Autophagy modulation alleviates cryoinjury in murine spermatogonial stem cell cryopreservation
| Autor: | Hui-Jo Oh, Jin Seop Ahn, Buom-Yong Ryu, Yong-Hee Kim, Sang-Eun Jung, Bang-Jin Kim, Sun-Uk Kim |
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| Rok vydání: | 2021 |
| Předmět: |
Cryopreservation
Male Cryoprotectant Adult Germline Stem Cells Chemistry Urology Endocrinology Diabetes and Metabolism Autophagy Pifithrin Spermatogonia Transplantation Andrology chemistry.chemical_compound Mice Endocrinology Cryoprotective Agents Reproductive Medicine In vivo Apoptosis Animals Stem cell |
| Zdroj: | AndrologyREFERENCES. 10(2) |
| ISSN: | 2047-2927 |
| Popis: | BACKGROUND Cryopreservation can expand the usefulness of spermatogonial stem cells (SSCs) in various fields. However, previous investigations that have attempted to modulate cryoinjury-induced mechanisms to increase cryoprotective efficiency have mainly focused on apoptosis and necrosis. OBJECTIVES This study aimed to establish an effective molecular-based cryoprotectant for SSC cryopreservation via autophagy modulation. MATERIALS AND METHODS To determine the efficacy of autophagy modulation, we assessed the recovery rate and relative proliferation rate and performed western blotting for the determination of autophagy flux, immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization, and spermatogonial transplantation for in vivo SSC functional activity. RESULTS The results showed that a basal level of autophagy caused a higher relative proliferation rate (pifithrin-μ 0.01 μM, 184.2 ± 11.2%; 3-methyladenine 0.01 μM, 175.3 ± 10.3%; pifithrin-μ 0.01 μM + 3-methyladenine 0.01 μM, P3, 224.6 ± 22.3%) than the DMSO control (100 ± 6.2%). All treatment groups exhibited normal characteristics, suggesting that these modulators could be used as effective cryoprotectants without changing the properties of the undifferentiated germ cells. According to the results of the in vivo spermatogonial transplantation assay, the colonies per total number of cultured SSCs was significantly higher in the pifithrin-μ 0.01 μM (1596.7 ± 172.5 colonies), 3-methyladenine 0.01 μM (1522.1 ± 179.2 colonies), and P3 (1727.5 ± 196.5 colonies) treatment groups than in the DMSO control (842.8 ± 110.08 colonies), which was comparable to that of the fresh control (1882.1 ± 132.1 colonies). DISCUSSION A basal level of autophagy is more essential for resilience in frozen SSCs after thawing, rather than the excessive activation or inhibition of autophagy. CONCLUSION A basal level of autophagy plays a critical role in the pro-survival response of frozen SSCs after thawing; herein, a new approach by which SSC cryoprotective efficiency can be improved was identified. |
| Databáze: | OpenAIRE |
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