Background and Aim: Cryopreservation of ovarian tissue is an important technique for preserving the reproductive potential of valuable and endangered species and plays a critical role in establishing genetic resource banks. However, the success of ovarian tissue cryopreservation depends largely on the choice of cryoprotective agent (CPA), which must balance protection against intracellular ice formation with potential cytotoxic effects. In feline ovarian tissue, the dense collagen-rich stromal architecture may further limit CPA diffusion, thereby influencing cryopreservation efficiency. This study aimed to evaluate the effects of two commonly used penetrating CPAs, dimethyl sulfoxide (Me₂SO) and ethylene glycol (EG), individually and in combination, on cellular apoptosis and the preservation of key oocyte-secreted factors (OSFs), growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15), following slow-freezing cryopreservation of feline ovarian tissue. Materials and Methods: Ovarian tissues were collected from six healthy domestic cats undergoing routine ovariohys-terectomy. Cortical fragments (approximately 2 × 2 × 3 mm³) were randomly assigned to five groups: Fresh control, Cryo-control (no CPA), 10% Me₂SO, 10% EG, and a combination of 5% Me₂SO + 5% EG. Tissues were cryopreserved using a programmable slow-freezing protocol with controlled cooling rates and stored in liquid nitrogen. Post-thaw tissue integrity was evaluated through histomorphological examination, apoptosis detection using the TUNEL assay, and protein expression analysis using Western blotting and immunohistochemistry. Statistical analysis was performed using analysis of variance with Bonferroni post hoc testing (p < 0.05). Results: All cryopreserved groups exhibited significantly higher apoptosis than fresh tissue. However, tissues preserved with Me₂SO alone or in combination with EG demonstrated apoptotic indices comparable to those of Fresh controls, whereas EG alone and Cryo-control groups showed significantly higher apoptosis. Western blot analysis revealed that both GDF9 and BMP15 protein levels were significantly reduced after cryopreservation. Nevertheless, GDF9 expression was partially preserved in CPA-treated groups compared with Cryo-control tissue. In contrast, BMP15 expression remained markedly reduced in all cryopreserved groups, indicating high cryosensitivity. Immunohistochemical analysis further showed that the combined Me₂SO + EG treatment better maintained follicular localization and intensity of GDF9 and BMP15 in primordial and primary follicles. Conclusion: Me₂SO-based cryopreservation protocols effectively reduce apoptosis and maintain structural integrity in feline ovarian tissue. However, significant depletion of critical OSFs, particularly BMP15, occurs despite preserved morphology. These findings highlight a molecular–structural discrepancy in cryopreserved ovarian tissue and emphasize the need to optimize cryopreservation strategies that preserve both cellular viability and functional molecular signaling essential for successful follicular development and fertility restoration.
Thatsanabunjong et al. (Sun,) studied this question.