Transcriptomics of Meiosis in the Male Mouse

Abstract

Molecular studies of meiosis in mammals have been long relegated due to some intrinsic obstacles, namely the impossibility to reproduce the process in vitro, and the difficulty to obtain highly pure isolated cells of the different meiotic stages. In the recent years, some technical advances, from the improvement of flow cytometry sorting protocols to single-cell RNAseq, are enabling to profile the transcriptome and its fluctuations along the meiotic process. In this mini-review we will outline the diverse methodological approaches that have been employed, and some of the main findings that have started to arise from these studies. As for practical reasons most studies have been carried out in males, and mostly using mouse as a model, our focus will be on murine male meiosis, although also including specific comments about humans. Particularly, we will center on the controversy about gene expression during early meiotic prophase; the widespread existing gap between transcription and translation in meiotic cells; the expression patterns and potential roles of meiotic long non-coding RNAs; and the visualization of meiotic sex chromosome inactivation from the RNAseq perspective.

Keywords: MSCI; RNAseq; lncRNAs; meiosis; meiotic prophase; spermatogenesis; spermatogenic cell sorting; transcriptomics.

FIGURE 1

(A) Examples from reports showing a switch in gene expression patterns, and the expression of spermiogenesis-specific programs during mouse meiotic prophase. (a) Heat map representing the clustering of 790 differentially expressed sequences derived from a microarray study in testes of animals of increasing age, from 7 days post-partum (d7) to 17 days post-partum (d17). Columns represent the expression profile of all the sequences for the indicated post-natal ages, while rows represent expression of specific genes over time (low expression levels are shown in purple, and high expression levels in yellow). Note the overall change from d12 to d14, coinciding with pachytene onset. A tree representing clustering by resemblance of expression profiles across the indicated post-natal ages is shown below. The figure is reproduced from Waldman Ben-Asher et al. (2010), with permission from John Wiley and Sons (license number 5014730359883). (b) Heat map showing the relative expression levels of 5,281 microarray probe sets divided into 8 K-means clusters, in different spermatogenic cell populations purified by FACS. Each horizontal line corresponds to a probe set (high expression in yellow, low expression in blue). The switch in the transcriptome between L/Z and mid-P is evident. Reprinted from Fallahi et al. (2010), under the Creative Commons Attribution License. (c) Heat map of expression levels and hierarchical clustering for the global differential gene expression in four FACS-sorted spermatogenic cell populations, profiled through RNAseq (2C, spermatogonia and somatic testicular cells; LZ, leptotene/zygotene; PS, pachytene spermatocytes; RS, round spermatids). High expression: red; low expression: green. To the right, the main enriched GO terms for biological process category (BP) of the upregulated genes in PS are shown. The heat map evidences a switch in gene expression patterns from L/Z to PS, while the GO analysis shows enrichment in spermiogenesis-related terms. Reproduced from da Cruz et al. (2016), under the Creative Commons Attribution License. (d) Pie charts showing enriched BP and cellular component (CC) GO terms among upregulated genes in the testes of animals at post-natal day 14 compared to those of post-natal day 7 (PND7/14), obtained from an RNAseq study. Note the upregulation of spermiogenesis-related terms both for BP and CC categories. The figure is reproduced from Laiho et al. (2013), under the Creative Commons Attribution License. (e) GO term enrichment analysis for downregulated transcripts in PS of Sox30^–/– mice compared to WT, as assessed through RNAseq of STA-PUT-isolated stage-specific spermatogenic cells. Reprinted from Bai et al. (2018), with permission from The Company of Biologists Ltd.; permission conveyed through Copyright Clearance Center, Inc. (B) Diagram representing the three main spermatogenic phases, and the timing of mouse spermatogenesis. The different cell types and the onset of some of them (days postpartum) are indicated. The substitution of histones – first by transition proteins (TNPs) and then by protamines – is shown as well. GC, gonocytes; SPG, spermatogonia; PL, preleptotene; L, leptotene; Z, zygotene; PS, pachytene; D, diplotene; M, meiotic divisions; 1–16 represent the different spermatid stages. Adapted from Trovero et al. (2020), under the Creative Commons Attribution License.

FIGURE 2

Heat maps from different transcriptomic studies evidencing MSCI in mouse. (A) Relative expression levels of 874 X-linked genes, as obtained by microarray analysis, and ordered relative to their chromosome location from centromere to telomere. The different spermatogenic cell populations were purified by FACS. High expression is shown in yellow, and low expression in blue. The figure is reproduced from Fallahi et al. (2010), under the Creative Commons Attribution License. (B) Relative expression levels of X-linked protein-coding genes for FACS-sorted testicular cell populations, resulting from RNAseq analysis (cell-type abbreviations are the same as in Figure 1A,c). The genes are ordered according to their position on the chromosome from p to q. High expression: red; low expression: green. The figure is reproduced from da Cruz et al. (2016), under the Creative Commons Attribution License. (C) Differentially expressed X- and Y-linked mRNAs and lncRNAs between spermatogonia vs. PS, and PS vs. RS, on a red-to-blue scale. The cell populations were purified by STA-PUT, and transcriptomic profiles were obtained through RNAseq. Reprinted from Wichman et al. (2017), with permission from Oxford University Press (license number 4933230295359). (D) Expression patterns of sex chromosome-linked genes along 20 developmental stages, obtained through scRNAseq of synchronized spermatogenic cells (Concerning meiotic cells, L, leptotene; Z, zygotene; eP, early pachytene; mP, middle pachytene; lP, late pachytene; D, diplotene; MI, metaphase I; MII, metaphase II). The genes are grouped according to: MSCI PMSC (post-meiotic sex chromatin silencing), MSCI/escape PMSC, escape MSCI, RS-specific, and other. Colors from yellow to blue represent high to low relative expression levels. The figure is reproduced from Chen et al. (2018), under the Creative Commons Attribution License.