Mechanism of abnormal specification of spermatogonial stem cells after <italic style="font-style: italic">Rb1</italic> knockout in mitotic-arrested prospermatogonia

CHEN Xinling; LONG Yixuan; DU Guihua

doi:10.16016/j.2097-0927.202407013

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Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia

Basic Medicine | 更新时间：2025-01-10

- Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia
- Journal of Army Medical University Vol. 47, Issue 1, Pages: 82-91(2025)
- 作者机构：
  
  1.南昌大学：，公共卫生学院劳动卫生与毒理学教研室，江西南昌
  2.南昌大学：，疾病预防与公共卫生江西省重点实验室，江西南昌
- 作者简介：
  
  DU Guihua， E-mail： guihua.du@ncu.edu.cn
- 基金信息：
  
  the Regional Project of National Natural Science Foundation of China(32260182);the Natural Science Foundation for Young Scholars of Jiangxi Province(20212BAB216074);the Innovation and Entrepreneurship Training Program for College Students of Nanchang University(202310403088)
- DOI：10.16016/j.2097-0927.202407013
  CLC： R321.1;R329.28;R394.2
- Published：15 January 2025，
  
  Received：02 July 2024，
  
  Revised：14 September 2024，
- 稿件说明：
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陈新玲,龙旖璇,杜桂花.静息期精原前体细胞Rb1基因敲除致精原干细胞特化异常机制初探[J].陆军军医大学学报,2025,47(01):82-91.

CHEN Xinling,LONG Yixuan,DU Guihua.Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia[J].J Army Med Univ,2025,47(1):82-91.
陈新玲,龙旖璇,杜桂花.静息期精原前体细胞Rb1基因敲除致精原干细胞特化异常机制初探[J].陆军军医大学学报,2025,47(01):82-91. DOI：10.16016/j.2097-0927.202407013.

CHEN Xinling,LONG Yixuan,DU Guihua.Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia[J].J Army Med Univ,2025,47(1):82-91. DOI：10.16016/j.2097-0927.202407013.

摘要

目的

探讨静息期精原前体细胞

Rb1

敲除致雄鼠精原干细胞（spermatogonial stem cells， SSCs）特化异常的调控机制。

方法

①用R对在基因表达综合数据库（gene expression omnibus， GEO）（GEO登记号：GSE124904）获取的出生后0.5 d（postnatal day 0.5， P0.5）雄鼠精原前体细胞单细胞测序数据进行分析。②用

Vasa

-Cre工具鼠与

Rb1

flox/flox

（或

Id4

gfp

；

Rb1

flox/flox

）小鼠配繁构建静息期精原前体细胞

Rb1

条件性敲除（conditional knock out， cKO）雄鼠或带ID4绿色荧光（ID4-GFP）的

Rb1

cKO雄鼠，通过PCR基因鉴定判断生殖细胞

Rb1

敲除情况以区分cKO雄鼠与对照（Con）。采集出生后数天内的小鼠（

=3～8）睾丸，利用流式细胞术基于细胞荧光强度将ID4‐GFP细胞均分为3个群落，检测各个群落细胞数量和细胞周期。③免疫荧光染色检测生殖细胞增殖（Ki67+）、SSCs特化（FOXO1胞核转换）和生殖细胞分化（STRA8+）情况。④TUNEL染色检测细胞凋亡。

结果

①单细胞测序数据分析显示：在P0.5精原前体细胞的2类细胞中，与细胞增殖相关的基因在将特化为精原干细胞的那一群细胞中富集表达。②生殖细胞增殖情况检测表明：P2.5时，对照［（11.22±3.27）%］与cKO小鼠［（46.10±6.21）%）］小鼠睾丸横切面生殖细胞Ki67+占比差异具有统计学意义（

0.01）。③流式细胞术分析表明：P2.5对照［（5.05±1.46）%］与cKO［（12.05±2.22）%］小鼠睾丸ID4‐GFP荧光强度最强的那群细胞处于S期的占比差异具有统计学意义（

0.05）。④TUNEL染色显示cKO组而非对照组小鼠睾丸横切面检测到细胞发生凋亡。⑤SSCs特化标志检测结果显示对照［（20.57±2.15）%］和cKO［（45.08±2.45）%］小鼠睾丸横切面生殖细胞FOXO1位于细胞质的占比差异具有统计学意义（

0.01）。

结论

静息期精原前体细胞

Rb1

敲除使其出生后细胞周期恢复紊乱、凋亡发生，导致SSCs特化异常。

Abstract

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

Rb1

flox/flox

Id4-gfp

；

Rb1

flox/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 （

=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）%

（11.22±3.27）%

，

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）%

（5.05±1.46）%，

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）%

（45.08±2.45）%，

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.

关键词

精原前体细胞Rb1精原干细胞特化细胞增殖细胞凋亡

Keywords

prospermatogoniaRb1spermatogonial stem cellsspecificationcell proliferationcell apoptosis

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⁰

Mechanism of abnormal specification of spermatogonial stem cells after Rb1 knockout in mitotic-arrested prospermatogonia

DOI：10.16016/j.2097-0927.202407013

摘要

Abstract

关键词

Keywords

references