Role of GPER in the anterior pituitary gland focusing on lactotroph function

in Journal of Endocrinology
Correspondence should be addressed to G Díaz-Torga: gdiaz@ibyme.conicet.gov.ar
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Ovarian steroids control a variety of physiological functions. They exert actions through classical nuclear steroid receptors, but rapid non-genomic actions through specific membrane steroid receptors have been also described. In this study, we demonstrate that the G-protein-coupled estrogen receptor (GPER) is expressed in the rat pituitary gland and, at a high level, in the lactotroph population. Our results revealed that ~40% of the anterior pituitary cells are GPER positive and ~35% of the lactotrophs are GPER positive. By immunocytochemical and immuno-electron-microscopy studies, we demonstrated that GPER is localized in the plasmatic membrane but is also associated to the endoplasmic reticulum in rat lactotrophs. Moreover, we found that local Gper expression is regulated negatively by 17β-estradiol (E2) and progesterone (P4) and fluctuates during the estrus cycle, being minimal in proestrus. Interestingly, lack of ovarian steroids after an ovariectomy (OVX) significantly increased pituitary GPER expression specifically in the three morphologically different subtypes of lactotrophs. We found a rapid estradiol stimulatory effect on PRL secretion mediated by GPER, both in vitro and ex vivo, using a GPER agonist G1, and this effect was prevented by the GPER antagonist G36, demonstrating a novel role for this receptor. Then, the increased pituitary GPER expression after OVX could lead to alterations in the pituitary function as all three lactotroph subtypes are target of GPER ligand and could be involved in the PRL secretion mediated by GPER. Therefore, it should be taken into consideration in the response of the gland to an eventual hormone replacement therapy.

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  • Supplemental figure 1 - GPER protein expression in GH3 cells by immunocytochemistry (ICC).

 

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    GPER expression in anterior pituitary gland. Anterior pituitary cells from 3-month-old female rats in diestrus were processed for GPER identification. White arrows show lactotrophs (PRL), somatotrophs (GH) and gonadotrophs (β-LH) expressing GPER. Nuclei were stained with DAPI. To validate the specificity of the immunostaining, negative controls were performed using blocking peptide or replacing primary antibody with the corresponding normal serum and then incubated with secondary antibody Alexa 594 or Alexa 488. Bar = 20 μm.

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    Flow cytometry analysis of GPER-positive cells in dispersed anterior rat pituitary cells. Dispersed anterior pituitary cells were incubated with anti-GPER antibody (ab39742, Abcam) and analyzed by flow cytometry, n = 8. Representative dot plots and histograms showing: (A and B) percentage of total anterior pituitary cells GPER-positive (GPER+) and (C and D) lactotrophs GPER-positive. Gray: isotype controls; gate: lactotrophs.

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    Regulation of Gper mRNA expression in the rat pituitary by E2 and P4. Alterations induced by OVX, and during the estrous cycle. (A) E2 regulation of pituitary Gper mRNA levels was assessed in vivo in female rats in diestrus (E2, 0.2 mg/kg BW, sc) 1, 2 and 24 h or vehicle (CTRL). Pituitary Gper expression was analyzed by qRT-PCR. One-way ANOVA followed by Tukey’s post hoc test, n = 5, **P < 0.0052 E2 2 h vs CTRL; ****P < 0.0001 E2 24 h vs CTRL; **P = 0.0059 E2 1 h vs 2 h; ****P < 0.0001 E2 1 h vs 24 h and *P = 0.0239 E2 2 h vs 24 h. (B) P4 regulation of Gper mRNA levels was studied similarly, in vivo (P4 6.5 mg/kg BW, sc, 1, 2 and 24 h) or castor oil (CTRL) in female rats in diestrus. One-way ANOVA followed by Tukey’s post hoc test, n = 5, *P = 0.0484 P4 24 h vs CTRL and *P = 0219 P4 1 h vs 24 h. (C) The effect of OVX (15 days post-OVX) on pituitary Gper mRNA levels, analyzed by qRT-PCR. Student’s t test, n = 6, **P = 0.0083 OVX vs control. (D) Gper mRNA levels in pituitaries from cycling rats. One-way ANOVA followed by Tukey’s test, n = 5; ***P < 0.0001 proestrus vs diestrus; *P < 0.0139 estrus vs diestrus.

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    Effect of OVX in GPER expression in lactotrophs. (A) Percentage of GPER+ pituitary cells measured by flow cytometry in OVX rats compared to their control in diestrus: Student’s t test, n = 6, **P = 0.0022. (B) Percentage of GPER+ lactotrophs (PRL+) population: Student’s t test, n = 6, **P = 0.0063. (C) Percentage of GPER+ non-lactotrophs (PRL−) pituitary cells, Student’s t test, n = 6, P > 0.05. (D) Percentage of lactotrophs in both groups. Student’s t test, n = 6, P > 0.05.

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    Immuno-electron-microscopy for GPER. (A, B and C) Subtype I lactotroph cells from female rat at diestrus with gold particles of 15 nm indicating the presence of GPER in plasmatic membrane (arrows), rough endoplasmic reticulum (RER) and free cytosol. Inset: Irregular, large and polymorphic secretory granules immunolabelled for PRL (5 nm gold particles). (D) Negative control. Bar = 0.5 μm. (E, F and G) Lactotroph cells from OVX female rat immunolabelled for GPER with gold particles of 15 nm (GPER) in plasmatic membrane (arrows). PRL was immunostained with 5 nm gold particles identifying lactotroph cells with large and irregular secretory granules (E: Subtype I lactotroph), lactotrophs with spherical granules about diameter 200–250 nm (F: Subtype II), and lactotroph cells with small spherical granules, between 100 and 200 nm(G: Subtype III). N, nucleus; pm, plasmatic membrane; g, secretory granules. Bar = 0.2 μm.

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    Effect of E2, G1 and/or G36 on PRL levels in vitro and ex vivo. (A) GH3 cells were incubated with DMEM containing vehicle (V) (ethanol, 1 μM) or G36 (1 μM) for 30 min and then estradiol (100 nM) or G1 (100 nM) were added alone or in combination with G36 for 15 min at 37°C. After treatments, medium was collected and rat PRL levels were measured by RIA. One-way ANOVA followed by Tukey’s post hoc test, n = 3, three replicates in each set of experiments, ***P < 0.001 E2 vs V, *P < 0.05 E2 + G36 vs V, ***P < 0.001 G1 vs V and ***P < 0.001 G1 vs G1 + G36. (B) SD rats were sacrificed and anterior pituitaries were collected. Explants were incubated 30 min with G36 (1 μM) or vehicle (ethanol, 1 μM) and then estradiol (100 nM) or G1 (100 nM) were added alone or in combination with G36 for 15 min at 37°C. After treatments, PRL levels were measured by RIA in secreted medium. One-way ANOVA followed by Tukey’s post hoc test, n = 3, three replicates in each set of experiments, **P < 0.01 E2 vs V, **P < 0.01 G1 vs V, ***P < 0.001 E2 vs E2 + G36 and ***P < 0.001 G1 vs G1 + G36.

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