<i>In vivo</i> T cell development with LSK-EVAi cells.

<p>(A, left panel) <i>Rag2/γc</i> mice were transplanted with either LSK cells infected with a non-interfering (LSK-CT) or with an Eva1-interfering lentivirus (LSK-EVAi). After 8 weeks, thymi were excised and compared: thymus from mice transplanted with LSK-EVAi were comparable to untreated ones for dimension and total cellularity (A, right panel). Untr., untreated control animal. Scale bar, 2 mm. (B) Time course of T cell development of LSK-CT (white bars) or LSK-EVAi (black bars) cells respectively, in recostitution experiments in <i>Rag2/γc</i> mice. Flow cytometric analysis of CD25/CD44 and CD4/CD8 stainings revealed that LSK-EVAi reconstituted thymus had a delay of T cell differentiation with an accumulation of DN cells at 6 weeks after trasplant (left panel). Percentage of DN cells in LSK-EVAi reconstituted thymus persists higher than in LSK-CT reconstituted thymi at 8 weeks after transplant (right panel). Mean and SD of three independent experiments are shown (** p<0.01). (C) Counts of total DN cells generated in the three different mouse groups at six and eight weeks post-treatment.</p

Analysis of <i>Eva1</i> expression.

<p><i>Eva1</i> real-time RT-PCR in fetal thymi (A), adult DN subpopulations (B, left panel) and thymocytes from mutant mice (B, right panel). mRNA from flow cytometrically purified TECs (CD45-) and intrathymic haematopoietic cells (CD45+) or DN1-3 subpopulations was reverse transcribed and used as the template for PCR with <i>Eva1</i>-specific primers. All samples were normalized to the geometric mean of the GAPDH housekeeping gene. NB, newborn; 2mth, two months; WT, wild type; ΔCAM, Tg-Calcineurin; <i>Prkdc</i>^scid, protein kinase, DNA-activated, catalytic polypeptide (C, upper panel) Eva1 interference in LSK cells by lentiviral vector was controlled by real time RT-PCR. LSK-CT cells shown a comparable expression of <i>Eva1</i> while LSK-EVAi cells shown a drastic decrease of <i>Eva1</i> expression, indicating that the interference has occurred. (C, lower panel), fluorescence microscopy analysis confirming <i>Eva1</i> interference in LSK-EVAi cells. LSK-WT, uninfected LSK; LSK-CT, LSK infected with a non-interfering lentiviral vector; LSK-EVAi, LSK infected with a <i>Eva1</i>-interfering lentiviral vector.</p

SM induces meiotic entry of Kit-positive spermatogonia.

<p>A) Morphology of Kit-positive spermatogonia from testes of 7dpp mice after 2 days of culture under SM and unit gravity seen at light microscopy. Scale bar, 100 µm. B) Representative immunofluorescence images showing Scp3 organization on nuclear spreads at different stages of meiotic prophase I made on cultured spermatogonia. Scale bar, 10 µm. C, D) Histograms representing the percentage of nuclei with a typical meiotic Scp3 organization (C) and relative amount of early leptotene/leptotene and zygotene meiotic figures (D) after 48 h of cultures kept at unit gravity and in SM. The percentage of Scp3-positive cells present in freshly collected Kit-positive cells is shown as T0. The values were obtained by counting 400 nuclei in each sample. Data are means ± SD of at least four independent experiments. P≤0.005. E, F) Representative semiquantitative RT-PCR (E) and western blots (F) for pre-meiotic markers Kit and Stra8 and meiotic markers Spo11, Scp3, Scp1 in spermatogonia under SM versus gravity cultures. Similar results were obtained in three separate experiments. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009064#pone-0009064-t001" target="_blank">Table 1</a> for quantitative data.</p

Isolation and characterization of Kit-positive Spermatogonia.

<p>A) Germ cells were obtained with sequential enzymatic digestions from testis of 7dpp animals and analyzed by FACS analysis for Kit expression (left panel). Kit-positive spermatogonia were purified using CD-117 conjugated beads, and Kit and Stra8 protein expression were detected by Western blot analysis. Tubulin was used as loading control (right panel). B) Representative images of DAPI (blue) and Scp3 (green) staining of nuclear spreads from isolated Kit-positive spermatogonia. Scp3-negative nuclei with decondensed chromatin (type A spermatogonia) or with heterochromatic nuclei (type B spermatogonia), and Scp3-positive nuclei with small and diffuse spots (Transition Spermatogonia) or with large and defined spots (Preleptotene) are shown. Scale bar, 10 µm.</p

Up-regulation of pro-meiotic markers in Kit-positive spermatogonia from 7dpp testes, cultured under SM with respect to cells kept at unit gravity.

<p>Up-regulation of pro-meiotic markers in Kit-positive spermatogonia from 7dpp testes, cultured under SM with respect to cells kept at unit gravity.</p

SM induces the last round of premeiotic DNA synthesis in Kit positive spermatogonia.

<p>A) Kit positive spermatogonia from 7 dpp mice were cultured for 24 h under gravity and SM conditions in the presence of BrdU and analysed by flow cytometry for BrdU incorporation and propidium iodide labeling. The upper quadrants show the limits for replicating cells. Percentages of cells in the different states are indicated. BrdU, bromodeoxyuridine. B) Representative immunofluorescence images of Scp3-positive (red) and BrdU-stained (green) premeiotic S-phase nuclei (preleptotene). Scale bar, 10 µm. C) Histogram showing the percentage of double positive cells respect to total BrdU stained nuclei after 24 of culture under gravity or SM condition. The values are means ± SD of three separate experiments. P≤0.05.</p