MOLECULAR CLONING, EXPRESSION AND FUNCTION ANALYSIS OF GREB1 IN GIBEL CARP, CARASSIUS GIBELIO

Greb1 (Growth regulation by estrogen in breast cancer cell 1), an estrogen stimulated gene, plays critical roles in estrogen response. Previous study indicated that Greb1 was highly expressed in gibel carp (Carassius gibelio) pituitary. The current study cloned and characterized the expression of Carassius gibelio Greb1 (CgGreb1), and investi-gated its function in early embryonic development. The full length cDNA of CgGreb1 was 954 bp, encoding a deduced protein of 255 amino acids. Based on the NCBI database, most vertebrates contained at least one Greb1 isoforms, whose amino acid length ranged from 386—1988 and contained conserved N-terminus. Among those Greb1 homologues, CgGreb1 was the shortest one, locating in the N-terminus of other Greb1 homologues and sharing high identi-ties over 60%. CgGreb1 was dominantly expressed in pituitary, brains, liver and gonads with the highest level in pitui-tary. The expression of CgGreb1 was gradually increased by inducing spawning gibel carp females, peaked at 6 hours post induction, and then decreased and maintained at a relatively high level. During embryogenesis, the expression of CgGreb1 started from 50% epiboly, increased as embryogenesis progression, peaked at 24 hours post fertilization (hpf), and then gradually decreased and was undetectable at 48 hpf. WISH analysis showed that CgGreb1 signal was specifi-cally located in the embryonic margin during gastrulation, gradually distributed in the central neural system during somitogenesis, and was strongly expressed in many brain regions and notochord, but was gradually weakened from 30 hpf and was disappeared at 48 hpf. Knockdown of CgGreb1 specifically decreased the expression of tshβ, prl and gthα but not pomca in producing cells, suggesting that CgGreb1 played an important role in early pituitary development in gibel carp. Our present study provides insights into the molecular mechanism underlying pituitary development and growth regulation in gibel carp.