Effects of cryopreservation on the viability and function of Larimichthys crocea ovarian tissue

Cryopreservation of mature fish eggs and embryos remain major barriers to the preservation of maternal genetic information because of their large size and high yolk content. This study developed a cryopreservation protocol for ovarian tissue from juvenile large yellow croaker (Larimichthys crocea) to preserve oogonial stem cells as an alternative approach. A screening of permeable and non-permeable cryoprotectants, alone and in combination, was performed using a simple two-step freezing procedure (-80°C for 1 h followed by liquid nitrogen immersion). Post-thaw cell viability was assessed by trypan blue exclusion, and the optimal combination of 10% Me₂SO, 15% PG, and 15% FBS was identified, yielding the highest viability of 72.12 ± 8.87%. Histology revealed that oogonia and Perinucleolus stage oocytes maintained intact morphology post-cryopreservation, whereas vitellogenic oocytes sustained significant cryodamage, including oolemma shrinkage, vacuolization, and rupture. qRT-PCR results further revealed upregulated germline stem cell markers of vasa, klf4, oct4, and nanos2, alongside downregulated sycp3 and nanog gene, suggesting that the high viability of oogonia, which consistent with the results of histology. In addition, ccnd1, rhoA, and cirbpa exhibited reduced transcript levels and hsp70 showed elevated levels, indicating that cryopreservation is associated with altered expression of genes involved in cell cycle regulation, cytoskeletal signaling, and stress response. Finally, functional validation via intraperitoneal transplantation into 12 dpf larvae confirmed that cryopreserved oogonia retained the abilities to migrate and colonize on the genital ridge at 15 days post-transplantation. The study established cryopreservation protocol of ovarian tissue that can effectively safeguard the oogonial stem cells, providing a feasible approach for conservation of maternal germplasm in L. crocea and serving as a valuable reference for germplasm conservation of other economically important and endangered fish species.