimulates prolactin secretion. The aim of the present study was to address whether Interleukin 6 is involved in the regulation of VIP, as well as other factors involved in the regulation of prolactin such as dopamine, TRH and estradiol. Accordingly, we performed an in vitro study on monolayer cultures of rat pituitary cells, neutralizing the possible paracrine effect of IL-6 by immunosuppressing the protein by treatment with polyclonal antibody against IL-6 over 1, 3, 6 or 24 hours and then determining the degree of proliferation of VIP cells using double immunocytochemical labelling for VIP or PRL and proliferating cell nuclear antigen (PCNA). As a control, the effects of immunosuppression on the proliferation of PRL-positive cells were analyzed. Immunosuppression of IL-6 induced modifications in the cellular and nuclear size of VIP-positive cells, indicating an inhibitory process. Moreover, immunosuppression induced a significant decrease in the proliferation rate of PRL-positive or VIP-positive cells for all time-points analyzed. Similar effects on the proliferation rate of PRL-positive cells were found. The results of the present study demonstrate that IL-6 is involved in the regulation of the activity and proliferation of pituitary VIP-producing cells and suggest that, without ruling out a direct effect of IL-6 on prolactin cells, IL-6 could regulate prolactin by acting on pituitary VIP.

Gonadal steroids regulate aromatase P450 expression in the rat pituitary

Rat aromatase immunohistochemical expression is different in male than in female adult rats. In order to analyze if such differences are related to the presence of gonadal steroids, a study was carried out on pituitaries of adult castrated rats and rats castrated and treated with gonadal steroids, using immunohistochemistry, Western blotting and in situ hybridization for rat aromatase P450. Rat aro-matase P450 mRNA was detected in the pituitary of male and female rats. Sex-related variations in the mRNA evidence were observed, the mRNA signal was more abundant in males than in females. Moreover, the male pituitaries showed more immunohistochemical positive cells than females and by Western blotting the enzyme was seen to be more abundant in males than in females. With the three methods assayed, ovariectomy elicited a considerable increase in the reaction to aromatase in females. In male rats, castration reduced the number of reactive cells, although the reaction persisted. Treatment with gonadal steroids after castration modified aro-matase expression in the sense that in testosterone-treated castrated males the expression of aromatase was increased while in ovariectomised females treated with oestradiol it decreased. Our results demonstrate the synthesis of aromatase in the pituitary gland and its immunohistochemical expression in the gland of adult rats and suggest that the expression of this enzyme is sex-dependent and that it can be modified by castration and gonadal steroid administration. This in turn suggests that aromatase may be involved in the regulation of adenohypophyseal cytology by gonadal steroids (AU)

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Immunocytochemical evidence for the coexistence of aromatase P450 and estrogen receptor α in the pituitary gland of the adult male rat

In previous studies we demonstrated the immunocytochemical expression of aromatase in rat pituitary cells. The aim of the present study is to demonstrate the synthesis of the enzyme through the expression of the mRNA of the enzyme in pituitary cells by in situ hybridization and the relationship between the immunocytochemical expression of aromatase and estrogen receptor α in adult male rats. In situ hybridization revealed that aromatase-mRNA is located in the cytoplasm of 41% of pituitary cells. By immunocytochemistry, different patterns of reaction for aromatase were found. Moreover, using immunocytochemistry and RT-PCR, all aromatase-positive tumors proved be estrogen receptor α-positive tumors. The immunocytochemical coexistence of aromatase and estrogen receptor α was found. These observations demonstrate the synthesis of aromatase in the pituitary gland and its functional ability to develop its estrogenic effects mediated by estrogen receptor α (AU)

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In vitro immunoblockade of VIP inhibits the proliferation of pituitary prolactin cells.

VIP is a peptide synthesised in the pituitary gland and is involved in the stimulation of prolactin secretion. However, to date it has not been determined whether VIP is able to regulate the proliferation of pituitary prolactin-producing cells, like other factors involved in the regulation of prolactin such as estradiol or dopamine. The aim of the present study was to address whether VIP is involved in regulating the proliferation of pituitary prolactin-secreting cells. Thus, we performed an in vitro study on monolayer cultures of rat pituitary cells, neutralising the possible paracrine effect of VIP by immunoblockade of the peptide and later determining the degree of proliferation of prolactin-secreting cells. The effects of immunoblockade were validated by determining the levels of VIP in the culture media, which were decreased (P < 0.01), and modifications in the patterns of the immunohistochemical reaction to prolactin-positive cells. Immunoblockade of VIP decreased the proliferation of pituitary prolactin-positive cells at all antibody concentrations analysed, mainly between 3 and 12 h (P < 0.01). Moreover, immunoblockade decreased the sizes of the cellular and nuclear areas, except at 1 h, at which point it only decreased the nuclear area of prolactin-positive cells. The results obtained suggest that-in the same way as it regulates the secretion of the hormone-VIP could be involved in regulating the proliferation of prolactin cells, like estradiol or dopamine.

Immunocytochemical evidence for growth hormone-releasing hormone in the tanycytes of the median eminence of the rat.

The current study was performed to analyse the potential existence and structure of a GHRH-transporting tuberoinfundibular system in the rat median eminence. The immunocytochemical analysis using anti-GHRH revealed an intense immunoreaction in the ependimary cells, tanycytes, at the level of the floor of the infundibular recess forming part of the median eminence. The basal processes of these cells course towards the external layer of the median eminence and reach the growth hormone-releasing hormone (GHRH) fibres of the tuberoinfundibular tract and this reaction was increased after intraventricular treatment with colchicine. Thus, these observations suggest the existence of a second or alternative cerebrospinal fluid-mediated route of GHRH transport to the median eminence and implicate the involvement of tanycytes in the regulation of this novel transport system.