Cloning and characterization of the follistatin gene from Crassostrea angulata and its expression during the reproductive cycle.

Follistatin is an activin-binding protein that prevents activin from binding to its receptor and neutralizes its activity. Follistatin plays a key role in regulating folliculogenesis and the development of ovary. However, limited information on follistatin genes from molluscs is available until now. By using Race, real-time PCR, in situ hybridization and in silico analysis, a full-length cDNA of follistatin of the Portuguese oyster Crassostrea angulata was acquired. The full-length (1297bp) cDNA of Ca-follistatin encodes a peptide of 241 amino acids. The similarity of its deduced amino acid sequence to these of other invertebrate species was about 60%. Ca-follistatin mRNA transcript was most abundantly expressed in ovary (p<0.05), and it was also expressed in testis, adductor muscle, mantle, gill and visceral mass. In situ hybridization revealed that the expression and distribution of Ca-follistatin gene were expressed exclusively in granulosa cells, neither in cumulus oophorus nor in oocytes. During the reproductive cycle of female oyster (initiation stage, maturation stage, ripeness stage and partially spent stage), the expression of Ca-follistatin in the ovary continuously increased from initiation to ripeness stages attaining its highest value (p<0.05), then the expression level decreased sharply to the lowest point in the partially spent stage (p<0.05), whereas the Ca-follistatin mRNA transcript of male oyster in the testis maintained a relatively stable low level during the first three stages, and also noticeably decreased thereafter (p<0.05). These findings suggest that follistatin is likely to play an important role in the ovary development of oysters by autocrine signaling.