In our study of differentially expressed miRNA all the significantly down-regulated miRNA molecules i.e. miR-293, 291a-5p, 290-5p and 294* belong to the miR-290-295 cluster and have previously been found to be highly enriched in the germ cell population of day 6 testis when compared to the somatic cell population 
. Additionally, the miR-290-295 cluster is highly expressed in mouse embryonic stem cells 
, with a role in promoting cell proliferation and maintaining pluripotency. This cluster is a target of pluripotency factors, and in turn targets the pluripotency factors OCT4, SOX2 and NANOG 
. The artificial expression of members of the miR-290 cluster (291a-3p, 294 and 295) restores the proliferation potential of ES cells lacking members of the miRNA processing machinery. These ES cells have a tendency to stall in the G1 stage of the cell cycle 
. More recently the miR-290 cluster has been found to have a pro-survival role by directly targeting pro-apoptosis genes, Caspase2 and Ei24 for mRNA degradation 
. The expression of the miR-290 cluster is high in multipotent adult germ cells, which are established upon culture of spermatogonial stem cells under ES cell conditions. We propose that the elevated expression of the miR-290 cluster maintains the expression of the pluripotency factor OCT4, and promotes the expression of early germ cell specific proteins in these cells for an extended period when compared to ES cells stimulated to differentiate 
. Additionally, knockout of the miR-290 cluster, besides inducing a partially penetrant embryonic lethality, leads to the defective migration of primordial germ cells to the genital ridges during development, resulting in reduced germ cell numbers in the nascent gonads. Male germ cells recovered their numbers by mitotic proliferation while female germ cells were unable to do this causing infertility in the surviving female knockouts 
. The reduction in the expression of the members of the miR-290 cluster in our spermatogonial analysis, we believe, indicates that these cells have initiated the process of differentiation i.e. spermatogenesis.
In contrast, little is known about the three miRNAs (miR-136, miR-743a and miR-463*) up-regulated in spermatogonia and their role in germ cell development remains unclear. The miR-136 has been found to be highly expressed in placental tissue as well as overexpressed in lung cancer, however knockdown has no effect on tumour growth, and its function has still not been elucidated 
. Conversely miR-743a has been identified to play a role in the oxidative stress response in mitochondria 
. The above miRNAs are only slightly enriched in germ cells (miR-743a and 463*) or preferentially expressed in the somatic tissue (miR-136) of day 6 testis 
indicating that these miRNA molecules may play a role in the differentiation process and not in maintaining pluripotency.
One pathway identified in spermatogonia by our bioinformatic analysis (Fig. S2
) as containing a significant number of differentially expressed miRNA targets was the Wnt signalling pathway (). Wnt signalling has been shown to play a role in both stem cell renewal and differentiation in response to differing signals from surrounding niche cells 
. Wnt signalling also participates in the maintenance of the undifferentiated spermatogonial cell population and promoting their proliferation 
. Treatment of isolated early male germ cells with Wnt3a and Wnt10b promotes morphological changes and cell migration, indicating Wnt signalling could have unique roles in addition to directing spermatogonial stem cell renewal 
. Knockout of naked cuticle 1 (Nkd1), an antagonist of the canonical Wnt signalling pathway (controlling novel gene expression), caused sub fertility in male mice. The elongating spermatid population is reduced in these mice, indicating a problem in the later stages of spermiogenesis 
. Furthermore, elements of the non-canonical Wnt signalling pathway i.e. Dishevelled1 (Dvl1) (which influences cell morphology and migration), are thought to control the morphological change(s) of spermatids that eventually permit their differentiation into mature spermatozoa 
. In addition to their essential role in germ cell development, Wnt signalling also directs the maturation of Sertoli cells and their ability to support the process of spermatogenesis 
. Wnt signalling is involved in the maintenance of “stemness” in multipotent and pluripotent stem cells including neural, mammary and embryonic stem cells 
. As components of the Wnt signalling pathway such as FZD7 and FZD4 are disrupted in many cancer types including colon, renal, brain and breast tumours 
, it is believed they promote the ability of the tumour to proliferate and invade the surrounding tissue. The multiple potential miRNA targets identified in the Wnt signalling pathway in our analysis indicates regulation by miRNA molecules is likely to play a significant role in modifying the Wnt signalling pathway and in turn germ cell development. By analogy it is possible that dysregulation of the miRNAs may contribute to germ cell tumour development.
Within the Wnt signalling pathway () the expression of Sox2 and Sox11 was examined and were found to be decreased in spermatogonial cells when compared to gonocytes (protein and/or mRNA) (). As described previously, Sox2 is involved in the maintenance of pluripotency 
. While the role of Sox11 is not as well defined as that of Sox2, particularly in postnatal life, it has been implicated in neuron development and differentiation in the embryo 
. Sox11 has also been found to be expressed in multipotent stromal stem cells, and Sox11 knockdown leads to reduced proliferation and promotes differentiation 
. In contrast, knockdown of Sox11 in mantle cell lymphoma cell lines actually increases cell proliferation and promotes tumorigenesis when injected into nude mice 
. Therefore, while it is clear that Sox11 is involved in the regulation of the cell cycle, its ability to promote or inhibit cellular proliferation may in fact be influenced by the specific cellular environment in which it is expressed. In the case of spermatogonia, the reduction in Sox11 expression may increase cell proliferation by up regulating the expression of the downstream target Cyclin D1 ( and ). Interestingly, this same protein also appears as a downstream target of the PTEN signalling cascade, suggesting convergence or cross-talk between these pathways may be important in driving the differentiation of gonocytes.
The PTEN tumour suppressor pathway was also predicted to contain members which were potential targets of differentially expressed miRNA (Fig. S2
and 6). One of the possible outcomes from the PTEN signalling pathway is the modification of the cell cycle via the post translational regulation of Cyclin D1. Cyclin D1 is known to promote cell cycle progression from G1
to S phase 
, and its expression has previously been shown to occur in spermatogonia at post natal day 4 when the cell cycle has resumed 
. After post natal day 4 in the mouse testis, we propose that post-translational control of the activity of Cyclin D1 could be required to fine tune the cell cycle. The seven significantly different miRNA molecules between gonocytes and spermatogonia identified in this study have the potential to influence the activity of Cyclin D1. One down-regulated miRNA molecule (miR-291a-5p) targets the negative regulator of Cyclin D1 (GSK3) allowing its expression and therefore limiting the effect of Cyclin D1. At the same time the positive regulator of Cyclin D1, ERK1/2, is also theoretically targeted by an up-regulated miRNA molecule (miR-136), which may result in a reduction of expression to limit the effect of Cyclin D1 on the cell cycle.
Cyclin D1 has previously been shown to be an indirect target of miRNA via a different pathway. Using epithelial cells transformed by c-Myc, Feng et al 
demonstrated miR-378 was a direct target of c-Myc. miR-378 in turn cooperates with either RAS or HER2 to target the cell cycle repressor TOB2 which directly controls the expression of Cyclin D1 
. As Cyclin D1 is frequently over expressed in testicular germ cell tumours which have become resistant to cisplatin therapies, we concur with the hypothesis that the high levels of Cyclin D1 cause an accelerated cell cycle transition and limit the cells sensitivity to the chemotherapy agent 
The KIT and EBBR2 receptors are frequently up regulated in testicular germ cell tumours. Both of these receptors activate the KRAS signalling pathway, which in turn is also commonly up-regulated in these tumours. KRAS activates both the MAP kinase pathway as well as the PI3K/AKT pathway to control cell proliferation and survival 
. The PI3K/AKT is a key signalling pathway promoting cell proliferation, survival and migration which is subject to modulation by several negative regulators such as PTEN and PI3kip1 
. Importantly, the ablation of PTEN is associated with the transformation of CIS cells, the precursor lesion for seminoma and nonseminoma into invasive cancer 
. PTEN loss allows these cells to survive and proliferate without the support of the Sertoli cells, a key step in the neoplastic development of CIS 
. In addition, the reduced expression of PI3kip1 in testicular germ cell tumours is associated with a high disease relapse rate 
. PI3K and AKT are activated by GDNF and have been shown to be key promoters of self- renewal and survival in spermatogonial stem cells 
as well as promoting the proliferation and survival of meiotic germ cells. However, despite PTEN being expressed in all stages of male germ cells and very highly expressed in spermatogonia, the PTEN knockout mouse has normal spermatogenesis, are fertile and have no testicular tumours, indicating that PTEN may have a functional redundancy in mice 
. Knockdown of ETV5 and POU3F1 (oct6), both transcription factors activated by GDNF, caused apoptosis in spermatogonial stem cells in part due to the overexpression of PI3kip1, further indicating the importance of PI3K/AKT signalling and its regulation in normal spermatogenesis 
In this study we identified seven miRNA molecules differentially expressed between postnatal gonocytes and spermatogonia. The miRNA molecules down-regulated (miR-293, 291a-5p, 290-5p and 294*) are located in a miRNA cluster previously found to be involved in the maintenance of pluripotency within stem cells. In contrast, the up-regulated miRNA molecules in spermatogonia (miR-136, MiR-743a and miR-463*) have mostly unknown functions. However, the abundance of predicted targets in the Wnt/β-catenin signalling pathway and the PTEN signalling which have previously been found to be important for male fertility indicates their possible role in maintaining germ cell differentiation and or pluripotency.