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 prospe...
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Editorial Office of Journal of Army Medical University
2025-01-01
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| Series: | 陆军军医大学学报 |
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| author | CHEN Xinling CHEN Xinling LONG Yixuan DU Guihua DU Guihua |
| author_facet | CHEN Xinling CHEN Xinling LONG Yixuan DU Guihua DU Guihua |
| author_sort | CHEN Xinling |
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| description | 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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| institution | Kabale University |
| issn | 2097-0927 |
| language | zho |
| publishDate | 2025-01-01 |
| publisher | Editorial Office of Journal of Army Medical University |
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| series | 陆军军医大学学报 |
| spelling | doaj-art-4f6e802ccb0046159e53a3d8e9f451562025-01-13T09:07:26ZzhoEditorial Office of Journal of Army Medical University陆军军医大学学报2097-09272025-01-01471829110.16016/j.2097-0927.202407013Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogoniaCHEN Xinling0CHEN Xinling1LONG Yixuan2DU Guihua3DU Guihua4Department of Occupational Health and Toxicology， School of Public Health, Nanchang University， Nanchang， Jiangxi， ChinaJiangxi Provincial Key Laboratory of Disease Prevention and Public Health, Nanchang University， Nanchang， Jiangxi， ChinaDepartment of Occupational Health and Toxicology， School of Public Health, Nanchang University， Nanchang， Jiangxi， ChinaDepartment of Occupational Health and Toxicology， School of Public Health, Nanchang University， Nanchang， Jiangxi， ChinaJiangxi Provincial Key Laboratory of Disease Prevention and Public Health, Nanchang University， Nanchang， Jiangxi， ChinaObjective 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. https://aammt.tmmu.edu.cn/html/202407013.htmlprospermatogoniarb1spermatogonial stem cellsspecificationcell proliferationcell apoptosis |
| spellingShingle | CHEN Xinling CHEN Xinling LONG Yixuan DU Guihua DU Guihua Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia 陆军军医大学学报 prospermatogonia rb1 spermatogonial stem cells specification cell proliferation cell apoptosis |
| title | Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia |
| title_full | Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia |
| title_fullStr | Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia |
| title_full_unstemmed | Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia |
| title_short | Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia |
| title_sort | mechanism of abnormal specification of spermatogonial stem cells after rb1 knockout in mitotic arrested prospermatogonia |
| topic | prospermatogonia rb1 spermatogonial stem cells specification cell proliferation cell apoptosis |
| url | https://aammt.tmmu.edu.cn/html/202407013.html |
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