O-254 ACTRT2 deficiency increases spermatogonia vulnerability to ferroptosis

Abstract

Abstract Study question How does the actin-related proteins T2, ACTRT2, which is specifically expressed in spermatogonia, regulate spermatogonia death? Summary answer ACTRT2 deficiency in spermatogonia leads to intracellular iron overload and damage to mitochondria, ultimately increasing spermatogonia vulnerability to ferroptosis. What is known already ACTRT2 is tightly related to spermatogenesis. On the one hand, ACTRT2 is involved in regulating iron uptake in spermatogonia. ACTRT2 deficiency leads to intracellular iron overload and mitochondrial damage, ultimately increasing the vulnerability of spermatogonia to ferroptosis. On the other hand, for ACTRT2-related spermatogenic impairment, the survival of spermatogonia could be maintained by inhibiting ferroptosis, and spermatogenesis of the testis could be improved. Study design, size, duration The expression of ACTRT2 was knocked down in GC-1 cells (spermatogonial cell line). Busulfan was used to induce GC-1 cells death. And then to explore which type of cell death ACTRT2 was involved in, GC-1 cells were treated with 50 μM Z-VAD-FMK (apoptosis inhibitor), 50 μM necrostin-1 (necroptosis inhibitor), or 0.1 μM ferrostatin-1 (ferroptosis inhibitor).The mechanism by which ACTRT2 regulates ferroptosis in spermatogonocytes was investigated in ACTRT2 knockout mice. Participants/materials, setting, methods ACTRT2 expression was knocked down in vitro in GC-1 cells using siRNA. Then, GC-1 cell death was induced using 150 μM busulfan. ROS levels, morphological changes in mitochondria, and the ferroptosis marker GPX4 were detected. ACTRT2 knockout mice were constructed in vivo using CRISPR/Cas9. The spermatogenesis of the mice was measured with H&E staining. After treatment with busulfan, ferrostatin-1 was used to inhibit ferroptosis in order to protect spermatogenesis. Main results and the role of chance ACTRT2 was specifically expressed in testicular tissue and was associated with spermatogenesis. In vitro, when the cells were treated with busulfan, the proportion of GC-1 cell death in the low-ACTRT2 group increased significantly compared with the si-NC group. Furthermore, ferrostatin-1 significantly improved the survival of busulfan-treated cells. In low-ACTRT2 GC-1 cells, ROS accumulation and typical mitochondrial changes associated with ferroptosis occurred, and GPX4 was downregulated. In vivo, the seminiferous tubules in ACTRT2-/- mice were significantly shrunken. In addition, after being treated with busulfan, spermatogenesis in ACTRT2+/- mice decreased significantly compared to that in wild-type mice. In ACTRT2+/- testes, the expression levels of ACSL4 and ALOX15 were upregulated, while the expression levels of SLC7A11 and GPX4 were downregulated. Then, ferrostatin-1 was administered, which resulted in improved spermatogenesis. Finally, we found that the expression of SLC11A2, IREB2, and TFRC increased significantly in the low-ACTRT2 group, which transports iron into the cell to increase the intracellular unstable iron pool. Meanwhile, the expression of SLC40A1, which transports excess iron out of the cell, significantly decreased. Limitations, reasons for caution We found that ferrostatin-1 can inhibit ferroptosis in ACTRT2-deficient spermatogonia, which is expected to be a new therapeutic strategy for male infertility. However, its safety and efficacy still need to be further evaluated, and the therapeutic schedule still needs to be further explored and optimized. Wider implications of the findings Ferrostatin-1 can inhibit ferroptosis in ACTRT2-deficient spermatogonia, which is expected to be a new therapeutic strategy for male infertility. Trial registration number not applicable