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. 2014 Jan 18;15:39.
doi: 10.1186/1471-2164-15-39.

Integrated Transcriptome Analysis of Mouse Spermatogenesis

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Free PMC article

Integrated Transcriptome Analysis of Mouse Spermatogenesis

Gennady Margolin et al. BMC Genomics. .
Free PMC article

Abstract

Background: Differentiation of primordial germ cells into mature spermatozoa proceeds through multiple stages, one of the most important of which is meiosis. Meiotic recombination is in turn a key part of meiosis. To achieve the highly specialized and diverse functions necessary for the successful completion of meiosis and the generation of spermatozoa thousands of genes are coordinately regulated through spermatogenesis. A complete and unbiased characterization of the transcriptome dynamics of spermatogenesis is, however, still lacking.

Results: In order to characterize gene expression during spermatogenesis we sequenced eight mRNA samples from testes of juvenile mice from 6 to 38 days post partum. Using gene expression clustering we defined over 1,000 novel meiotically-expressed genes. We also developed a computational de-convolution approach and used it to estimate cell type-specific gene expression in pre-meiotic, meiotic and post-meiotic cells. In addition, we detected 13,000 novel alternative splicing events around 40% of which preserve an open reading frame, and found experimental support for 159 computational gene predictions. A comparison of RNA polymerase II (Pol II) ChIP-Seq signals with RNA-Seq coverage shows that gene expression correlates well with Pol II signals, both at promoters and along the gene body. However, we observe numerous instances of non-canonical promoter usage, as well as intergenic Pol II peaks that potentially delineate unannotated promoters, enhancers or small RNA clusters.

Conclusions: Here we provide a comprehensive analysis of gene expression throughout mouse meiosis and spermatogenesis. Importantly, we find over a thousand of novel meiotic genes and over 5,000 novel potentially coding isoforms. These data should be a valuable resource for future studies of meiosis and spermatogenesis in mammals.

Figures

Figure 1
Figure 1
A time course of spermatogenesis. Primordial germ cells (PGC’s) are the germline progenitors. They give rise to various types of spermatogonia, which proliferate and differentiate into spermatocytes. Meiosis and genetic recombination occur in spermatocytes, which become haploid spermatids after the completion of meiosis. Spermatids undergo the process of spermiogenesis until their maturity, which includes elongation, genome condensation and formation of a flagellum. The approximate timeline of the first wave of spermatogenesis in mice, in days after birth (dpp) is indicated. The figure is adopted from [45] by the authors holding the copyright.
Figure 2
Figure 2
Temporal gene expression clustering. Genes with maximal expression above 2 RPKM, at least two-fold expression change and a mature transcript length ≥100b were clustered. Given these criteria, 12,895 genes were selected for clustering into 8 clusters. The cluster sizes are 1313, 3227, 2826, 1302, 1087, 900, 895 and 1345 for clusters from 1 through 8, respectively. The heatmap (A) and normalized expression profiles of cluster centroids (B) are shown. As clusters 3, 4, 6 and 7 show a rising profile during meiosis, 8 to 20dpp, these clusters were designated as meiotic clusters.
Figure 3
Figure 3
Schematics of the deconvolution algorithm to estimate cell type-specific gene expression. We have measured gene expression by dpp (S), and have estimates of cell type fractions by dpp from the literature (F). Our goal is to estimate gene expression by cell type (C), as well as to re-estimate cell type fractions, or cell type contributions to gene expression (F). The iterative procedure is depicted on the figure, and the details are in Materials and Methods. Due to both biological and mathematical considerations, 5 combined cell types were considered in our analysis.
Figure 4
Figure 4
Results of the deconvolution algorithm to estimate cell type-specific gene expression. (Left) Cell type fractions used in the first iteration (A; based on Bellve et al. [25]) and obtained after 10 iterations (B). (C) Cell type-expression heatmap of genes selected for deconvolution, obtained after 10 iterations after genes were clustered in 5 clusters, corresponding to the cell types considered. Somatic expression (cell type A) is more ubiquitous, and cell types D and E share many expressed genes.
Figure 5
Figure 5
Gene and isoform predictions. (Top) Novel alternative splicing of transcription elongation factor Supt5h differentially expressed in spermatogenesis. We see an ORF-preserving exon skipping event (A) that is frequently detected in samples after 12 dpp (B). (C) Predicted transcripts (variant 1 and variant 2 differ only by one retained intron). This prediction is based on our RNA-Seq data in combination with UCSC and Ensembl annotations and Genscan computational gene predictions. Only the protein coding part of the predicted transcripts is shown. The respective lengths of the predicted proteins are 566aa and 589aa. Both of these variants have a HMG box, which is a DNA-binding domain, towards their C-terminus, which is completely missed by the UCSC and Ensembl gene models. Expression at this locus peaks sharply at 12dpp. Note that there are a few differences in the boundaries of some exons between the predicted transcripts and the known annotation, which are invisible here.
Figure 6
Figure 6
Detection of polyadenylation. The proportion of the polyadenylation sites perfectly matching the annotated 3′ ends of known genes is shown in the center of the diagram. Subsequent rings going outwards show the percentage of tentative polyadenylation sites successively further away from the known 3′ ends. The percentages without brackets are based on the total of 5,229 sites found with zero mismatches, while bracketed percentage values correspond to the total of 6,801 sites allowing up to one mismatch – see text for details.
Figure 7
Figure 7
The X chromosome is transcriptionally silenced in spermatogenesis as a result of Meiotic Sex Chromosome Inactivation. (A) The average expression of X-linked genes decreases after 12dpp, and eventually drops by a factor of ~3 in adult testes. (B), (C) Heatmaps of X-linked genes. Only genes selected for temporal clustering are shown (B). The drop in gene expression, relative to genome-wide expression, is evident starting at around 16dpp, the pachytene stage. In the adult testes, at 38dpp, many genes that have not been active during the earlier stages of the first wave of spermatogenesis are turned on. This is also evident from the deconvolution calculations (C).
Figure 8
Figure 8
Example of mRNA-Seq and Pol II ChIP-Seq coverage in a 164 kb genomic window on chromosome 8 (chr8:97,939,000-98,103,500). mRNA expression is high in blue and absent in grey. There is Pol II accumulation at promoters of genes, and an elevated Pol II signal can be seen along gene bodies as well. Here, some genes are expressed throughout the whole time course, while others are initially virtually silent. Consistent with these expression profiles, the presence of Pol II at corresponding promoters occurs at different time points.

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