Germ Granule-Mediated RNA Regulation in Male Germ Cells

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

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