Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage

Abstract

Background: Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis.

Results: We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation.

Conclusions: This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals.

Keywords: Flow cytometry; RNAseq; Spermatogenesis; Transcriptome.

Fig. 1

Flow cytometric purification and immunocytochemical analysis of sorted testicular cell populations with VDG. Mice aging 10–11 dpp (a, b) and 24–25 dpp (c, d) were used. a, c. Dot plots depicting forward scatter (FSC-H) vs VDG fluorescence intensity and their corresponding histograms showing the gated cell populations. b, d. Confocal immunocytochemical analysis with anti-SYCP3 antibody (red) as a marker of the LZ (b) and PS (d) sorted fractions. Nuclei were counterstained with DAPI. Bars correspond to 10 μm

Fig. 2

Venn diagram showing all the expressed genes in 2C, LZ, PS, and RS cell populations. Separate and overlapping expression between samples are shown. Only transcripts with a level of expression of RPKM ≥ 2 were considered

Fig. 3

Representation of DEG between pairwise sample comparisons of the four populations in chronological order. The following comparisons were performed: 2C/LZ; LZ/PS; PS/RS (|FC| ≥ 2; Kal’s test p ≤ 0.01). a. Heat map of expression levels and hierarchical clustering for the global gene expression in the four samples. All genes detected as differential in at least one sample were included. Z-score values are coded on the green-to-red scale (high expression: red; low expression: green). b. Venn diagram of up-regulated and down-regulated genes. Separate and overlapping expression between samples is shown. c. Temporal expression profiles of DEG, ordered based on the p-value significance of the number of assigned vs expected genes. Only the 10 most significant profiles are shown. The p-value (bottom of each panel) and number of genes (below) for each profile are shown

Fig. 4

Enriched GO categories and differential expression of genes in early meiotic prophase (LZ). a. Enrichment analysis of biological process GO terms of up-regulated genes in 2C/LZ comparison. The fold enrichment shows the ratio of observed vs expected genes for each category, with an adjusted p-value <0.01. b. Venn diagram showing separate and overlapping expression between comparisons of the lists “2C/LZ up” (i.e. genes that are up-regulated in LZ compared to 2C) and “LZ/PS down” (i.e. genes that are down-regulated in PS compared to LZ). The intersection contains a subset of DEG whose expression peaks in early meiotic prophase. c. Graphical representation of the expression levels (RPKM) of the genes within the LZ peak in the four cell populations. The fifteen top genes are listed to the right in decreasing order according to their expression levels in LZ. d. Heat maps showing relative expression levels of the genes contained within GO categories “reciprocal meiotic recombination” (GO# 0007131) and “meiotic chromosome segregation” (GO# 0045132). High expression levels are indicated in red and low expression levels in green

Fig. 5

Enriched GO categories and differential expression of genes in PS and RS. a. Enriched biological process GO terms of up-regulated genes in the PS population compared to LZ. b. Enriched GO terms of up-regulated genes in RS compared to PS. c. Heat maps of the GO categories “sperm motility” (GO# 003017) and “sperm-egg recognition” (GO# 0035036). High expression levels are indicated in red and low expression levels in green

Fig. 6

Dynamic expression patterns of 13 selected genes representative of the different expression profiles. The analyses were carried out by RNAseq (CLC bio and edgeR) and qPCR. a. Expression profiles obtained by RNAseq analysis (CLC bio: orange; edgeR: blue) and qPCR analysis (yellow). Col1a1: collagen, type I, alpha 1; Sycp3: synaptonemal complex protein 3; Dazl: deleted in azoospermia-like; Tex15: testis expressed 15; Top2a: DNA topoisomerase II, alpha isozyme; Dnahc8: dynein, axonemal, heavy chain 8; Tcte3: T-complex-associated-testis-expressed 3; Atp8b3: ATPase, aminophospholipid transporter, class I, type 8B, member 3; Clgn; calmegin; Spa17: sperm autoantigenic protein 17; Ldhc: lactate dehydrogenase c; Tnp1: transition protein 1; Prm1: protamine 1. b. Correlation between the expression levels in RNAseq analysis (RPKM values of CLC bio analysis) and those obtained by qPCR analysis for the 13 selected genes in the four testicular cell populations

Fig. 7

Heat maps showing relative expression levels of X-linked protein-coding genes in the four cell populations. a. The genes were ordered according to their position on the chromosome from p to q. Chromosome bands are indicated to the left of the figure. b. Hierarchical clustering. High expression levels are indicated in red and low expression levels in green for both heat maps

Fig. 8

Representation of the transcription and execution times of four selected GO categories. The diagram represents the time when the biological processes shown in the heat maps in Figs. 4c and 5c are transcriptionally activated, and when these processes are executed along the first spermatogenic wave in mouse. The onset (in dpp) for the different stages along the first spermatogenic wave is denoted on top. The time of histone substitution - first by TNP and then by PRM - is also represented. PGC: primordial germ cells; Spg: spermatogonia; PL: preleptotene; L: leptotene; Z: zygotene; P: pachytene; D: diplotene; M: meiotic divisions