Production of functional European sea bass recombinant anti-müllerian hormone

Trabajo presentado en la 26th Conference of European Comparative Endocrinologists celebrada en Zúrich (Suiza) del 21 al 25 de agosto de 2012Anti-Müllerian hormone (Amh), also called Müllerian-inhibiting substance, is a dimeric glycoprotein member of the TGF-ß family. In mammals, Amh is processed to yield a bioactive C-terminal homodimer 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 (AmhRII) with serine-threonine kinase activity, which recruits and phosphorylates one or more of the activin-receptor kinases (ALK), mediating stimulatory (ALK2 and 3) or inhibitory (ALK6) effects and involving Smad proteins in the downstream signalling cascade. Teleost fish lack Müllerian ducts, but Amh has been identified in several species including sea bass. However, AmhRII has only been reported in medaka and black porgy. Here, we report on the production in CHO cells of a recombinant, internally His-tagged form of the sea bass Amh with a modified cleavage motif sequence. Western-blot analysis of the recombinant hormone in up-concentrated cell culture media using a C-terminally directed antibody revealed proteins in the range of 66-79 KDa corresponding to the full length Amh and disulfide linked dimmers in the range of 140-160 KDa. Treatment with increasing concentrations of plasmin produced proteins in the range of 12-15 KDa corresponding to the mature TGF-ß. In prepubertal sea bass testes, Amh was detected by immunohistochemistry in Sertoli cells surrounding early germ-cell generations. Experiments using COS-7 cells, co-transfected with sea bass AmhRII cDNA and Bre-luciferase expression plasmids, and stimulated with recombinant sea bass Amh showed that only plasmin-treated Amh was able to induce luciferase expression to a level higher than controls. In conclusion, using a mammalian expression system we have produced a sea bass recombinant Amh that once proteolytically treated is fully bioactive.Supported by EU Project LIFECYCLE (FP7-222719-1), GV REPROBASS (PROMETEO/2010/003) and MICINN (AGL2011-28890)