Fig 2: Elevated Mitochondrial/ER Activities and MFN Expression during Spermatogonial Development(A and B) ROS (A) and cellular Ca2+ levels (B) were measured in CD90+/KIT- undifferentiated spermatogonia and KIT+ differentiating spermatogonia collected from wild-type P12 testes by flow cytometry.(C) Real-time RT-PCR analyses on the expression of ER chaperons in sorted CD9+/KIT- undifferentiated spermatogonia and KIT+ differentiating population from wild-type P12 testes.(D) Protein levels of MFN1 and MFN2 were measured by western blots in testes collected at various time points during spermatogenesis.(E) Real-time RT-PCR analyses on Mfn1 and Mfn2 expression in sorted CD9+/KIT- and KIT+ cells from wild-type P12 testes.(A–C and E) Data are presented as mean ± 1 SEM from three or more biological replicates. Fold changes or expression levels were compared with mean value of CD90+/KIT- or CD9+/KIT- cells. *p < 0.05, **p < 0.01, ***p < 0.001; N.S., no statistical significance.

Fig 3: TCF3 Occupies Developmental-Stage-Specific Enhancer-Promoter Loops and Affects Cell-Cycle Phase and Lineage Potential(A) FL HSC-specific TCF3 targets have significantly higher expression.(B) ChIP-qPCR confirmation of TCF3 binding to enhancers involved in FL HSC-specific EP loops.(C) Enriched GO terms among genes targeted by FL HSC-specific EP loops occupied by TCF3.(D) Western blot showing knock down of TCF3 by CRISPR-Cas9.(E and F) Cell cycle phase analysis by co-staining with propidium iodide and anti-Ki-67 antibody.(E) Representative fluorescence-activated cell sorting (FACS) plots of wild-type and Tcf3 knockout HPC-7 cells.(F) Quantification of cell cycle phases, mean ± SD of three biological replicates. p values were computed using t test.(G) Limiting dilution assay showing significantly reduced lymphoid potential in Tcf3 knockout HPC-7 cells. y axis, frequencies of CD45+CD25+CD90+ T progenitors and CD45+B220+CD19+ B progenitors produced by wild-type and Tcf3 knockout HPC-7 cells after 10–12 days of co-culturing with OP9/OP9-DL1 cells. Values are mean of 2 biological replicate experiments. Error bar, SD.

Fig 4: G6P formation from glucose in glycolysis is required for spermatogonial differentiation.a Overview of inhibitors used to block various steps of glycolysis. b–f The percentages of CD90+ and c-Kit+ cells were determined using flow cytometry on spermatogonia in the absence (-RA) or presence (+RA) of RA for 24 h. Inhibitors include enoblock (Eno; b), oxamate (c), 2-DG (d), loniamine (Lon; e), and 6-aminonicotinamide (6-AN; f). g Gene expression levels were measured by real-time RT-PCR on spermatogonia treated with inhibitors for 24 h. h The numbers of spermatogonia were counted in the presence of 10 mM 2-DG for three days (D3), and fold changes of cell numbers after 2-DG treatment was calculated in comparison to mock controls. g, h Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; N.S., no statistical significance; n = 3.

Fig 5: Identification of mouse mesenchymal stem cells (mMSCs) and mMSCs-derived exosomes. A Flow cytometric analysis for cell surface markers of CD44, Sca-1, CD90.2, CD34, CD11b, and CD45 on mMSCs. Percentage of positive cells shown in upper right corner as standardized with the isotype control antibody-incubated cells. B Assessment of differentiating capacity of mMSCs into osteocytes, chondrocytes, and adipocytes stained by Alizarin Red (left), Alcian Blue (middle), and Oil Red (right), respectively. Scale bar = 100 µm. C Representative image of transmission electron microscopy of mMSCs-Exo, and red arrows indicate typical exosomes. Scale bar = 200 nm. D Western Blot analysis for exosomal markers CD63, TSG101, and CD9, as well as the negative marker GM130 in mMSCs-Exo. The culture medium was used as control. E Nanoparticle tracking analysis of mMSCs-Exo. F Confocal images shown that PKH67-labeled exosomes (green) were taken up by MLE-12 cells at indicated time points in vitro. Nuclei were counterstained with DAPI. Scale bar = 40 µm