DA increases apoptosis in PR1-D2S cells incubated in the presence of E2.

<p>PR1-V, PR1-D2L or PR1-D2S cells were cultured with DMEM-F12-SS in the absence (A) or presence of E2 (1 nM, B) for 48 h. Then the cells were incubated with DA (1 µM-100 µM) in the presence or absence of E2 for 4 h and apoptosis was detected by ELISA. Each column represents the mean ± SE expressed of OD as the percentage of control without DA (n = 5 wells/group). Data were analyzed by ANOVA followed by Dunnett's test. * p<0.05 vs control without DA.</p

DA increases the percentage of apoptotic PR1-D2S cells incubated in the presence of E2.

<p>PR1-D2S cells were cultured with DMEM-F12-SS in the absence (VEH) or presence of E2 (1 nM) for 48 h. Then the cells were incubated with DA (1 µM) with or without E2 for 4 h. Each column represents the percentage of TUNEL-positive cells ± CI (>2,000 cells/group). Data from at least two separate experiments were analyzed by χ². ** p<0.01 vs respective control without DA. ∧∧ p<0.01 vs respective control without E2.</p

A p38 MAPK inhibitor blocks the apoptosis of anterior pituitary cells induced by D2R activation.

<p>Anterior pituitary cells were cultured with E2 (1 nM) for 48 h. Then the cells were preincubated with SB203580 (1 µM), a p38 MAPK inhibitor, for 30 min and then incubated with DA (1 µM, A and B) or CAB (1 µM, C and D) in the presence or absence of SB203580 for 4 h. Each column represents the percentage of TUNEL-positive cells ± CI (>2,000 cells/group, A, C) or the percentage of TUNEL positive lactotropes ± CI (>500 cells/group B, D). Data from at least two separate experiments were analyzed by χ². ** p<0.01 vs respective control without DA or CAB. ∧ p<0.05 vs respective control without SB203589.</p

p38 MAPK is involved in DA-induced apoptosis of PR1-D2S cells.

<p>A: PR1-D2S cells were cultured with E2 (1 nM) for 48 h. Then the cells were preincubated with SB203580 (1 µM), a p38 MAPK inhibitor, for 30 min and then incubated with DA (1 µM) in the presence or absence of SB203580 for 4 h. Each column represents the percentage of TUNEL-positive cells ± CI (>2,000 cells/group). Data from at least two separate experiments were analyzed by χ². ** p<0.01 vs respective control without DA. ∧∧ p<0.01 vs respective control without SB203580. B: PR1-D2S cells were cultured with or without E2 (1 nM) for 48 h. Then the cells were incubated with DA (1 µM) in the presence of E2 or VEH for 15 min, 30 min or 4 h. Proteins were extracted and p38 MAPK and phospho-p38 MAPK were detected by western blot.</p

CAB induces apoptosis of anterior pituitary cells in an E2-dependent manner.

<p>OVX and E2 treated rats were injected with CAB (1 mg/kg, ip) or vehicle (CONTROL) and euthanized 16 h later. Anterior pituitary cells were dispersed and apoptosis was detected by Annexin-V and flow cytometry. Each column represents the mean ± SE of the percentage of apoptotic cells (n = 8 rats/group). Data were analyzed by two-way ANOVA, followed by Tuckey's test. *p<0.05 vs. respective control without CAB, ∧p<0.05 vs respective control without E2.</p

The percentage of lactotropes and somatotropes expressing mERα varies during the estrous cycle.

<p>Dispersed anterior pituitary cells from rats euthanized at diestrus I or proestrus were immunostained for both mERα and PRL or GH and analyzed by flow cytometry. Each column represents the mean ± SE of anterior pituitary cells (A), lactotropes (B) or somatotropes (C) expressing mERα (n = 3–4 animals per group). *p<0.05, **p<0.01 vs diestrus I, Student’s <i>t</i> test.</p

An ERα-antagonist (MPP) blocks E2-BSA-induced apoptosis of lactotropes.

<p>Anterior pituitary cells in culture were pre-incubated with MPP (100 nM) for 30 min prior to the addition of E2-BSA (1 nM) or VEH for 120 min. Apoptosis was analyzed by TUNEL and lactotropes and somatotropes were identified by immunocytochemistry. Each column represents the percentage ± CI (95%) of TUNEL-positive cells (n≥2600) (A), lactotropes (n≥1500 cells/group) (B) and somatotropes (n≥1100 cells/group) (C). *p<0.05 vs respective control without MPP; ∧p<0.05, <0.01 vs respective control without E2-BSA, χ² test.</p

Cell surface biotinylation of anterior pituitary cells.

<p>Cultured anterior pituitary cells from cycling rats were processed for cell surface biotinylation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041299#s2" target="_blank"><i>Material and Methods.</i></a> Western blots from biotinylated (membrane) and non-biotinylated (intracellular) protein fractions were probed with anti-rat ERα and β-actin antibodies (A). PVDF membranes from gels loaded with membrane protein fractions were stained with reversible Ponceau S to ensure equal loading of proteins (B). M: MW marker.</p

The expression of mERα isoforms varies during the estrous cycle.

<p>Anterior pituitary cells from cycling female rats killed at diestrus I or proestrus were cultured for 24 h to allow attachment to the culture plate and then processed for cell surface biotinylation. Expression of full-length ERα (A) and its 39 kDa (B) and 22 kDa (C) isoforms was evaluated by Western blot in membrane (<i>left panels</i>) and intracellular (<i>right panels</i>) protein fractions. Densitometric data from 3–6 animals per group were normalized by the corresponding Ponceau staining (<i>left panels</i>) or β-actin value (<i>right panels</i>) and analyzed by paired Student’s <i>t</i> test, *p<0.05 vs diestrus I. Each column represents the mean ± SE of the relative increment of proestrus versus corresponding diestrus I.</p

17β-estradiol increases expression of 66 kDa (full-length) and 39 kDa mERα isoforms in a time-dependent manner.

<p>Anterior pituitary cells from OVX rats in culture were incubated with E2 (1 nM) for 0–120 min and then processed for cell surface biotinylation. Expression of full-length ERα and its isoforms was evaluated by Western Blot in membrane (A) and intracellular (B) protein fractions. Densitometric data from 3–5 experiments were normalized by the corresponding Ponceau staining (A) or β-actin value (B) and analyzed by repeated measures one-way ANOVA followed by Dunnett’s multiple comparisons test, *p<0.05, **p<0.01 vs 0 min. Each point represents the mean ± SE of the relative increment of each time compared to corresponding time 0 min for each ERα isoform.</p