Fig 2: Heterochronic fecal microbiota transfer affects spermatogenesis of young and old mice. A The experimental workflow for fecal microbiota transplantation (FMT) and multi-omics examination of young (6-week), old (18–20-month), and FMT mice. For FMT groups, y FMT y and o FMT y (damaged comparison) indicate young mice received young or old donor microbiota, respectively; o FMT o and y FMT o (rescued comparison) indicate old mice received old or young donor microbiota, respectively. B–D Sperm quality including sperm concentration (B), progressive motility (grade A + B sperm) (C), and total motility (grade A + B + C sperm) (D). E–L Representative images of H&E staining (E, F, I, J) and immunofluorescence staining of testis tissues (G, H, K, L) for FMT mice. DAZL (spermatogonia marker), SYCP3 (spermatocyte marker), TNP1 (spermatid marker), PGK2 (spermatozoa marker), and WT1 (Sertoli cell marker) were stained. Scale bar = 100 μm for E, F, I, J or 25 μm for G, H, K, L. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Data are analyzed by two-tailed unpaired Student’s t test. n = 7–8 mice per group

Fig 3: The expression levels of Dazl in glioblastoma tissues and cell lines. a Dazl expression was examined by immunohistochemical analysis in human glioblastoma tissues and adjacent normal tissues. Strong cytoplasmic expression of Dazl (brown staining) was detected in stage III/IV glioblastoma cells, and the nucleus was stained blue with hematoxylin. Nor: Normal. Images were taken from the inverted microscope (bars = 50 μm, magnification × 200). b The correlation of Dazl expression and glioblastoma grade was analyzed from the Chinese Glioblastoma Genome Atlas (CGGA) data (**P < 0.01, ****P < 0.001). c The correlation of Dazl expression and overall survival of glioblastoma patients was determined from the CGGA data (**P < 0.01). d The lysates of glioblastoma cells from A172, U251, and LN229 cell lines were harvested and examined for Dazl expression, the cell lysates of NHA cell line were used as the negative control (**P < 0.01, ****P < 0.001). e Detection of gene expression by agarose gel electrophoresis after RT-PCR. f Dazl expression in glioblastoma cell lines was detected by western blotting. g Relative Dazl expression was quantified by Image J software using Gapdh as an internal control. (*P < 0.05)

Fig 4: Changes of spermatogenesis-related indexes were observed from gene expression and protein level, respectively. The expression of the following genes, such as Dazl, Vasa, Plzf, c-Kit, H2ax, Stra8, Sycp1, Sycp3, Tnp2, and Piwil1, in the testis tissue of the two groups were detected by real-time PCR (qRT-PCR) technology (A). Also, the changes of protein levels such as VASA, DAZL, and γH2AX between the two groups of mice were observed by Western Blot (B,C) and immunofluorescence staining experiments (D–G). * Significant differences compared to the control (p < 0.05).

Fig 5: Gene expression analyses of pluripotency and GC markers during bovine testis development and immunodetection of DAZL, STRA8, and BOULE in bovine fetal testis. (A) Levels of mRNA of pluripotent OCT4 and NANOG and GC genes c-KIT, DAZL, STRA8, BOULE, and PIWIL2 were higher (P < 0.05) in adult compared to fetal testis. (B) Sertoli (arrow) and pre-spermatogonial (arrowhead) cells are indicated in epifluorescence and H&E staining of bovine fetal testis (M9). An intense immunosignal associated to DAZL located in Sertoli and pre-spermatogonial cells was detected in the fetal testis. STRA8 immunosignal was observed in the spotted areas mainly in the basal compartment of the seminiferous tubules. NANOG immunosignal was observed across the spermatogenic epithelium with high intensity in Sertoli and pre-spermatogonial cells. (*) indicates significant differences (P < 0.05) for gene expression levels between the developmental stages for each gene. Scale bars: 200 μm.