The translationally active <i>Prm2</i> mRNA associates with the p42 CBF-A isoform.

<p>(<b>A–B</b>) Fractionation of adult mouse testicular lysates on a 15–50% continuous sucrose gradient in the absence (A) or presence (B) of EDTA. Fractions were analyzed on Northern blots for the <i>Prm2</i> mRNA and on immunoblots for CBF-A (SAK22) and hnRNP A2. (<b>C–D</b>) The <i>Prm2</i> mRNA is translationally inhibited in postnatal day −28 and −30 mouse testis. (<b>C</b>) Developmental expression of <i>Prm2</i> mRNA in mouse testes. Total RNA was collected from mouse testis of 20, 28, 32 postnatal day of age (20–32 d), and analyzed by RT-PCR. RT, reverse transcriptase. (<b>D</b>) Developmental expression of PRM2 and CBF-A in mouse testes. Cytoplasmic testicular extracts from 20, 28, 30, 32 d mice were analyzed on immunoblots for PRM2, CBF-A (SAK22) and tubulin. (<b>E</b>) RIPs from adult and 30 d mouse testicular extracts. Cytoplasmic fractions were incubated with SAK22, ICCI or control anti-mouse IgGs. Immunoprecipitated fractions were analyzed on immunoblots for CBF-A (SAK22) and hnRNP A2, and by RT-PCR with primers amplifying the <i>Prm2</i>, α-tubulin or clusterin cDNAs. (<b>F–G</b>) The CBF-A p42 isoform associates with the 5′ mRNA cap complex. Cytoplasmic fraction from adult mouse testis was incubated with 7-methyl-GTP (m⁷GTP)-Sepharose or protein G-Sepharose (control). Bound proteins were analyzed on immunoblots for CBF-A (SAK22), hnRNP A2 and eIF4E and α-tubulin. (F) Lanes 1, 2 respectively 2% and 1% input. (G) Lane 1 and 2 are 2% input. Where indicated, testis lysates were pre-incubated with RNase A prior to incubation with the beads.</p

The <i>Prm2</i> mRNA expression is regulated by CBF-A at the translational level during spermatogenesis.

<p>(<b>A</b>) Immunoblots of CBF-A (Sak22), PRM2 and tubulin on Hnrnpab^+/− and Hnrnpab^−/− testis lysates. Total proteins were stained with Coomassie brilliant blue and shown as loading control. Densitometric analysis of the signals in the immunoblots was performed on 3 individual animals per genotypes. Data represent mean +/−SE ^*<i>p</i><0.05 student t-test. (<b>B</b>) Immunostaining for CBF-A on Hnrnpab^+/− and Hnrnpab^−/− testis sections. Bar, 50 µm. (<b>C</b>) Northern blotting analysis for the <i>Prm2</i> mRNA on total RNA from Hnrnpab^+/− and Hnrnpab^−/− mouse testis. Densitometric analysis was on 3 individual animals of each genotype, and shown as mean +/− SE. (<b>D</b>) <i>In situ</i> hybridization on Hnrnpab^−/− and Hnrnpab^+/− testis sections showed no differences on <i>Prm2</i> mRNA expression. Bar, 50 µm. The inset shows a magnified image of the marked area. (<b>E</b>) Fractionation of testicular lysates from adult Hnrnpab^+/+ and Hnrnpab^−/− mice on a 15–50% continuous sucrose gradient. Fractions were analyzed on Northern blots for the <i>Prm2</i> mRNA. (<b>F</b>) Testis section of Hnrnpab^+/− stained with a PRM2 antibody (Green) and DAPI (gray). PRM2 was not detected in the seminiferous tubules of stage IX–XII, in which pachytene spermatocytes (P) and elongating spermatids (EL) are contained. (<b>G</b>) Testis section from Hnrnpab^−/− stained with a PRM2 antibody (Green) and DAPI (gray). PRM2 signal was detected in some nuclei of elongating spermatids. (<b>H and I</b>) Magnified image of marked area in (G). DAPI staining shows that chromatin of PRM2-positive nuclei (white arrow head) appears more condensed than in PRM2-negative nuclei (blank arrow head). (<b>J</b>) Testis section of Hnrnpab^−/− stained with a PRM2 antibody (Green) and DAPI (gray). The seminiferous tubule was categorized as stage IX–XII, because it does not contain round-spermatid layer (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858.s007" target="_blank">Figure S7</a>), but nuclei of elongating spermatids appear prematurely condensed and positive for PRM2 signals. * denotes background staining of Leydig cells with the PRM2 antibody. Bars, 50 µm.</p

Speculative model for the role of CBF-A in translation regulation of the <i>Prm2</i> mRNA.

<p>We hypothesize that in early-step spermatids, p37 mainly binds to the RTS in the <i>Prm2</i> mRNA 3′UTR, leading to a translationally repressed configuration of the <i>Prm2</i> mRNA. In the cytoplasm of later step spermatids, p37 is released from the RTS through yet unknown mechanisms, and p42 is able to interact with the RTS and with the 5′ mRNA cap binding complex. This exchange may be regulated through cooperative association with hnRNP A2. We propose that the p37–p42 relay contributes to the temporal regulation of the <i>Prm2</i> mRNA translation during spermatogenesis.</p

CBF-A is in cytoplasmic RNPs in complex with the <i>Prm2</i> mRNA in testicular cells.

<p>(<b>A</b>) Immunoblots of nuclear and cytoplasmic fractions of testis lysates. Anti-fibrillarin (nucleolar protein) and anti-Tom20 (mitochondrial protein) antibodies were used to characterize the fractionation. (<b>B</b>) Untreated or RNase-treated cytoplasmic fractions of testis lysates were incubated with anti-CBF-A antibodies (SAK22 or ICCI) or control non-specific IgGs and the immunoprecipitates were probed as indicated. * marks a testis-specific variant of hnRNP A2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858-Kamma1" target="_blank">[56]</a>. (<b>C</b>) RNA immunoprecipitation (RIP) assays. Cytoplasmic fractions of testis lysates were incubated with anti-CBF-A antibodies (SAK22 or ICCI), control anti-mouse IgGs, or without antibodies (mock). In all cases, total RNA was extracted from the immunoprecipitates and analyzed by RT-PCR with primers amplifying <i>Prm2</i>, α-tubulin or clusterin cDNA. (<b>D</b>) Densitometric quantifications of the RIP experiments. The signal intensities of the RT-PCR bands were calculated from 3 independent experiments and shown as % of input (mean+/−SE).</p

Transmission electron micrographs of sperm heads from the epididymis from Hnrnpab^−/− and Hnrnpab^+/− mice.

<p>(A) Normal sperm from Hnrnpab^−/− mouse showing the nucleus (n) revealed like an evenly stained black mass (due to high electron density) that contains the highly compacted DNA, surrounded by the nuclear envelope. The acrosome (a) located in the anterior half of the head and the proximal centriole (pc) in the neck of the sperm. (B) Abnormal sperm of Hnrnpab^−/− mice with fibers extending out of the main mass of local regions of nucleus (inset in B and B′). (C) Abnormal sperm of Hnrnpab^−/− mice with fibers extending out of the main mass around the nucleus (inset in C and C′). (D) Elongated spermatid at step 13 of spermatogenesis of wild-type mice. Posterior part of DNA, which is less packed than in anterior region, was observed as fibers (inbox in D and D′). Scale bar is 2 µm in A–D and 500 nm in B′–D′.</p

Immunostaining on tubule squash preparations of testicular cells at different steps of spermatogenesis.

<p>Cells were triple-stained with the antisense probe for the <i>Prm2</i> mRNA (red), an anti-PRM2 antibody (green) and with the anti-MVH antibody (white) as well as DAPI. The <i>Prm2</i> mRNA was co-localized with MVH to chromatoid bodies as from step 7–8 spermatids. In late step spermatids (step 9–12 and step 13–15), the <i>Prm2</i> mRNA becomes more diffusely localized to the cytosol. Scale bars, 10 µm (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858.s003" target="_blank">Figure S3</a> for more examples).</p

<i>In vivo</i> localization of <i>Prm2</i> mRNA in adult mouse testes.

<p>(<b>A</b>) <i>In situ</i> hybridization on testis cryosections (10 µm) using a <i>Prm2</i> mRNA antisense probe. Nuclei were stained with DAPI and shown in blue. Roman numbers indicate stages of seminiferous epithelium cycle. (<b>B</b>) Diagram of the 12-stage growth cycle of mouse spermatogenesis <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858-Russell1" target="_blank">[55]</a>, showing the stages of the <i>Prm2</i> mRNA expression during spermatogenesis. The <i>Prm2</i> mRNA is detected from step 7 round spermatids to step 14 elongated spermatids (highlighted in magenta). (<b>C</b>) Subcellular localization of the <i>Prm2</i> mRNA in spermatids during spermatogenesis. Testis cryosections were double stained with the <i>Prm2</i> mRNA antisense probe (red, upper panels) and the anti-MVH antibody (white, lower panels). The <i>Prm2</i> mRNA was localized to perinuclear structures positive for the chromatoid body marker MVH in the spermatids at stage VII–VIII and dispersed into cytosol at later stages. Scale bar, A; 100 µm, C; 20 µm.</p

<i>In vivo</i> distribution of CBF-A in mouse testes.

<p>(<b>A–B</b>) Analysis of steady state expression levels of CBF-A on immunoblots of total (A) or cytoplasmic lysates (B) using the anti-CBF-A antibody SAK22. 20 µg proteins/lane. (<b>C</b>) Developmental expression profiles of CBF-A on immunoblots of mouse testis lysates prepared from juvenile mouse testes from days postpartum 8 (8 d) to 32 d, using the SAK22 antibody. AB Sg, AB type spermatogonia, Zg Sc, zygoten spermatocyte, Pachy Sc, Pachytene spermatocyte, Rs, Round spermatids, Es, Elongate spermatids. (<b>D</b>) Overview of mouse testes cryosections immunostained with the anti-CBF-A antibody (ICCI) and DAPI, and analyzed by confocal microscopy (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858.s001" target="_blank">Figure S1</a> for analysis with the SAK22 antibody). (<b>E–F</b>) Higher magnification images of mouse testes cryosections. (E) CBF-A is localized to nuclei of round spermatids (see arrowheads). In elongating spermatids CBF-A was preferentially found in the cytoplasm (see arrows). (F) Nuclei were stained with DAPI and shown in blue. (<b>G</b>) Overview of mouse testis cryosections subjected to antigen retrieval and immunostained using the anti-CBF-A antibody (ICCI) and DAPI. (<b>H–I</b>) Higher magnification images of a testis section stained with the ICCI antibody and DAPI. (H) The ICCI antibody labels nuclei of round spermatids as well as perinuclear compartments (see arrowheads). (<b>I</b>) Nuclei were stained with DAPI and shown in blue. (<b>J</b>) Immunostaining on squash preparations of testicular cells. The signals from the immunolabeling with the anti-CBF-A antibody (ICCI, green) and the anti-MVH antibody (magenta) were found to co-localize in chromatoid bodies of round spermatids (see arrowhead). Scale bars, D and G; 50 µm, H–J; 10 µm.</p

The RTS in the 3′ UTR of the <i>Prm2</i> mRNA is primarily targeted by p37.

<p>(<b>A</b>) Location and sequence of the <i>Prm2</i> mRNA RTS and control probes. For the pull-down assays all probes were biotinylated and individually coupled to streptavidin Sepharose. (<b>B–C</b>) <i>In vitro</i> RNA pull-down assays with recombinant (B) p37 and p42 or (C) p37 and hnRNP A2, incubated with wtRTS-Prm2 beads. As revealed on immunoblots with CBF-A (SAK22) or hnRNP A2 antibodies, all proteins were specifically co-precipitated with the beads. However, upon co-incubation with p37, precipitations of (B) p42 and (C) hnRNP A2 were significantly impaired. (<b>D</b>) p42 and hnRNP A2 are co-precipitated with wtRTS-Prm2 beads. (<b>E</b>) p37 specifically targets the RTS-containing <i>Prm2</i> mRNA 3′UTR. As indicated biotinylated oligonucleotides and <i>in vitro</i> transcribed full-length or Δ3′UTR <i>Prm2</i> mRNA were conjugated to Streptavidin beads and incubated with cytoplasmic testicular lysate. Bound proteins were analyzed by immunoblotting for CBF-A (SAK22 antibody) and hnRNP A2. Densitometric measurements of the bound CBF-A (<b>F</b>) and hnRNP A2 fractions (<b>G</b>) to biotinylated transcripts in panel (E) over three independent experiments are plotted as signal intensities from the respective immunoblots. The bar diagrams include standard deviations.</p