A curiosity of germ cell formation and differentiation across species is the conservation of genes that encode components of germ plasm in diverse species regardless of the mode of germ cell specification. For example, in Drosophila
, the disruption of genes such as Oskar
, Germ cell-less
results in the lack of a germ line (35
). The function of these genes is to assemble germ plasm, which contains highly conserved, interacting RNA-binding proteins such as Pumilio and Nanos which are thought to act to repress translation and indirectly silence gene transcription in nascent germ cells (35
). In Pumilio
mutants, nascent germ cells may divide prematurely, migrate abnormally and subsequently die during early embryonic development (35
). Several years ago, homologs of several germ plasm components were identified in mammalian germ cells, including those of humans (24
). In a screen for proteins that interact with DAZ and DAZL, Moore et al
. identified a set of genes that included PUMILIO homologs, PUM1 and PUM2 (39
). In other studies, it was shown that a human NANOS homolog interacts with PUMILIO homologs and is expressed in germ cells as well (24
). These interactions are intriguing given that, in humans, deletions and variants of DAZ homologs are associated with the production of very few or no germ cells and, in diverse model organisms, these genes are required solely in the development of the germ cell lineage (24
). Thus, it has been hypothesized that the protein complex containing human DAZ, PUMILIO and NANOS homologs likely functions in germ cell formation and/or differentiation by regulating RNA localization, transcription, translation or stability (39
). However, previously, it was not possible to directly test this hypothesis in humans. Indeed, human germ cell development remains very poorly understood in spite of the fact that defects in germ cell development contribute to a majority of infertility in the human population (57
Here, we report our analysis of a highly conserved family of proteins that is implicated in germ cell development in diverse species (22
). We show that, in human ovary and testis tissues, NANOS3 is expressed in human germ cells, with the protein localized to the germ cell nucleus. Nuclear localization of the NANOS3 protein and co-expression with known germ cell proteins BLIMP1, VASA and STELLA were also observed in hESC-derived germ cells. Moreover, we found that, in the absence of NANOS3, the molecular program(s) which drive human germ cell development were altered, thereby altering gene expression and quantifiably reducing the total germ cell number in active cell division. Taken together, these findings suggest that NANOS3 functions during human germ cell development in a cell-autonomous fashion and provides the first direct experimental evidence that NANOS3
functions in human germ cell development. Thus, NANOS3
is one of just two genes that has been shown directly to function in germ cell development from flies and worms to frogs, mice and now, humans (the other is BOULE,
a member of the DAZ
The decreased mRNA levels of NANOS3 following 7 days of differentiation are somewhat surprising, given that MOs should only affect protein levels. It is possible, however, that a negative-feedback loop exists whereby NANOS3 mRNA levels are regulated based on the NANOS3 protein levels. Alternatively, NANOS3 mRNA may have been converted to protein following the decreased effectiveness of the MOs after 7 days. Given that so little is known regarding the mechanism of NANOS3 expression regulation, this is an area of study which warrants further investigation.
Due to the lack of an internal reporter system when using MOs, we were unable to track each cell in which NANOS3 expression levels were decreased. Despite this inability to track individual cells in such a heterogeneous cell population, we were able to quantify that fewer hESCs differentiated to germ cells in culture when NANOS3 expression was decreased.
Based on our data, we propose a model for NANOS3 expression in human hESC-derived germ cells (Fig. ). The inner cell mass (ICM) of a human blastocyst can be extracted, and, in concert with the loss of positive and/or negative signals, gives rise to hESCs which can be maintained in vitro. ES cells, which express DAZL, are capable of differentiating to both germ and somatic cellular lineages. With the addition of extracellular signaling, PGCs can be derived in vitro from hESCs. PGCs express numerous factors required for germ cell development, including (but not limited to) VASA, STELLA and BLIMP1, and give rise to the germ cell lineage exclusively. In this study, we show that PGCs also express the highly conserved germ cell factor NANOS3. Based on our observations, we propose that down-regulation of NANOS3 results in decreased expression of genes required for several processes required for the development of human germ cells including (i) decreased expression of germ cell genes, (ii) decreased expression of genes required for the maintenance of pluripotency and (iii) reduced DAZL protein levels. The resultant down-regulation of the germ cell molecular profile ultimately disrupts the process of human germ cell development.
Figure 6. Proposed model. The ICM of a human blastocyst can be dissected, and in concert with the removal of positive and/or negative signals yields hESCs which can be maintained in vitro indefinitely. ES cells have previously been shown to express DAZL and can (more ...)
While the mechanism of germ cell regulation is as yet unknown, based on the results described here, it is likely that NANOS3 modulates essential aspects of human germ cell development perhaps during the cell cycle. Furthermore, we present the novel discovery that NANOS3 localizes to the nuclei of human germ cells and may regulate human germ cell development at both mRNA and DNA levels in contrast to the exclusive regulation at the mRNA level that has been previously reported (32
). Further investigation of the specific mechanism and potential targets of NANOS3 would undoubtedly increase our knowledge of human germ cell development and could potentially provide information for the purpose of advancing human fertility studies as well as treatments.