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European sea bass (Dicentrarchus labrax) anti-müllerian hormone: production in a yeast system and functional studies

AuthorsZapater, Cinta ; Rocha, Ana; Molés, Gregorio ; Ibáñez, Soledad; Zanuy, Silvia ; Gómez, Ana
Issue Date4-Sep-2017
Citation6th International Workshop on the Biology of Fish Gametes (2017)
AbstractIn higher vertebrates, anti-Müllerian hormone (AMH) is required for involution of the Müllerian ducts during male sexual differentiation and for negatively regulating gonadal development in both sexes. AMH signals through a transmembrane AMH type II receptor (AMHR2) with serine-threonine kinase activity. Despite the absence of Müllerian ducts in teleosts, orthologues of mammalian AMH and AMHR2 have been described in some fish species, and a role of this hormone in sex determination and gonad differentiation has been demonstrated. In addition, it seems that Amh inhibits germ cell proliferation and differentiation, as well as steroidogenesis, in adult gonads of both sexes. However, the mechanism of Amh signaling and its implication in gonad development are poorly investigated. In European sea bass, a recent study showed higher expression levels of amh and amhr2 during early and final stages of spermatogenesis, and immunolocalization of Amh in Sertoli cells surrounding early germ-cell generations, in line with its role in germ cell proliferation and differentiation. Available information of amh and amhr2 expression during sea bass ovarian development shows an opposite profile for these two genes. Although this information may suggest a role of Amh in early stages of oogenesis, the specific mechanisms of Amh signaling during ovarian development still have to be investigated. As tool for this kind research recombinant sea bass Amh would be needed. We know from previous studies that this hormone is processed in a similar way to mammalian AMH, becoming a biologically active protein able to bind and activate the sea bass Amhr2. Here, we report the production of an His-tagged recombinant sea bass Amh in a Pichia pastoris expression system, where Amh is endogenously cleaved and secreted as a mature peptide into the culture media. Bioactivity of this recombinant sea bass Amh has been demonstrated using COS7 cells co-transfected with the sea bass amhr2 cDNA and the BRE-luc reporter plasmid. Localization of Amh in granulosa cells of vitellogenic ovarian follicles was shown using a specific antibody. To investigate how Amh regulates sea bass ovarian development, we performed an in-vitro tissue culture of vitellogenic ovaries treated with recombinant Amh. First, we analyze steroid release in the medium by using a specific EIA. Finally, several candidate genes regulated by Amh were analyzed by real time qPCR.
DescriptionTrabajo presentado en el 6th International Workshop on the Biology of Fish Gametes, celebrado en Vodňany (República Checa), del 4 al 7 de septiembre de 2017
Appears in Collections:(IATS) Comunicaciones congresos
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