Journal: PLoS Genetics

Article Title: Functional robustness of adult spermatogonial stem cells after induction of hyperactive Hras

doi: 10.1371/journal.pgen.1008139

Figure Lengend Snippet: A. Schematic showing workflow for collection of the A undiff population from dissociated testicular cells of genetically engineered mice with induced germ cell-restricted tdTomato expression with or without the Hras G12V allele. After 3–4 months of tamoxifen administration to Gfra1-creER T2 ; tdTom fl/- (WT) mice, testes were dissociated, and the tdTomato+ cells were sorted based on MCAM expression levels (high, medium, dim, and negative). B. Flow cytometry analysis of dissociated testicular cells from induced WT mice. After exclusion of doublet and dead (DAPI+) cells, tdTomato+ gated cells (top) were sorted to obtain MCAM high, medium, dim, and negative populations (bottom). C. Gfra1 and Hras transcripts in different germ line populations of the wild-type testis. Using tamoxifen-induced Gfra1-creER T2 ; tdTom fl/- animals (n = 3), the tdTomato+ population which contains only germline cells) was sorted into MCAM-high, medium, dim, and negative populations. The MCAM-high population yielded elevated Gfra1 mRNA (left graph), indicating that this population is enriched for undifferentiated spermatogonia (A undiff ). The level of Hras transcript (relative to Actb ) was similar among these four different germline populations. Inset: the final qPCR products were run on agarose gel and shown on the top. D. Strategy to induce Hras G12V exclusively in undifferentiated spermatogonia. After tamoxifen administration, the wild-type Hras allele is excised as it is flanked by two loxP sites (black triangle). Both the wild-type Hras allele (before recombination) and the recombined Hras G12V alleles are under the regulatory control of the endogenous Hras promoter. After tamoxifen administration, we designated the animals as induced WT (iWT) or induced FR (iFR). E. Sanger sequence trace of reverse-transcribed PCR product from tdTomato+ germ line cells in testis of iWT and iFR three months after a four-day tamoxifen regimen. Note that the mutant A peak is smaller than the C peak, indicating that not all the tdTomato+ cells were positive for Hras G12V . F. Similar Hras mRNA levels in A undiff from iWT and iFR (n = two littermates; right and left). RT-qPCR primers were designed to recognize a common region for iWT & iFR. Animals were treated with two courses of tamoxifen (one course = 4 consecutive days) over two weeks. G. Reporter expression in the undifferentiated spermatogonia after one course of tamoxifen. Top panels are whole-mount immunofluorescence using anti-GFRA1 (pseudocolored magenta). On day 6 (day 0 = first day of tamoxifen administration), tdTomato (pseudocolored green) efficiently labeled undifferentiated spermatogonia positive for GFRA1 (magenta) both in iWT and iFR (representative images from n = 2 animals for each group). A representative A single cell positive for GFRA1 is magnified from the white box. After 3 months, whole segments were occupied by tdTomato+ germline cells. Bottom panels are cross-sectioned tubules showing that tdTomato+ A undiff differentiate and produce spermatids both in iWT and iFR. Scale bars: 100 μm.

Article Snippet: After incubation with anti-Gfra1 antibody (1:200, BD) overnight, an anti-rabbit biotinylated secondary antibody, followed by Alexa647-conjugated streptavidin was used for detection.

Techniques: Expressing, Flow Cytometry, Agarose Gel Electrophoresis, Sequencing, Mutagenesis, Quantitative RT-PCR, Immunofluorescence, Labeling

Journal: PLoS Genetics

Article Title: Functional robustness of adult spermatogonial stem cells after induction of hyperactive Hras

doi: 10.1371/journal.pgen.1008139

Figure Lengend Snippet: A. Experimental strategy for lineage tracing. At 6–8 weeks of age, FR mice (n = 4 mice at each timepoint) were treated with tamoxifen (100 mg/kg) for 4 days and sacrificed to obtain tdTomato+ germline cells by FACS and sperm analysis at 3, 7, and 14 months after tamoxifen. The two independent methods, Sanger sequencing and gDNA qPCR (described in ), were employed to calculate the Hras G12V -positive cell fraction. B. (left) Proportion of Hras G12V -positive cells in the labeled (tdTomato+) germline population over time (obtained by FACS). B. (right) Hras G12V -positive sperm proportion. C. Experimental strategy for serial sperm collection and a representative image of collected sperm (right). Microsurgical epididymis sperm aspiration was repeated twice in the same individuals at 3–4 months and 11–13 months. At each time point, gDNA qPCR was performed to measure the Hras G12V + cell fraction and the reference allelic fraction. For qPCR of SV40 PA , Gfra1-creER T2 ; FR fl/- mice were also used (*). Additionally, GFP+ and tdTomato+ spermatids were counted to calculate the ratio of GFP+ sperm to total sperm (GFP+ plus tdTom+). The green and red arrowheads indicate representative GFP+ and tdTomato+ spermatids, respectively. D. Scatter plot showing the correlation between the cell fraction of the recombined mTmG and GFP+ sperm ratio. One dot corresponds to one sample. A strong correlation was found for the qPCR-measured AF of recombined mTmG and the ratio of manually counted GFP+ sperm to total sperm (r 2 = 0.9008, p<0.0001). Black = WT. Red = MUT. E. Fraction of Hras G12V -positive population in sperm at 4 and 12 months after tamoxifen (paired t-test, ***p<0.001, n = 13 pairs). Sperm was collected twice within the same animals, and the gDNA was extracted for qPCR. Black dots represent animals treated with tamoxifen for 4 days; gray dots for 2 days; and light gray dots for 1 day (same for F, G, and H). F. Fraction of mTmG -recombined allele in iWT. No change was observed in the fraction over time (paired t-test, P = 0.82, n = 10 pairs). G. Fraction of GFP+ sperm out of total sperm from iFR. No change was observed from 4 to 12 months (paired t-test, P = 0.94, n = 8 pairs). H. Fraction of GFP+ sperm out of total sperm from iWT. No change was observed from 4 to 12 months (paired t-test, P = 0.12, n = 10 pairs).

Article Snippet: After incubation with anti-Gfra1 antibody (1:200, BD) overnight, an anti-rabbit biotinylated secondary antibody, followed by Alexa647-conjugated streptavidin was used for detection.

Techniques: Sequencing, Labeling