Efecto del 17a-etinilestradiol sobre el sistema inmunitario y reproductor de la dorada (Sparus aurata L.). Caracterización funcional del receptor de estrógenos asociado a proteína G = Effect of 17a-ethinylestradiol on inmune and reproductive system of the gilthead seabream (Sparus aurata L.). Functional characterization of the G protein-couplet estrogen receptor

Abstract

"Effect of 17?-ethinylestradiol on immune and reproductive system of the gilthead seabream (Sparus aurata L.). Functional characterization of the G protein-coupled estrogen receptor" Dª. Isabel Cabas Sánchez Directores: Dª Alfonsa García Ayala, D. Victoriano Mulero Méndez y D. José Meseguer Peñalver. OBJETIVES: 1. To evaluate the ability of EE2 to provoke an estrogenic response in vivo in male gilthead seabream. 2. To evaluate the ability of EE2 to alter the physiology and the local immune response of the gonad. 3. To evaluate the ability of EE2 to alter the systemic immune response, analysing immune activities of head kidney leukocytes, the expression profile of macrophages and the in vivo capabilities to respond to an immune challenge. 4. To perform a functional characterization of the G protein-coupled estrogen receptor, GPER, both in vitro and in vivo. METHODOLOGY: In vivo and in vitro experimentation was carried out. For this, healthy specimens of gilthead seabream (Sparus aurata L.) were bred and kept at the Centro Oceanográfico de Murcia (IEO, Mazarrón, Murcia). In vivo experiments were carried out by exposing the specimens to EE2 or G1 at different doses and times. In some cases, the exposure was simultaneous to a scheduled immunization. Afterwards, analysis of survival, sperm quality, histological, determination of hormones and IgM in serum, gene expression and flow cytometry were performed. In general, for the in vitro experiments, leukocytes from head kidney (bone marrow equivalent) were treated with several doses of EE2 and G1 (to specifically activate GPER signalling) at different time points, and analyzed immune activities such as phagocytosis and respiratory burst and gene expression profile. CONCLUSIONS: 1. Exposure to EE2 in vivo promotes an evident estrogenic response characterized by decreased GSI, altered serum levels of E2, T and 11KT, and induced hepatic expression of vtg. These effects are more evident when EE2 is administered in the diet than when is administered in bath water. In addition, its effects slightly depend on the development stage of the specimens. 2. EE2 increases the sensitivity to estrogens in the gonad and the head kidney by increasing the expression of the gene coding for ER?. 3. Exposure to EE2 in vivo disrupts spermatogenesis and induces a characteristic morphology of the post-spawning in the testis. However, the seminiferous tubules were filled with sperm, causing a reduction in the volume and sperm motility. Moreover, EE2 also generates a pro-inflammatory process in the gonad, promoting a massive infiltration of leukocytes and an increased expression of the genes encoding cytokines and molecules involved in antigen recognition and presentation. 4. EE2 decreases the ability of specimens to respond to an immune stimulus in vivo by inhibiting the production of pro-inflammatory cytokines after immunization but does not behave as an immunosuppressor. Moreover, EE2 inhibits in vitro the immune activities of head kidney leukocytes and direct primary macrophages towards an anti-inflammatory phenotype. 5. GPER is expressed in reproductive and immune tissues in gilthead seabream and its expression is not modulated by its activation. 6. Acidophilic granulocytes are the head kidney cells with a higher level of expression of GPER. They express a functional GPER whose activation in vitro leads, in general, to an anti-inflammatory phenotype. These effects are regulated, in part, by activation of the cAMP/PKA/CREB signalling pathway. 7. GPER activation in vivo does not promote an estrogenic response, although in general, provokes an anti-inflammatory effect and slightly modulates the adaptive immune response.