Chromosomes 21 and 18 displaying altered crossover distributions in NOA men.

<p>Chromosome arms were divided into 10% intervals, and the crossover frequency in each interval was calculated. The Y-axis represents the frequency of crossovers in each interval. The X-axis represents the relative crossover position from the centromere with the values representing the upper limit of each interval. The centromere is labeled ‘C’ with the p-arm to the left and q-arm to the right. As crossovers in the p-arm of chromosome 21 are extremely rare, the p-arm is not shown. The black bars indicate the control group and the white bars indicate the individual NOA man. The crossover frequencies in each interval were compared to the control group and significant differences are indicated by asterisks (P < 0.05, Fisher test).</p

Immunofluorescence and FISH analysis of pachytene spermatocytes.

<p>(A) Spermatocytes were immunostained using antibodies against SYCP3/SYCP1, MLH1 and CREST to visualize the SC (red), crossover sites (green) and centromeres (blue). A spermatocyte with 45 crossovers from patient OA20 is shown. Although patient OA20 displayed normal rates of recombination, the crossover distribution on chromosome 18 was altered. We observed an increase in crossovers near the centromere and telomere on 18q and 18p, respectively. (B) Subsequent FISH was performed to identify chromosomes 13 (green, LSI 13), 18 (blue, CEP 18) and 21 (red, LSI 21) in the previously immunostained spermatocytes. (C) A spermatocyte from patient OA19 is shown. Although the patient showed normal rates of recombination, we observed an increased rate of synaptic errors compared to controls (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156817#pone.0156817.t001" target="_blank">Table 1</a>). The unsynapsed region along a bivalent is indicated by the white arrow, where there is an absence of staining for SYCP3/SYCP1.</p

Temporal mapping of the metabolic checkpoints from G0.

<p>(<b>A</b>) Schematic representation of the experiment shown in (<b>B</b>). (<b>B</b>) BJ cells were plated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074157#pone-0074157-g001" target="_blank">Figure 1A</a> for 24 hr. Cells were synchronized in G0 by shifting to DMEM+1mM Q lacking GF for 48 hr. The cells were released from G0 by shifting to CM containing DMEM (1mM Q) and 1 µCi/ml [³H]-TdR. Various blocking conditions along with [³H]-TdR were applied at indicated time points. After 36 hr from the release from G0, cells were collected and the incorporated label was determined. This experiment utilized DMEM with reduced Q (1 mM vs. 4 mM) because Q withdrawal following DMEM with high Q did not give strong G1 arrest. Error bars represent the standard error for experiments repeated three times.</p

Analysis of crossover frequencies on chromosome 13, 18 and 21 in control and OA men.

<p>Analysis of crossover frequencies on chromosome 13, 18 and 21 in control and OA men.</p

Analysis of recombination and synaptic errors in control and OA men.

<p>Analysis of recombination and synaptic errors in control and OA men.</p

Analysis of crossover frequencies on chromosome 13, 18 and 21 in control and NOA men.

<p>Analysis of crossover frequencies on chromosome 13, 18 and 21 in control and NOA men.</p

Diagram depicting meiotic crossovers in regions along a chromosome.

<p>The p and q arms of the chromosome are divided into 10% intervals, with the centromere (C) at 0%, and telomeres at 100%. The subtelomere is shaded in the 80–100% intervals. The pericentromere, the region surrounding the centromere is shaded in the 10–30% intervals. This region attracts high levels of cohesin which are protein complexes that hold sister chromatids together until they separate during meiosis. Crossover formation in each region of the chromosome, and the resulting recombinant chromosomes are illustrated: a) a single crossover near the telomere on the p-arm; b) a single crossover near the centromere on the q-arm; c) a single crossover near the telomere on the q-arm; d) double crossovers on the q-arm.</p

Growth factor and amino acid deprivation, as well as mTOR inhibition induce G1 cell cycle arrest.

<p>(<b>A</b>) BJ hTERT cells were plated at 20% confluence in DMEM containing 10% FBS for 24 hr at which time they were shifted to complete medium (CM) or various blocking conditions [-GF, -EAA, -Q, +Rapamycin (20 µM)] for 24 or 48 hr. The blocking conditions for Q used DMEM lacking Q; and for EAA, DMEM lacking Leu, Lys, and Arg as described in Material and Methods. The CM contained 10% dialyzed FBS (DFBS) instead of 10% FBS. Cells were labeled with [³H]-TdR for the final 24 hr of treatment, after which the cells were collected and the incorporated label was determined by scintillation counting as described in Materials and Methods. Values were normalized to the cpm for CM, which was given a value of 100%. Total cpm for the CM controls was 60,512 +/- 6529 for the 24 hr time point and 80,427 +/- 3567 for the 48 hr time point. Error bars represent the standard deviation for experiments repeated at least two times. (<b>B</b>) BJ cells were plated and shifted to CM or various blocking conditions for 48 hr as in (<b>A</b>), after which the cells were harvested and analyzed for cell cycle distribution by measuring DNA content/cell as described in Materials and Methods. Error bars represent the standard error from independent experiments repeated four times. (<b>C</b>) To investigate the kinetics for progression into S-phase, BJ cells were plated and shifted to blocking conditions for 48 hr as in (<b>A</b>). Cells were subsequently released by shifting to complete medium, and pulsed with [³H]-TdR at the indicated time points for 1 hr – after which the cells were collected and the incorporated label was determined. Error bars represent the standard error of mean for experiments repeated three times.</p

Restriction point and metabolic checkpoint arrest lead to differential patterns of cell cycle regulator expression and phosphorylation.

<p>(<b>A</b>) Cells were plated at 30% confluence in 10-cm plates in DMEM containing 10% FBS. After 24 hr, the cells were shifted to CM or blocking conditions for 4 hr, at which time the cells were harvested and the levels of the indicated protein or phosphoprotein was determined by Western blot analysis. The data shown are representative of experiments repeated at least two times. (<b>B</b>) Quantitative analysis of relative protein levels for Western blots shown in (<b>A</b>) using ImageJ software. (<b>C</b>–<b>F</b>) BJ cells were plated and shifted to various blocking conditions for 48 hr as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074157#pone-0074157-g001" target="_blank">Figure 1A</a>. The cells were subsequently released by shifting to CM, and the cells were harvested and lysates collected at indicated time points. The levels of the indicated protein or phosphoprotein were determined by Western blot analysis. The data shown are representative of experiments repeated at least two times. Also shown in the line graphs are the kinetic analyses of relative protein levels normalized to actin and quantitated using ImageJ.</p

GF, EAA, Q, and rapamycin mediated G1 cell cycle arrests are distinct and distinguishable.

<p>(<b>A</b>–<b>D</b>) BJ hTERT cells were plated and shifted to various first blocking conditions for 48 hr as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074157#pone-0074157-g001" target="_blank">Figure 1A</a>. The cells were subsequently shifted to CM or different second block conditions containing [³H]-TdR for 24 hr, after which the cells were collected and the incorporated label was determined. Error bars represent the standard error for the experiment repeated at least four times. (<b>E</b>) Schematic model showing relative positions of different metabolic checkpoints relative to R (not drawn to represent precise time scales). G1-pm is post-mitotic phase in G1, G1-ps is pre-S phase of G1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074157#B4" target="_blank">4</a>].</p