REPRODUCTIONREVIEW

Germ granule-mediated RNA regulation in male germ cells

Tiina Lehtiniemi and Noora Kotaja Institute of Biomedicine, University of Turku, Turku, Finland Correspondence should be addressed to N Kotaja; Email: [email protected]

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. Reproduction (2018) 155 R77–R91

Introduction then rapidly undergo the second meiotic division (meiosis II) resulting in haploid spermatids. The final Spermatogenesis is a highly specialized process that phase of spermatogenesis is haploid differentiation aims to transmit correct paternal genetic and epigenetic (spermiogenesis), which includes dramatic information to the next generation. At the embryonic morphological changes through which spermatozoa stage, the mammalian germ cell lineage is specified reach their dynamic sleek shape (Fig. 1A). After they are when primordial germ cells (PGCs) are separated from released from the seminiferous epithelium, spermatozoa somatic lineages. PGCs are a transient cell population pass through the epididymis where they undergo of which a part migrates to the genital ridges to initiate further maturation. germ cell differentiation. They become gonocytes Throughout male germ cell differentiation, non- (prospermatogonia) that rest at G0 phase until 3–4 days membrane-bound organelles packed with RNA and postpartum. They then resume mitotic divisions and RNA-binding proteins are known to appear in the become postnatal spermatogonial stem cells (SSC) that cytoplasm of germ cells (Eddy 1970, Parvinen 2005, are the source material for the cyclic production of sperm Chuma et al. 2009, Meikar et al. 2011). These RNP for the entire duration of a male’s reproductive age. granules, better known as germ granules, are present Upon a specific signal, SSCs enter the differentiation in the germline of most if not all sexually reproducing pathway that begins with the mitotic proliferation of metazoans. They are known to participate in the type A, intermediate and type B spermatogonia. The regulation of RNA localization, stability and translation, final mitotic division of type B spermatogonia produces as well as in the production and function of small primary spermatocytes that subsequently undergo non-coding RNAs and in the control of chromatin meiosis. The prophase of meiosis I is the longest phase organization (Voronina et al. 2011). Different types of of meiosis. During the leptotene stage of prophase germ granules have been described in several organisms I, chromosomes condense. Then, synapsis between with diverse nomenclature. Germ granules are known homologous chromosomes begins at the zygotene stage. as germinal granules in Xenopus laevis, polar granules The formation of synaptonemal complex facilitates or nuage in Drosophila melanogaster and P granules synapsis by holding the paired chromosomes together. in Caenorhabditis elegans. In these organisms, germ At the pachytene stage, synapsis is completed and granules contain maternal mRNAs required for germ crossing over can occur until the diplotene stage when cell specification and direct the timing of mRNA the synaptonemal complex degenerates. This is followed translation to promote the establishment of the germline by the completion of the first meiotic division and the in the early embryo (Leatherman & Jongens 2003). separation of the homologous chromosomes between In contrast, the most prominent mammalian germ two cells. The newly formed secondary spermatocytes granules appear in the male germline long after the

© 2017 Society for Reproduction and Fertility https://doi.org/10.1530/REP-17-0356 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/26/2021 08:49:31AM via free access

10.1530/REP-17-0356 R78 T Lehtiniemi and N Kotaja germ lineage has been established, and therefore, are Chuma et al. 2009, Meikar et al. 2011). Just before meiotic not functioning in the germline specification but rather divisions, CB precursor granules are disintegrated, in male germ cell differentiation. Nevertheless, germ but aggregate again into larger (0.5 µm) structures in granules in different species share many homologous secondary spermatocytes. After meiosis in step 1 round components indicating that they all function in RNA spermatids, these granules aggregate to form a single big regulation and are likely to share similar mechanisms (~1 µm) CB (Fig. 1B). Mitochondria are dispersed during of action (Chuma et al. 2009). In this review article, we and after meiotic divisions, and the IMC is no longer will mainly focus on the best-characterized mammalian detectable in haploid cells. germ granules: the chromatoid body (CB) in haploid The CB can be detected during all steps of round spermatids and the intermitochondrial cement (IMC) in spermatid differentiation (steps 1–8). The largest CBs are pachytene spermatocytes. 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 Morphological features of germ granules et al. 1970, Parvinen 2005). Although the CB size is The CB was the first germ granule discovered likely due dramatically reduced at the end of the round spermatid to its unusually large size, which makes it detectable phase, CB-like structures are still observed in elongating even with light microscopy (von Brunn 1876, Yokota spermatids. During spermatid elongation, this so-called 2008). Early light microscopic studies on the cytoplasm ‘late CB’ splits into two separate structures. One of these of spermatogenic cells identified granules that stain CB fragments forms a dense sphere that is discarded deeply with basic dyes. ‘Protoplasmaanhäufungen’ with most of the cytoplasm in the residual body (Fawcett was a name given to these granules in rat spermatids et al. 1970, Susi & Clermont 1970, Shang et al. 2010). described by von Brunn as early as 1876 (von Brunn The second CB fragment forms a ring around the basis of 1876). In 1891, Benda observed similar granules in the flagellum, and then moves together with the annulus guinea pig spermatocytes and called them ‘chromatoide (a ringed barrier structure between the middle piece Nebenkörper’ (Benda 1891). In 1901, Regaud named and principal piece of the sperm tail) distally along the the granules he observed in rat spermatids as ‘corps flagellum. Behind the migrating ring-shaped late CB, the chromatoides’ (Regaud 1901). In 1955, the structures mitochondria become associated with the axoneme. It were studied by electron microscopy for the first time has been suggested that the late CB is indeed involved the in Felis domestica, and an irregular mass of osmiophilic formation of the mitochondrial sheath in the midpiece granular material near the Golgi was termed ‘chromatic of the sperm tail (Fawcett et al. 1970, Shang et al. 2010). body’ (Burgos & Fawcett 1955). Under electron In step 16 elongating spermatids, the CB is no longer microscopy, the CB is a conspicuous, intensively stained visible, but the details of the timing and mechanisms of structure of highly irregular shape (Fig. 1B). As Fawcett the CB disappearance are not yet understood (Fig. 1A). and coworkers described in 1970, ‘the CB appears to In addition to the IMC and CB, Russell and Frank be composed of exceedingly thin filaments that are (1978) described distinct types of germ granules in consolidated into a compact mass or into dense strands spermatogenic cells of rat (Russell & Frank 1978, Yokota of varying thickness that branch and anastomose to form 2012). In pachytene spermatocytes, 70- to 90-nm an irregular network’ (Fawcett et al. 1970). particles are present at stages VIII–XII of the seminiferous The use of electron microscopy enabled the epithelial cycle both close to the nuclear envelope and identification of other types of germ granules that scattered in the cytoplasm. Clusters of 30-nm particles, are less pronounced in size. The IMC appears as an whose profile is similar to that of free ribosomes, are few electron-dense, CB-like material that is found among in number and observed in pachytene spermatocytes clusters of mitochondria in many cell types during during meiosis. Irregularly shaped perinuclear granules male germ cell differentiation, including in fetal (ISPGs; also known as clusters of 60- to 90-nm particles) prospermatogonia, postnatal spermatogonia and mid- are also observed in pachytene spermatocytes at stage to-late pachytene spermatocytes (Fig. 1B) (Fawcett VIII–XII. They disappear during meiotic divisions, but a et al. 1970, Russell & Frank 1978, Yokota 2012). IMC few particles adhere to mitochondria that seem to be is particularly evident in pachytene spermatocytes. distributed to daughter cells. Satellite bodies (SB; also In very late pachytene spermatocytes, another type of known as sponge bodies) appear as a network of fibrils germ granule appears and co-exists for a short time with overlaid by denser patches of amorphous material, and the IMC. These small granules are associated with the they can be detected in pachytene spermatocytes and nuclear envelope and intermingled with small vesicles, also closely associated and contiguous with the CB. All but not with mitochondria. They are thought to provide these granules are poorly characterized at the molecular the precursor material for the CB, therefore, often level, but they seem to share some components with the named ‘CB precursors’ (Fig. 1A) (Russell & Frank 1978, CB and the IMC (Yokota 2012).

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Figure 1 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. Germ granules and the requirements of RNA are governed by transcriptional, posttranscriptional and regulation in male germ cells epigenetic mechanisms (Kimmins et al. 2004, Kimmins & Sassone-Corsi 2005). The progress of spermatogenesis Male germ cell differentiation relies on spatially and is accompanied by the orchestrated waves of gene temporally regulated gene expression programs that expression that generate specific transcriptome profiles www.reproduction-online.org Reproduction (2018) 155 R77–R91

Downloaded from Bioscientifica.com at 09/26/2021 08:49:31AM via free access R80 T Lehtiniemi and N Kotaja for each differentiating cell type (Chalmel et al. 2007, coordination of the complex transcriptome of haploid Laiho et al. 2013). These cell type-specific transcripts male germ cells (Meikar et al. 2014). not only include mRNAs and their isoforms, hundreds of them being testis-specific (Chalmel & Rolland 2015), Molecular composition of germ granules but also a diverse set of non-coding RNAs that have also been shown to be differentially regulated during Our understanding of the molecular features of spermatogenesis (Bao et al. 2013a, Laiho et al. 2013, Sun germ granules, particularly the CB and the IMC has et al. 2013). In fact, male germ cells, particularly meiotic dramatically increased during recent years. Several spermatocytes and postmeiotic round spermatids, have mutant mouse models deficient in genes encoding an exceptionally diverse transcriptome containing germ granule components have been created, each a large number of different non-coding RNAs that of them providing important insight into the function mostly derive from uncharacterized, poorly conserved and importance of germ granules (Fig. 2). In fact, intergenic regions (Soumillon et al. 2013). It is currently the loss-of-function mutation of germ granule core unclear if these non-coding genomic regions are components generally leads to spermatogenesis defects transcribed for a reason or whether their transcription and compromised male fertility, strongly supporting the is a secondary consequence of the dramatic chromatin critical role that germ granules have in postnatal male remodeling taking place during spermatogenesis. Either germ cell differentiation. way, the control of this complex transcriptome requires Many of the protein components of germ granules special posttranscriptional mechanisms that ensure that are evolutionarily conserved. For example, a DEAD- all transcripts meet their intended fate. box RNA helicase DDX4/MVH/VASA (DEAD (Asp-Glu- A notable posttranscriptional challenge is faced in Ala-Asp) box polypeptide 4) is consistently found in all late steps of spermatogenesis when the chromatin of germ granules, including polar granules in Drosophila elongating spermatids condenses. In these cells, most and all forms of germ granules in mouse germ cells ( histones are replaced first by transition proteins and Chuma et al. 2009). Other examples of conserved germ subsequently by protamines, which enables genomic granule components include some of the Tudor domain- material to become particularly tightly packed. This containing proteins, MAEL (homolog to Drosophila compaction of chromatin effectively halts transcription. maelstrom) and PIWI (P-element-induced wimpy testis) The final haploid differentiation phase is characterized proteins. The conservation of these proteins implies by major changes in cellular morphology, including that they have a central role in germ granules and the construction of sperm-specific structures such as germ cells. Interestingly, loss-of-function mutations of the acrosome and the flagellum. Because condensing these components have clearly demonstrated that germ spermatids are transcriptionally incapacitated, these granules have distinct biological roles in different species; genes are transcribed earlier and translationally while the loss-of-function mutations of vasa, tudor and repressed until needed. It has been shown that majority mael in Drosophila show defects in germline formation of meiotic and postmeiotic mRNAs are transcribed and female fertility (Boswell & Mahowald 1985, Raz without immediate translation during spermatogenesis 2000, Findley et al. 2003), mutations of their homologs (Kleene & Kleene 2003, Idler & Yan 2012). For example, Ddx4 or Tdrd1 or Mael in mice cause fertility defects mRNAs for transition proteins (Tnp1 and Tnp2) and only in males even though the genes are expressed in protamines (Prm1 and Prm2) are transcribed already in both male and female germlines (Tanaka et al. 2000, late pachytene spermatocytes, but are translated only Chuma et al. 2006, Soper et al. 2008). Furthermore, during elongation, about a week later in mice (Kleene in Drosophila, Piwi is required for the maintenance of 1989, Fajardo et al. 1997). both male and female germline stem cells, while mouse The fate of RNAs is controlled mainly by their PIWI family genes PIWIL1/MIWI and PIWIL2/MILI are association with RNA-binding proteins that bind their essential only for the postnatal germ cell differentiation targets either non-specifically or through specific motifs in males (Deng & Lin 2002, Kuramochi-Miyagawa to form ribonucleoprotein (RNP) complexes (Ascano et al. 2004). et al. 2013). High requirements for posttranscriptional The full molecular composition of the CB was control during spermatogenesis are indeed reflected by a recently discovered. The successful isolation and mass high number of RNA-binding proteins expressed in male spectrometric analysis of murine CBs revealed a list of germ cells (Kleene & Kleene 2003, Paronetto & Sette around 90 CB-localized proteins (Fig. 3) (Meikar et al. 2010, Idler & Yan 2012). RNP complexes can form larger 2010, 2014, Meikar & Kotaja 2014). The majority of the RNP granules that have the ability to compartmentalize CB components are RNA-binding proteins and other different RNA regulatory pathways. The largest RNP proteins involved in RNA regulation, which support granule, the CB, emerges in the cytoplasm of male its central role in the posttranscriptional control. The germ cells when the level and complexity of the gene CB proteins include ubiquitously expressed proteins expression is at its very peak. Therefore, it is tempting that are involved, for example, in pre-mRNA binding to think that the CB could have a pivotal role in the and processing, and also a substantial number of

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Figure 2 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. www.reproduction-online.org Reproduction (2018) 155 R77–R91

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Figure 3 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. germline-specific proteins. The most prominent species of RNAs. In fact, it has been shown that the molecular pathway localized in the CB is the piRNA morphology of the CB depends on active transcription. pathway (discussed below), and the most abundant This was demonstrated by inhibiting transcription proteins in the CB were shown to be the piRNA-binding with actinomycin D, which dramatically affected PIWI proteins MILI and MIWI and the Tudor domain- the appearance of the CB as observed by electron containing proteins TDRD1, TDRD6 and TDRD7, as microscopy (Parvinen et al. 1978). Furthermore, well as DEAD-box helicases DDX4 and DDX25 (Tanaka experiments tracing labeled RNAs in round spermatids et al. 2000, 2011, Deng & Lin 2002, Kuramochi- demonstrated that there is a constant flow of RNA into Miyagawa et al. 2004, Tsai-Morris et al. 2004, Chuma the CB, and the labeled RNA accumulates in the CB et al. 2006, Vasileva et al. 2009, Meikar et al. 2014). (Meikar et al. 2014). The RNA sequencing of isolated The CB contains several different Tudor domain- CBs revealed that the CB retains a considerable portion containing proteins, including TDRD1, TDRD3, of the round spermatids mRNA transcriptome (Meikar TDRD5, TDRD6, TDRD7, RNF17 and STK31 (Pan et al. et al. 2014). In addition to mRNAs, the CB accumulates 2005, Chuma et al. 2006, Vasileva et al. 2009, Tanaka piRNAs and piRNA precursors, as well as thousands et al. 2011, Yabuta et al. 2011, Bao et al. 2013b, Meikar of non-annotated intergenic non-coding transcripts. et al. 2014, Zhou et al. 2014). Considering the well- However, it is currently unclear how RNAs are targeted characterized role of the Tudor proteins as molecular to the CB. De novo motif prediction revealed a presence scaffolds (Pek et al. 2012) and given the disrupted of possible CB-exclusion signals (Meikar et al. 2014), CB morphology in the absence of TDRD6 or TDRD7 but the details of the CB-targeting mechanisms remain (Vasileva et al. 2009, Tanaka et al. 2011), it is likely to be characterized. that Tudor proteins serve as a structural mesh for the In contrast to the CB, the full molecular composition CB architecture. Tudor proteins are known to interact of the IMC has not been identified. Only a few proteins with proteins that contain symmetric dimethylarginines have been shown to localize to the IMC and some of (Kirino et al. 2009), a modification found to be enriched them, including TDRD1, TDRD6 and MILI, localize in the CB (Vagin et al. 2009). Among other proteins to the CB as well. However, some proteins appear to that contain this modification, PIWI proteins are known be IMC specific (Chuma et al. 2006, Hosokawa et al. binding targets of Tudor proteins, and Tudor proteins 2007, Ma et al. 2009, Bao et al. 2013b, Zhou et al. could therefore have a critical role in their recruitment 2014). For example, GASZ (germ cell-specific protein to germ granules (Vagin et al. 2009, Siomi et al. 2010). with four Ankyrin repeats, a sterile alpha motif and a As can be predicted from the CB protein composition basic leucine Zipper domain), localizes predominantly and in particular from the presence of a large number to the IMC (Ma et al. 2009), which is largely explained of RNA-binding proteins, the CB accumulates various by its functional mitochondrial targeting signal and its

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Downloaded from Bioscientifica.com at 09/26/2021 08:49:31AM via free access Germ granule-mediated RNA regulation R83 function in promoting mitofusion (Zhang et al. 2016). by an enzyme called trimmer with the help of Papi Another mitochondria-interacting IMC protein is PLD6/ and to take place on mitochondrial membranes (Izumi MitoPLD, a phospholipase that facilitates mitofusion, et al. 2016). In mouse, the 3′ trimming of piRNAs participates in nuage formation and piRNA biosynthesis is predicted to follow a similar route and in fact, the during spermatogenesis (Huang et al. 2011, Watanabe mouse Papi homolog TDRD2/TDRKH was shown to et al. 2011). The structural and functional relationship be required for the production of correct size piRNAs between the IMC and the CB is still unclear, even though (Saxe et al. 2013). piRNA biogenesis is also dependent their similar protein composition indicates that they may on mitochondrial membrane proteins GPAT2 and GASZ share similar functions. Some studies have even suggested (Ma et al. 2009, Shiromoto et al. 2013, Zhang et al. that the CB could originate from the IMC. However, 2016). Trimmed piRNAs are finally ′2 -O-methylated at TDRD1-null mice lack the IMC in spermatocytes, and their 3′ ends by HENMT1 resulting in primary piRNAs yet, the CB can be clearly detected in round spermatids with a predominant preference for a 5′ uridine (Horwich (Chuma et al. 2006), thus demonstrating that the IMC is et al. 2007, Saito et al. 2007, Lim et al. 2015). not a fundamentally important source or a precursor for In the ping-pong amplification cycle, piRNAs are the CB. amplified by PIWI proteins to silence transposon expression, and therefore, to protect the genome integrity by preventing harmful transposon insertions (Siomi Germ granules as platforms for piRNA pathway et al. 2011). The ping-pong amplification mechanism is The piRNA pathway is one of the main molecular particularly predominant in fetal male germ cells that are pathways localized to germ granules, and the production challenged by the global resetting of the male germline of piRNAs has been linked to germ granules in mouse and epigenome, which causes an induction of the transposon lower organisms (Hirakata & Siomi 2016). piRNAs are a expression. To initiate the amplification cycle in mice, unique class of small (26–31 nucleotides) non-coding MILI associates with primary piRNAs and operates in RNAs that form piRNP complexes with PIWI proteins. the ping-pong cycle together with other factors to yield A functional piRNA pathway is required for normal antisense secondary piRNAs that in turn associate with spermatogenesis and mutation of piRNA pathway genes MIWI2 (De Fazio et al. 2011, Pandey et al. 2013, Yang in mice, including the genes for PIWI proteins MIWI, et al. 2015). MIWI2 then translocates to the nucleus MILI and MIWI2, as well as other proteins necessary for where it initiates transcriptional transposon silencing piRNA production or function, leads to male sterility via DNA methylation at the target loci (Aravin et al. (Deng & Lin 2002, Kuramochi-Miyagawa et al. 2004, 2008, Kuramochi-Miyagawa et al. 2008, De Fazio et al. Carmell et al. 2007, Weick & Miska 2014). Two different 2011). Therefore, piRNA amplification cycle functions piRNA biogenesis pathways exist. One produces on transposon silencing both by directly detecting primary piRNAs from genomic clusters. These piRNAs and slicing transposon sequences, and by silencing operate as silencing triggers with multiple functions transposon expression at the transcriptional level. including keeping resident mobile elements repressed During mammalian spermatogenesis, two populations and switching off genic transcripts after meiosis. The of piRNAs are distinguished and named as pre-pachytene second is the adaptive pathway that is used to amplify and pachytene piRNAs according to the developmental piRNAs by a so-called ping-pong cycle. This produces timing of their appearance (Weick & Miska 2014). Pre- secondary piRNAs that function by repressing active pachytene piRNAs are co-expressed and associate with transposons (Czech & Hannon 2016). MILI and MIWI2 in fetal male germ cells and in early For primary processing, piRNAs are transcribed postnatal germ cells. As mentioned earlier, these piRNAs from the genome as long single-stranded piRNA are amplified by the ping-pong cycle and are critical for precursor transcripts that are subsequently transported transposon silencing. Pachytene piRNAs, on the other to the cytoplasm for further processing. Mitochondria- hand, are co-expressed in pachytene spermatocytes anchored endonuclease PLD6/MITOPLD first cleaves and round spermatids with MILI and MIWI, and they piRNA precursor transcripts to produce the 5′ ends mainly associate with MIWI. The sequence diversity of piRNAs (Huang et al. 2011, Watanabe et al. 2011, of pachytene piRNAs is remarkably high (Aravin et al. Ipsaro et al. 2012, Nishimasu et al. 2012). MOV10L1, 2006, Girard et al. 2006, Grivna et al. 2006). They a testis-specific RNA helicase, supports the piRNA are produced in vast quantities from discrete genomic primary processing by recognizing and resolving loci, pachytene piRNA clusters. The transcription of the secondary structures within the piRNA precursors piRNA clusters is regulated by a conserved transcription (Zheng et al. 2010, Vourekas et al. 2015, Fu et al. 2016). factor A-MYB in mice (Li et al. 2013). A BTB domain- piRNA intermediate transcripts are then loaded on PIWI containing protein BTB18 was also shown to participate proteins that stabilize their 5′ ends. Subsequently, the 3′ in the expression of pachytene piRNA precursors by ends have to be trimmed. This final trimming includes promoting transcription elongation (Zhou et al. 2017). endonucleolytic cleavage as well as 3′–5′ exonuclease Although MILI and MIWI have been shown to be activity that in silkworm has been shown to be mediated capable of engaging in the ping-pong cycle in meiotic www.reproduction-online.org Reproduction (2018) 155 R77–R91

Downloaded from Bioscientifica.com at 09/26/2021 08:49:31AM via free access R84 T Lehtiniemi and N Kotaja cells (Wasik et al. 2015), the general analysis of and acting therefore as an adhesive trap that captures pachytene piRNAs in adult testes do not show strong mRNAs to the germ plasm (Vourekas et al. 2016). It ping-pong signatures (Beyret et al. 2012). Therefore, the remains to be characterized whether the CB-localized ping-pong cycle seems to be rather inactive in these MIWI- or MILI-loaded piRNAs could share a similar cells and pachytene piRNAs are produced mainly by function in tethering mRNAs. the primary processing pathway. In fact, a Tudor protein RNF17 has been shown to repress secondary piRNA Chromatoid body and nonsense-mediated amplification in adult gonads, and the deletion of RNF17 in mice unleashed ping-pong during meiosis and mRNA decay caused aberrant production of piRNAs and degradation In addition to the piRNA pathway, the CB also harbors of genic transcripts (Wasik et al. 2015). Pachytene components of another RNA regulatory pathway, known piRNAs were originally reported to be depleted from as the nonsense-mediated mRNA decay (NMD) (Meikar transposon sequences (Girard et al. 2006). However, et al. 2014). Historically NMD has been perceived as a it has later been demonstrated that young, potentially quality control system that evolved to eliminate aberrant, active elements are enriched in pachytene piRNA premature termination codon (PTC)-containing mRNAs. clusters. These elements show a bias in orientation to However, recently, it has become clear that in addition enable the production of antisense piRNAs that play a to its quality control function, NMD participates in role in suppressing transposons during meiosis (Reuter regulating and fine-tuning gene expression by targeting et al. 2011, Hirano et al. 2014, Wasik et al. 2015). In many seemingly ‘normal’ mRNAs that code for full-length addition to the transposon silencing, pachytene piRNAs functional proteins. Therefore, this pathway can influence have been shown to be involved in the degradation of a wide variety of biological processes potentially in a mRNAs and lncRNAs in late meiotic and haploid cells tissue- and cell type-specific manner (Chang et al. 2007, (Reuter et al. 2011, Goh et al. 2015, Gou et al. 2015, Rebbapragada & Lykke-Andersen 2009). Watanabe et al. 2015, Zhang et al. 2015). The characteristics that render a particular mRNA Germ granules provide important platforms for to become degraded by NMD are not yet fully the biosynthesis and function of piRNAs. In fetal understood, but several NMD-inducing contexts are prospermatogonia, MILI and MIWI2 localize to the known. In mammals, the best-characterized NMD germ granules that cooperate in the production and triggering mRNA contains exon–exon junction located amplification of piRNAs; IMC-resembling pi-bodies sufficiently downstream (50–55 nt) of a PTC (Lykke- that contain MILI and TDRD1, and processing body- Andersen & Jensen 2015). In addition, upstream open resembling piP-bodies that contain MIWI2, TDRD9, reading frames (uORFs) that are located 5′ of the main MAEL and signature components of processing bodies ORF and long 3′ untranslated regions (UTR) are known (Aravin et al. 2009, Shoji et al. 2009). In postnatal to induce NMD (Mendell et al. 2004, Singh et al. 2008, meiotic cells, the IMC is likely to function as a site Yepiskoposyan et al. 2011). After target recognition, for pachytene piRNA primary processing. This is mRNA degradation can be initiated by several supported by its close association with mitochondria different mechanisms: endonucleolytic cleavage by and the localization of many proteins involved in SMG6, SMG5–SMG7-mediated recruitment of the piRNA processing, including MILI, MITOPLD and CCR4-NOT complex to NMD targets, which induce GASZ, to the IMC. In contrast to the IMC, the CB is not their deadenylation-dependent decapping, or by associated with mitochondria (Fawcett et al. 1970), direct deadenylation-independent decapping of and consequently, it does not contain the key piRNA- an mRNA target (Lykke-Andersen & Jensen 2015). processing proteins, such as MITOPLD that localizes How different degradation pathways contribute to to mitochondrial membranes. Therefore, the CB’s role the total NMD activity is not yet understood, nor do in the piRNA pathway appears to be unrelated to the we know whether certain pathways target distinct biosynthesis of piRNAs. The current model is that the subpopulations of mRNAs for example in a tissue- or initial steps of pachytene piRNA processing occur in time-specific manner. mitochondria-associated germ granules such as the The current consensus is that NMD begins as a IMC, and the PIWI protein-loaded piRNAs are then ribosome stalls in a favorable context and leads to the transferred to the CB for downstream action (Fig. 4). assembly of a multiprotein complex that includes an The potential role of the CB in piRNA-mediated target ATP-dependent RNA helicase up frameshift 1 (UPF1). recognition or degradation is supported by the high UPF1 is a central component of NMD that can be accumulation of piRNAs and, on the other hand, phosphorylated by SMG1, which promotes the ability the presence of a wide range of cellular transcripts of UPF1 to activate mRNA decay (Yamashita et al. 2001, in the CB. Interestingly, Aub-loaded piRNAs (Aub; Amrani et al. 2004, Kurosaki et al. 2014, He & Jacobson protein aubergine, one of the PIWI proteins in fly) 2015, Lykke-Andersen & Jensen 2015, Karousis et al. in the Drosophila germ plasm have been shown to 2016). In addition to SMG1, the phosphorylation use partial base-pairing to bind mRNAs randomly, of UPF1 has been shown to require the presence of

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Figure 4 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. additional NMD factors, UPF2 and UPF3 (Kashima The conditional knockout of Upf2 gene in male et al. 2006). Interestingly, two UPF3 paralogs, UPF3A germ cells revealed the critical role of UPF2 in and UPF3B, were recently shown to have antagonistic spermatogenesis (Bao et al. 2016). The deletion of roles in NMD. While UPF3B is a potent NMD factor, Upf2 in fetal germ cells just before birth resulted UPF3A was shown to be a NMD repressor that can in small testes with almost complete absence of all sequester UPF2 away from the EJC and the NMD germ cells indicating that UPF2 is required for the machinery (Shum et al. 2016). Both UPF3A and UPF3B development of prospermatogonia. When Upf2 was are highly expressed during spermatogenesis. However, deleted in postnatal spermatogonia, spermatogenesis while the expression of X-chromosomal UPF3B is progressed to meiotic and postmeiotic phases. downregulated in spermatocytes likely due to the However, a massive depletion of both spermatocytes meiotic sex chromosome silencing, the expression of and spermatids was observed, which resulted in the autosomal UPF3A peaks in these cells (Shum et al. the absence of spermatozoa in the epididymis. 2016). In spermatocytes, where UPF3A is particularly Interestingly, the deletion of NMD repressor Upf3a, abundantly expressed, the expression of UPF1 and UPF2 but not Upf3b, led to spermatogenic failure in mice is upregulated as well (Bao et al. 2016). While we do (Shum et al. 2016). Therefore, the correct balance not know the reason behind the expression alterations between the NMD factors and NMD repressors of different NMD components nor how NMD activity is appears to be important for the proper progression of regulated during different stages of spermatogenesis, it spermatogenesis. Interestingly, UPF1, UPF2, UPF3B is tempting to speculate that they may reflect the unique and SMG1 all localize to the CB and the CB-localized requirements of transcriptome control in meiotic and UPF1 has been shown to be at least partially postmeiotic cells. phosphorylated (Meikar et al. 2014, Bao et al. 2016, www.reproduction-online.org Reproduction (2018) 155 R77–R91

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Shum et al. 2016, MacDonald & Grozdanov 2017) Interplay of the chromatoid body with other (our unpublished observation). However, although cellular compartments the studies by Shum and coworkers and Bao and coworkers reported the essential role of NMD in The CB is not a stagnant granule but rather a dynamic spermatogenesis, the importance of CB-localization structure with its shape under continuous reformations, for the function of these NMD factors remains to be and its cellular location continuously changing (Parvinen investigated (Bao et al. 2016, Shum et al. 2016). & Jokelainen 1974, Parvinen & Parvinen 1979, Parvinen Important support for the involvement of CB in the et al. 1997, Parvinen 2005). The movement of the CB has NMD pathway comes from a study by Fanourgakis and been shown to be microtubule dependent and can be cowkorkers (Fanourgakis et al. 2016). These investigators disturbed by microtubule-depolymerizing drugs, which studied Tdrd6 mutant mice, that were previously also cause CB fragmentation (Ventela et al. 2003, Da Ros shown to exhibit developmental arrest in haploid germ et al. 2017). One possible link between the microtubules cells (Vasileva et al. 2009). TDRD6 is essential for the and the CB could be the microtubule-binding kinesin formation of the CB because in Tdrd6 mutant mice, motor protein, KIF17b, which accumulates in the CB the CB morphology is disrupted. Therefore, Tdrd6- (Kotaja et al. 2006). The CB is usually located just next null germ cells provide a valuable model to study the to the nucleus where it continuously moves along the in vivo role of the CB and importance of the proper nuclear envelope and frequently makes contacts with it. CB structure in biological pathways and processes. In There seems to be active transfer of materials between the absence of TDRD6, UPF1, but not UPF2, failed the nucleus and the CB, which is also supported by to localize to the remnants of the CB. Furthermore, the observation that nuclear pores tend to be more the UPF1/UPF2 interaction was shown to be largely concentrated in the area adjacent to the CB (Fawcett affected in Tdrd6-null testes. Considering the known et al. 1970, Soderstrom & Parvinen 1976, Da Ros et al. dependence of UPF1 helicase activity on the presence 2015). While moving, the CB constantly sends out and of UPF2, NMD is likely to be significantly defected in receives small particles, and it has been thought to be these mice. The analysis of UPF2 mutant mice revealed involved in the transport of RNAs or other components in the cytoplasm (Soderstrom & Parvinen 1976, Parvinen that long 3′ UTR-containing transcripts derived from ubiquitous genes were upregulated in the absence of & Parvinen 1979). During later steps of round spermatid UPF2, while the levels of PTC-containing transcripts development, the CB often dissociates from the nuclear were not affected (Bao et al. 2016). Importantly, the envelope and moves more widely in the cytoplasm. transcriptome analysis of Tdrd6-null germ cells were Sometimes the CB can be found in association with in accordance with these results, showing that the long cytoplasmic bridges, and it has been shown that the CB-derived particles can even be transported from one 3′ UTR-stimulated NMD pathway was in fact impaired in these cells, likely due to disrupted interactions haploid cell to another through the bridges (Ventela within the NMD machinery (Fanourgakis et al. 2016). et al. 2003). Considering these results, it is possible that NMD The CB is not a membrane-bound organelle, but interestingly, it appears to communicate closely with in male germ cells preferably targets long 3′ UTR- containing transcripts. the endomembrane system. It frequently associates The active role of the CB in NMD is supported by with the Golgi complex and the multivesicular bodies the presence of NMD factors and, on the other hand, (MVBs). In addition, it is always surrounded by small the absence of the NMD repressor UPF3A. This model vesicle structures that are also seen embedded inside the comes with the caveat that NMD is a translation- lobes of the CB (Da Ros et al. 2015). The abundance of dependent pathway and the CB appears not to be vesicles associated with the CB is even more prominent associated with an active translation apparatus (Kleene in elongating spermatids where the late CB is completely & Cullinane 2011). One intriguing hypothesis is that the covered with vesicles (Fujii et al. 2016). Recently, these UPF3A-mediated NMD repression in spermatocytes and CB-associated vesicles were shown to be involved in the round spermatids is not complete or that UPF3A in fact autophagy-lysosomal pathway (Fujii et al. 2016, Da Ros represses only certain branches of the NMD pathway. If et al. 2017). Autophagy (‘to eat oneself’ from Greek) is so, mRNAs that have been recognized by NMD factors a conserved mechanism where various macromolecules during translation in the cytoplasm could be discarded and organelles are delivered to lysosomes for from the translation machinery and, still bound to NMD degradation (Mizushima & Komatsu 2011). A double- factors, targeted to the CB for degradation. This model membrane structure, the autophagosome, forms to is supported by the localization of the endonuclease sequester a part of the cytosol that subsequently fuses SMG6 to the CB (Meikar et al. 2014). Altogether, the with a lysosome. The vesicles associated with the CB accumulating evidence suggests that active NMD- contain autophagosomal and lysosomal markers, and mediated RNA regulation takes place in germ cells and the induction of autophagy leads to the dramatic increase that dynamic germ granules such as the CB are central in the accumulation of lysosomes onto the CB (Da Ros for these processes. et al. 2017). Therefore, autophagy seems to provide

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Downloaded from Bioscientifica.com at 09/26/2021 08:49:31AM via free access Germ granule-mediated RNA regulation R87 additional catabolic activity for the CB-dependent the CB suggests that germ granules may function as processes. Autophagy could, for example, be involved sites for active RNA degradation. However, it is not in the assembly and clearance of the CB material in an known if any functional cooperation between these analogous way that has been demonstrated for stress pathways exists. Furthermore, it is still unclear whether granules that are stress-responsive somatic RNP granules the piRNA and the NMD pathways need to locate to (Buchan et al. 2013, Ryu et al. 2014, Seguin et al. 2014). germ granules in order to function properly or whether Interestingly, a CB component FYCO1 (FYVE and germ granules in fact form as a consequence of the coiled-coil domain-containing protein 1) was shown to high activity of these pathways in germ cells. be responsible for the recruitment of autophagosomes All in all, existing evidence supports the immense and lysosomes onto the CB (Da Ros et al. 2017). FYCO1 importance of germ granule-associated activities in binds phosphatidylinositol 3-phosphate (PtdIns3P) male germ cell differentiation and the maintenance and MAP1LC3B/LC3B (microtubule-associated of male fertility. Interestingly, in addition to their proteins 1A/1B light chain 3B) that is anchored on the essential role in the control of spermatogenesis, autophagosomal membrane (Pankiv et al. 2010, Olsvik germline-derived non-coding RNAs such as germ et al. 2015). FYCO1 has been shown to participate in granule-accumulating piRNAs have been implicated the plus end-directed transport of autophagosomes in RNA-mediated epigenetic inheritance of acquired along microtubules and is also implicated in various traits. Future studies will be required to uncover processes connected with the maturation and formation the potential role of germ granules in epigenetic of phagophores/lysosomes in somatic cells (Pankiv et al. inheritance-related processes, for example in the 2010, Mrakovic et al. 2012, Ma et al. 2014, Raiborg preparation of the pool of RNAs that is retained in et al. 2015). In male germ cells, FYCO1 was shown to spermatozoa and transmitted to next generations localize in the peripheral regions of the CB (Da Ros et al. in fertilization. 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 Declaration of interest (Da Ros et al. 2017). The defective vesicle recruitment did not appear to have a marked effect in the round The authors declare that there is no conflict of interest spermatid transcriptome or to cause any spermatogenic that could be perceived as prejudicing the impartiality of this review. 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 Funding between different catabolic processes in haploid male This work was supported by the Academy of Finland, the germ cells. Sigrid Jusélius Foundation and Turku Doctoral Programme in Molecular Medicine. Future perspectives Germ granules provide a powerful means to Acknowledgements concentrate different RNA regulatory pathways in The authors thank all the Kotaja group members for their discrete cytoplasmic foci thereby maximizing the insights and critical reading of the manuscript. accuracy and effectiveness of RNA regulation. Though germ granules are under intense scrutiny and recent research has revealed much about their molecular References composition and function, exact mechanistic details Amrani N, Ganesan R, Kervestin S, Mangus DA, Ghosh S & Jacobson A are still lacking. Furthermore, the molecular and 2004 A faux 3′-UTR promotes aberrant termination and triggers functional relationship of different types of germ nonsense-mediated mRNA decay. Nature 432 112–118. (https://doi. granules requires further clarification. The best- org/10.1038/nature03060) Aravin A, Gaidatzis D, Pfeffer S, Lagos-Quintana M, Landgraf P, Iovino N, characterized germ granules are the IMC and the CB Morris P, Brownstein MJ, Kuramochi-Miyagawa S, Nakano T et al. 2006 that seem to have distinct functions in the piRNA A novel class of small RNAs bind to MILI protein in mouse testes. Nature pathway. The primary processing of piRNAs is likely to 442 203–207. (https://doi.org/10.1038/nature04916) Aravin AA, Sachidanandam R, Bourc’his D, Schaefer C, Pezic D, Toth KFKF, take place in the IMC while the role of the CB seems Bestor T & Hannon GJ 2008 A piRNA pathway primed by individual to be associated with the processes downstream of transposons is linked to de novo DNA methylation in mice. Molecular piRNA processing. In addition, the CB is packed with Cell 31 785–799. (https://doi.org/10.1016/j.molcel.2008.09.003) the components of the NMD pathway that act by Aravin AA, van der Heijden GW, Castaneda J, Vagin V V, Hannon GJ & Bortvin A 2009 Cytoplasmic compartmentalization of the fetal piRNA recognizing and degrading specific mRNA targets. The pathway in mice. PLoS Genetics 5 e1000764. (https://doi.org/10.1371/ presence of both the piRNA and the NMD pathway in journal.pgen.1000764)

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