Abstract
Germ cells have exceptionally diverse transcriptomes. Furthermore, the progress of spermatogenesis is accompanied by dramatic changes in gene expression patterns, the most drastic of them being near-to-complete transcriptional silencing during the final steps of differentiation. Therefore, accurate RNA regulatory mechanisms are critical for normal spermatogenesis. Cytoplasmic germ cell-specific ribonucleoprotein (RNP) granules, known as germ granules, participate in posttranscriptional regulation in developing male germ cells. Particularly, germ granules provide platforms for the PIWI-interacting RNA (piRNA) pathway and appear to be involved both in piRNA biogenesis and piRNA-targeted RNA degradation. Recently, other RNA regulatory mechanisms, such as the nonsense-mediated mRNA decay pathway have also been associated to germ granules providing new exciting insights into the function of germ granules. In this review article, we will summarize our current knowledge on the role of germ granules in the control of mammalian male germ cell’s transcriptome and in the maintenance of fertility.
Introduction
Spermatogenesis is a highly specialized process that aims to transmit correct paternal genetic and epigenetic information to the next generation. At the embryonic stage, the mammalian germ cell lineage is specified when primordial germ cells (PGCs) are separated from somatic lineages. PGCs are a transient cell population of which a part migrates to the genital ridges to initiate germ cell differentiation. They become gonocytes (prospermatogonia) that rest at G0 phase until 3–4 days postpartum. They then resume mitotic divisions and become postnatal spermatogonial stem cells (SSC) that are the source material for the cyclic production of sperm for the entire duration of a male’s reproductive age.
Upon a specific signal, SSCs enter the differentiation pathway that begins with the mitotic proliferation of type A, intermediate and type B spermatogonia. The final mitotic division of type B spermatogonia produces primary spermatocytes that subsequently undergo meiosis. The prophase of meiosis I is the longest phase of meiosis. During the leptotene stage of prophase I, chromosomes condense. Then, synapsis between homologous chromosomes begins at the zygotene stage. The formation of synaptonemal complex facilitates synapsis by holding the paired chromosomes together. At the pachytene stage, synapsis is completed and crossing over can occur until the diplotene stage when the synaptonemal complex degenerates. This is followed by the completion of the first meiotic division and the separation of the homologous chromosomes between two cells. The newly formed secondary spermatocytes then rapidly undergo the second meiotic division (meiosis II) resulting in haploid spermatids. The final phase of spermatogenesis is haploid differentiation (spermiogenesis), which includes dramatic morphological changes through which spermatozoa reach their dynamic sleek shape (Fig. 1A). After they are released from the seminiferous epithelium, spermatozoa pass through the epididymis where they undergo further maturation.
Appearance and morphology of the IMC and the CB. (A) A schematic diagram of male germ cell differentiation from pachytene spermatocytes to mature sperm in mice. The IMC (green) is prominent in mid and late pachytene spermatocytes where it appears as granular material intermingled with mitochondria. The CB precursors (pink) co-exist with the IMC in late spermatocytes. The CB (pink) is condensed to its final form in early round spermatids. In step 8 round spermatids, the CB is found at the basis of the flagellum. In elongating spermatids, the late CB splits into two separate structures. One fragment stays in the cytoplasm and the other forms a ring around the basis of the flagellum and migrates distally along the flagellum during the mitochondrial sheath formation. In step 16 elongating spermatids, the CB can no longer be detected. The steps of spermatid differentiation are indicated (steps 1–16). A transient microtubular structure, manchette, is shown in blue. The Golgi complex is depicted as a darker gray structure in all cells. (B) Immunofluorescence image (left) shows a typical germ granule staining of a stage VII–VIII seminiferous tubule cross-section. MILI antibody (yellow) labels the IMC in pachytene spermatocytes and DDX25 (pink) labels the CB in round spermatids. Nuclei are stained with DAPI. Scale bar: 10 µm. The electron microscopy images visualize the electron-dense appearance of the CB and the IMC. The IMC (blue arrows in the image with blue frames) appears as a dense material between the mitochondrial clusters. The CB (image with purple frames) has a lobulated structure, and it is surrounded by small vesicles and membranes (annotated by purple arrows) that are found even inside the CB lobes. The CB is usually detected right by the nuclear membrane, and it is not associated with mitochondria. Scale bars for electron microscopy images: 250 nm. M, mitochondria; Nu, nucleus.
Citation: Reproduction 155, 2; 10.1530/REP-17-0356
Throughout male germ cell differentiation, non-membrane-bound organelles packed with RNA and RNA-binding proteins are known to appear in the cytoplasm of germ cells (Eddy 1970, Parvinen 2005, Chuma et al. 2009, Meikar et al. 2011). These RNP granules, better known as germ granules, are present in the germline of most if not all sexually reproducing metazoans. They are known to participate in the regulation of RNA localization, stability and translation, as well as in the production and function of small non-coding RNAs and in the control of chromatin organization (Voronina et al. 2011). Different types of germ granules have been described in several organisms with diverse nomenclature. Germ granules are known as germinal granules in Xenopus laevis, polar granules or nuage in Drosophila melanogaster and P granules in Caenorhabditis elegans. In these organisms, germ granules contain maternal mRNAs required for germ cell specification and direct the timing of mRNA translation to promote the establishment of the germline in the early embryo (Leatherman & Jongens 2003). In contrast, the most prominent mammalian germ granules appear in the male germline long after the germ lineage has been established, and therefore, are not functioning in the germline specification but rather in male germ cell differentiation. Nevertheless, germ granules in different species share many homologous components indicating that they all function in RNA regulation and are likely to share similar mechanisms of action (Chuma et al. 2009). In this review article, we will mainly focus on the best-characterized mammalian germ granules: the chromatoid body (CB) in haploid spermatids and the intermitochondrial cement (IMC) in pachytene spermatocytes.
Morphological features of germ granules
The CB was the first germ granule discovered likely due to its unusually large size, which makes it detectable even with light microscopy (von Brunn 1876, Yokota 2008). Early light microscopic studies on the cytoplasm of spermatogenic cells identified granules that stain deeply with basic dyes. ‘Protoplasmaanhäufungen’ was a name given to these granules in rat spermatids described by von Brunn as early as 1876 (von Brunn 1876). In 1891, Benda observed similar granules in guinea pig spermatocytes and called them ‘chromatoide Nebenkörper’ (Benda 1891). In 1901, Regaud named the granules he observed in rat spermatids as ‘corps chromatoides’ (Regaud 1901). In 1955, the structures were studied by electron microscopy for the first time in Felis domestica, and an irregular mass of osmiophilic granular material near the Golgi was termed ‘chromatic body’ (Burgos & Fawcett 1955). Under electron microscopy, the CB is a conspicuous, intensively stained structure of highly irregular shape (Fig. 1B). As Fawcett and coworkers described in 1970, ‘the CB appears to be composed of exceedingly thin filaments that are consolidated into a compact mass or into dense strands of varying thickness that branch and anastomose to form an irregular network’ (Fawcett et al. 1970).
The use of electron microscopy enabled the identification of other types of germ granules that are less pronounced in size. The IMC appears as an electron-dense, CB-like material that is found among clusters of mitochondria in many cell types during male germ cell differentiation, including in fetal prospermatogonia, postnatal spermatogonia and mid-to-late pachytene spermatocytes (Fig. 1B) (Fawcett et al. 1970, Russell & Frank 1978, Yokota 2012). IMC is particularly evident in pachytene spermatocytes. In very late pachytene spermatocytes, another type of germ granule appears and co-exists for a short time with the IMC. These small granules are associated with the nuclear envelope and intermingled with small vesicles, but not with mitochondria. They are thought to provide the precursor material for the CB, therefore, often named ‘CB precursors’ (Fig. 1A) (Russell & Frank 1978, Chuma et al. 2009, Meikar et al. 2011). Just before meiotic divisions, CB precursor granules are disintegrated, but aggregate again into larger (0.5 µm) structures in secondary spermatocytes. After meiosis in step 1 round spermatids, these granules aggregate to form a single big (~1 µm) CB (Fig. 1B). Mitochondria are dispersed during and after meiotic divisions, and the IMC is no longer detectable in haploid cells.
The CB can be detected during all steps of round spermatid differentiation (steps 1–8). The largest CBs are observed in step 4, 5 and 6 round spermatids (Fig. 1A). In step 7 round spermatids, the CB starts decreasing in size and moving toward the basis of the flagellum (Fawcett et al. 1970, Parvinen 2005). Although the CB size is dramatically reduced at the end of the round spermatid phase, CB-like structures are still observed in elongating spermatids. During spermatid elongation, this so-called ‘late CB’ splits into two separate structures. One of these CB fragments forms a dense sphere that is discarded with most of the cytoplasm in the residual body (Fawcett et al. 1970, Susi & Clermont 1970, Shang et al. 2010). The second CB fragment forms a ring around the basis of the flagellum, and then moves together with the annulus (a ringed barrier structure between the middle piece and principal piece of the sperm tail) distally along the flagellum. Behind the migrating ring-shaped late CB, the mitochondria become associated with the axoneme. It has been suggested that the late CB is indeed involved the formation of the mitochondrial sheath in the midpiece of the sperm tail (Fawcett et al. 1970, Shang et al. 2010). In step 16 elongating spermatids, the CB is no longer visible, but the details of the timing and mechanisms of the CB disappearance are not yet understood (Fig. 1A).
In addition to the IMC and CB, Russell and Frank (1978) described distinct types of germ granules in spermatogenic cells of rat (Russell & Frank 1978, Yokota 2012). In pachytene spermatocytes, 70- to 90-nm particles are present at stages VIII–XII of the seminiferous epithelial cycle both close to the nuclear envelope and scattered in the cytoplasm. Clusters of 30-nm particles, whose profile is similar to that of free ribosomes, are few in number and observed in pachytene spermatocytes during meiosis. Irregularly shaped perinuclear granules (ISPGs; also known as clusters of 60- to 90-nm particles) are also observed in pachytene spermatocytes at stage VIII–XII. They disappear during meiotic divisions, but a few particles adhere to mitochondria that seem to be distributed to daughter cells. Satellite bodies (SB; also known as sponge bodies) appear as a network of fibrils overlaid by denser patches of amorphous material, and they can be detected in pachytene spermatocytes and also closely associated and contiguous with the CB. All these granules are poorly characterized at the molecular level, but they seem to share some components with the CB and the IMC (Yokota 2012).
Germ granules and the requirements of RNA regulation in male germ cells
Male germ cell differentiation relies on spatially and temporally regulated gene expression programs that are governed by transcriptional, posttranscriptional and epigenetic mechanisms (Kimmins et al. 2004, Kimmins & Sassone-Corsi 2005). The progress of spermatogenesis is accompanied by the orchestrated waves of gene expression that generate specific transcriptome profiles for each differentiating cell type (Chalmel et al. 2007, Laiho et al. 2013). These cell type-specific transcripts not only include mRNAs and their isoforms, hundreds of them being testis-specific (Chalmel & Rolland 2015), but also a diverse set of non-coding RNAs that have also been shown to be differentially regulated during spermatogenesis (Bao et al. 2013a, Laiho et al. 2013, Sun et al. 2013). In fact, male germ cells, particularly meiotic spermatocytes and postmeiotic round spermatids, have an exceptionally diverse transcriptome containing a large number of different non-coding RNAs that mostly derive from uncharacterized, poorly conserved intergenic regions (Soumillon et al. 2013). It is currently unclear if these non-coding genomic regions are transcribed for a reason or whether their transcription is a secondary consequence of the dramatic chromatin remodeling taking place during spermatogenesis. Either way, the control of this complex transcriptome requires special posttranscriptional mechanisms that ensure that all transcripts meet their intended fate.
A notable posttranscriptional challenge is faced in late steps of spermatogenesis when the chromatin of elongating spermatids condenses. In these cells, most histones are replaced first by transition proteins and subsequently by protamines, which enables genomic material to become particularly tightly packed. This compaction of chromatin effectively halts transcription. The final haploid differentiation phase is characterized by major changes in cellular morphology, including the construction of sperm-specific structures such as the acrosome and the flagellum. Because condensing spermatids are transcriptionally incapacitated, these genes are transcribed earlier and translationally repressed until needed. It has been shown that majority of meiotic and postmeiotic mRNAs are transcribed without immediate translation during spermatogenesis (Kleene & Kleene 2003, Idler & Yan 2012). For example, mRNAs for transition proteins (Tnp1 and Tnp2) and protamines (Prm1 and Prm2) are transcribed already in late pachytene spermatocytes, but are translated only during elongation, about a week later in mice (Kleene 1989, Fajardo et al. 1997).
The fate of RNAs is controlled mainly by their association with RNA-binding proteins that bind their targets either non-specifically or through specific motifs to form ribonucleoprotein (RNP) complexes (Ascano et al. 2013). High requirements for posttranscriptional control during spermatogenesis are indeed reflected by a high number of RNA-binding proteins expressed in male germ cells (Kleene & Kleene 2003, Paronetto & Sette 2010, Idler & Yan 2012). RNP complexes can form larger RNP granules that have the ability to compartmentalize different RNA regulatory pathways. The largest RNP granule, the CB, emerges in the cytoplasm of male germ cells when the level and complexity of the gene expression is at its very peak. Therefore, it is tempting to think that the CB could have a pivotal role in the coordination of the complex transcriptome of haploid male germ cells (Meikar et al. 2014).
Molecular composition of germ granules
Our understanding of the molecular features of germ granules, particularly the CB and the IMC has dramatically increased during recent years. Several mutant mouse models deficient in genes encoding germ granule components have been created, each of them providing important insight into the function and importance of germ granules (Fig. 2). In fact, the loss-of-function mutation of germ granule core components generally leads to spermatogenesis defects and compromised male fertility, strongly supporting the critical role that germ granules have in postnatal male germ cell differentiation.
Many germ granule and piRNA pathway components are required for normal spermatogenesis. The figure summarizes the spermatogenic phenotypes of selected mouse models with mutated germ granule or piRNA pathway components. The deletion of some causes a block of spermatogenesis at meiotic phase, while other factors are critical for haploid differentiation as spermatogenic defects arise either at round spermatid or elongating spermatid phase. The molecular function of these proteins or the biological process they participate in is described. The figure also includes information about their localization to germ granules. P-bodies, processing bodies.
Citation: Reproduction 155, 2; 10.1530/REP-17-0356
Many of the protein components of germ granules are evolutionarily conserved. For example, a DEAD-box RNA helicase DDX4/MVH/VASA (DEAD (Asp-Glu-Ala-Asp) box polypeptide 4) is consistently found in all germ granules, including polar granules in Drosophila and all forms of germ granules in mouse germ cells ( Chuma et al. 2009). Other examples of conserved germ granule components include some of the Tudor domain-containing proteins, MAEL (homolog to Drosophila maelstrom) and PIWI (P-element-induced wimpy testis) proteins. The conservation of these proteins implies that they have a central role in germ granules and germ cells. Interestingly, loss-of-function mutations of these components have clearly demonstrated that germ granules have distinct biological roles in different species; while the loss-of-function mutations of vasa, tudor and mael in Drosophila show defects in germline formation and female fertility (Boswell & Mahowald 1985, Raz 2000, Findley et al. 2003), mutations of their homologs Ddx4 or Tdrd1 or Mael in mice cause fertility defects only in males even though the genes are expressed in both male and female germlines (Tanaka et al. 2000, Chuma et al. 2006, Soper et al. 2008). Furthermore, in Drosophila, Piwi is required for the maintenance of both male and female germline stem cells, while mouse PIWI family genes PIWIL1/MIWI and PIWIL2/MILI are essential only for the postnatal germ cell differentiation in males (Deng & Lin 2002, Kuramochi-Miyagawa et al. 2004).
The full molecular composition of the CB was recently discovered. The successful isolation and mass spectrometric analysis of murine CBs revealed a list of around 90 CB-localized proteins (Fig. 3) (Meikar et al. 2010, 2014, Meikar & Kotaja 2014). The majority of the CB components are RNA-binding proteins and other proteins involved in RNA regulation, which support its central role in the posttranscriptional control. The CB proteins include ubiquitously expressed proteins that are involved, for example, in pre-mRNA binding and processing, and also a substantial number of germline-specific proteins. The most prominent molecular pathway localized in the CB is the piRNA pathway (discussed below), and the most abundant proteins in the CB were shown to be the piRNA-binding PIWI proteins MILI and MIWI and the Tudor domain-containing proteins TDRD1, TDRD6 and TDRD7, as well as DEAD-box helicases DDX4 and DDX25 (Tanaka et al. 2000, 2011, Deng & Lin 2002, Kuramochi-Miyagawa et al. 2004, Tsai-Morris et al. 2004, Chuma et al. 2006, Vasileva et al. 2009, Meikar et al. 2014).
The CB proteome. The figure contains the majority of the proteins identified by mass spectrometry (MS) of isolated CBs (Meikar et al. 2014), as well as some proteins that have been identified as CB components in other studies by immunostaining experiments. The nine CB proteins that were found to represent around 70% of the CB mass by MS analysis are in bold (MILI, MIWI, TDRD7, TDRD6, DDX4, DDX25, D1PAS1, PABP1 and HSPA2). The proteins are grouped in functional pathways, including piRNA pathway, NMD pathway, RNA-binding proteins/translation-related proteins, proteins involved in microtubule-mediated transport and vesicles and other uncategorized proteins.
Citation: Reproduction 155, 2; 10.1530/REP-17-0356
The CB contains several different Tudor domain-containing proteins, including TDRD1, TDRD3, TDRD5, TDRD6, TDRD7, RNF17 and STK31 (Pan et al. 2005, Chuma et al. 2006, Vasileva et al. 2009, Tanaka et al. 2011, Yabuta et al. 2011, Bao et al. 2013b, Meikar et al. 2014, Zhou et al. 2014). Considering the well-characterized role of the Tudor proteins as molecular scaffolds (Pek et al. 2012) and given the disrupted CB morphology in the absence of TDRD6 or TDRD7 (Vasileva et al. 2009, Tanaka et al. 2011), it is likely that Tudor proteins serve as a structural mesh for the CB architecture. Tudor proteins are known to interact with proteins that contain symmetric dimethylarginines (Kirino et al. 2009), a modification found to be enriched in the CB (Vagin et al. 2009). Among other proteins that contain this modification, PIWI proteins are known binding targets of Tudor proteins, and Tudor proteins could therefore have a critical role in their recruitment to germ granules (Vagin et al. 2009, Siomi et al. 2010).
As can be predicted from the CB protein composition and in particular from the presence of a large number of RNA-binding proteins, the CB accumulates various species of RNAs. In fact, it has been shown that the morphology of the CB depends on active transcription. This was demonstrated by inhibiting transcription with actinomycin D, which dramatically affected the appearance of the CB as observed by electron microscopy (Parvinen et al. 1978). Furthermore, experiments tracing labeled RNAs in round spermatids demonstrated that there is a constant flow of RNA into the CB, and the labeled RNA accumulates in the CB (Meikar et al. 2014). The RNA sequencing of isolated CBs revealed that the CB retains a considerable portion of the round spermatids mRNA transcriptome (Meikar et al. 2014). In addition to mRNAs, the CB accumulates piRNAs and piRNA precursors, as well as thousands of non-annotated intergenic non-coding transcripts. However, it is currently unclear how RNAs are targeted to the CB. De novo motif prediction revealed a presence of possible CB-exclusion signals (Meikar et al. 2014), but the details of the CB-targeting mechanisms remain to be characterized.
In contrast to the CB, the full molecular composition of the IMC has not been identified. Only a few proteins have been shown to localize to the IMC and some of them, including TDRD1, TDRD6 and MILI, localize to the CB as well. However, some proteins appear to be IMC specific (Chuma et al. 2006, Hosokawa et al. 2007, Ma et al. 2009, Bao et al. 2013b, Zhou et al. 2014). For example, GASZ (germ cell-specific protein with four Ankyrin repeats, a sterile alpha motif and a basic leucine Zipper domain), localizes predominantly to the IMC (Ma et al. 2009), which is largely explained by its functional mitochondrial targeting signal and its function in promoting mitofusion (Zhang et al. 2016). Another mitochondria-interacting IMC protein is PLD6/MitoPLD, a phospholipase that facilitates mitofusion, participates in nuage formation and piRNA biosynthesis during spermatogenesis (Huang et al. 2011, Watanabe et al. 2011). The structural and functional relationship between the IMC and the CB is still unclear, even though their similar protein composition indicates that they may share similar functions. Some studies have even suggested that the CB could originate from the IMC. However, TDRD1-null mice lack the IMC in spermatocytes, and yet, the CB can be clearly detected in round spermatids (Chuma et al. 2006), thus demonstrating that the IMC is not a fundamentally important source or a precursor for the CB.
Germ granules as platforms for piRNA pathway
The piRNA pathway is one of the main molecular pathways localized to germ granules, and the production of piRNAs has been linked to germ granules in mouse and lower organisms (Hirakata & Siomi 2016). piRNAs are a unique class of small (26–31 nucleotides) non-coding RNAs that form piRNP complexes with PIWI proteins. A functional piRNA pathway is required for normal spermatogenesis and mutation of piRNA pathway genes in mice, including the genes for PIWI proteins MIWI, MILI and MIWI2, as well as other proteins necessary for piRNA production or function, leads to male sterility (Deng & Lin 2002, Kuramochi-Miyagawa et al. 2004, Carmell et al. 2007, Weick & Miska 2014). Two different piRNA biogenesis pathways exist. One produces primary piRNAs from genomic clusters. These piRNAs operate as silencing triggers with multiple functions including keeping resident mobile elements repressed and switching off genic transcripts after meiosis. The second is the adaptive pathway that is used to amplify piRNAs by a so-called ping-pong cycle. This produces secondary piRNAs that function by repressing active transposons (Czech & Hannon 2016).
For primary processing, piRNAs are transcribed from the genome as long single-stranded piRNA precursor transcripts that are subsequently transported to the cytoplasm for further processing. Mitochondria-anchored endonuclease PLD6/MITOPLD first cleaves piRNA precursor transcripts to produce the 5′ ends of piRNAs (Huang et al. 2011, Watanabe et al. 2011, Ipsaro et al. 2012, Nishimasu et al. 2012). MOV10L1, a testis-specific RNA helicase, supports the piRNA primary processing by recognizing and resolving secondary structures within the piRNA precursors (Zheng et al. 2010, Vourekas et al. 2015, Fu et al. 2016). piRNA intermediate transcripts are then loaded on PIWI proteins that stabilize their 5′ ends. Subsequently, the 3′ ends have to be trimmed. This final trimming includes endonucleolytic cleavage as well as 3′–5′ exonuclease activity that in silkworm has been shown to be mediated by an enzyme called trimmer with the help of Papi and to take place on mitochondrial membranes (Izumi et al. 2016). In mouse, the 3′ trimming of piRNAs is predicted to follow a similar route and in fact, the mouse Papi homolog TDRD2/TDRKH was shown to be required for the production of correct size piRNAs (Saxe et al. 2013). piRNA biogenesis is also dependent on mitochondrial membrane proteins GPAT2 and GASZ (Ma et al. 2009, Shiromoto et al. 2013, Zhang et al. 2016). Trimmed piRNAs are finally 2′-O-methylated at their 3′ ends by HENMT1 resulting in primary piRNAs with a predominant preference for a 5′ uridine (Horwich et al. 2007, Saito et al. 2007, Lim et al. 2015).
In the ping-pong amplification cycle, piRNAs are amplified by PIWI proteins to silence transposon expression, and therefore, to protect the genome integrity by preventing harmful transposon insertions (Siomi et al. 2011). The ping-pong amplification mechanism is particularly predominant in fetal male germ cells that are challenged by the global resetting of the male germline epigenome, which causes an induction of the transposon expression. To initiate the amplification cycle in mice, MILI associates with primary piRNAs and operates in the ping-pong cycle together with other factors to yield antisense secondary piRNAs that in turn associate with MIWI2 (De Fazio et al. 2011, Pandey et al. 2013, Yang et al. 2015). MIWI2 then translocates to the nucleus where it initiates transcriptional transposon silencing via DNA methylation at the target loci (Aravin et al. 2008, Kuramochi-Miyagawa et al. 2008, De Fazio et al. 2011). Therefore, piRNA amplification cycle functions on transposon silencing both by directly detecting and slicing transposon sequences, and by silencing transposon expression at the transcriptional level.
During mammalian spermatogenesis, two populations of piRNAs are distinguished and named as pre-pachytene and pachytene piRNAs according to the developmental timing of their appearance (Weick & Miska 2014). Pre-pachytene piRNAs are co-expressed and associate with MILI and MIWI2 in fetal male germ cells and in early postnatal germ cells. As mentioned earlier, these piRNAs are amplified by the ping-pong cycle and are critical for transposon silencing. Pachytene piRNAs, on the other hand, are co-expressed in pachytene spermatocytes and round spermatids with MILI and MIWI, and they mainly associate with MIWI. The sequence diversity of pachytene piRNAs is remarkably high (Aravin et al. 2006, Girard et al. 2006, Grivna et al. 2006). They are produced in vast quantities from discrete genomic loci, pachytene piRNA clusters. The transcription of the piRNA clusters is regulated by a conserved transcription factor A-MYB in mice (Li et al. 2013). A BTB domain-containing protein BTB18 was also shown to participate in the expression of pachytene piRNA precursors by promoting transcription elongation (Zhou et al. 2017).
Although MILI and MIWI have been shown to be capable of engaging in the ping-pong cycle in meiotic cells (Wasik et al. 2015), the general analysis of pachytene piRNAs in adult testes do not show strong ping-pong signatures (Beyret et al. 2012). Therefore, the ping-pong cycle seems to be rather inactive in these cells and pachytene piRNAs are produced mainly by the primary processing pathway. In fact, a Tudor protein RNF17 has been shown to repress secondary piRNA amplification in adult gonads, and the deletion of RNF17 in mice unleashed ping-pong during meiosis and caused aberrant production of piRNAs and degradation of genic transcripts (Wasik et al. 2015). Pachytene piRNAs were originally reported to be depleted from transposon sequences (Girard et al. 2006). However, it has later been demonstrated that young, potentially active elements are enriched in pachytene piRNA clusters. These elements show a bias in orientation to enable the production of antisense piRNAs that play a role in suppressing transposons during meiosis (Reuter et al. 2011, Hirano et al. 2014, Wasik et al. 2015). In addition to the transposon silencing, pachytene piRNAs have been shown to be involved in the degradation of mRNAs and lncRNAs in late meiotic and haploid cells (Reuter et al. 2011, Goh et al. 2015, Gou et al. 2015, Watanabe et al. 2015, Zhang et al. 2015).
Germ granules provide important platforms for the biosynthesis and function of piRNAs. In fetal prospermatogonia, MILI and MIWI2 localize to the germ granules that cooperate in the production and amplification of piRNAs; IMC-resembling pi-bodies that contain MILI and TDRD1, and processing body-resembling piP-bodies that contain MIWI2, TDRD9, MAEL and signature components of processing bodies (Aravin et al. 2009, Shoji et al. 2009). In postnatal meiotic cells, the IMC is likely to function as a site for pachytene piRNA primary processing. This is supported by its close association with mitochondria and the localization of many proteins involved in piRNA processing, including MILI, MITOPLD and GASZ, to the IMC. In contrast to the IMC, the CB is not associated with mitochondria (Fawcett et al. 1970), and consequently, it does not contain the key piRNA-processing proteins, such as MITOPLD that localizes to mitochondrial membranes. Therefore, the CB’s role in the piRNA pathway appears to be unrelated to the biosynthesis of piRNAs. The current model is that the initial steps of pachytene piRNA processing occur in mitochondria-associated germ granules such as the IMC, and the PIWI protein-loaded piRNAs are then transferred to the CB for downstream action (Fig. 4). The potential role of the CB in piRNA-mediated target recognition or degradation is supported by the high accumulation of piRNAs and, on the other hand, the presence of a wide range of cellular transcripts in the CB. Interestingly, Aub-loaded piRNAs (Aub; protein aubergine, one of the PIWI proteins in fly) in the Drosophila germ plasm have been shown to use partial base-pairing to bind mRNAs randomly, and acting therefore as an adhesive trap that captures mRNAs to the germ plasm (Vourekas et al. 2016). It remains to be characterized whether the CB-localized MIWI- or MILI-loaded piRNAs could share a similar function in tethering mRNAs.
Updated model of the IMC and CB function. Pachytene piRNA primary processing is likely to take place in the IMC that is closely associated with mitochondrial membranes. The IMC (green) is prominent in mid pachytene spermatocytes. CB precursors (purple) appear in late pachytene spermatocytes, and they fuse to form one single big CB right after meiotic divisions. The current hypothesis is that piRNAs produced in the IMC are bound by PIWI proteins and transferred to the CB. The CB then serves as site for piRNA target recognition and possibly degradation. The CB function is also linked to the nonsense-mediated decay (NMD) pathway. In the immunofluorescence images, MILI (green) stains the IMC (yellow arrows), CB precursors (white arrow in late pachytene spermatocytes) and CBs (white arrows) in early round spermatids, while DDX25 (magenta) is found only in CB precursors as well as in CBs during all steps of round spermatid differentiation. Co-localization of MILI and DDX25 in CB precursors and early CBs is shown as white color. Scale bar: 10 µm.
Citation: Reproduction 155, 2; 10.1530/REP-17-0356
Chromatoid body and nonsense-mediated mRNA decay
In addition to the piRNA pathway, the CB also harbors components of another RNA regulatory pathway, known as the nonsense-mediated mRNA decay (NMD) (Meikar et al. 2014). Historically NMD has been perceived as a quality control system that evolved to eliminate aberrant, premature termination codon (PTC)-containing mRNAs. However, recently, it has become clear that in addition to its quality control function, NMD participates in regulating and fine-tuning gene expression by targeting many seemingly ‘normal’ mRNAs that code for full-length functional proteins. Therefore, this pathway can influence a wide variety of biological processes potentially in a tissue- and cell type-specific manner (Chang et al. 2007, Rebbapragada & Lykke-Andersen 2009).
The characteristics that render a particular mRNA to become degraded by NMD are not yet fully understood, but several NMD-inducing contexts are known. In mammals, the best-characterized NMD triggering mRNA contains exon–exon junction located sufficiently downstream (50–55 nt) of a PTC (Lykke-Andersen & Jensen 2015). In addition, upstream open reading frames (uORFs) that are located 5′ of the main ORF and long 3′ untranslated regions (UTR) are known to induce NMD (Mendell et al. 2004, Singh et al. 2008, Yepiskoposyan et al. 2011). After target recognition, mRNA degradation can be initiated by several different mechanisms: endonucleolytic cleavage by SMG6, SMG5–SMG7-mediated recruitment of the CCR4-NOT complex to NMD targets, which induce their deadenylation-dependent decapping, or by direct deadenylation-independent decapping of an mRNA target (Lykke-Andersen & Jensen 2015). How different degradation pathways contribute to the total NMD activity is not yet understood, nor do we know whether certain pathways target distinct subpopulations of mRNAs for example in a tissue- or time-specific manner.
The current consensus is that NMD begins as a ribosome stalls in a favorable context and leads to the assembly of a multiprotein complex that includes an ATP-dependent RNA helicase up frameshift 1 (UPF1). UPF1 is a central component of NMD that can be phosphorylated by SMG1, which promotes the ability of UPF1 to activate mRNA decay (Yamashita et al. 2001, Amrani et al. 2004, Kurosaki et al. 2014, He & Jacobson 2015, Lykke-Andersen & Jensen 2015, Karousis et al. 2016). In addition to SMG1, the phosphorylation of UPF1 has been shown to require the presence of additional NMD factors, UPF2 and UPF3 (Kashima et al. 2006). Interestingly, two UPF3 paralogs, UPF3A and UPF3B, were recently shown to have antagonistic roles in NMD. While UPF3B is a potent NMD factor, UPF3A was shown to be a NMD repressor that can sequester UPF2 away from the EJC and the NMD machinery (Shum et al. 2016). Both UPF3A and UPF3B are highly expressed during spermatogenesis. However, while the expression of X-chromosomal UPF3B is downregulated in spermatocytes likely due to the meiotic sex chromosome silencing, the expression of the autosomal UPF3A peaks in these cells (Shum et al. 2016). In spermatocytes, where UPF3A is particularly abundantly expressed, the expression of UPF1 and UPF2 is upregulated as well (Bao et al. 2016). While we do not know the reason behind the expression alterations of different NMD components nor how NMD activity is regulated during different stages of spermatogenesis, it is tempting to speculate that they may reflect the unique requirements of transcriptome control in meiotic and postmeiotic cells.
The conditional knockout of Upf2 gene in male germ cells revealed the critical role of UPF2 in spermatogenesis (Bao et al. 2016). The deletion of Upf2 in fetal germ cells just before birth resulted in small testes with almost complete absence of all germ cells indicating that UPF2 is required for the development of prospermatogonia. When Upf2 was deleted in postnatal spermatogonia, spermatogenesis progressed to meiotic and postmeiotic phases. However, a massive depletion of both spermatocytes and spermatids was observed, which resulted in the absence of spermatozoa in the epididymis. Interestingly, the deletion of NMD repressor Upf3a, but not Upf3b, led to spermatogenic failure in mice (Shum et al. 2016). Therefore, the correct balance between the NMD factors and NMD repressors appears to be important for the proper progression of spermatogenesis. Interestingly, UPF1, UPF2, UPF3B and SMG1 all localize to the CB and the CB-localized UPF1 has been shown to be at least partially phosphorylated (Meikar et al. 2014, Bao et al. 2016, Shum et al. 2016, MacDonald & Grozdanov 2017) (our unpublished observation). However, although the studies by Shum and coworkers and Bao and coworkers reported the essential role of NMD in spermatogenesis, the importance of CB-localization for the function of these NMD factors remains to be investigated (Bao et al. 2016, Shum et al. 2016).
Important support for the involvement of CB in the NMD pathway comes from a study by Fanourgakis and cowkorkers (Fanourgakis et al. 2016). These investigators studied Tdrd6 mutant mice, that were previously shown to exhibit developmental arrest in haploid germ cells (Vasileva et al. 2009). TDRD6 is essential for the formation of the CB because in Tdrd6 mutant mice, the CB morphology is disrupted. Therefore, Tdrd6-null germ cells provide a valuable model to study the in vivo role of the CB and importance of the proper CB structure in biological pathways and processes. In the absence of TDRD6, UPF1, but not UPF2, failed to localize to the remnants of the CB. Furthermore, the UPF1/UPF2 interaction was shown to be largely affected in Tdrd6-null testes. Considering the known dependence of UPF1 helicase activity on the presence of UPF2, NMD is likely to be significantly defected in these mice. The analysis of UPF2 mutant mice revealed that long 3′ UTR-containing transcripts derived from ubiquitous genes were upregulated in the absence of UPF2, while the levels of PTC-containing transcripts were not affected (Bao et al. 2016). Importantly, the transcriptome analysis of Tdrd6-null germ cells were in accordance with these results, showing that the long 3′ UTR-stimulated NMD pathway was in fact impaired in these cells, likely due to disrupted interactions within the NMD machinery (Fanourgakis et al. 2016). Considering these results, it is possible that NMD in male germ cells preferably targets long 3′ UTR-containing transcripts.
The active role of the CB in NMD is supported by the presence of NMD factors and, on the other hand, the absence of the NMD repressor UPF3A. This model comes with the caveat that NMD is a translation-dependent pathway and the CB appears not to be associated with an active translation apparatus (Kleene & Cullinane 2011). One intriguing hypothesis is that the UPF3A-mediated NMD repression in spermatocytes and round spermatids is not complete or that UPF3A in fact represses only certain branches of the NMD pathway. If so, mRNAs that have been recognized by NMD factors during translation in the cytoplasm could be discarded from the translation machinery and, still bound to NMD factors, targeted to the CB for degradation. This model is supported by the localization of the endonuclease SMG6 to the CB (Meikar et al. 2014). Altogether, the accumulating evidence suggests that active NMD-mediated RNA regulation takes place in germ cells and that dynamic germ granules such as the CB are central for these processes.
Interplay of the chromatoid body with other cellular compartments
The CB is not a stagnant granule but rather a dynamic structure with its shape under continuous reformations, and its cellular location continuously changing (Parvinen & Jokelainen 1974, Parvinen & Parvinen 1979, Parvinen et al. 1997, Parvinen 2005). The movement of the CB has been shown to be microtubule dependent and can be disturbed by microtubule-depolymerizing drugs, which also cause CB fragmentation (Ventela et al. 2003, Da Ros et al. 2017). One possible link between the microtubules and the CB could be the microtubule-binding kinesin motor protein, KIF17b, which accumulates in the CB (Kotaja et al. 2006). The CB is usually located just next to the nucleus where it continuously moves along the nuclear envelope and frequently makes contacts with it. There seems to be active transfer of materials between the nucleus and the CB, which is also supported by the observation that nuclear pores tend to be more concentrated in the area adjacent to the CB (Fawcett et al. 1970, Soderstrom & Parvinen 1976, Da Ros et al. 2015). While moving, the CB constantly sends out and receives small particles, and it has been thought to be involved in the transport of RNAs or other components in the cytoplasm (Soderstrom & Parvinen 1976, Parvinen & Parvinen 1979). During later steps of round spermatid development, the CB often dissociates from the nuclear envelope and moves more widely in the cytoplasm. Sometimes the CB can be found in association with cytoplasmic bridges, and it has been shown that the CB-derived particles can even be transported from one haploid cell to another through the bridges (Ventela et al. 2003).
The CB is not a membrane-bound organelle, but interestingly, it appears to communicate closely with the endomembrane system. It frequently associates with the Golgi complex and the multivesicular bodies (MVBs). In addition, it is always surrounded by small vesicle structures that are also seen embedded inside the lobes of the CB (Da Ros et al. 2015). The abundance of vesicles associated with the CB is even more prominent in elongating spermatids where the late CB is completely covered with vesicles (Fujii et al. 2016). Recently, these CB-associated vesicles were shown to be involved in the autophagy-lysosomal pathway (Fujii et al. 2016, Da Ros et al. 2017). Autophagy (‘to eat oneself’ from Greek) is a conserved mechanism where various macromolecules and organelles are delivered to lysosomes for degradation (Mizushima & Komatsu 2011). A double-membrane structure, the autophagosome, forms to sequester a part of the cytosol that subsequently fuses with a lysosome. The vesicles associated with the CB contain autophagosomal and lysosomal markers, and the induction of autophagy leads to the dramatic increase in the accumulation of lysosomes onto the CB (Da Ros et al. 2017). Therefore, autophagy seems to provide additional catabolic activity for the CB-dependent processes. Autophagy could, for example, be involved in the assembly and clearance of the CB material in an analogous way that has been demonstrated for stress granules that are stress-responsive somatic RNP granules (Buchan et al. 2013, Ryu et al. 2014, Seguin et al. 2014).
Interestingly, a CB component FYCO1 (FYVE and coiled-coil domain-containing protein 1) was shown to be responsible for the recruitment of autophagosomes and lysosomes onto the CB (Da Ros et al. 2017). FYCO1 binds phosphatidylinositol 3-phosphate (PtdIns3P) and MAP1LC3B/LC3B (microtubule-associated proteins 1A/1B light chain 3B) that is anchored on the autophagosomal membrane (Pankiv et al. 2010, Olsvik et al. 2015). FYCO1 has been shown to participate in the plus end-directed transport of autophagosomes along microtubules and is also implicated in various processes connected with the maturation and formation of phagophores/lysosomes in somatic cells (Pankiv et al. 2010, Mrakovic et al. 2012, Ma et al. 2014, Raiborg et al. 2015). In male germ cells, FYCO1 was shown to localize in the peripheral regions of the CB (Da Ros et al. 2017). Interestingly, a germline-specific deletion of the Fyco1 gene in mice resulted in the complete absence of CB-associated vesicles and the fragmentation of the CB (Da Ros et al. 2017). The defective vesicle recruitment did not appear to have a marked effect in the round spermatid transcriptome or to cause any spermatogenic problem since Fyco1-knockout males were fertile. While the exact role of autophagy in the regulation of CB homeostasis remains to be characterized, these studies revealed an intriguing functional interplay between different catabolic processes in haploid male germ cells.
Future perspectives
Germ granules provide a powerful means to concentrate different RNA regulatory pathways in discrete cytoplasmic foci thereby maximizing the accuracy and effectiveness of RNA regulation. Though germ granules are under intense scrutiny and recent research has revealed much about their molecular composition and function, exact mechanistic details are still lacking. Furthermore, the molecular and functional relationship of different types of germ granules requires further clarification. The best-characterized germ granules are the IMC and the CB that seem to have distinct functions in the piRNA pathway. The primary processing of piRNAs is likely to take place in the IMC while the role of the CB seems to be associated with the processes downstream of piRNA processing. In addition, the CB is packed with the components of the NMD pathway that act by recognizing and degrading specific mRNA targets. The presence of both the piRNA and the NMD pathway in the CB suggests that germ granules may function as sites for active RNA degradation. However, it is not known if any functional cooperation between these pathways exists. Furthermore, it is still unclear whether the piRNA and the NMD pathways need to locate to germ granules in order to function properly or whether germ granules in fact form as a consequence of the high activity of these pathways in germ cells.
All in all, existing evidence supports the immense importance of germ granule-associated activities in male germ cell differentiation and the maintenance of male fertility. Interestingly, in addition to their essential role in the control of spermatogenesis, germline-derived non-coding RNAs such as germ granule-accumulating piRNAs have been implicated in RNA-mediated epigenetic inheritance of acquired traits. Future studies will be required to uncover the potential role of germ granules in epigenetic inheritance-related processes, for example in the preparation of the pool of RNAs that is retained in spermatozoa and transmitted to next generations in fertilization.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work was supported by the Academy of Finland, the Sigrid Jusélius Foundation and Turku Doctoral Programme in Molecular Medicine.
Acknowledgements
The authors thank all the Kotaja group members for their insights and critical reading of the manuscript.
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