Differential Expression of Non-Coding RNAs and Continuous Evolution of the X Chromosome in Testicular Transcriptome of Two Mouse Species

BACKGROUND: Tight regulation of testicular gene expression is a prerequisite for male reproductive success, while differentiation of gene activity in spermatogenesis is important during speciation. Thus, comparison of testicular transcriptomes between closely related species can reveal unique regulatory patterns and shed light on evolutionary constraints separating the species. METHODOLOGY/PRINCIPAL FINDINGS: Here, we compared testicular transcriptomes of two closely related mouse species, Mus musculus and Mus spretus, which diverged more than one million years ago. We analyzed testicular expression using tiling arrays overlapping Chromosomes 2, X, Y and mitochondrial genome. An excess of differentially regulated non-coding RNAs was found on Chromosome 2 including the intronic antisense RNAs, intergenic RNAs and premature forms of Piwi-interacting RNAs (piRNAs). Moreover, striking difference was found in the expression of X-linked G6pdx gene, the parental gene of the autosomal retrogene G6pd2. CONCLUSIONS/SIGNIFICANCE: The prevalence of non-coding RNAs among differentially expressed transcripts indicates their role in species-specific regulation of spermatogenesis. The postmeiotic expression of G6pdx in Mus spretus points towards the continuous evolution of X-chromosome silencing and provides an example of expression change accompanying the out-of-the X-chromosomal retroposition

DNA Demethylation and USF Regulate the Meiosis-Specific Expression of the Mouse Miwi

Miwi, a member of the Argonaute family, is required for initiating spermiogenesis; however, the mechanisms that regulate the expression of the Miwi gene remain unknown. By mutation analysis and transgenic models, we identified a 303 bp proximal promoter region of the mouse Miwi gene, which controls specific expression from midpachytene spermatocytes to round spermatids during meiosis. We characterized the binding sites of transcription factors NF-Y (Nuclear Factor Y) and USF (Upstream Stimulatory Factor) within the core promoter and found that both factors specifically bind to and activate the Miwi promoter. Methylation profiling of three CpG islands within the proximal promoter reveals a markedly inverse correlation between the methylation status of the CpG islands and germ cell type–specific expression of Miwi. CpG methylation at the USF–binding site within the E2 box in the promoter inhibits the binding of USF. Transgenic Miwi-EGFP and endogenous Miwi reveal a subcellular co-localization pattern in the germ cells of the Miwi-EGFP transgenic mouse. Furthermore, the DNA methylation profile of the Miwi promoter–driven transgene is consistent with that of the endogenous Miwi promoter, indicating that Miwi transgene is epigenetically modified through methylation in vivo to ensure its spatio-temporal expression. Our findings suggest that USF controls Miwi expression from midpachytene spermatocytes to round spermatids through methylation-mediated regulation. This work identifies an epigenetic regulation mechanism for the spatio-temporal expression of mouse Miwi during spermatogenesis

Discovery of Potential piRNAs from Next Generation Sequences of the Sexually Mature Porcine Testes

Piwi- interacting RNAs (piRNAs), a new class of small RNAs discovered from mammalian testes, are involved in transcriptional silencing of retrotransposons and other genetic elements in germ line cells. In order to identify a full transcriptome set of piRNAs expressed in the sexually mature porcine testes, small RNA fractions were extracted and were subjected to a Solexa deep sequencing. We cloned 6,913,561 clean reads of Sus Scrofa small RNAs (18–30 nt) and performed functional characterization. Sus Scrofa small RNAs showed a bimodal length distribution with two peaks at 21 nt and 29 nt. Then from 938,328 deep-sequenced small RNAs (26–30 nt), 375,195 piRNAs were identified by a k-mer scheme and 326 piRNAs were identified by homology searches. All piRNAs predicted by the k-mer scheme were then mapped to swine genome by Short Oligonucleotide Analysis Package (SOAP), and 81.61% of all uniquely mapping piRNAs (197,673) were located to 1124 defined genomic regions (5.85 Mb). Within these regions, 536 and 501 piRNA clusters generally distributed across only minus or plus genomic strand, 48 piRNA clusters distributed on two strands but in a divergent manner, and 39 piRNA clusters distributed on two strands in an overlapping manner. Furthermore, expression pattern of 7 piRNAs identified by homology searches showed 5 piRNAs displayed a ubiquitous expression pattern, although 2 piRNAs were specifically expressed in the testes. Overall, our results provide new information of porcine piRNAs and their specific expression pattern in porcine testes suggests that piRNAs have a role in regulating spermatogenesis

Deletion of the Pluripotency-Associated Tex19.1 Gene Causes Activation of Endogenous Retroviruses and Defective Spermatogenesis in Mice

As genetic information is transmitted through successive generations, it passes between pluripotent cells in the early embryo and germ cells in the developing foetus and adult animal. Tex19.1 encodes a protein of unknown function, whose expression is restricted to germ cells and pluripotent cells. During male spermatogenesis, Tex19.1 expression is highest in mitotic spermatogonia and diminishes as these cells differentiate and progress through meiosis. In pluripotent stem cells, Tex19.1 expression is also downregulated upon differentiation. However, it is not clear whether Tex19.1 has an essential function in germ cells or pluripotent stem cells, or what that function might be. To analyse the potential role of Tex19.1 in pluripotency or germ cell function we have generated Tex19.1−/− knockout mice and analysed the Tex19.1−/− mutant phenotype. Adult Tex19.1−/− knockout males exhibit impaired spermatogenesis. Immunostaining and histological analysis revealed defects in meiotic chromosome synapsis, the persistence of DNA double-strand breaks during meiosis, and a loss of post-meiotic germ cells in the testis. Furthermore, expression of a class of endogenous retroviruses is upregulated during meiosis in the Tex19.1−/− testes. Increased transposition of endogenous retroviruses in the germline of Tex19.1−/− mutant mice, and the concomitant increase in DNA damage, may be sufficient to disrupt the normal processes of recombination and chromosome synapsis during meiosis and cause defects in spermatogenesis. Our results suggest that Tex19.1 is part of a specialised mechanism that operates in the germline to repress transposable genetic elements and maintain genomic stability through successive generations

Identification of Piwil2-Like (PL2L) Proteins that Promote Tumorigenesis

PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G0/1 into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development

Expression of HIWI in human esophageal squamous cell carcinoma is significantly associated with poorer prognosis

<p>Abstract</p> <p>Background</p> <p>HIWI, the human homologue of Piwi family, is present in CD34⁺hematopoietic stem cells and germ cells, but not in well-differentiated cell populations, indicating that HIWI may play an impotent role in determining or maintaining stemness of these cells. That HIWI expression has been detected in several type tumours may suggest its association with clinical outcome in cancer patients.</p> <p>Methods</p> <p>With the methods of real-time PCR, western blot, immunocytochemistry and immunohistochemistry, the expression of HIWI in three esophageal squamous cancer cell lines KYSE70, KYSE140 and KYSE450 has been characterized. Then, we investigated HIWI expression in a series of 153 esophageal squamous cell carcinomas using immunohistochemistry and explored its association with clinicopathological features.</p> <p>Results</p> <p>The expression of HIWI was observed in tumour cell nuclei or/and cytoplasm in 137 (89.5%) cases, 16 (10.5%) cases were negative in both nuclei and cytoplasm. 86 (56.2%) were strongly positive in cytoplasm, while 49 (32.0%) were strongly positive in nuclei. The expression level of HIWI in cytoplasm of esophageal cancer cells was significantly associated with histological grade (<it>P </it>= 0.011), T stage (<it>P </it>= 0.035), and clinic outcome (<it>P </it>< 0.001), while there was no correlation between the nuclear HIWI expression and clinicopathological features.</p> <p>Conclusion</p> <p>The expression of HIWI in the cytoplasm of esophageal cancer cells is significantly associated with higher histological grade, clinical stage and poorer clinical outcome, indicating its possible involvement in cancer development.</p

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Microarray-Based Approach Identifies Differentially Expressed MicroRNAs in Porcine Sexually Immature and Mature Testes

MicroRNAs (miRNAs) are short non-coding RNA molecules which are proved to be involved in mammalian spermatogenesis. Their expression and function in the porcine germ cells are not fully understood.We employed a miRNA microarray containing 1260 unique miRNA probes to evaluate the miRNA expression patterns between sexually immature (60-day) and mature (180-day) pig testes. One hundred and twenty nine miRNAs representing 164 reporter miRNAs were expressed differently (p<0.1). Fifty one miRNAs were significantly up-regulated and 78 miRNAs were down-regulated in mature testes. Nine of these differentially expressed miRNAs were validated using quantitative RT-PCR assay. Totally 15,919 putative miRNA-target sites were detected by using RNA22 method to align 445 NCBI pig cDNA sequences with these 129 differentially expressed miRNAs, and seven putative target genes involved in spermatogenesis including DAZL, RNF4 gene were simply confirmed by quantitative RT-PCR.Overall, the results of this study indicated specific miRNAs expression in porcine testes and suggested that miRNAs had a role in regulating spermatogenesis

Dicer1 Depletion in Male Germ Cells Leads to Infertility Due to Cumulative Meiotic and Spermiogenic Defects

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis