REPRODUCTIONREVIEW

Regulation of long non-coding RNAs and circular RNAs in spermatogonial stem cells

Fan Zhou2, Wei Chen1, Yiqun Jiang1 and Zuping He1,2

1Hunan Normal University School of Medicine, Changsha, Hunan, China and 2Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

Correspondence should be addressed to Z He; Email: zupinghe@hunnu.edu.cn

Abstract

Spermatogonial stem cells (SSCs) are one of the most significant stem cells with the potentials of self-renewal, differentiation, transdifferentiation and dedifferentiation, and thus, they have important applications in reproductive and regenerative medicine. They can transmit the genetic and epigenetic information across generations, which highlights the importance of the correct establishment and maintenance of epigenetic marks. Accurate transcriptional and post-transcriptional regulation is required to support the highly coordinated expression of specific genes for each step of spermatogenesis. Increasing evidence indicates that non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), play essential roles in controlling gene expression and fate determination of male germ cells. These ncRNA molecules have distinct characteristics and biological functions, and they independently or cooperatively modulate the proliferation, apoptosis and differentiation of SSCs. In this review, we summarized the features, biological function and fate of mouse and human SSCs, and we compared the characteristics of lncRNAs and circRNAs. We also addressed the roles and mechanisms of lncRNAs and circRNAs in regulating mouse and human SSCs, which would add novel insights into the epigenetic mechanisms underlying mammalian spermatogenesis and provide new approaches to treat male infertility.Reproduction (2019) 158 R15–R25

Introduction

Spermatogenesis is crucial for the transmission of genetic information and the fertility of male mam-mals. This sophisticated process involves the delicate balance between the self-renewal and differentiation of spermatogonial stem cells (SSCs), which is tightly modulated by growth factors (Meng et al. 2000, Wang et al. 2009, Hai et al. 2014, Chen & Liu 2015), genes (Reinke et al. 2000) and epigenetic factors. Originated from the primordial germ cells (PGCs), SSCs have the potential of progressively differentiating into the differ-entiated spermatogonia, spermatocytes and eventually mature spermatozoa.

ncRNAs emerge as important epigenetic factors that determine the fate decision of SSCs. The ncRNAs, including miRNAs, piRNAs, lncRNAs and circRNA, are transcripts of genomic sequences that contribute to biological modification without DNA sequence change. Increasing studies have shown thousands of ncRNAs with indispensable functions (Santosh et  al. 2015). Among different kinds of ncRNAs mentioned above, lncRNAs and circRNAs emerge as novel regulators in a myriad of biological processes. LncRNAs are a large class of transcribed RNA molecules with a length of more than 200 nucleotides (nt). Recent studies suggest

that lncRNAs are highly diverse in regulating gene expression. Meanwhile, lncRNAs may interact with miRNAs by acting as endogenous sponges or competing endogenous RNA (ceRNA) and further affect expression of targeting genes. CircRNAs are a newly found type of ncRNAs in the endogenous transcriptome with a specific structure of continuous loop, and they could function as sponges to affect relevant miRNAs and their targets (Qu et  al. 2015). Certain regulatory networks consist of miRNAs, piRNAs, lncRNAs and circRNAs with significant roles in the regulation of physiology and disease. In this review, we delineated the biological features of mouse and human SSCs, and we further discussed the functions and mechanisms of the lncRNAs and circRNAs in controlling fate determination of SSCs.

Morphological and phenotypic features and fate decision of mouse and human SSCs

Adult germ cells from SSCs to spermatids are able to transmit genetic information across generations via fertilization (Oatley & Brinster 2008), and SSCs undergo self-renewal to maintain the pool of stem cells and differentiation to spermatocytes and spermatids (Phillips et al. 2010). Much progress has been achieved in uncovering the biology of SSCs in rodents. Notably,

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cell types and phenotypic characteristics of SSCs are distinct between rodents and primates (De Rooij & Russell 2000). Spermatogonia are categorized into A-single (As), A-paired (Apr), A-aligned (Aal), A1-A4, Intermediate (In) and type B cells in rodents according to their morphology and phenotype. SSCs are defined as a subpopulation of type A spermatogonia, and type As spermatogonia are generally regarded as the actual SSCs in rodents (Huckins 1971, Oakberg 1971, de Rooij 1973). Additionally, type Apr and Aal spermatogonia have been suggested to be potential SSCs since these cells are able to self-renew in case of stem cell deficiency and/or the niche depletion (Nakagawa et al. 2007, Yoshida et al. 2007, De Rooij & Griswold 2012). SSCs self-renew or generate Apr spermatogonia which then produce the Aal spermatogonia that give rise to other daughter cells, including type A1-A4, In and type B spermatogonia (de Rooij & Grootegoed 1998). SSCs of rodents have the following morphological features, including large nuclei, a high ratio of nucleus to cytoplasm and few organelles in the cytoplasm, which reflects their undifferentiated status (Bellve et  al. 1977). A number of markers, for example α6-integrin (CD49f), β1-intergrin (CD29), Thy1 (CD90), GFRA1, RET, PLZF (ZBTB16), POU5F1 (Oct4), GPR125, neurogenin 3 and TNAP (tissue-nonspecific alkaline phosphatase), have been identified for rodent SSCs and/or their progenitors (He et al. 2009). Moreover, ID4, PAX7, BMI1 and EOMES have been shown to be restricted to rodent actual SSCs (Oatley et al. 2011, Aloisio et al. 2014, Komai et al. 2014), while UTF1, NANOS2, ZBTB16 (PLZF), SALL4, LIN28, CDH1 and FOXO1 are expressed in actual and potential SSCs (Tokuda et  al. 2007, van Bragt et al. 2008, Suzuki et al. 2009, Goertz et al. 2011, Eildermann et al. 2012, Hobbs et al. 2012, Fayomi & Orwig 2018). However, no specific marker is available for rodent SSCs, and thus, the combination of two or more biochemical markers should be utilized to characterize and identify these cells.

Based upon the difference in morphology, human spermatogonia are classified into three distinct sub-populations, including Apale, Adark and B spermatogonia (Clermont 1966). It has been suggested that the undif-ferentiated Apale spermatogonia and Adark spermatogonia are human SSCs. The Apale spermatogonia are the renew-ing stem cells that support spermatogenesis, while Adark spermatogonia are the reserve stem cells (Clermont 1963, 1969, Clifton & Bremner 1983). In morphology, Apale sper-matogonia are characterized by their relatively larger, oval or round, pale and elliptical nucleus containing coarser or granular chromatin. Compared to Apale spermatogo-nia, Adark spermatogonia are relatively smaller, spherical or ovoid cells with dark, dense chromatins in the nuclei. Due to a limited access to human testis tissues, there is rare information of biochemical markers for human SSCs. In 2010, we reported that human SSCs share some but not all phenotypic characteristics with rodent SSCs, and we identified GPR125, ITGA6, ZBTB16, UCHL1,

GFRA1 and THY1 as markers for human SSCs (He et al. 2010). However, POU5F1 (also known as Oct4), a hallmark for rodent SSCs, is undetectable in human SSCs (He et al. 2010). Human SSCs exhibit diversity of expression pattern which is not correlated with the classical subtypes of Apale and Adark spermatogonia (von Kopylow et  al. 2012a,b). Other hallmarks, for example SALL4 (Hobbs et al. 2012), LIN28 (Aeckerle et al. 2012), SSEA4 (Izadyar et al. 2011), UTF1 (Valli et  al. 2014, Di Persio et  al. 2017), ENO2 (Valli et al. 2014) and ID4 (Sachs et al. 2014), have been reported to be present in the undifferentiated Apale and Adark spermatogonia. Most of these markers are expressed in rodent SSCs (Fayomi & Orwig 2018).

SSCs have various kinds of potentials in vivo and in vitro. In addition to self-renewal to maintain the pool of stem cells and differentiation into mature and functional spermatids, SSCs are able to dedifferentiate to embryonic stem (ES)-like cells to acquire pluripotency in vitro (Guan et al. 2006, Conrad et al. 2008), and they can also be reprogrammed to transdifferentiate to cell lineages of other tissues (Chen et al. 2017). Therefore, SSCs have significant applications in treating male infertility and regenerative medicine for various kinds of human diseases.

Properties, categories and mechanisms of lncRNAs

Properties and categories of lncRNAs

Recent studies have indicated the new and indispensable roles of ncRNAs including lncRNAs in regulating SSCs. LncRNAs are defined as transcribed RNA molecules that are typically more than 200 nucleotides (nt) in size, and they have distinct features of protein-coding transcripts, for example a 5′ cap, poly (A) tail and introns (Carninci et al. 2005). The properties of lncRNAs are summarized in Table  1. It has been estimated that 75–90% of the mammalian genome is transcribed as ncRNAs, while lncRNAs account for most of the transcription (Derrien et al. 2012, Harrow et al. 2012). In total, 19,175 lncRNAs with potential functions have been identified in human genome (Hon et al. 2017). Significantly, testis has been regarded as one of the tissues which express the greatest amount of lncRNAs (Necsulea et  al. 2014). LncRNAs are transcribed by polymerase II, and they have tissue-specific expression patterns (Cabili et al. 2011, Derrien et al. 2012).

LncRNAs can be categorized pursuant to their position in the genome relative to protein-coding genes. Stand-alone lncRNAs are distinct transcription units located in sequence space that do not overlap protein-coding genes (Guttman et al. 2009). Most of these lncRNAs have been identified through chromatin signatures for actively transcribed genes, including H3K4me3 at the promoter and H3K36me3 along the transcribed length (Kung et al. 2013). The stand-alone lncRNAs usually have an average length of 1 kb, and they are transcribed by RNA

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polymerase II, polyadenylated and spliced. Divergent transcripts are produced from the vicinity of transcription start sites (TSSs) in both sense and antisense directions, corresponding to peaks of polymerase II occupancy due to pausing, and these transcripts are associated with histone H3K56 acetylation and polymerase II phosphorylation. They usually have low abundance, capped and polyadenylated (Core et al. 2008). A class of promoter-associated RNAs, in addition to short (<2 kb) bidirectional transcripts, namely enhancer RNAs (eRNAs) produced by enhancers, has also been found to influence the transcription of protein-coding genes by stabilizing loop formation and transcription of the associated genes or interfering with enhancer–promoter contact (Wang et al. 2011). Natural antisense transcripts (NATs) are lncRNAs whose transcription opposites the sense DNA strand of annotated transcription units (Katayama et al. 2005, He et al. 2008). There is complete overlap between these sense–antisense (SAS) pairs, but natural antisense transcripts tend mostly to be enriched around the 5′ or 3′ ends of the sense transcript. As a result, there are a number of dual lncRNA SAS pairs or coding/non-coding SAS pairs. Another class of lncRNAs is called pseudogenes that are extra gene copies under no selective pressure but bear coding potential (Balakirev & Ayala 2003, Pink et  al. 2011). This portion of pseudogenes (2–20%) can be transcribed and even translated, and sometimes they have high levels of sequence conservation. In some cases, these transcribed pseudogenes have been found to regulate gene expression by epigenetic or post-transcriptional mechanisms (Elisaphenko et  al. 2008). Being long

intronic ncRNAs, their transcripts are encoded within the introns of annotated genes. There is a special lncRNA, namely the fragment of p15AS, which has been proven to mediate the chromatin architecture (Louro et al. 2009).

Molecular mechanisms of lncRNAs

LncRNAs play critical roles in regulating gene and genome activities by affecting chromatin modifications and their structure, and they act as regulators by their transcriptional, translational, or epigenetic mechanisms. As illustrated in Fig.  1 and summarized in Table  1, lncRNAs act via different ways, which includes the increase or decrease of mRNA stability, inhibition of translation and sequestration of miRNA.

LncRNAs are differentially expressed in various tissues, and they have important functions in controlling cellular processes, for example cell proliferation, motility and apoptosis. In addition to certain lncRNAs that are located within intergenic sequences, a majority of lncRNAs is transcribed as interlaced networks of overlapping sense and antisense transcripts that usually include protein-coding genes (Kapranov et al. 2007). LncRNAs can recruit certain complexes, for example chromatin-modifying complexes and polycomb complexes, for the regulation of chromatin states. They function in cis or trans, interact with chromatin-modifying complexes and recruit them to specific genomic loci or further regulate the levels of other lncRNAs and genes (Zhao et al. 2008, 2010, Khalil et al. 2009). LncRNAs can also recruit their protein interaction partners to specific genomic loci

Table 1 Properties and mechanisms of lncRNAs and circRNAs.

LncRNAs CircRNAs

Properties Size More than 200 nt More than 200 nt Structure Linear RNAs with 5′ cap, polyadenylated tail, introns and a fewer

number of exons than mRNAsCircular RNAs with covalently closed loop

structures with neither 5′–3′ polarities nor polyadenylated tails and exons

Biogenesis Canonical splicing and transcribed by Polymerase II Backsplicing. Inverted and repeated Alu pairs (IRAlus) and exon skipping are essential for circRNA formation

Category LncRNAs, divergent transcripts, convergent transcripts, bidirectional transcripts (eRNA), NAT, pseudogene and long intronic ncRNA

circRNA, EIciRNA and ciRNA

Cellular localization Nucleus or associated with chromatin Cytoplasm; certain EIciRNAs and ciRNAs are primarily located in the nucleus

Conversation across different species

Poorly conserved Evolutionarily conserved, whereas some ciRNAs are less conserved

Expression level Very low. Expressed in spatially and temporally patterns Generally at low levels Translation Little or no protein-coding potential Majority of circRNAs are endogenous ncRNAsMechanisms Interaction with target

DNA and RNAPrevent DNA methylation, play roles in chromatin remodeling.

Function in pre-mRNA splicing and cytoplasmic transport. Influence the stability of mRNAs, regulation of transcription and translation

Regulating alternative splicingInfluence transcription of linear RNAs.

Resource of pseudogenes

Interaction with miRNA MiRNA sponge, inhibition of miRNA function, ceRNA regulation and precursor of some miRNAs

MiRNA sponge, inhibit miRNA functionCeRNA regulation

Interaction with target protein

Recruiting proteins, acting as decoys, scaffolds and tethers Protein sponge, acting as scaffolds, translating proteins

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to form a DNA-RNA triplex and direct chromatin or transcriptional modulators. Sometimes, the lncRNAs act as a scaffold onto which multiple protein complexes can assemble (Pandey et al. 2008, Bertani et al. 2011). By acting as recruiters or scaffolds in histone modifications, lncRNAs have the repressive effect or they are involved in gene activation. Additionally, lncRNAs interact with a subunit of chromatin remodeling complexes and lead to a depression of gene activities (Prensner et  al. 2013). It has been proposed that lncRNAs can act via transcription factors and recruit them to the promoter, which subsequently synergizes the gene expression (Jiang et al. 2015).

LncRNAs oppose the epigenetic mark of DNA methylation. The transcription of lncRNAs or binding of lncRNAs to target genes usually inhibits gene locus methylation, whereas knockdown of extra coding transcripts leads to downregulation of genes and increases DNA methylation of the locus (Di Ruscio et  al. 2013, O’Leary et  al. 2015). LncRNAs have the dual ability to function as ligands for proteins and mediate base-pairing interaction that guides lncRNA-containing complexes to specific RNA or DNA targets. This dual activity is always shared with small ncRNAs. In these mechanisms, lncRNAs have been suggested to function as molecular scaffolds and assemble diverse combinations of regulatory proteins with flexible and modular nature. Since lncRNAs tend to be localized in the nucleus or are associated with chromatin, they are involved in post-transcriptional RNA processing (Herman et  al. 1976, Derrien et  al. 2012, Werner &

Ruthenburg 2015). LncRNAs have been reported to regulate various aspects of post-transcriptional RNA processing, including pre-mRNA splicing, cytoplasmic transport, translation and degradation, which involves base pairing between lncRNAs and the target mRNAs. By binding to primary RNA transcripts, lncRNAs influence splicing patterns (Tripathi et al. 2010).

LncRNAs are the precursors for small RNAs. Short RNAs, including miRNAs and piRNAs, are produced from introns or exons of longer host RNAs, for example protein-coding genes and many lncRNAs (Ha et  al. 2014, Watanabe et al. 2015). Since lncRNAs are largely indistinguishable from mRNAs by molecular structure (e.g. a cap, a polyA tail and introns), they are regulated by small RNAs in the same way as mRNAs. It has been reported that miRNAs bind to the transcribed lncRNAs through miRNA response elements (MREs), which act as molecular sponges or decoys and suppress the targeting mRNAs by miRNAs (Cesana et  al. 2011). The RNAs compete for the binding of miRNAs to their mRNA-binding sites, and thus, they are named as competitive endogenous RNAs (ceRNAs). The ceRNAs regulate diverse cellular development and diseases, and lncRNA–miRNA interaction forms the intertwined and complex regulatory networks. Otherwise, extensive complementarity between miRNA and lncRNA result in lncRNA cleavage, since lncRNAs can be integrated into the RISC (RNA-induced silencing complex) complex and they are potentially cleaved by Argonaute proteins. Numerous lncRNAs are targeted by miRNAs through conventional seed sites (Yamamura et al. 2018),

Figure 1 Molecular mechanisms of lncRNAs. LncRNAs recruit certain complexes for the regulation of chromatin states and some proteins to specific genomic loci. lncRNAs act as scaffolds onto which multiple protein complexes can assemble or as recruiters in histone modifications. LncRNAs prevent the epigenetic mark of DNA methylation, and they are involved in inhibiting transcription and act as protein decoys by which influence the DNA-protein binding. LncRNAs participate in post-transcriptional RNA processing, including pre-mRNA splicing and translation inhibition. The stability of mRNAs can be controlled by lncRNA-mRNA binding. LncRNAs are the precursors for small RNAs, and they can also compete for the binding of miRNAs to their mRNA-binding sites.

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while the functional importance of lncRNA–miRNA interaction remains to be elucidated. Small nucleolar RNAs (snoRNAs) are ncRNAs of approximately 60–200 nucleotides (nt), and they chemically modify other RNAs (e.g. ribosomal RNAs, transfer RNAs and small nuclear RNAs) and are primarily required for maturation of ribosomal RNAs. SnoRNAs have been reported to have non-canonical functions, including splicing and editing (Dupuis-Sandoval et al. 2015), and lncRNAs have found to encode snoRNAs.

The roles and mechanisms of lncRNAs in fate determination of SSCs

Recently, it has been demonstrated that lncRNAs have important functions in controlling mammalian SSCs. LncRNAs have been shown to be intrinsically functional, and increasing evidence highlights the roles of lncRNAs as the determinant of stem cell fate, specifically as regulators of self-renewal and differentiation (Guttman et al. 2011, Wang et al. 2013). LncRNA Mrhl is negatively regulated by Wnt signaling activation through its protein partner Ddx5/p68, which leads to the differentiation of mouse spermatogonia (Arun et  al. 2012). As a key transcription factor in canonical Wnt signaling, TCF4 is upregulated by Mrhl RNA silencing. Interestingly, Wnt signaling is suppressed in SSCs, but it is activated in differentiated spermatocytes (Yeh et  al. 2011). Mrhl decreases the expression level of Sox8, which is essential for spermatogonial differentiation (Kataruka et al. 2017). It is speculated that Mrhl recruits corepressor Sin3A at the promoter site of Sox8 through a direct interaction. Wnt signaling-mediated downregulation of Mrhl RNA and upregulation of Sox8 are essential for the regulation of various premeiotic and meiotic marker genes (Akhade et  al. 2014), which is crucial for meiotic entry or spermatogonial differentiation. NLC1-C has been shown to be downregulated in the cytoplasm and accumulated in the nuclei of spermatogonia and primary spermatocytes in human testes of non-obstructive azoospermia (NOA) patients with maturation arrest compared to normal men, and it inhibits miR-320a and miR-383 transcripts and promotes cell proliferation of testicular embryonal carcinoma by binding to nucleolin (Lu et  al. 2015). GDNF (glial cell-derived neurotrophic factor) is crucial for maintaining SSC self-renewal (proliferation and survival). After GDNF withdrawal from culture medium of mouse SSCs, the level of lncRNA033862 is reduced. It has been reported that lncRNA033862 is preferentially expressed in gonocytes and spermatogonial progenitors, and it is essential for the maintenance of SSC self-renewal by regulation of Gfra1 (cell-surface receptor of GDNF) (Li et al. 2016). Knockdown of lncRNA033862 decreases the transcripts of gene hallmarks for SSCs, including Bcl6b, Ccnd2 and Pou5f1 (Oct4), while transcripts of genes associated with differentiation, for example Stra8, Sypc1 and Kit, are unaffected (Li et al. 2016), indicating

that lncRNA033862 is associated with the maintenance of mouse SSCs rather than differentiation. In addition, lncRNA AK015322 is expressed at a higher level in mouse SSCs than other types of male germ cells (Hu et al. 2017), and notably, LncRNA AK015322 promotes the proliferation of mouse SSC line C18-4 cells in vitro and serves as a decoy of miR-19b-3p via the repression of transcriptional factor ETV5 (E-twenty-six variant gene 5), which is crucial for SSC self-renewal (Wu et al. 2011, Hu et al. 2017). AK015322 may neutralize the suppressive effect of miR-19b-3p on expression level of ETV5, and it promotes the proliferation of C18-4 cells via acting as a competing endogenous RNA for miR-19b-3p, reflecting that the lncRNA AK015322-miR-19b-3p-ETV5 pathway is involved in the fate determination of SSCs. Recently, lncRNA Gm2044 has been shown to inhibit the proliferation of GC-1 cells (mouse spermatogonial cell line) by binding to Utf1 (undifferentiated transcription factor 1) (Hu et al. 2018). Overexpression of Gm2044 in GC-1 cells leads to the suppression of cellular proliferation and Utf1 protein. LncRNA Gm2044 is highly expressed in pachytene spermatocytes and it inhibits Utf1 mRNA in spermatogenesis. DMRT1 directly represses Stra8 transcription in mouse spermatogonia and activates transcription of Sohlh1, a marker for spermatogonial differentiation (Matson et al. 2010). It has been demonstrated that Dmr (Dmrt1-related gene), a ncRNA gene from mouse chromosome 5, suppresses protein level of DMRT1 through a trans-splicing mechanism (Zhang et al. 2010), suggesting that Dmr acts as a lncRNA in regulating the differentiation of SSCs. We summarized the expression profiles and the roles of lncRNAs in regulating the self-renewal and differentiation of SSCs in Table  2. These studies mentioned above illustrate the important roles of lncRNA in the fate decision of mammalian SSCs.

Properties, biogenesis and mechanisms of circRNAs

Properties and biogenesis of circRNAs

CircRNAs are a novel type of ncRNAs by virtue of their unique loop structure resulting from a 3′ to 5′ end-joining event (back splice or head-to-tail splice), which distinguishes from linear transcripts. Since the circRNAs do not have susceptible 5′ and 3′ ends, they are resistant to endonuclease enzymatic degradation and more stable than linear RNAs. CircRNAs are often derived from exons close to the 5′ end of a protein-coding gene, and they consist of a single or multiple exons (Guo et al. 2014). Meanwhile, circRNAs can be originated from introns, intergenic regions, untranslated regions (UTRs), ncRNA loci and location antisense to known transcripts (Memczak et al. 2013). The properties of circRNAs are summarized in Table 1.

Spliceosome has been implicated in the generation of circRNAs, while the canonical splice signals flank the junction site in these RNA molecules (Ashwal-Fluss

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et  al. 2014). Reversed complementary sequences, for example, the inverted and repeated Alu pairs (IRAlus) and exon skipping, are involved in circRNA formation. RNA-binding proteins (RBPs) also participate in the generation of circRNAs. The circularization can be induced through several non-exclusive pathways (Ebbesen et  al. 2016). As illustrated in Fig. 2 and summarized in Table 1, two models of circRNA formation, namely ‘lariat-driven circularization’ or ‘exon skipping’ and ‘intron-pairing-driven circularization’ or ‘direct back splicing’, have been proposed (Jeck et al. 2013). In the model of lariat-driven circularization, one or more exons of the transcript are skipped and spliced out of the transcripts, which results in an exon-containing lariat by the covalent junction of splice donor and acceptor. The lariat-containing skipped exons are subsequently recognized and joined by the spliceosome. Intron-pairing-driven circularization pathway is a model by which introns flank the exons to be circularized and forms a circular structure, and introns are removed or retained to form circRNA or exon–intron circRNA (EIciRNA). An additional biogenesis pathway has been found by discovering a new type of circRNA, namely circular intronic RNAs (ciRNAs), which is derived from introns. This mechanism of circRNA biogenesis depends on a motif containing GU-rich

sequence near the 5′ splice site and C-rich sequence close to the branch point site. By trimming the 3′ ‘tail’ downstream from the branch point, a stable ciRNA is formed (Zhang et al. 2013). Inducing of some circRNAs has also been suggested to count on RBP pathways, and quaking and muscle blind are two splicing factors that are capable of inducing circularization from genes containing binding motifs for RBPs (Ashwal-Fluss et al. 2014, Conn et al. 2015). Binding of the specific RBP to the flanking intronic sequence motifs brings the exons close together due to the interaction between the bound RBPs, and circRNAs are formed after junction of circle and removal of the introns.

Molecular mechanisms of circRNAs

With regards to the action mechanisms, some circRNAs have been demonstrated to interact with the miRNA–Argonaute 2 (Ago2) complex and inhibit miRNA function by redirecting the miRNAs, and thus, the circRNAs possess binding sites away from other targets. CircRNAs containing the selectively conserved target sites for miRNAs can form a mismatch at the central part of the target sites, which prevents miRNA-mediated cleavage. Therefore, circRNAs act as the sponge for miRNAs and

Figure 2 Biogenesis and mechanisms of circRNAs. CircRNAs are induced by the model of lariat-driven circularization. Intron-pairing-driven circularization is another pathway by which circRNAs or EIciRNAs are generated. CiRNAs are derived from introns depend on a motif containing GU-rich sequence near the 5′ splice site and C-rich sequence. CircRNAs act as sponges for miRNAs and function as scaffolds to facilitate protein interactions. CircRNAs are identified as resources for derivation of pseudogenes and associated with RNA translation.

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increase levels of miRNA targets (Hansen et  al. 2013, Memczak et al. 2013). This is also a ceRNA regulation similar to the way by which miRNAs are mediated by lncRNAs. The production of the circRNA may function to regulate linear RNA expression from the same gene. The majority of circRNAs is derived from exonic regions within protein-coding genes, and exons that are incorporated into circRNAs are unable to become the part of any potential linear skipped transcripts derived from the same primary transcript as the circRNAs (Jeck & Sharpless 2014). Depending on the specific circRNA–protein combination, circRNAs have also been shown to interact with RNA-binding proteins, and they might function as a scaffold to facilitate protein interactions, regulate protein function or sequester the bound protein.

CircRNAs can assume putative roles, for example acting as miRNA sponges, modulating the expression of parental genes, regulating alternative splicing (Ashwal-Fluss et al. 2014) and RNA–protein interactions, acting as scaffolds in the assembly of protein complexes, sequestering proteins from their native subcellular localization, and functioning as templates for protein synthesis. Additionally, circRNAs are identified as resources for derivation of pseudogenes, and a small subset of endogenous circRNAs are associated with RNA translation (Li et al. 2018). As novel molecules of ncRNAs, expression levels and functions of circRNAs in mammalian reproductive systems are relatively limited. It has recently been demonstrated that circRNAs function in mammalian germline stem cells at an early stage of expression profiles (Li et al. 2017).

The expression profile of lncRNAs and circRNAs in mammalian SSCs and their relationship with the spermatogenesis and male infertility

Infertility affects 10–15% of couples in the world, and male factors account for about half of these disorders (Agarwal et al. 2015). The molecular mechanisms underlying fate determination of SSCs need to be well understood, since there is a delicate balance between the self-renewal and differentiation of SSCs, which is significantly related to normal spermatogenesis. Increasing ncRNA expression profiles have been identified, and the establishment of these databases will be invaluable resources for subsequent

functional and mechanism studies. Azoospermia comprises approximately 15% of male infertility (Esteves 2015), and thus, it is important to elucidate the factors of pathology and physiology in human testis and male germ cells including SSCs. Azoospermia can be classified into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). OA patients have been defined as having an obstruction in any region of the sperm excurrent ductal system with normal spermatogenesis in the seminiferous tubule, while NOA patients usually are subject to spermatogenesis failure (Ezeh 2000, Hu et al. 2011).

The profiles of lncRNAs and mRNAs have been compared among mouse SSCs, type A spermatogonia, pachytene spermatocytes and round spermatids by microarray analysis (Liang et  al. 2014). The lncRNAs/mRNAs profiles have been divided into three groups of specific expression in each type of germ cells, and expression in several types of male germ cells is summarized in Table  2. With the further correlation analysis of the lncRNAs/mRNAs expression trends, a high correlation coefficient of lncRNAs/mRNAs has been found, which indicates that transcribed lncRNAs/mRNAs gene pairs may be coordinately regulated during mouse spermatogenesis. In another study, the patterns of lncRNAs, circRNAs and mRNAs have been uncovered in mouse primitive type A spermatogonia, preleptotene spermatocytes, pachytene spermatocytes and round spermatids (Lin et  al. 2016). Among these male germ cells, mRNAs are generally more abundant than lncRNAs, while linear transcripts are more abundant than circRNAs. A considerable fraction of lncRNAs from functional screenings of mouse SSCs has been found to be conserved across species, and these lncRNAs are expressed in spermatogonia at higher levels than in other types of male germ cells (Table 2). The expression profiles of lncRNAs and circRNAs have also been unveiled in mouse male and female germline stem cells by high-throughput sequencing (Li et  al. 2017). The whole gene expression profiles of male and female germline stem cells showed that certain genes have similar gene expression patterns at both the lncRNA and mRNA levels. In addition, the sex-biased lncRNA and circRNA profiles are related to the self-renewal and the sex-specific properties, as evidenced

Table 2 Function of lncRNAs in mouse and human SSCs.

LncRNAs Species and cells Function References

NLC1-C Human spermatogonia It stimulates the proliferation of germ cells and is associated with male infertility

Lu et al. (2015)

LncRNA033862 Mouse SSCs It promotes SSC self-renewal and survival Li et al. (2016)Mrhl Mouse spermatogonial cell line

(Gc1-Spg cells)Downregulation of Mrhl leads to spermatogonial

differentiationAkhade et al. (2014)

LncRNA16120, 90867, 24617 and 56293

Mouse SSCs Knockdown of these lncRNAs facilitates the differentiation of SSCs into spermatogonia

Lin et al. (2016)

LncRNA AK015322 Mouse SSC line C18-4 cells It promotes the proliferation of mouse SSC line C18-4 cells

Hu et al. (2017)

LncRNA Gm2044 Mouse spermatogonia (GC-1 cell line) It inhibits the proliferation of mouse spermatogonia Hu et al. (2018)

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by the findings that hundreds of lncRNAs and circRNAs assume sex-biased expression patterns associated with acquisition of germline stem cells and that lncRNAs Gm11851, Gm12840, 4930405O22Rik and Atp10d are correlated with sex differences. These findings implicate that lncRNAs and circRNAs play essential roles in controlling the maintenance and fate determination of mammalian SSCs.

Perspectives and summary

Male germ cells including SSCs have the ability of transmitting their genetic and epigenetic information across generations, which highlights the significance of establishment and maintenance of epigenetic hallmarks. SSCs are the foundation for spermatogenesis, and they promise great applications in reproduction and regenerative medicine. NcRNAs, including lncRNAs and circRNAs, have major roles in the control of gene expression and fate determination in male germ cells. The functions of ncRNAs are diverse and require further investigation. Human and mouse share numerous hallmarks that are specifically expressed in SSCs. Increasing studies have revealed indispensable roles of lncRNAs and circRNAs in regulating physiological and pathological conditions of mammalian germ cells including SSCs. These ncRNA molecules may have important functions to independently or cooperatively modulate the self-renewal (proliferation and survival), apoptosis and differentiation of SSCs. We address the features, biogenesis and functions and mechanisms of lncRNAs and circRNAs in human and mouse SSCs. LncRNAs are very similar in their structure and modifications to mRNAs, and they interact with ceRNAs. CircRNAs belong to a novel subclass of ncRNA, and their profiles assume to be distinct in different types of male germ cells, reflecting that they play important roles in controlling the self-renewal and differentiation of SSCs. It is essential to identify the regulatory networks governing lncRNA and circRNAs and unveil their new roles and mechanisms in the control of mammalian SSCs, which could shed novel insights into the epigenetic regulation of male germline stem cells and offer new targets for the treatment of male fertility.

Declaration of interest

Zuping He is on the Editorial Board of Reproduction. Zuping He was not involved in the review or editorial process for this paper, on which he is listed as an author. The other authors have nothing to disclose.

Funding

This work was supported by grants from National Nature Science Foundation of China (31671550, 31872845, 31230048), Chinese Ministry of Science and Technology (2016YFC1000606), High Level Talent Gathering Project in

Hunan Province (2018RS3066), The Open Fund of the NHC Key Laboratory of Male Reproduction and Genetics (KF201802) and Shanghai Hospital Development Center (SHDC12015122).

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Received 30 September 2018First decision 16 November 2018Revised manuscript received 16 March 2019Accepted 2 April 2019

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