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

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

Gonadal steroids differentially regulate the expression of mERα in lactotropes and somatotropes <i>in vivo</i>.

<p>OVX female rats were injected for two consecutive days with vehicle (VEH), 17β-estradiol (E2), progesterone (P4) or E2+P4 and euthanized on the third day. Dispersed anterior pituitary cells 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 vs respective control without P4; ∧p<0.05 vs respective control without E2, two-way ANOVA followed by Tukey’s test.</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

N-terminal PRL fragments content in the anterior pituitary varies along the estrous cycle.

<p>A: Recombinant 23 kDa PRL (r23-PRL), recombinant 16 kDa prolactin (r16-PRL) or pituitary protein extracts treated with β-mercaptoethanol (P+β, reducing conditions) or without β-mercaptoethanol (P, non-reducing conditions) were incubated with anti-recombinant rat PRL antibody (anti rrPRL, 1∶25000). The antibody recognizes both PRL forms. B: Anterior pituitaries from rats euthanized at diestrus I or proestrus were processed for western blot. Upper panel: Each column represents the mean ± SE of the relative increment of N-terminal PRL fragments content with respect to diestrus I. Data of each column were normalized to β-actin expression (n = 4–7 animals per group). *p<0.01 vs. diestrus I, Student's t test. Lower panel: Representative blot of pituitary proteins from rats euthanized at diestrus I or proestrus.</p

Estradiol increases the secretion and content of N-terminal PRL fragments from anterior pituitary cells in culture.

<p>Anterior pituitary cells from OVX rats were incubated in the presence of VEH or E2 for 24 h. A: Culture media and B: cells were obtained and processed for western blot analysis. Each column represents the mean ± SE of the relative increment of N-terminal PRL fragments with respect to VEH. In B, data of each column were normalized to β-actin expression (A, n = 6 wells/group; B, n = 6 wells/group), representative of three independent experiments. *p<0.05, **p<0.01 vs. VEH without E2, Student's t test. Lower panels: Representative blots of media (A) or cells (B) cultured with VEH or E2.</p

16 kDa PRL induces apoptosis of anterior pituitary cells.

<p>A and B: Anterior pituitary cells from OVX rats were incubated with VEH or E2 for 24 h and with or without 16 kDa PRL (10 nM) for the last 4 h. A: Each column represents the percentage ± CI (95%) of TUNEL-positive cells (n≥1500 cells/group, left) or TUNEL-positive lactotropes (n≥300 cells/group, right), representative of four independent experiments. Data were analyzed by χ². **p<0.01 vs. respective control without 16 kDa PRL. ∧∧p<0.01, ∧p<0.05 vs. respective control without E2. B: Representative images of anterior pituitary cells showing immunoreactivity for prolactin (red) counterstained with DAPI (blue, upper panels) and DNA fragmentation determined by TUNEL method (green, lower panels). Arrowheads indicate apoptotic lactotropes. Scale bar: 50 µm. C: Anterior pituitary cells from OVX rats were incubated with E2 for 24 h, and with or without 16 kDa PRL (10 nM) for the last 4 h. The percentage of hypodiploid cells was determined by flow cytometry using PI. Left: Each column represents the mean ± SE of the percentage of sub-G1 cells (n = 3 wells/group), representative of three independent experiments. Data were analyzed by Student's t test. **p<0.01 vs. respective control without 16 kDa PRL. Right: Representative histograms of fluorescence intensity of DNA content of anterior pituitary cells incubated in the presence or absence of 16 kDa PRL.</p

16 kDa PRL inhibits anterior pituitary cell proliferation.

<p>A and B: Anterior pituitary cells from OVX rats were incubated for 24 h with VEH and FSK, and with or without 16 kDa PRL (10 nM) for the last 4 h. D and E: Anterior pituitary cells from OVX rats were incubated for 24 h with E2, then for 1 h with E2 and FSK, and for the last 4 h in the same media with or without 16 kDa PRL (10 nM). Proliferation was determined by BrdU incorporation (3 h) and fluorescence microscopy. A and D: Each column represents the percentage ± CI (95%) of BrdU-positive cells (n≥1000 cells/group, left) or BrdU-positive lactotropes (n≥300 cells/group, right), representative of four independent experiments. Data were analyzed by χ². **p<0.01 vs. respective control without 16 kDa PRL. B and E: Representative images of anterior pituitary cells showing immunoreactivity for prolactin (red) counterstained with DAPI (blue) and BrdU (green). Arrowheads indicate proliferating lactotropes. Scale bar: 50 µm. C and F: Anterior pituitary cells in culture were incubated with VEH (C) or E2 (F) for 24 h, and with or without 16 kDa PRL (10 nM) for the last 4 h. Cell cycle was analyzed by flow cytometry using PI. Left: Each column represents the mean ± SE of the percentage of S-phase cells (n≥4 wells/group), representative of three independent experiments. Data were analyzed by Student's t test. *p<0.05 vs. respective control without 16 kDa PRL. Right: Representative histograms of DNA content of anterior pituitary cells incubated in the presence or absence of 16 kDa PRL.</p