Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia
Objective To investigate the underlying mechanism of abnormal specification of spermatogonial stem cells(SSCs)in male mice following Rb1 conditional knockout in mitotic-arrested prospermatogonia.Methods ① R language was used to analyze the single-cell RNA sequencing(scRNA-seq)data of prospermatogonia derived from postnatal day 0.5(P0.5)male mice through the gene expression omnibus(GEO)public database(accession number:GSE124904).② Mitoti-arrested prospermatogonia Rb1 conditional knockout(cKO)mice as well as Rb1 cKO mice with Id4-gfp transgene were generated using Vasa-Cre mice crossed with Rb1flox/flox or Id4-gfpTg;Rb1flox/flox mice.PCR was employed to detect the deletion of Rb1 in order to distinguish the control and cKO male mice.The testes of male mice(n=3～8)within a few days after birth were collected.After that,flow cytometry was applied to divide the ID4-GFP cells into 3 communities based on the GFP fluorescence intensity,and then detect the number of cells and cell cycle in each community.③ Germ cell proliferation(Ki67 positive,Ki67+),SSCs specification(FOXO1 nuclear transition),and germ cell differentiation(STRA8+)were detected with immunofluorescence staining.④ TUNEL staining was utilized to detect cell apoptosis.Results ① The results of scRNA-seq showed that in the two set clusters of prospermatogonia,the prospermatogonia that further specifies to generate SSCs had enriched genes that are associated with cell proliferation.② Germ cell proliferation assay indicated that the average ratio of Ki67+germ cells in the testicular cross-section of the cKO mice was significantly higher than that of the control mice at P2.5[(46.10±6.21)%vs(11.22±3.27)%,P<0.01].③ Flow cytometry revealed that,among the brightest community of ID4-GFP cells,the percentage of the cells at S phase was obviously higher in the testicular cells derived from the cKO mice when compared to the control mice at P2.5[(12.05±1.22)%vs(5.05±1.46)%,P<0.05].④ TUNEL staining displayed that cell apoptosis was detected in the testicular cross-section of cKO mice rather than that of the control mice.⑤ The results of SSCs specification exploration showed that statistical difference was observed in the percentage of cytoplasmic FOXO1 in the testicular cross-section between the control and cKO mice[(20.57±2.15)%vs(45.08±2.45)%,P<0.01].Conclusion Rb1 knockout in mitotic-arrested prospermatogonia disrupts their postnatal cell cycle re-enter and induces cell apoptosis,which further results in abnormal SSC specification.
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维普
