Induction of ovulation in sexually mature zebrafish by injection of gap junction protein CX43 blocker Gap27.

In aquaculture, ovulation is commonly induced using exogenous hormones, which are associated with broodstock stress, variable responses, and environmental concerns. Gap junctions formed by connexin 43 (Cx43) in ovarian granulosa cells play a critical role in maintaining meiotic arrest by facilitating the transfer of meiosis-inhibiting substances from granulosa cells to oocytes. The present study investigated whether pharmacological blockade of Cx43 using the specific inhibitor Gap27 could induce ovulation in zebrafish as a non-hormonal alternative. Sexually mature female zebrafish were divided into four groups: control (saline), low-dose (50 μM Gap27), medium-dose (150 μM Gap27), and high-dose (250 μM Gap27), with injections administered through the genital pore. Ovulation was assessed at 14 days post-injection by gentle abdominal pressure. The high-dose group achieved an ovulation rate of 77.78% (7/9), whereas no ovulation was observed in the control or low-dose groups, and only one female ovulated in the medium-dose group (11.11%). Histological examination revealed that the high-dose group exhibited loose ovarian tissue with abundant stage V (mature) follicles, while the control and low-dose groups were dominated by stage III and IV follicles. Transcriptomic analysis indicated dose-dependent transcriptional remodeling, with the high-dose group displaying a distinct expression profile. Comparative analysis between the medium-dose and high-dose groups identified 255 differentially expressed genes, from which 17 ovulation-related genes were screened, including mmp9, nos2b, fosb, igf3, nppc, il17ra1a, and plk2b, which are involved in meiotic arrest release, extracellular matrix remodeling, inflammatory signaling, and cell cycle regulation. Based on these findings, a "bypass activation" model is proposed, in which Gap27-mediated blockade of Cx43 triggers four coordinated molecular modules-brake release, inflammation and tissue remodeling, stress and proliferation, and alternative growth signals-ultimately leading to ovulation. Collectively, these results suggest that targeted Cx43 blockade by Gap27 effectively induces ovulation in zebrafish, providing a proof-of-concept for non-hormonal ovulation induction strategies in aquaculture.