To investigate the underlying mechanism of abnormal specification of spermatogonial stem cells (SSCs) in male mice following Rb1 conditional knockout in mitotic-arrested prospermatogonia.

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 or Id4-gfp^Tg; 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 (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.

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].

Rb1 knockout in mitotic-arrested prospermatogonia disrupts their postnatal cell cycle re-enter and induces cell apoptosis, which further results in abnormal SSC specification.