TNF-alpha is involved in the regulation of normal tissue homeostasis affecting cell proliferation, differentiation, and death. We previously reported that TNF-alpha reduces anterior pituitary cell proliferation and PRL release in an estrogen-dependent manner. In the present project we studied the induction of apoptosis by TNF-alpha in anterior pituitary cells from female rats. TNF-alpha (50 ng/ml) decreased the viability of anterior pituitary cells. Incubation with TNF-alpha for 24 h increased the percentage of terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive cells. TNF-alpha increased the percentage of somatotropes and lactotropes with apoptotic nuclear morphology without affecting the proportion of apoptotic corticotropes or gonadotropes. TNF-alpha increased the percentage of apoptotic lactotropes in cultured cells from rats killed in proestrus and estrus, but not in diestrus. This effect was significantly higher in cells from rats in proestrus than in estrus. In anterior pituitary cells from ovariectomized rats, TNF-alpha significantly increased the percentage of apoptotic lactotropes only when the cells were incubated in the presence of 17beta-estradiol. These results indicate that TNF-alpha induces apoptosis in somatotropes and lactotropes from female rats. The apoptotic effect of TNF-alpha on lactotropes is dependent on estrogens and could be involved in the regulation of anterior pituitary cell renewal during the estrous cycle.

The Fas/FasL system provides the major apoptotic mechanism for many cell types, participating in cell turnover in hormone-dependent tissues. In the present study, we localized both Fas and FasL in anterior pituitary cells, mainly in lactotropes and somatotropes. The percentage of anterior pituitary cells showing immunoreactivity for Fas or FasL was higher in cells from rats killed in proestrus than in diestrus. Also, the proportion of pituitary cells from ovariectomized (OVX) rats expressing Fas or FasL increased in the presence of 17beta-estradiol (10(-9) M). This steroid increased the percentage of lactotropes with immunoreactivity for Fas or FasL and the percentage of somatotropes expressing Fas. Activation of Fas by an agonist anti-Fas antibody (Mab-Fas) decreased the vi-ability-3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT assay)-of anterior pituitary cells from OVX rats cultured in the presence of 17beta-estradiol. Also, membrane-bound FasL decreased cell viability-[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (MTS assay)-only when anterior pituitary cells from OVX rats were incubated with 17beta-estradiol. Moreover, FasL increased the percentage of hypodiploid anterior pituitary cells (flow cytometry). Mab-Fas increased the percentage of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive pituitary cells and lactotropes from OVX rats only when cells were incubated in the presence of 17beta-estradiol. Also, Mab-Fas triggered apoptosis of anterior pituitary cells from rats killed at proestrus but not at diestrus. Our results show that 17beta-estradiol up-regulates the expression of the Fas/FasL system in anterior pituitary cells and increases Fas-induced apoptosis in lactotropes, suggesting that Fas-induced apoptosis could be involved in the pituitary cell renewal during the estrous cycle.

It is now accepted that estrogens not only stimulate lactotrope proliferation but also sensitize anterior pituitary cells to proapoptotic stimuli. In addition to their classical mechanism of action through binding to intracellular estrogen receptors (ERs), there is increasing evidence that estrogens exert rapid actions mediated by cell membrane-localized ERs (mERs). In the present study, we examined the involvement of membrane-initiated steroid signaling in the proapoptotic action of estradiol in primary cultures of anterior pituitary cells from ovariectomized rats by using estren, a synthetic estrogen with no effect on classical transcription and a cell-impermeable 17beta-estradiol conjugate (E2-BSA). Both compounds induced cell death of anterior pituitary cells after 60 min of incubation as assessed by flow cytometry and the [3-(4,5-dimethylthiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Estren, E2, and E2-BSA induced apoptosis of lactotropes and somatotropes as evaluated by the deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and immunodetection of prolactin (PRL) and growth hormone (GH). The proapoptotic effect of E2-BSA was abrogated by ICI-182,780, an antagonist of ERs. The expression of membrane-associated ERalpha was observed in PRL- and GH-bearing cells. Our results indicate that estradiol is able to exert a rapid apoptotic action in anterior pituitary cells, especially lactotropes and somatotropes, by a mechanism triggered by mERs. This mechanism could be involved in anterior pituitary cell turnover.

We previously reported that TNF-alpha-induced apoptosis of lactotropes is estrogen dependent and predominant at proestrus. Here we observed that TNF-alpha (50 ng/ml) failed to induce apoptosis of anterior pituitary cells from ovariectomized rats cultured in the presence of progesterone (10(-6) m). However, progesterone blocked the apoptotic effect of TNF-alpha in anterior pituitary cells and lactotropes cultured with 17beta-estradiol (10(-9) m). In addition, 17beta-estradiol induced apoptosis of somatotropes and triggered the proapoptotic action of TNF-alpha in these cells, effects completely blocked by ICI 182 780 (10(-6) m), an estrogen receptor antagonist. Progesterone reverted the permissive effect of 17beta-estradiol on TNF-alpha-induced apoptosis of somatotropes. TNF-alpha induced apoptosis of somatotropes from rats killed at proestrus but not at diestrus. The antiprogestine ZK 98,299 (10(-6) m) completely inhibited the protective action of progesterone on TNF-alpha-induced apoptosis of anterior pituitary cells, lactotropes, and somatotropes. Although progesterone can interact with glucocorticoid receptors, dexamethasone (10(-6) m) had no effect on TNF-alpha-induced apoptosis of anterior pituitary cells, lactotropes, and somatotropes. Our results show that progesterone, by interacting with progesterone receptors, antagonizes the permissive action of estrogens on TNF-alpha-induced apoptosis of lactotropes and somatotropes. These observations suggest that the steroid milieu may modulate the apoptotic response of anterior pituitary cells during the estrous cycle.

homeostasis results from a balance between cell proliferation and cell death by apoptosis. Estradiol affects proliferation as well as apoptosis in hormone-dependent tissues. In the present study, we investigated the apoptotic response of the anterior pituitary gland to lipopolysaccharide (LPS) in cycling female rats, and the influence of estradiol in this response in ovariectomized (OVX) rats. The OVX rats were chronically estrogenized with implanted Silastic capsules containing 1 mg of 17␤-estradiol (E2). Cycling or OVX and E2-treated rats were injected with LPS (250 g/rat ip). Apoptosis was determined by the terminal deoxynucleotidyl-mediated dUTP nickend labeling (TUNEL) method in sections of the anterior pituitary gland and spleen. Chronic estrogenization induced apoptosis in the anterior pituitary gland. Acute endotoxemia triggered apoptosis of cells in the anterior pituitary gland of E2-treated rats but not of OVX rats. No differences were observed in the apoptotic response to LPS in spleen between OVX and E2-treated rats. The apoptotic response of the anterior pituitary to LPS was variable along the estrous cycle, being higher at proestrus than at estrus or diestrus I. Approximately 75% of the apoptotic cells were identified as lactotropes by immunofluorescence. In conclusion, our results indicate that estradiol induces apoptosis and enables the proapoptotic action of LPS in the anterior pituitary gland. Also, our study suggests that estrogens may be involved in anterior pituitary cell renewal during the estrous cycle, sensitizing lactotropes to proapoptotic stimuli. estrous cycle; estradiol; lactotropes; lipopolysaccharide LIPOPOLYSACCHARIDE (LPS), an endotoxin of gram-negative bacteria, is commonly used to study neuroendocrine-immune interactions. After systemic administration of LPS, the plasma concentrations of several cytokines, such as TNF-␣, IL-1, and IL-6 rise in a temporal-sequential manner (14, 27). Systemic cytokines, as well as those locally released in the central nervous system and pituitary, contribute to the neuroendocrine response to endotoxemia, such as hypothalamic-pituitary-adrenal axis (HPA) activation and hypothalamic-pituitary-gonadal axis inhibition (27,28,29). We have reported that systemic LPS and central TNF-␣ administration exert inhibitory effects on prolactin secretion in male rats by stimulating dopaminergic activity in the hypothalamic-pituitary axis (4), and that LPS (25) and TNF-␣ (26) reduce in vitro prolactin release from anterior pituitary cells of female rats.Gonadal steroid hormones modulate the neuroendocrine response to inflammatory stimuli. It has been reported that endotoxin-induced HPA activation is higher in female than in male rats. This sexual difference is abolished by gonadectomy and restored by administration of estradiol to ovariectomized (OVX) rats (28). We have observed that estradiol stimulates basal and LPS-induced TNF-␣ secretion from anterior pituitary cells of OVX rats (25). Also, TNF-␣ release from anterior pituitary cells varies along the estrous...