Production of European sea bass recombinant anti-Müllerian hormone proteins using two different expression systems

Trabajo presentado en el 9º Congreso de la Asociación Ibérica de Endocrinología Comparada, celebrado en Barcelona (España), los días 13 y 14 de julio de 2013Anti-Müllerian hormone (Amh) is a dimeric glycoprotein member of the TGF-ß family. In mammals, Amh is required for Müllerian duct regression in foetal males. Amh is also produced during postnatal life in both male and female regulating steroidogenesis and early stages of folliculogenesis. Amh signals through a transmembrane Amh type II receptor (Amhr2) with serine-threonine kinase activity. Teleost fish lack Müllerian ducts, but Amh has been identified in several species including European sea bass (Dicentrarchus labrax). However, Amhr2 has only been reported in medaka, fugu and black porgy. Here, we report on the production of recombinant sea bass Amh (sbAmh) in two expression systems: Pichia pastoris and CHO cells. Using the CHO cells we have produced a recombinant form of the sbAmh that once proteolytically treated with plasmin generates a mature protein. Using the yeast P. pastoris expression system we have produced recombinant sbAmh proteins that are endogenously cleaved by the Kex2p enzyme and secreted into the media. Western-blot analysis of up-concentrated culture media using an antibody directed against the C-terminal end of sbAmh revealed proteins in the range of 66-79 KDa corresponding to the full length Amh and dimers in the range of 140-160 KDa. The mature TGF-ß proteins have a size of 12-15 KDa. In prepubertal sea bass testes, Amh was detected by immunohistochemistry in Sertoli cells surrounding early germ-cell generations. During spermatogenesis, staining in Sertoli cells surrounding spermatocytes is weaker. Confirmation of this data came from quantitative PCR experiments showing higher expression levels of amh and amhr2 during early and final stages of spermatogenesis. In conclusion, using two different expression systems we have produced recombinant sbAmh that will be used to study how this growth factor regulates sea bass gametogenesis and steroid release in tissue culture.Financial support: LIFECYCLE (FP7-222719-1), MICINN (AGL2011-28890) and REPROBASS (PROMETEO/2010/003)