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. 2014 Apr;20(4):483-95.
doi: 10.1261/rna.043729.113. Epub 2014 Feb 19.

An atlas of chromatoid body components

Oliver Meikar, Vasily V Vagin, Frédéric Chalmel, Karin Sõstar, Aurélie Lardenois, Molly Hammell, Ying Jin, Matteo Da Ros, Kaja A Wasik, Jorma Toppari, Gregory J Hannon, Noora Kotaja

An atlas of chromatoid body components

Oliver Meikar et al. RNA. 2014 Apr.

Abstract

The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells.

Keywords: chromatoid body; male germ cells; piRNA; post-transcriptional; ribonucleoprotein granule.

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Figures

FIGURE 1.
FIGURE 1.
CBs accumulate RNA. (A) Pieces of the seminiferous tubules (stage II–V) were incubated in the absence (Control) or presence of 5-ethynyl uridine (EU) to detect the newly synthesized, EU-labeled RNA (green). RNA accumulated in the nuclei and in the CBs (arrows). The CB localization was confirmed by immunostaining with anti-MVH antibody (red) and phase contrast microscopy. The nuclei were stained blue with DAPI. Scale bar, 10 µm. (B) EU-containing RNA retained inside the CB for at least 12 h after the removal of EU from the culture media. (C) The general CB RNA profile. CB RNA was separated by agarose and polyacrylamide gel electrophoresis and stained by SYBR Gold. The CB RNA can be grouped by size into ∼30 nt (small), ∼50–300 nt (medium), and >300 nt (large) RNAs. (D) Small RNAs in the CB mainly correspond to pachytene piRNAs. piRNAs derived from the pachytene piRNA clusters were enriched in the CB compared with the round spermatids (RS).
FIGURE 2.
FIGURE 2.
Proteomic analysis of the CB. (A) The main CB components MVH/DDX4, MIWI/ PIWIL1, TDRD6, TDRD7, GRTH/ DDX25, PABP, HSP72/HSPA2, DDX3L, and MILI/PIWIL2 are presented as the mole percentage of the total CB protein content. (B) Pie chart of the most represented domains in the 88 CB proteins. (C) Selection of the main Gene Ontology (GO) terms of the molecular functions, biological processes, and cellular components of the 88 CB proteins. The numbers on the bars represent the CB-protein hits in the corresponding GO term. (FDR) False discovery rate of a given GO term.
FIGURE 3.
FIGURE 3.
CB long RNA analysis. (A) CB RNA reads mapped to five main categories: exons, introns, structural RNA, transposable elements (TE), and other intergenic regions. The majority of the reads were from the exonic regions of the genes. (CB-IP) Isolated CBs; (LYS) total testis lysate; (RS) round spermatid lysate. (B) Transcript abundances in the CB vs. the RS. (Gene) mRNAs; (TE) transposable elements; (rRNA) ribosomal RNAs; (snoRNA) small nucleolar RNAs; (lincRNAs) annotated long intergenic RNAs. (C) Transcripts from pachytene piRNA clusters were enriched during the CB isolation procedure. (LYS) Whole testis lysate after sonication, (SUP) supernatant fraction after centrifugation, (PEL) CB-containing pellet fraction after centrifugation, (PF) pellet fraction after additional filtration, (CB) immunoprecipitation of the PF fraction using anti-MVH antibody. (D) Precursors of the pachytene piRNA clusters were specifically enriched in the CB compared with the round spermatid transcriptome (RS). (E) Correlation between the abundance of long RNA transcripts and small RNAs in the CB.
FIGURE 4.
FIGURE 4.
Novel long noncoding RNAs were enriched in the CB. (A) Distribution of the CB-enriched noncoding transcript genes on mouse chromosomes. (B) Relative expression levels of five novel transcripts in different mouse tissues as detected by RT-qPCR. (C) Expression levels of four transcripts were compared in the spermatocytes (Spc), round spermatids (RS), and elongating spermatids (ES). Note that two of the transcripts were mainly expressed in the round spermatids, whereas the other two transcripts have higher expression in the spermatocytes.
FIGURE 5.
FIGURE 5.
CBs accumulate proteins involved in mRNA maturation and regulation. (A) The CB proteins that have known functions in mRNA maturation, processing, splicing, and mRNA binding are listed in the table. (B) Confocal fluorescence microscopy to show the localization of the NMD components UPF, phosphorylated UPF1, SMG1, and SMG6 (red) in round spermatids. Costaining with DDX25 (green) confirmed their localization in the CB. DDX25 has earlier been reported as a CB component (Sato et al. 2010) and we further validated its CB localization by coimmunostaining with anti-MVH antibody (Supplemental Fig. S5B). Nuclei are stained with DAPI (blue). White arrows point to some selected CBs. Scale bar, 10 µm.
FIGURE 6.
FIGURE 6.
Expression profiling of the mRNAs localized in the CBs. (A) The round spermatid (rSpt+) mRNAs that were found in the CB at high levels (CB-High+) or at low levels (CB-Low+), or were not found in the CB (CB−) were compared with the list of genes shown to be differentially expressed in the somatic cells of the testis (SO), mitotic spermatogonia (MI), meiotic spermatocytes (ME), or post-meiotic spermatids (PM) (Chalmel et al. 2007). Not-DET represents the group of genes that are expressed in round spermatids but are not differentially expressed in the testis. The table summarizes the intersections with the corresponding P-values (hypergeometric). The number of total nonredundant gene IDs for each category is provided in parentheses. (B) Pie charts representing the distribution of the CB-High+ and CB− genes from the previous table.
FIGURE 7.
FIGURE 7.
GO term enrichment analysis. Forty-two significantly enriched GO terms are listed, followed by the total number of genes associated with the term. The genes expressed in the round spermatids (rSpt+) were divided into those detectable in high abundance in the CB (CB-High+) or undetectable in the CB (CB−). These classes were further divided into the clusters of differentially expressed genes during spermatogenesis with peak expression during the meiosis (ME) or post-meiotic phases (PM), or not differentially expressed during spermatogenesis (Not-DET). For each cluster of genes, the numbers of genes observed vs. expected by chance for each GO term are indicated. The total number of genes associated with a biological process term and the genes in the classes are shown at the top of the column. A color scale of P-values for enriched (red) and depleted (blue) terms is shown at the bottom. To allow a direct comparison of the CB-High+ and CB− classes, the enrichment calculations were computed by considering only the genes expressed in rSpt, instead of all the genes in the genome.
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References

    1. Aravin A, Gaidatzis D, Pfeffer S, Lagos-Quintana M, Landgraf P, Iovino N, Morris P, Brownstein MJ, Kuramochi-Miyagawa S, Nakano T, et al. 2006. A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442: 203–207 - PubMed
    1. Aravin AA, van der Heijden GW, Castaneda J, Vagin VV, Hannon GJ, Bortvin A 2009. Cytoplasmic compartmentalization of the fetal piRNA pathway in mice. PLoS Genet 5: e1000764. - PMC - PubMed
    1. Armour CD, Castle JC, Chen R, Babak T, Loerch P, Jackson S, Shah JK, Dey J, Rohl CA, Johnson JM, et al. 2009. Digital transcriptome profiling using selective hexamer priming for cDNA synthesis. Nat Methods 6: 647–649 - PubMed
    1. Barchi M, Geremia R, Magliozzi R, Bianchi E 2009. Isolation and analyses of enriched populations of male mouse germ cells by sedimentation velocity: The centrifugal elutriation. Methods Mol Biol 558: 299–321 - PubMed
    1. Bono F, Gehring NH 2011. Assembly, disassembly and recycling: The dynamics of exon junction complexes. RNA Biol 8: 24–29 - PMC - PubMed

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