(219) Regulatory Mechanisms of SKP1 in DSB Homeostasis in Mouse Germ Cells

Disturbances in the double-strand break (DSB) homeostatic regulation can severely impair germ cell genomic stability and normal meiotic progression, ultimately resulting in male infertility. The SCF (SKP1–CULLIN–F-box) is a family of ubiquitin E3 ligases, in which SKP1 associates with various F-box proteins to form distinct E3 ligase complexes targeting specific substrates for proteasome-mediated degradation, which play critical roles in numerous biological processes, including spermatogenesis. Previous results showed that meiosis-specific knockout of Skp1 in mice causes early meiotic arrest and an abnormal distribution of DSBs in early meiotic cells. Therefore, we aimed to investigate the mechanisms by which Skp1 regulates DSB homeostasis in mouse germ cells and proposed the hypothesis that the primary cause of germ cell DSB dysregulation is the DNA replication stress in S-phase germ cells induced by skp1 deficiency.
To test this hypothesis, we first generated the Skp1 Spo11 double_knockout mice. Immunofluorescence analyses revealed that the abnormal DSB distribution remained, consistent with that in the Skp1 single-knockout model, thereby ruling out SPO11 involvement in DSB abnormality. Next, we performed micronucleus assays and immunofluorescent staining of DNA damage response (DDR) markers (RPA, γH2AX) to examine DDR signaling in germ cells. We found that DDR signaling in Skp1-deficient germ cells was markedly higher than in control cells, with significantly elevated DDR signals detected in pre-meiotic germ cells. Furthermore, via BrdU incorporation experiments, we labeled S-phase germ cells in the Skp1 knockout model and assessed DNA replication capacity. We observed a significant reduction of the BrdU-positive cells in the testes of Skp1-deficient mice, and at the same time, we detected aggregated RPA foci in a part of BrdU-positive S-phase germ cells.
These findings suggest that Skp1_deficiency may trigger DNA replication stress responses, closely linked to the regulation of DSB homeostasis in germ cells. Given the ubiquitination activity of the SCF E3 ligase complexes and building on prior studies, we are currently working to identify downstream substrates and novel pathways involved in SKP1-mediated regulation of germ cell DNA replication stress. Notably, we have detected an aberrant increase in Cyclin E levels in Skp1-deficient germ cells. In the future, we will employ immunofluorescence assays, flow cytometry, and in vitro interaction analyses to further validate the relevance between Cyclin E and SKP1-mediated replication stress regulatory pathway in germ cells, aiming to elucidate the underlying molecular mechanisms.