Germ cells of many animals exhibit characteristic cytoplasmic structures—termed germ
granules or nuage—which are ribonucleoprotein (RNP) amorphous aggregates without limiting
membranes and are often closely associated with nuclei or mitochondria [1]. In several
model animals, such as Drosophila, Caenorhabditis elegans, and Xenopus, studies on
germ granules have mainly focused on their asymmetric partitioning to prospective
germ cells in early embryogenesis, leading to a supposition that the RNP structures
contribute to the establishment of the germline. In mammals, in contrast, germ granules
become discernible at later stages of germ cell differentiation, i.e., in spermatogenesis
and oogenesis, and are not asymmetrically segregated. Thus, their possible function
seems different from those in early embryos of other species. Despite the difference,
germ granules of diverse animals, including mammals, not only share morphological
similarities, but their molecular compositions are also conserved [2], suggesting
a common and essential function in the germline, which however remained unanswered
for many years.
Recently, almost a century after the first description of germ granules [1], this
longstanding enigma, or at least a part of it, is finally being unraveled. Accumulating
evidence now points to a close association of germ granules with retrotransposon control
and, especially, the piwi-small RNA pathway. Germ cells of many animals, from worms
to mammals, are equipped with specific members of the argonaute subfamily, the piwi
proteins, which associate with piwi-interacting small RNAs (piRNAs), and this small
RNA pathway is critical for retrotransposon silencing in the germline (and gonadal
soma in Drosophila) [3]. In mice, Mili/Piwil2 and Miwi2/Piwil4, two mouse piwi members,
are central to the feed-forward or ping-pong production of piRNAs from retrotransposon
and other cellular transcripts in the male germline, and their loss-of-function mutations
lead to deregulation of Line-1 and IAP retrotransposons, resulting in male sterility
with meiotic spermatocyte defects [4]–[6].
In this issue of PLoS Genetics, Aravin and colleagues in the Bortvin and Hannon groups
report the remarkable finding that both piwi proteins exhibit distinct subcellular
compartmentalization in fetal prospermatogonia/gonocytes, wherein retrotransposon
silencing is established during male germline development [7]. MILI localizes to inter-mitochondrial
cement (also called inter-mitochondrial material/bar/cloud etc.), a form of germ granules
commonly observed in divergent animals, and MIWI2 accumulates at processing bodies
(P-bodies), an mRNP assembly widely conserved in eukaryotes from yeast to humans and
whose presumed function is general RNA degradation/translational control, including
those mediated by miRNAs and siRNAs [8]. These distinct MILI and MIWI2 granules contain
tudor domain containing proteins TDRD1 and TDRD9, respectively, which also operate
in the piwi pathway to suppress retrotransposons [9]–[12]. The discrete localization
of MILI-TDRD1 to germ granules and MIWI2-TDRD9 to processing bodies indicates that
the two RNP complexes, which are often found in close proximity, represent functionally
separate assemblies of the small RNA machineries that likely co-operate and interdependently
function in piRNA biogenesis and retrotransposon silencing.
Aravin et al. add another key player, Maelstrom (MAEL), in the piwi-small RNA pathway.
MAEL has a HMG box and a domain homologous to DnaQ-H 3′-5′ exonuclease, and is conserved
from protists to mammals [13]–[16]. The authors show that the MAEL protein in mice
is specifically colocalized with MIWI2-TDRD9 granules in the cytoplasm of prospermatogonia,
in addition to their accumulation in the nucleus. The MIWI2-TDRD9-MAEL complex was
shown to correspond to a subpopulation of processing bodies as identified by P-body
markers DCP1a, DDX6, XRN1, and GW182. They named this subpopulation of processing
bodies containing MIWI2-TDRD9-MAEL “piP-bodies” and inter-mitochondrial cement localized
with MILI-TDRD1 “pi-bodies”. The latter term was recently also proposed for Drosophila
nuage enriched with piwi pathway components, retroelement transcripts, and processing
body components [17]. In mice, the MAEL localization is dependent on the Mili function,
but not vice versa, similarly to the requirement of Mili for MIWI2 and TDRD9 localizations
[10],[18], and then Mael regulates the assembly of MIWI2 and TDRD9 onto piP-bodies.
Thus, MAEL acts downstream of MILI and upstream of MIWI2-TDRD9 with respect to the
subcellular compartmentalization in fetal prospermatogonia in mice.
Previously, the authors reported that Mael gene–targeted mice are male-sterile and
show a strong activation of Line-1 retrotransposon in postnatal testes [16]. Now,
they extend their analysis of Mael mutants to fetal prospermatogonia and uncover a
striking finding that piRNA production is severely impaired at embryonic day 16.5
(E16.5) with transposon-derived piRNAs being virtually absent in Mael mutants, but
the defect largely recovers at postnatal day 2 (P2), while secondary piRNAs, which
preferentially load onto MIWI2, are under-represented by several-fold. Together with
the precise colocalization of MAEL with MIWI2 at piP-bodies and their epistatic relationship,
the authors argue that the Mael mutation affects the MIWI2 pathway in the ping-pong
production of piRNAs, resulting in a delayed accumulation of piRNAs with decreased
secondary piRNA signatures. In Mili, Miwi2, and Tdrd1 mutants, the biogenesis and/or
sequence profile of piRNAs are also significantly impacted, and de novo DNA methylation
of retrotransposon loci, which usually takes place in fetal prospermatogonia in the
male germline, is severely impaired [4]–[6],[10],[12],[19]. However, in Mael mutants,
DNA methylation at Line-1 retrotransposon loci examined is only moderately decreased
in prospermatogonia at E16.5, which recovers by E18.5 and then the difference is not
detectable at neonatal P2. The authors correlate this unexpected lag and recovery
of de novo DNA methylation with delayed nuclear accumulation of MIWI2 in Mael mutant
prospermatogonia and suggest that MAEL “facilitates” MIWI2-dependent steps of the
piRNA pathway. These observations provide unprecedented insights that the coordinated
and ordered operations of the piwi pathway components at around embryonic day E16.5—when
or just after fetal prospermatogonia become arrested at the G1/G0 stage and DNA methylation
reprogramming commences—are essential in the establishment of retrotransposon silencing
in the male germline in mice. In these processes, Maelstrom is a critical modulator
that acts in the MIWI2 pathway. Meanwhile, one key question that arises from this
study is, given the recovery of DNA methylation in the Mael mutant, what then triggers
the later retrotransposon activation in postnatal spermatogenesis as was reported
previously [16]? One possibility might be that histone modifications are affected
independent of DNA methylation at the locus examined. Alternatively, Mael might have
an additional function separate from other piwi pathway components so far identified.
A recent study in Drosophila actually showed that Mael regulates Bag-of-marbles via
repression of miR-7 and ensures proper differentiation of spermatocytes [20]. It remains
to be addressed whether this novel function of Mael in the miRNA pathway is retained
across species, in addition to its conserved role in the piwi machinery.
The study by Aravin et al. reveals that germ granules, namely pi-bodies, and a germline
analogue of processing bodies, piP-bodies, are cytoplasmic compartments where piwi
pathway components assemble. The next questions are how and why these components are
differently sorted-out into distinct subcellular domains, and what is the underlying
molecular mechanism wherein the two RNP complexes cooperate in the piRNA biogenesis,
which is intimately linked to retrotransposon silencing at both transcriptional and
post-transcriptional levels. It is important to note that current experiments performed
with fixed tissue sections give us a static image of potentially dynamic interactions
between the two RNP complexes. Development of suitable cell culture systems that recapitulate
the piRNA pathway and the use of live-cell imaging techniques will help explore this
further. It is also currently unclear whether these RNP assemblies are functional
prerequisites for the piwi-small RNA pathway operation, or such cytoplasmic aggregations
are consequences and by-products of normal cellular metabolism. Indeed, in somatic
cells, microscopically visible processing bodies are not required for proper functioning
of the small RNA pathway [21]. Another evident but untouched issue is that “pi-bodies”
in prospermatogonia correspond to “inter-mitochondrial cement” located in the midst
of mitochondrial clusters (Figure 1). At present, we do not have any experimental
clues to discuss whether there would possibly be any correlation between the piwi
pathway and mitochondria. The physiological function(s) of germ granules is one of
the classic but still enigmatic problems in developmental and cell biology and remains
to be fully determined. Further characterization of germline RNPs and the piwi-small
RNA pathway associated there would uncover an intriguing molecular mechanism(s) that
is present but still hidden within the germline.
10.1371/journal.pgen.1000770.g001
Figure 1
Germinal granules/nuage in mouse germ cells.
Electron microscopy of a fetal prospermatogonium (A) and postnatal spermatid (B).
In (A), inter-mitochondrial cement structures are seen as fine electron-dense material
among mitochondria (arrowheads). Inter-mitochondrial cement is also seen in postnatal
spermatogonia, spermatocytes and in growing oocytes. In (B), a chromatoid body (arrowhead),
a specialized form of germinal granules/nuage, is seen in the cytoplasm independently
of mitochondria. Chromatoid bodies are much larger in size than inter-mitochondrial
cement and are observed mostly as one or two solitary aggregates in haploid spermatids.
Both inter-mitochondrial cement and chromatoid bodies contain piwi-pathway components.
Nuc, nucleus; Cyt, cytoplasm; Mit, mitochondria.