Decreased sperm production in <i>Arf</i>-null mice is compensated by loss of <i>Ink4a</i> or <i>p53</i>.

<p>(A, C) Testes were dissected from adult (2–6 month old) mice of the indicated genotypes and weighed as pairs. (B, D) Caudal epididymides were collected from corresponding mice, and recovered sperm were enumerated using a hemocytometer. Relative testes weights (A) and sperm counts (B) are reduced in <i>Arf</i>-null males but increased in <i>Ink4a</i>-null mice. <i>Ink4a-Arf</i> double-null and <i>p53</i>^−/−;<i> Arf</i>^−/− double null mice exhibit increased testes weights (A) and sperm counts (B). While testes weights (C) are not significantly reduced in <i>Arf</i>^Cre/Flox males, reduced sperm counts (D) mimic the <i>Arf</i> loss-of-function phenotype. [N = 20–32 mice (A, B) and 5 mice (C, D)]. Bars represent standard deviations from the mean. P values were determined using a Student's t-test (*p<0.001, **p<0.0001) and designate significant differences from the wild type genotype.</p

Levels of p53 detected in wild-type and <i>Arf</i>-null testis.

<p>Immunoblotting analysis was performed using whole testis lysates from four representative 3–4 month-old mice of each genotype. Actin was used as a loading control.</p

Diminished Dmc1/Rad51 focus formation in <i>Arf</i>-deficient spermatocytes.

<p>(A, B) Surface spread spermatocytes from three month old WT (left panels) and <i>Arf</i>-null (right panels) mice were immunostained for SCP3 (A, B; green) and Dmc1 (A; red) or Rad51 (B; red). Images were captured with the same exposure time using a Marianas spinning disc confocal microscope. (C, D) Histograms showing the distribution of the number of Dmc1 (C) and Rad51 (D) foci found in wild-type (blue bars) and <i>Arf</i>-null (red bars) spermatocytes. Foci were enumerated from one hundred zygotene spermatocytes immunostained for SYCP3 and either Dmc1 (C) or Rad51 (D) using Slidebook 5.0 SDC software. Actual values of foci per cell are plotted within bin ranges to display the distribution of frequencies. (E, F) Histograms showing the distribution of average intensities of Dmc1 (C) and Rad51 (D) foci found in wild-type (blue bars) and <i>Arf</i>-null (red bars) spermatocytes analyzed in panels C and D.</p

<i>Arf</i> deficiency provokes elevated and persistent γ-H2AX foci.

<p>(A) Testis sections from 3 month-old mice of the indicated genotypes were immunostained for γ-H2AX, as visualized here at low magnification to demonstrate overall relative intensities of staining. The brightest γ-H2AX-positive cells are primary spermatocytes. (B) Immunoblotting analysis of whole testis lysates from 3 month-old mice showing an accumulation of γ-H2AX in <i>Arf</i>-deficient testis. (C) Surface spread spermatocytes from three month old mice were immunostained for SYCP3 (red), γ-H2AX (green), and with DAPI to visualize nuclei (blue). Representative spreads from each stage of prophase I are shown for wild type (top) and <i>Arf</i>-null (bottom) strains, demonstrating persistence of autosome-associated γ-H2AX in pachytene spermatocytes from <i>Arf</i>-deficient mice. Staining of sex bodies persists throughout prophase in both genotypes. Images were captured with the same exposure times using a Zeiss Axioscope fluorescence microscope (A) or Intelligent Imaging Innovations Marianas spinning disc confocal microscope (C). Scale bars: (A) 100 µm.</p

<i>Arf</i>-null pachytene spermatocytes exhibit synaptic defects.

<p>(A–C) Staining of surface spread spermatocytes for SYCP3 marks the lateral elements of bivalents. (A) In wildtype pachytene spermatocytes, fully synapsed autosomal bivalents are observed, as judged by continuous SYCP3 staining along the axes. (B–C) Representative synaptic defects in <i>Arf</i>-null pachytene spermatocytes include (B) unsynapsed ends (arrow) and interstitial asynaptic “bubbles” (arrow heads), and (C) asynapsis of the X and Y chromosomes (arrow). Synapsed X and Y chromosomes are shown in (A, B). Synaptonemal complexes with segmental disruption of SYCP3 staining were more frequently observed (p = 0.051) in <i>Arf</i>-null cells (C, arrowheads) versus their wild-type counterparts (191 versus 53 such segments in 300 pachytene chromosomal spreads from each genotype). (D) Quantification of synaptic defects observed in wild-type and <i>Arf</i>-null pachytene spermatocytes. “Wide open” ends are illustrated in (B); less extensive asynapsis at telomeres was categorized as “slight” open ends. 100 pachytene spermatocytes from each of three mice of different genotypes yielded highly significant differences in overall defects (p = 0.0017 by Student's t-test). Error bars indicate standard deviation from the mean.</p

<i>Arf</i> deficiency leads to increased apoptosis of primary spermatocytes.

<p>(A) During spermatogenesis, germ cells move intraluminally as they differentiate, initially yielding primary meiotic spermatocytes (immunostained for Dmc1, green) that have detached from the basement membrane (represented by a dashed line). Dmc1-positive cells are visualized in a longitudinal section of a tubule from a two month-old <i>Arf</i>-null mouse. Spermatocytes within the tubular lumen are TUNEL-positive (red). DAPI (blue) marks the nuclei of all developing germ cells within the tubule. Neither DAPI-positive spermatogonia residing on the basement membrane nor more mature intraluminal cells express Dmc1 or are TUNEL-positive. (B–D) Surface spread spermatocytes from three month old wild-type (B), <i>Arf^−/−</i> (C), and <i>p53</i>^−/−;<i> Arf</i>^−/− (D) mice were immunostained for SYCP3 (green) and assayed for TUNEL (red). Although pachytene and diplotene spermatocytes from <i>Arf^−/−</i> mice were TUNEL-positive, few such cells were identified in spreads from age-matched wildtype or <i>p53^−/−</i>;<i> Arf^−/−</i> mice. (E) TUNEL assays were performed on testis sections from 2–3 month old mice of the indicated genotypes. The total numbers of TUNEL-positive cells were enumerated in 50 tubules within three different sections taken from 3 different mice of each genotype. (F) One hundred surface spread spermatocytes (n = 3 mice) were categorized into the four phases of prophase I based on SYCP3 and γ-H2AX immunostaining patterns, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002157#s4" target="_blank">Materials and Methods</a>. Errors bars represent standard deviations from the mean. ** p<0.0001 and *p<0.003 vs wild-type levels by Student's t-test.</p

<i>Arf</i> deficiency leads to increased γ-H2AX foci in spermatogonia.

<p>The number of A and intermediate-type spermatogonia with γ-H2AX foci in 150 seminiferous tubules and the total number of foci contained within these cells were quantified in wild-type and <i>Arf</i>-null mice at P17. Enumeration of γ-H2AX foci within 150 tubules in each of three different sections from each genotype was performed at high magnification for foci limited to spermatogonia distinguished by morphology and position along the basement membrane. P values were determined using a Student's t- test; **p value<0.0001 vs wild-type. Error bars indicate standard deviations from the mean.</p

Loss of expression of p19^Arf in <i>Arf</i>^Cre/Flox testes.

<p>Immunoblotting analysis was performed using an antibody against p19^Arf. Whole testis lysates were prepared from two month old mice of the indicated genotypes. Immunoblotting with an antibody to actin was used to control for equal protein loading per lane.</p

Increased frequency of BrdU-incorporating spermatogonia in <i>Ink4a</i>-null mice.

<p>Quantification of BrdU-positive spermatogonia in five month-old wild-type, <i>Arf</i>-null, <i>Ink4</i>a-null, and doubly <i>Ink4a</i> and <i>Arf</i>-null mice was determined two hours after intraperitoneal BrdU administration. BrdU-labeled cells were scored in 100 tubules in testis sections from seven different mice. Error bars indicate standard deviations from the mean. ** p<0.0001 vs wild-type by Student's t-test.</p

Arf protein expression in mitotically dividing spermatogonia.

<p>Protein expression in sections of seminiferous tubules were determined by immunofluorescence analysis. (A) p19^Arf (green, left panel) is expressed in spermatogonia that intervene between Sox9-expressing Sertoli cells (red, middle panel) in the seminiferous tubules of adult 4 month-old mice. The right panel shows merged images documenting no overlap in expression of the two proteins. Unlabeled cells within the lumina of the tubules are visualized with DAPI. (B) After a 2 hour <i>in vivo</i> pulse of bromodeoxyuridine (BrdU) in adult 5 month old mice, p19^Arf expression (green, left panel) was revealed in spermatogonia that had incorporated BrdU (red, middle panel). The right panel shows merged images documenting co-expression of both markers in many spermatogonia at the tubular periphery (yellow). (C) In seminiferous tubules from P12 mice, cells expressing p19^Arf (green, left panel) co-express cyclin D1 (red, middle panel), a protein expressed in actively cycling spermatogonia; a merged image is shown at the right. (D, E) Spermatogonia within the seminiferous tubules of P15 mice express p19^Arf (green) during mitosis. Examples of p19^Arf expression during metaphase (D) and telophase (E) are shown. DAPI (blue) highlights the nuclei of intratubular germ cells and somatic Leydig cells that occupy the intertubular space. Scale bars: (A, D, E) 50 µm; (B, C) 100 µm.</p