A Unique Combination of Male Germ Cell miRNAs Coordinates Gonocyte Differentiation

The last 100 years have seen a concerning decline in male reproductive health associated with decreased sperm production, sperm function and male fertility. Concomitantly, the incidence of defects in reproductive development, such as undescended testes, hypospadias and testicular cancer has increased. Indeed testicular cancer is now recognised as the most common malignancy in young men. Such cancers develop from the pre-invasive lesion Carcinoma in Situ (CIS), a dysfunctional precursor germ cell or gonocyte which has failed to successfully differentiate into a spermatogonium. It is therefore essential to understand the cellular transition from gonocytes to spermatogonia, in order to gain a better understanding of the aetiology of testicular germ cell tumours. MicroRNA (miRNA) are important regulators of gene expression in differentiation and development and thus highly likely to play a role in the differentiation of gonocytes. In this study we have examined the miRNA profiles of highly enriched populations of gonocytes and spermatogonia, using microarray technology. We identified seven differentially expressed miRNAs between gonocytes and spermatogonia (down-regulated: miR-293, 291a-5p, 290-5p and 294*, up-regulated: miR-136, 743a and 463*). Target prediction software identified many potential targets of several differentially expressed miRNA implicated in germ cell development, including members of the PTEN, and Wnt signalling pathways. These targets converge on the key downstream cell cycle regulator Cyclin D1, indicating that a unique combination of male germ cell miRNAs coordinate the differentiation and maintenance of pluripotency in germ cells

miRNA and mammalian male germ cells

Background: Achieving the correct spatial and temporal expression of germ-cell-specific genes is fundamental to the production of viable healthy spermatozoa. Notably, post-transcriptional gene regulation resulting in the repression of protein translation is central to many embryonic processes, and is particularly active during spermatogenesis. In this review, we discuss microRNA (miRNA) regulation of target gene expression in relation to mammalian spermatogenesis, the establishment of testicular germ cell tumours (TGCT) and the potential use of miRNA manipulation for cancer therapy and fertility regulation. Methods: Journal databases such as PubMed were searched using key words, including miRNA, testis, spermatogenesis, germ cell, testicular cancer and cancer. Results: In the past decade, the deployment of small non-coding RNA molecules, including miRNA, by the cell, has been recognized as among the most important mechanisms of fine-tuning translational regulation in differentiating cell types. For key regulators of male gametogenesis, high levels of gene expression do not always correspond to elevated levels of protein expression. Cumulatively this indicates that enhancement and repression of post-transcriptional regulatory mechanisms are essential to the success of spermatogenesis. There is also growing evidence that this form of regulation contributes to the aetiology of both TGCT and spermatocytic tumours. Conclusions: miRNA plays an essential role in regulation of genes during the process of spermatogenesis. Disruption of this regulation has the ability to contribute to the neoplastic development of germ cell tumours. However, targeted knockdown of specific miRNA molecules has the potential to form both anti-oncogenic reagents and underpin the basis for novel contraceptive technologies

The rise of testicular germ cell tumours: the search for causes, risk factors and novel therapeutic targets

Since the beginning of the 20th century there has been a decline in the reproductive vitality of men within the Western world. The declining sperm quantity and quality has been associated with increased overt disorders of sexual development including hypospadias, undescended testes and type II testicular germ cell tumours (TGCTs). The increase in TGCTs cannot be accounted for by genetic changes in the population. Therefore exposure to environmental toxicants appears to be a major contributor to the aetiology of TGCTs and men with a genetic predisposition are particularly vulnerable. In particular, Type II TGCTs have been identified to arise from a precursor lesion Carcinoma in situ (CIS), identified as a dysfunctional gonocyte; however, the exact triggers for CIS development are currently unknown. Therefore the transition from gonocytes into spermatogonia is key to those studying TGCTs. Recently we have identified seven miRNA molecules (including members of the miR-290 family and miR-136, 463* and 743a) to be significantly changed over this transition period. These miRNA molecules are predicted to have targets within the CXCR4, PTEN, DHH, RAC and PDGF pathways, all of which have important roles in germ cell migration, proliferation and homing to the spermatogonial stem cell niche. Given the plethora of potential targets affected by each miRNA molecule, subtle changes in miRNA expression could have significant consequences e.g. tumourigenesis. The role of non-traditional oncogenes and tumour suppressors such as miRNA in TGCT is highlighted by the fact that the majority of these tumours express wild type p53, a pivotal tumour suppressor usually inactivated in cancer. While treatment of TGCTs is highly successful, the impact of these treatments on fertility means that identification of exact triggers, earlier diagnosis and alternate treatments are essential. This review examines the genetic factors and possible triggers of type II TGCT to highlight target areas for potential new treatments

Exosome complex orchestrates developmental signaling to balance proliferation and differentiation during erythropoiesis

Abstract Since the highly conserved exosome complex mediates the degradation and processing of multiple classes of RNAs, it almost certainly controls diverse biological processes. How this post-transcriptional RNA-regulatory machine impacts cell fate decisions and differentiation is poorly understood. Previously, we demonstrated that exosome complex subunits confer an erythroid maturation barricade, and the erythroid transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the cognate genes. While dissecting requirements for the maturation barricade in Mus musculus, we discovered that the exosome complex is a vital determinant of a developmental signaling transition that dictates proliferation/amplification versus differentiation. Exosome complex integrity in erythroid precursor cells ensures Kit receptor tyrosine kinase expression and stem cell factor/Kit signaling, while preventing responsiveness to erythropoietin-instigated signals that promote differentiation. Functioning as a gatekeeper of this developmental signaling transition, the exosome complex controls the massive production of erythroid cells that ensures organismal survival in homeostatic and stress contexts

The PTEN signalling pathway.

<p>The PTEN signalling pathway was chosen from the pathways identified in the IPA analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035553#pone.0035553.s002" target="_blank">Figure S2</a>) for closer examination. The signalling pathway was outlined in IPA and the genes targeted by miRNA molecules are identified on the pathway alongside their targeting miRNA molecules (red unregulated in spermatogonia, green down-regulated in spermatogonia). Targets examined for gene and protein expression levels are colour coded according to their expression in gonocytes and spermatogonia, up-regulated molecules red, unchanged molecules yellow and down-regulated molecules green. In the PTEN pathway the expression of PTEN, AKT, ERK and MEK remained unchanged between gonocytes and spermatogonia while the expression of the growth receptor BMPR1a was significantly reduced in spermatongia when compared to gonocytes. The target protein Cyclin D1 is identified by blue highlighting.</p

Wnt/β catenin signalling pathway.

<p>The Wnt/β catenin signalling pathway was chosen from the pathways identified in the IPA analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035553#pone.0035553.s002" target="_blank">Figure S2</a>) for closer examination. The signalling pathway was outlined in IPA and the genes targeted by miRNA molecules are identified on the pathway alongside their targeting miRNA molecules (red: up-regulated in spermatogonia, green: down-regulated in spermatogonia). Targets examined for gene and protein expression levels are colour coded according to their expression in gonocytes and spermatogonia, up-regulated molecules red, unchanged molecules yellow and down-regulated molecules green. In this pathway the expression of AKT, fzd4 and fzd7 was unchanged. Sox2 and sox11 expression were down-regulated at both the gene and protein level. The target protein Cyclin D1 is identified by blue highlighting.</p

Confirmation of miRNA microarray analysis using qPCR.

<p>Total RNA from isolated germ cell populations (five biological replicates) was reverse transcribed with specific primers and specific miRNA molecules were amplified by QPCR. The resultant data was analysed using the 2e-ΔΔT calculation to determine the expression in spermatogonia relative to gonocytes. A) All the down-regulated miRNA (miR-293, 291a-5p, 294* and 290-5p (green)) identified by the SAM analysis in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035553#pone-0035553-g002" target="_blank">Figure 2</a> were found to be expressed in spermatogonia at approximately 20% of the level they were in gonocytes (p<0.0001). B) All the down-regulated miRNA (miR-136, 743a and 463* (red)) identified by SAM were found to be expressed at significantly higher levels in spermatogonia than in gonocytes. miR-136 was up-regulated 2 fold (p<0.001), miR743a was up-regulated 4 fold (p<0.0001) while miR-463* was up-regulated 50 fold (p<0.01).</p

Characterisation of isolated gonocyte and spermatogonia cells.

<p>Haematoxylin and eosin stained two day old testis section (1A) demonstrates that gonocytes are found in the centre of the seminiferous tubules of mice. Gonocytes differentiate and migrate to the basement membrane of the seminiferous tubules where they become established in their niche and begin the process of spermatogenesis as seen in the eight day old testis section stained with haematoxylin and eosin (1B). C) Total RNA from isolated germ cell populations (three biological replicates) reverse transcribed and subjected to qPCR. It was found that the gene expression of the germ cell markers <i>Oct3/4</i> and <i>Ngn3</i> were significantly higher (p<0.05) in spermatogonia, twice and four times the amount of gonocytes respectively. The gene expression of the ES cell markers <i>Nanog</i> and PLZF was significantly lower (p<0.05 and 0.001) in spermatogonia. Isolated gonocytes (D,E,F) and spermatogonia (G,H,I) were fixed on slides and stained with germ cell markers. 95% of both gonocytes (1D) and spermatogonia (1G) expressed oct4, while less than 5% of gonocytes (1E) and spermatogonia (1H) expressed PLZF. UCHL1 was expressed in over 95% of gonocytes (1F) and spermatogonia (1I). The expression of PLZF, OCT3/4 and UCHL1 are consistent with previous reports for germ cells indicating our isolated cell populations contained 95% germ cells.</p

Data analysis of the miRNA microarray.

<p>Total RNA was isolated from gonocytes and spermatogonia (three biological replicates) and analysed using the Illumina mouse miRNA microarray (version 1 revision 2). A) Significance Analysis of Microarrays (SAM) statistical software output (academic version 2.23 <a href="http://www-stat.stanford.edu/_tibs/SAM/" target="_blank">http://www-stat.stanford.edu/_tibs/SAM/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035553#pone.0035553-Tusher1" target="_blank">[44]</a> was used to identify significantly different miRNA molecules between gonocytes and spermatogonia. SAM expression analysis was performed with a two-class unpaired Wilcoxon test on unlogged data using 500 permutations. Significant miRNA species had a q-value<4 (false discovery rate <4%). Seven miRNA molecules were identified by SAM; 3 were up-regulated in spermatogonia (red) while 4 were down-regulated (green). B) The expression of the significant miRNAs in the individual replicates used for the array was examined using a heat map (Java Treeview) red staining indicating higher expression. miRs 136, 743a and 463* were expressed at consistently higher levels in the spermatogonia samples and the expression of miRs 293, 291a-5p, 294* and 290-5p were expressed at consistently lower levels in spermatogonia.</p

The expression of Cyclin D1 in gonocytes and spermatogonia.

<p>A) Total RNA from isolated gonocytes and spermatogonia (three biological replicates) was reverse transcribed before gene specific amplification. The resultant data was analysed using the 2e-ΔΔT calculation to determine mRNA expression in spermatogonia relative to gonocyte expression. Cyclin D1 miRNA expression in spermatogonia was found to be 6 times higher than in gonocytes (p<0.0001). B) SDS protein extractions of isolated gonocyte and spermatogonia cells were analysed by SDS-PAGE and immunoblotting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035553#pone.0035553-Baleato1" target="_blank">[39]</a>. Densitometry analysis of immunoblots (n = 2) indicates that the expression of Cyclin D1 was four times higher in spermatogonia compared to gonocytes. Immunohistochemistry of Cyclin D1 (red) on day 2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035553#pone-0035553-g007" target="_blank">Figure 7C</a>) and day 8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035553#pone-0035553-g007" target="_blank">Figure 7D</a>) indicates that Cyclin D1 was not expressed in day 2 testis but was expressed in the spermatogonia cells of day 8 testis.</p