(82) MIT-001 Mitigates Vitrification-Induced Mitochondrial Stress and Regulates F-actin Reorganization to Enhance the Survival and Development of Vitrified-Warmed Bovine Blstocysts

MIT-001 Mitigates Vitrification-Induced Mitochondrial Stress and Regulates F-actin Reorganization to Enhance the Survival and Development of Vitrified-Warmed Bovine Blstocysts

Seul-Gi Yang^2,3, Hyo-Jin Park^1,2, Dae-Wook Kim^1,2, Eun-Seo Kim^1,2, Hun-Wook Ha^1,2, Geun-Hwi Jo^1,2 and Deog-Bon Koo^1,2,3

1. Department of Biotechnology, Daegu University, 201 Daegudae-ro, Jillyang, Gyeongsan, Gyeongbuk 38453, Republic of Korea

2. DU Center for Infertility, Daegu University, 201 Daegudae-ro, Jillyang, Gyeongsan, Gyeongbuk 38453, Republic of Korea

3. Department of Companion Animal Industry, Daegu University, 201 Daegudae-ro, Jillyang, Gyeongsan 38453, Republic of Korea

Abstract Text:

This study investigated whether MIT-001 as a small molecule reactive oxygen species (ROS) scavenger targeting mitochondria improves the re-developmental ability and viability of bovine embryos in response to vitrification-induced mitochondrial dysfunction and stress. In this experiment, MIT-001 (0.1 μM) was allocated to three culture conditions based on treated period: (I) only warming (WARM), (II) only vitrification (VITR), and (III) both vitrification and warming compared with control (Non-treated). The survival analysis of cryopreserved bovine blastocysts revealed that MIT-001 supplementation during warming period (WARM group) significantly improved (p < 0.01; Non-treated: 57.3 ± 2.3% vs WARM: 74.2 ± 7.3%) post-thaw survival rates. Notably, surviving blastocysts in the WARM group exhibit significantly lower TUNEL-positive cells (p < 0.05) and a higher expanded blastocyst development ratio compared to other groups. Intracellular reactive oxygen species (ROS), as well as mitochondrial and nuclear superoxide levels, were significantly reduced (p < 0.001) in the MIT-001-treated WARM group accompanied by enhanced mitochondrial activation (MitoTracker Orange staining). Simultaneously, mitochondrial membrane potential (MMP) using JC-1 staining was elevated, whereas reduction of the mitochondrial fission marker dynamin-related protein 1 (DRP1) expression showed in surviving blastocysts from the MIT-001-supplemented WARM group (p < 0.01). In addition, MIT-001 improved cytoskeletal stability through the decrease in aggregation ratio of filamentous actin (F-actin, p < 0.001; Non-treated: 12.04 μm vs WARM: 4.07 μm) in bovine blastocyst of only WARM group. Finally, we confirmed that the developmental potential of vitrified-warmed blastocysts was linked to increased phospho-p38 mitogen-activated protein kinase (MAPK) expression exclusively in the WARM group compared to the other groups. In conclusion, we propose that MIT-001 alleviates cryopreservation-induced cellular stress through its mitochondrial functions and promotes F-actin stabilization, thereby enhancing the viability and developmental potential of vitrified-warmed bovine blastocysts. 

Funding

This research was supported by the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) (NRF-2021R1C1C2009469 and NRF-2022R1A2C1002800) and the Basic Science Research Program through the NRF funded by the Ministry of Education (RS-2023-00246139), Republic of Korea.