Autophagy modulation alleviates cryoinjury in murine spermatogonial stem cell cryopreservation

Abstract

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.

Elucidating the mechanisms and mitigation strategies for six-phthalate-induced toxicity in male germ cells

“…PTL and 3-MA were dissolved in DMSO and NAC was dissolved in the basal medium to reach the following desired final concentrations: PTL (1 μM), 3-MA (0.1 μM), and NAC (1 mM). The concentration range of the inhibitors was based on previous studies ( Li-Weber et al, 2005 ; Xiao et al, 2016 ; Jung et al, 2022 ; Kim et al, 2024 ). To address potential antagonism when combining the inhibitors, concentrations were set lower than those typically used individually.…”

Advances in the molecular regulation mechanism of tumor dormancy and its therapeutic strategy

“…Autophagy typically serves a protective role, acting as a dynamic circulatory system to mitigate cellular damage and maintain in vivo homeostasis [ 82 ]. In cancer, autophagy plays a crucial role in sustaining cancer cells during adverse conditions such as starvation, hypoxia, fozen and chemotherapy/radiotherapy [ 83 , 84 ]. This process offers protection against cell death and promotes cancer cell growth.…”

New strategies for germ cell cryopreservation: Cryoinjury modulation

“…In a similar vein, Gao et al [ 55 ] demonstrated that inhibiting autophagy triggered intrinsic apoptosis in immature oocytes during the vitrification-warming process, while Bang et al [ 56 ] depicted autophagic activation as a natural adaptive response to cold stress in vitrified-warmed oocytes. Conversely, Jung et al [ 9 ] suggested that autophagy could contribute to cryoinjury during SSC cryopreservation, indicating that excessive autophagy could result in cell death after freeze-thawing.…”

“…As such, the necroptosis inhibitor necrostatin-1 has shown beneficial effects on the proliferation rate while reducing necroptosis and apoptosis after thawing in SSC cryopreservation; it exerts these impacts by reducing ROS generation, apoptosis, and RIP1 activity [ 10 ]. The addition of pifithrin µ (a p53 inhibitor) or 3-methyladenine, which blocks autophagosome formation by inhibiting type III phosphatidylinositol 3-kinase, has been found to enhance cryoprotective effects and help maintain normal stem cell characteristics after thawing during SSC freezing [ 9 ]. Mitophagy, a selective autophagy that removes aged and damaged mitochondria, is also activated in frozen oocytes to improve survival by eliminating damaged mitochondria [ 55 ].…”

“…Unsurprisingly, various cell death pathways (including necroptosis, autophagy-dependent cell death [ADCD], NETosis, ferroptosis, parthanatos, and the mitochondrial permeability transition pore) have been discovered in recent decades in addition to apoptosis, which is the most widely recognized cell death pathway [7]. While necroptosis and autophagy have been associated with cryoinjury, our under-standing of the contributions of these pathways is still incomplete [8][9][10][11]. Consequently, gaining insight into the molecular mechanisms of cryoinjury could offer new strategies to enhance freezing efficacy.…”