Expression characterization and functional analysis of oocyte-specific Spindlin in gibel carp

Spindlin, an associated protein with meiotic spindle, had been suggested to regulate the transition from gamete to embryo in mouse. In previous studies, we had confirmed that gibel carp (Carassius auratus gibelio) Spindlin (CagSpin) interacted with tubulin and co-localized with meiotic spindle, and found that CagSpin was phosphorylated during oocyte maturation and played important roles in egg fertilization and the oocyte-to-embryo transition. In this study, we further investigated its expression pattern and function role during the hormone-induced oocyte maturation. Firstly, the maturing and matured oocytes were obtained from ovary every two hour interval from the initially induced time until the moment of spawning naturally. RT-PCR analysis revealed that CagSpin transcripts were abundantly, and CagSpin was actively transcribed in these developing oocytes during the whole maturation. Western blot detection demonstrated the corresponding expression pattern of CagSpin in different stages of oocyte maturation. Then, CagSpin-GFP-pCS2+ expression construct was made and used to detect the specific expression and localization of CagSpin in nucleus in CAB cells. Moreover, the CagSpin-GFP-pCS2+ construct was further used to transcribe CagSpin RNA in vitro for subsequent microinjection analysis. Additionally, the hormone-induced maturation processes could be clearly traced and observed through the dynamic changes of germinal vesicle. Thereby, the combined observation of in vitro oocyte maturation and microinjection assay demonstrated that the injected oocytes with CagSpin RNA could not undergo germinal vesicle broken down (GVBD), and kept in immature status during the whole induced process, which implicates that over-expression of CagSpin inhibits the normal oocyte meiotic maturation. Therefore, the data provide further evidence for CagSpin function, and confirm its significant role during oocyte maturation.