Translational repression and expression of high confidence targets.

<p>(A) Ratio of polysome to mRNP occupancy across genotypes as compared to wildtype demonstrating substantially higher polysome occupancy in compound heterozygotes when compared to single heterozygotes. Data represented as average across biological duplicates. (B) Expression of transcripts with altered mRNP and polysome occupancy compared across genotypes demonstrating minimal change in overall expression demonstrating altered polysome occupancy is not driven by differences in total expression. (C) Cell-type expression of high confidence targets demonstrating enriched expression in YBX2 and 3 positive cell types. Expression—log₂(average TPM). Black dot—median value. (D) Polysome to mRNP ratio of transcripts relevant to spermatogenesis showing increased polysome occupancy in compound heterozygotes relative to wildtype. Note the only transcript with a well-characterized YRS is <i>Spata18</i>. Data is represented as the average polysome to mRNP ratio ± SD. (E) Western blot detection of MAEL, NSUN2, and GAPDH in whole adult testis protein lysates from wildtype, single heterozygote, and compound heterozygous mutants demonstrating increase MAEL and NSUN2, but not GAPDH, abundance in the compound heterozygous mutant testis relative to wildtype.</p

Reproductive parameters of compound heterozygous mutant mice.

<p>(A) Body weight and (B) testis weight remained unchanged, while (C) epididymal sperm count is significantly decreased in compound heterozygous relative to single heterozygous (<i>Ybx2</i>^+/- or <i>Ybx3</i> ^+/-) or wildtype mice. Values are mean ± SD, N = 3, * indicates p < 0.05. Fertility assessed by (D) embryos produced from mating with C3H/HeJ fertile females and sperm function quantified by (E) motile sperm and (F) progressive sperm motility assessed by computer-assisted sperm analysis demonstrating defects in sperm motility in compound heterozygotes. The data are expressed as percentage of motile sperm ± SD. ** p < 0.01 relative to wildtype sperm. N = 3. (G) 2-cell embryos derived from <i>in vitro</i> fertilization (IVF) of oocytes from superovulated C3H/HeJ females demonstrating infertility in compound but not single heterozygous males. The data are expressed as number of embryos ± SD per dam or percentage ± SD of 2-cell embryos. N = 3, *** p < 0.001.</p

Testis transcript mRNP and polysome occupancy in single and compound heterozygotes.

<p>(A) Comparison of mutant versus wildtype mRNP or polysome abundance of expressed transcripts demonstrating global decreases in mRNP abundance and global increases in polysome abundance in compound heterozygotes but not single heterozygotes relative to wildtype. Data represented as mRNP or polysome abundance normalized by total RNA abundance and averaged across biological replicates. Red points—transcripts with significant differences between wildtype and mutant (t-test, N = 2, p-value < 0.05) within either the mRNP or polysome fractions. Yellow points—transcripts with significant differences between wildtype and mutant in both mRNP and polysome fractions. (B) Intersect of transcripts with differential mRNP or polysome occupancy in the three mutant models demonstrating limited overlap between the single and compound heterozygotes. (C) Intersect of transcripts with differential mRNP and polysome occupancy in compound heterozygotes. Transcripts within the overlap represent high confidence targets for abnormal translation repression.</p

Morphological analysis of spermatogenesis and sperm chromatin condensation in compound heterozygous mutant mice.

<p>(A) Periodic acid–Schiff–stained sections of adult testis shows tubule cross-sections containing abnormal elongating spermatids (asterisks). Step 9 spermatids demonstrate defective elongation and abnormal condensation within tubule cross-sections (arrowheads). (B) The presence of single stranded DNA indicative of DNA damage was evaluated by sperm acridine orange staining. In compound heterozygous mutant sperm acridine orange fluoresced in a manner consistent with single-stranded DNA. Quantification of acridine orange–stained sperm demonstrating increases in compound heterozygous relative to wildtype sperm. Values expressed as a percentage ± SD. N = 3. p = 0.052. (C). Western blot analysis confirming the presence of PRM2* in compound heterozygous mutant sperm, while mature PRM2 levels were decreased (Lane 4). A representative blot is shown (N = 3). PRM1 levels remained unchanged (Lower panel). (D) Quantitation of the PRM2*/PRM2 ratio by densitometric scan of the PRM2 western blot revealed a significant increase in PRM2*/PRM2 ratio in compound heterozygous mutant relative to wildtype or <i>Ybx2</i>^+/- or <i>Ybx3</i>^+/- mutants. p< 0.05, N = 3.</p

YRS-independent regulation in compound heterozygote testes.

<p>(A) RNA-sequencing derived polysome to mRNP ratio of known YRS-containing transcripts across wildtype and heterozygote testes demonstrating no impact on polysome occupancy in compound heterozygotes. Data is represented as the average polysome to mRNP ratio ± SD. (B) Stage specific expression of PRM1 and (C) PRM2 in elongating spermatids (arrowhead) in wildtype and compound heterozygous testis. <i>Prm1</i> and <i>Prm2</i> mRNAs are not prematurely translated in compound heterozygous mutant mice as shown by the absence of PRM1 staining in stage IX (step 9 elongating spermatids). Scale bar = 100 μm. egs: elongating spermatid. (D) Significance of degenerate YRS motifs identified in the 3’ UTR of transcripts with altered mRNP abundance across genotypes. Comparison with known YRS-containing UTRs demonstrates substantial under-representation of high confidence YRSs in mis-regulated transcripts.</p

Histology comparing WT and GCKO seminiferous tubules and epididymides at 5, 8 and 10 weeks.

<p>(A) In comparison to WT seminiferous tubules at 5 weeks, GCKO sections show tubules with increased lumen diameters and few elongating and elongated spermatids (*). By 8 weeks, cross sections from GCKO testes show prominent pynotic cells (arrows) and reduced numbers of elongating and elongated spermatids. By 10 weeks, cross sections of GCKO testes show further enlargement of tubule lumens and absence of elongating spermatids in the majority of tubules. (B) Epididymides of WT mice at 5 weeks and GCKO mice at 5, 8 and 10 weeks showing reduced sperm numbers in epididymides of knockout mice by 5 weeks and the absence of mature spermatozoa in GCKO epididymides at the 8 and 10 week timepoints.</p

Number of miRNAs and mRNAs up- or down-regulated for each chromosome in GCKO testes on P18. Data for miRNA and mRNA are expressed on a per chromosome basis and reported for those probe sets with intensities that could be scored as increased, decreased or no change in GCKO when compared to WT testes.

<p>Number of miRNAs and mRNAs up- or down-regulated for each chromosome in GCKO testes on P18. Data for miRNA and mRNA are expressed on a per chromosome basis and reported for those probe sets with intensities that could be scored as increased, decreased or no change in GCKO when compared to WT testes.</p

Meiotic spreads show deletion of <i>Dicer1</i> disrupts progression of meiosis I.

<p>(A) It was possible to identify the 5 sub-stages of meiosis I by combining the reactivity patterns of two antibodies against synaptonemal complex protein 3 (SYCP3) and γH2AX. SYCP3 is a protein essential for synapsis of homologous chromosomes and γH2AX localizes to double-strand breaks and XY bodies during meiosis. A total of 200 spreads were counted in testis cell preparations from WT and GCKO mice at P22. (B) Chi square analysis shows GCKO testes contained significantly higher numbers of germ cell spreads at the leptotene and zygotene stages of meiosis I and fewer spreads at pachytene, diplotene and metaphase I stages (<i>P</i><0.001), suggesting that the loss of <i>Dicer1</i> lead to disruptions in progression through meiosis I.</p

Levels of <i>Dicer1</i> transcripts and DICER1 protein in GCKO testis samples are significantly reduced by P18.

<p>(A) In comparison to WT testes, real-time qRT-PCR shows a 6-fold reduction in <i>Dicer1</i> RNase III endonuclease transcripts in GCKO testes by P18 (*<i>P</i>  = 0.018). (B) Western blot analysis using rabbit antibody against the N-terminal helicase domain of DICER1 and HRP-conjugated goat anti-rabbit IgG antibody shows reduced protein expression in GCKO testes by P18. The arrows point to the 217 kDa DICER1 protein and to the equivalent loading of 50 kDa protein from WT and GCKO testes. (C) Immunofluorescence detection of DICER1 protein in WT and GCKO testes at P18 using the same antibody used for western blotting. In WT sections, DICER1 localizes to the cytoplasm of most cell types populating the P18 testis, including Sertoli cells, spermatogonia and spermatocytes. At this developmental time point, secondary spermatocytes and spermatids are not present. In the P18 GCKO testis sections, DICER1 appears primarily in the cytoplasm of Sertoli cells located near the basement membrane with scant detection of DICER1 protein in the cytoplasm of spermatogonia and pachytene spermatocytes (magnification  = 40x). (D) Cross sections of WT and GCKO testes on P18 stained with HE show no major differences in cellular composition suggesting that the changes in in <i>Dicer1</i> transcript and protein levels in the GCKO testes were not explained by differences in cell populations.</p

Failure of meiotic sex chromosome inactivation (MSCI) in P18 <i>Dicer1</i> GCKO testes.

<p>A) Pie charts show the number of genes that are normally silenced during MSCI that were detected by array overlaid with the percentages of X- and Y-genes in GCKO testes with expression levels that were either significantly up-regulated (83.8% and 100%, respectively) or not changed (16.2 and 0%, respectively. B) Bar graph showing the number of overexpressed (>1.5 RFC) X- and Y-genes with or without binding sites for miRNAs shown to be deregulated in P18 GCKO testes. The majority of overexpressed genes (102/123 = 82.9%) contain recognition sites for deregulated miRNAs.</p