Expression Analysis of ZPB2a and Its Regulatory Role in Sperm-Binding in Viviparous Teleost Black Rockfish

Black rockfish is a viviparous teleost whose sperm could be stored in the female ovary for five months. We previously proposed that zona pellucida (ZP) proteins of black rockfish play a similar sperm-binding role as in mammals. In this study, SsZPB2a and SsZPB2c were identified as the most similar genes with human ZPA, ZPB1 and ZPB2 by Blastp method. Immunohistochemistry showed that ovary-specific SsZPB2a was initially expressed in the cytoplasm of oocytes at stage III. Then it gradually transferred to the region close to the cell membrane and zona pellucida of oocytes at stage IV. The most obvious protein signal was observed at the zona pellucida region of oocytes at stage V. Furthermore, we found that the recombinant prokaryotic proteins rSsZPB2a and rSsZPB2c could bind with the posterior end of sperm head and rSsZPB2a was able to facilitate the sperm survival in vitro. After knocking down Sszpb2a in ovarian tissues cultivated in vitro, the expressions of sperm-specific genes were down-regulated (p < 0.05). These results illustrated the regulatory role of ZP protein to the sperm in viviparous teleost for the first time, which could advance our understanding about the biological function of ZP proteins in the teleost.