Functions and signaling mechanisms of the membrane androgen receptor ZIP9 in Atlantic croaker (Micropogonias undulatus) and zebrafish (Danio rerio) ovaries

The overall goal of this research was to further characterize and verify the role of ZIP9 in mediating androgen-induced apoptosis of teleost granulosa/theca (G/T) cell co-culture. In addition, a global ZIP9 knockout zebrafish model was used to infer other roles of the receptor in the ovary, in vivo. While ZIP9 was first identified and found to mediate apoptosis of croaker G/T cells, little was known on the signaling mechanism mediated by the receptor. Therefore, stimulatory G protein alpha subunit-mediated signaling, MAP kinase (MAPK) activity, and the role of extracellular zinc were investigated in ZIP9-mediated apoptosis of croaker G/T cells. Additionally, the role of ZIP9 in mediation of testosterone-induced anti-apoptotic response observed in croaker G/T cells from fish outside of the reproductive peak was examined. To determine if ZIP9 mediates androgen-induced apoptosis in G/T cells of other teleosts, a zebrafish primary G/T culture system was used to examine androgen-mediated events. Finally, a global ZIP9 knockout zebrafish strain was developed to examine the effects of global ZIP9 knockout on female reproduction. The ZIP9-mediated apoptotic pathway in croaker G/T cells was found to require stimulatory G protein signaling and MAPK activity. Additionally, extracellular zinc is essential for the induction of ZIP9-mediated apoptosis in this model, and downstream responses to these signaling cascades include increased mRNA expression of pro-apoptotic protein members and caspase 3 activity. Pro- and anti-apoptotic effects of testosterone on croaker G/T cells was confirmed, and both responses were found to be mediated by ZIP9 in an ovarian follicle stage-dependent manner. ZIP9 activation of stimulatory and inhibitory G proteins elicits the pro- and anti-apoptotic responses, respectively. In pooled zebrafish G/T cells, testosterone treatment results in an increased incidence of apoptosis, caspase 3 activity, upregulation of pro-apoptotic protein member mRNA, and a rapid rise in intracellular free zinc. This testosterone-mediated response was attributed to ZIP9 activity, thus, ZIP9 mediates apoptosis through a similar mechanism in zebrafish G/T cells as observed in croaker. Finally, examination of ZIP9 global knockout zebrafish demonstrated that ZIP9 mutation results in reduced fecundity and abnormal egg phenotypes, even though no abnormalities in ovarian morphology were apparent through histological examination.Marine Scienc

Participation of membrane progesterone receptor α in the inhibitory effect of progesterone on prolactin secretion

The membrane progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ and mPRε) are known to mediate rapid nongenomic progesterone functions in different cell types. However, the functions of these receptors in the pituitary have not been reported to date. In the present study, we show that the expression of mPRα was the highest among the mPRs in the rat anterior pituitary gland. Immunostaining of mPRα was detected in somatotrophs, gonadotrophs and lactotrophs. Interestingly, 63% of mPRα-positive cells within the pituitary were lactotrophs, suggesting that mPRα is involved in controlling prolactin (PRL) secretion in the pituitary. To test this hypothesis, rat pituitaries were incubated (1 hour) with either progesterone (P4) or the mPRα-specific agonist Org OD 02-0. PRL secretion was then measured by radioimmunoassay. The results of this experiment revealed that both P4 and Org OD 02-0 decreased PRL secretion. Moreover, the results from the GH3 cell line (CCL-82.1) showed that P4 and Org OD 02-0 inhibited PRL release, although the nuclear PR agonist R5020 was ineffective. Our investigation of the cellular mechanisms behind mPRα activity indicated that both P4 and Org OD 02-0 decreased cAMP accumulation, whereas R5020 was ineffective. In addition, the Org OD 02-0-effect on PRL release was blocked by pretreatment with pertussis toxin, an inhibitor of Go/Gi proteins. Because transforming growth factor (TGF)β1 is a potent inhibitor of PRL secretion in lactotrophs, we lastly evaluated whether TGFβ1 was activated by progesterone and whether this effect was mediated by mPRα. Our results showed that P4 and Org OD 02-0, but not R5020, increased active TGFβ1 levels. This effect was not observed when cells were transfected with mPRα-small interfering RNA. Taken together, these data provide new evidence suggesting that mPRα mediates the progesterone inhibitory effect on PRL secretion through both decreases in cAMP levels and activation of TGFβ1 in the lactotroph population.Fil: Camilletti, María Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ferraris, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Abeledo Machado, Alejandra Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Converse, Aubrey. Marine Science Institute, University Of Texas At Austin; Estados UnidosFil: Faraoni, Erika Yanil. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pisera, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Gutiérrez, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Thomas, Peter. University of Texas at Austin; Estados UnidosFil: Díaz Torga, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin