Expression profiling reveals developmentally regulated lncRNA repertoire in the mouse male germline

Abstract

In mammals, the transcriptome of large noncoding RNAs (lncRNAs) is believed to be greater than that of messenger RNAs (mRNAs). Some lncRNAs, especially large intergenic noncoding RNAs (lincRNAs), participate in epigenetic regulation by binding chromatin-modifying protein complexes and regulating protein-coding gene expression. Given that epigenetic regulation plays a critical role in male germline development, we embarked on expression profiling of both lncRNAs and mRNAs during male germline reprogramming and postnatal development using microarray analyses. We identified thousands of lncRNAs and hundreds of lincRNAs that are either up- or downregulated at six critical time points during male germ cell development. In addition, highly regulated lncRNAs were correlated with nearby (<30 kb) mRNA gene clusters, which were also significantly up- or downregulated. Large ncRNAs can be localized to both the nucleus and cytoplasm, with nuclear lncRNAs mostly associated with key components of the chromatin-remodeling protein complexes. Our data indicate that expression of lncRNAs is dynamically regulated during male germline development and that lncRNAs may function to regulate gene expression at both transcriptional and posttranscriptional levels via genetic and epigenetic mechanisms.

Keywords: epigenetics; fertility; germ cell; reproduction; spermatogenesis.

FIG. 1

Microarray-based lncRNA expression profiling analyses on murine testes at six time points during male germline development. A) Schematic of the experimental workflow for lncRNA microarray analyses. B) Diagram of major cellular events occurring at the six time points during male germ cell development in mice. E, embryonic day; P, postnatal day; PGC, primordial germ cell; Spg, spermatogonia; Spc, spermatocytes; Rspd, round spermatids; Ad, adult; 1N, haploid; 2N, diploid; 4N, tetraploid. C) Summary of the total number of lncRNAs that were up- or downregulated at each of the six time windows during male germline development in mice. The regulated lncRNAs were subdivided into five groups based on fold change (FC). D) Distribution of the four types of lncRNAs (sense, antisense, bidirectional, and intergenic) in the regulated lncRNAs at each of the six time windows analyzed.

FIG. 2

Quantitative PCR validation of lncRNA expression in developing testes and in purified testicular cell types of mice. A) Heat map showing relative expression levels of 28 lncRNAs in developing testes at E12.5, E15.5, P1.5, P7, P14, P21, and adulthood (Ad). The lncRNAs analyzed include 16 intergenic (gene symbols in black), six antisense (in red), three sense (in green), and three bidirectional (in blue). B) Heat map representing relative expression levels of 13 lncRNAs in purified testicular cell types, including spermatogonia (Spg), spermatocytes (Spc), round spermatid (Rspd), Sertoli cells (Ser), and Leydig cells (Ley). C) Northern blot analyses of the expression of Tsx, a lincRNA, in developing testes at P7, P14, P21, P35, and adulthood (Ad). The left panel is a representative Northern blot, and the right panel represents the quantitative analyses of Northern blot results from three independent experiments. Data are presented as mean ± SEM (n = 3).

FIG. 3

RNA immunoprecipitation followed by real-time qPCR analyses demonstrating that many lncRNAs are associated with key components of chromatin-modifying complexes. A–J) Ten out of 26 lncRNAs analyzed were associated with EZH2, a H3K27 methylase that binds the core components Suz12 and EED to form polycomb repressive complex 2 (PRC2). The 10 lncRNAs include six lincRNAs, three antisense lncRNAs, and one bidirectional lncRNA. K) One antisense lncRNA, uc009cxn.1, interacted with LSD1, a demethylase that mediates enzymatic demethylation of H3K4me2 in the CoREST/REST complexes. Data were obtained from experiments in biological triplicates and presented as mean ± SEM. *P < 0.05.

FIG. 4

Subcellular localization of two lncRNAs in adult murine testes by ISH assays. A–D) ISH-based localization of a bidirectional lncRNA (AK082424) in adult murine testes. Brown, punctuated dots representing specific hybridization signals are confined to the nuclei of spermatocytes and round spermatids in seminiferous tubules at stages I–III, VII, and XI. Arrows point to representative positive cells (B–D). Sections hybridized to control probes show no specific hybridization signals (A). Insets show the digitally magnified view of the framed regions. Panels B–D are in the same magnification. Bar = 50 μm. E) Schematic of the localization of lncRNA AK082424 during spermatogenesis in adult murine testes. Cells expressing this lncRNA are framed, and the height of the frame represents relative expression levels. Note that specific hybridization signals are confined to the nuclei of spermatocytes and round spermatids (red dots). Roman numerals (I–XII) mark stages of the seminiferous epithelial cycle, and steps of spermatid differentiation (i.e., spermiogenesis) are labeled with Arabic numerals (1–16). Sc, Sertoli cells; As, single type A spermatogonia; As-pr, paired type A spermatogonia; Aal, aligned type A spermatogonia; A1-4, type A1-A4 spermatogonia; In, intermediate spermatogonia; B, type B spermatogonia; Pl, preleptotene spermatocyte; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; Di, diplotene spermatocyte; M, meiotically dividing. F–I) ISH-based localization of an antisense lncRNA (AK016507) in adult mouse testes. Brown, punctuated dots representing specific hybridization signals are confined mainly to the cytoplasm of spermatocytes and round and elongating spermatids in seminiferous tubules at stages IV, V, VII, X, and XI. Arrows point to representative positive cells (G–I). Sections hybridized to control probes show no specific hybridization signals (F). Insets show the magnified views of the framed regions. Panels F–I are all in the same magnification. Bar = 50 μm. J) Schematic of localization of lncRNA AK016507 during spermatogenesis in adult murine testes. Cells expressing this lncRNA are framed, and the height of the frame represents relative expression levels. Note that specific hybridization signals are mainly confined to the cytoplasm of spermatocytes and round and elongating spermatids (red dots). Labels are the same as in E.

FIG. 5

Many lncRNAs and their neighboring mRNA genes (<30 kb) are either co-regulated or display inverse expression patterns during the six time windows of male germline development. Up and down arrows denote up and downregulation, respectively, throughout. A) An example of an inverse correlation between a significantly upregulated lincRNA (MM9LINCRNAEXON11331) and three of its neighboring mRNA genes (Cthrc1, Slc25a32, and Dcaf13) that were drastically downregulated during PGC reprogramming in the fetal testes (E15.5 vs. E12.5). B) Two Hoxd genes (Hoxd10 and Hoxd9) are <30 kb away from four lincRNAs (MM9LINCRNAEXON-10590, −10960, −10964, and −10965) and are co-regulated with two distal lincRNAs (MM9LINCRNAEXON-10964 and −10965), but inversely regulated with one proximal (−10960) and one distal (−10590) lincRNA during transition from the mitotic to meiotic phases of spermatogenesis (P14 vs. P7). C) Co-regulation of five X-linked mRNA genes known to be suppressed during MSCI and six neighboring X-linked lncRNAs during late meiotic and early haploid phases of spermatogenesis (P21 vs. P14). D) While some X-linked mRNA genes (e.g., Uxt) remained suppressed after meiosis, some (e.g., Pdk3) started to be activated, and expression levels, thus, increased postmeiotically (Adult vs. P21). X-linked lncRNAs associated with these mRNA genes displayed similar changes in expression patterns. E) Co-regulation of distal and proximal lncRNAs with the Tnp-Prm gene cluster on chromosome 16 during spermiogenesis (Adult vs. P21). F) Dynamic correlations in expression patterns of seven Rhox genes and their neighboring lncRNAs during the four time windows of testicular development.

FIG. 6

Many lncRNAs appear to be associated with genomic regions enriched with miRNA clusters and/or under epigenetic regulations. A) Schematic representation of the distal chromosome 12 in mice, which is a parentally imprinted region enriched with miRNAs and lncRNAs. More than 10 lncRNAs (black oval shapes) derived from this region were significantly upregulated during fetal testicular development, and their levels remained high but became gradually downregulated with the progression of postnatal testicular development. Solid black bars represent active alleles, whereas gray bars denote inactive alleles. The transcriptional direction is marked with an arrow. Five major miRNA clusters containing >100 miRNAs are believed to be transcribed mainly from the maternal allele and, thus, are listed in rectangular boxes next to the maternal copy of chromosome 12. Some of the lncRNAs (e.g., 6430411K18Rik, Rian, Mirg, and Gtl2/Meg3) contain sequences overlapping with miRNAs, and some (e.g., AK003491, AK046809, and AC121784.1) are derived from sequences in between those large miRNA clusters. B) Several of the X-linked miRNA clusters (rectangular boxes) known to escape the MSCI have lncRNAs (black oval shapes) expressed from their neighboring regions.