Given the fundamental importance of gamete production for the perpetuation of a species, it comes as no surprise that spermatogenesis is a process tightly regulated at multiple levels, including the transcriptional and post-transcriptional level. An issue that has not yet been addressed is whether post-transcriptional regulation of spermatogenesis also occurs in the somatic compartment of the testis. Here, we report that Dicer, a central component of the RNAi machinery, is essential for SC maturation, function and survival. Specific deletion of Dicer in SCs leads to infertility due to absence of mature spermatozoa and testis degeneration, thus highlighting the absolute necessity of Dicer for the development of fully competent SCs.
The first histological signs of the phenotype appeared a few days after birth, and led already before puberty to a gradual degeneration of the seminiferous epithelium’s architecture. In fact, our expression profiling analyses revealed that although P0 control and mutant testes are morphologically indistinguishable, miRNA levels in SCs are reduced by approximately 50% and on the same time significant transcriptional alterations have already occurred in mutants. At P0, SC loss of Dicer affected the expression of not only numerous mRNAs specifically expressed in SCs, but also mRNAs in gonocytes (e.g. Serpin5a, TSLC1) and in LCs (e.g. Amhr2, PNMT). This suggests that despite no apparent testicular phenotype at birth, damages in SCs have already had important secondary effects on gene expression in the adjacent cell lineages, but more importantly, it confirms the long-standing notion of a close crosstalk between SCs and germ cells [reviewed in (Jegou, 1993
)]. From P5 onwards, numerous testicular abnormalities appeared, the most prominent being a delay in SC maturation and a delayed entry in meiosis. Testicular degeneration worsened upon aging; by 6 months of age, mutant testes were composed almost exclusively of Leydig and fibroblast-like cells interspersed with rare –completely disrupted- tubes. Overall, our results suggest that the striking testis size reduction is very likely to have been the result of both (i) SC inability to support germ cell survival and spermatogenesis and (ii) SC death.
Increased cell death has in fact been observed in numerous conditional Dicer
knockouts (Chen et al., 2008
; Harfe et al., 2005
; Harris et al., 2006
), thereby raising the possibility that Dicer might be a “universal” regulator of cell survival. Specific ablation of Dicer
in SCs was no exception since increased levels of SC apoptosis were detected as early as P5. At first glance, the dramatic phenotype of aging mutant testes could be simply explained by massive SC apoptosis leading to subsequent germ cell death. However, a closer examination of the phenotype suggests that Dicer has additional roles in regulating SC function, independent of its requirement for cell survival. Several of our findings are in support of this notion: (1) The significant alterations in gene expression at a time (P0) when histological defects and apoptosis are not yet detectable in mutant testes indicate that expression of genes essential for SC function is already affected and is not a consequence of cell death. (2) Apoptosis in mutant SCs appears gradually: some SCs die as early as P5, while some remain viable for several months and are at some level capable of supporting spermatogenesis, although they display other abnormalities (defective maturation, abnormal cellular architecture and polarization -as evidenced by TJP1 staining). Finally, (3) by 2 months of age, most remaining tubes are SCOs and composed of mutant, yet viable
SCs which are Dicer
-deficient, as confirmed by recombination of the Rosa26 stop LacZ marker (SI Fig. 1
), thus showing that mutant SCs have lost their capacity to support germ cell survival, but remain viable themselves. These data reinforce our belief that Dicer should not be merely viewed as a global regulator of cell survival, and that the effects caused by its absence should not be interpreted solely on the base of cell death.
Among the genes whose expression was downregulated in P0 and/or P5 mutant testes, were Gdnf, Kitl, Man2a2, Gata1, Dhh, Serpin5a, Wt1, Sox9
, all known to result in diverse spermatogenic defects when deleted in vivo
. The significant reduction of Kitl
levels was of particular interest; a balance between the Kitl/c-kit and GDNF/Ret signaling pathways is known to control the choice between spermatogonial differentiation and renewal (reviewed in (Wong et al., 2005
)). It is thereby reasonable to assume that deregulation of this balance, notably a 5- and 3-fold reduction of Gdnf
respectively, could perturb the initial phase of spermatogenesis. It is possible that around P5, when spermatogonia resume proliferation and either renew themselves or differentiate, the reduction of Gdnf impairs their capacity to renew, whereas the reduction of Kitl negatively affects their capacity to differentiate. Defective spermatogonial renewal could lead to gradual germ cell loss, and could thereby explain the tubular degeneration we observed upon aging.
An essential question that emerges with the findings presented here is which biological activities mediated by Dicer are essential for SC function. Dicer is involved in a variety of gene-silencing phenomena at the transcriptional or translational level –through the activity of small RNAs-, but is also required for the maintenance of chromatin structure (Kanellopoulou et al., 2005
). Therefore, it is likely that the Dcrfx/fx;MisCre
phenotype is the result of (1) the deregulation of genetic elements that are directly under the control of Dicer itself, and/or (2) the deregulation of –direct or indirect- miRNA target genes. In the first case, loss of Dicer could lead to the up-regulation of genetic elements that are normally transcriptionally silent, which in its turn could affect the expression of other genes essential for spermatogenesis. In the second case, loss of miRNAs -subsequent to the loss of Dicer- could result in the up-regulation of direct miRNA target genes, which in their turn could deregulate the expression of other downstream factors. For the moment, our data favor the second option, since we found no significant change in the expression of a selected
set of repetitive elements in mutant testes. A tempting hypothesis is raised by the fact that most genes that could actually be responsible for the observed phenotype were down-regulated in mutant testes. Taken together, our data suggest a model in which Dicer
deletion leads to the gradual -but ultimately complete-disappearance of miRNAs in Sertoli cells, followed by a major transcriptome deregulation that could be the result of an alteration in the fine tuning of protein synthesis, such as the upregulation of potential transcriptional repressors. This hypothesis is supported by recent high-throughput proteomic analyses revealing that a single miRNA can repress the production of hundreds of proteins but that this repression is relatively mild, rarely exceeding 4-fold (Baek et al., 2008
; Selbach et al., 2008
). We therefore hypothesize that the sterility phenotype observed in Dcrfx/fx;MisCre
mice is the indirect consequence of the downregulation of genes essential for Sertoli cell capacity to support germ cell survival and differentiation, such as Gdnf, Kitl, Wt1, Sox9
In conclusion, a better understanding of spermatogenesis is essential in order to become able to treat a rapidly increasing number of cases of male infertility. By demonstrating with our study that Dicer is essential for spermatogenesis, not only do we unravel a novel role for this gene, but we also provide new insights on the mechanisms controlling SC function and germ stem cell niche regulation in mammals.