Epigenetics of nonobstructive azoospermia

Abstract

Nonobstructive azoospermia (NOA) is a severe and heterogeneous form of male factor infertility caused by dysfunction of spermatogenesis. Although various factors are well defined in the disruption of spermatogenesis, not all aspects due to the heterogeneity of the disorder have been determined yet. In this review, we focus on the recent findings and summarize the current data on epigenetic mechanisms such as DNA methylation and different metabolites produced during methylation and demethylation and various types of small noncoding RNAs involved in the pathogenesis of different groups of NOA.

INTRODUCTION

Nonobstructive azoospermia (NOA) is the absence of spermatozoa in semen and influences about 10%–15% of all infertile men.1 NOA is a severe and heterogeneous form of male factor infertility caused by dysfunction of spermatogenesis. NOA results from a wide variety of causes affecting both somatic and germ cells, including gene mutations, incomplete testis development, somatic cell immaturity, hormonal imbalance, increased apoptosis, and inflammation or varicocele.2 Nevertheless, not all the factors of reduced spermatogenesis have been identified yet.

Recently, the association between NOA and aberrant DNA methylation of the whole genome, some genes, or male or female imprinted genes in spermatozoa was shown in many studies. In addition, many studies have indicated a role for short noncoding RNAs (sncRNAs) and different histone tail modifications in infertility. In this review, we focus on the recent findings of the current data on epigenetic mechanisms such as DNA methylation and different metabolites produced during methylation and demethylation. In addition, various types of sncRNAs involved in the pathogenesis of different groups of NOA are summarized.

DNA METHYLATION IN NOA

Methylation and demethylation of DNA are fundamental epigenetic rearrangements in the human genome, and errors in this process play a role in the pathogenesis of many diseases, including NOA. DNA methylation is the transfer of a methyl group from S-adenosyl methionine (SAM), a methyl donor substrate in cells, by DNA methyltransferases (DNMTs) to the 5^th carbon atom of the cytosine nucleotide, resulting in the formation of 5-methylcytosine (5mC).3,4 5mC is the most frequently detected nucleotide modification and constitutes approximately 1% of the nucleotides of the human genome.5 DNA methylation occurs mainly in cytosine-phosphate-guanine (CpG) islands and primarily results in the gene of interest silencing. CpG islands are evolutionarily conserved rich in CpG regions approximately in one kilobase length, formed by a G residue following the C nucleotide on the same strand of DNA.5 CpG islands are mostly found in gene promoters and lead to silencing of the gene (Figure 1). Gene expression regulation after DNA methylation is regulated either by preventing the binding of transcription factors to their target sequences or by recruiting methyl-CPG-binding proteins involved in gene repression to the target location.6 DNA methylation is required for the silencing of retroviral elements, genomic imprinting, X chromosome inactivation, and regulation of tissue-specific gene expression in germ cell development.3,5

Figure 1.

5-methylcytosine promoter methylation and transcriptional silencing. CpG: cytosine-phosphate-guanine.

Active methylation and demethylation of DNA are critical for male germ cell development, and studies have shown that one of the causes of NOA is aberrant DNA methylation.6,7,8,9 Members of the DNMT family catalyze the methylation of DNA. DNMT1, also known as maintenance DNMT, binds to newly synthesized DNA immediately following replication and copies the previous methylation marks to the new sequence.3,4 It can also repair DNA methylation and thus preserve the original methylation marks.5 In testicular biopsy specimens with hypospermatogenesis (HS), maturation arrest (MA), and Sertoli cell-only syndrome (SCOS), DNMT1 expression and global DNA methylation levels were found to decrease in correlation with the severity of NOA.10 The other two DNMTs, DNMT3A and DNMT3B, are known as de novo methyltransferases because they can add methyl groups to previously unmethylated DNA without the need for hemimethylation.6 Expressions of DNMT3A and DNMT3B in testicular biopsies of patients with NOA were found to show variation in HS, MA, and SCOS groups, with the lowest in the HS group.10 Despite their structural and functional similarities, DNMT3A is expressed in almost all tissue, while DNMT3B is more abundant in specific tissues, including testis.5,11 DNMT3L, on the other hand, is the last member of the DNMT family and plays a role in methylation-mediated silencing of retrotransposons with P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) in the male germ cell line.6 DNMT3L, which has no complete catalytic domain, cannot bind to DNA. However, it interacts with DNMT3A and DNMT3B and significantly increases their activity.12 DNMT3L is required for spermatogenesis, and animal studies have shown that Dnmt3L null males cannot progress beyond the spermatocyte stage; therefore, they are entirely azoospermic.13 DNMT3L is involved in genomic imprinting and silencing of retrotransposons by methylation. Approximately 45% of the human genome is formed from transposable elements localized in intergenic regions and controlled by methylation.5 Studies have revealed that errors in silencing of retrotransposons, which constitute the vast majority of transposable elements, may cause lack of spermatozoa in ejaculate.7,14,15

DNA methylation is a reversible process, active demethylation of DNA occurs through a series of enzymatic reactions catalyzed by members of the ten-eleven translocation (TET) enzyme family.6 TET1, TET2, and TET3 with the same catalytic domains carry out oxidation reactions starting from 5mC.16 The oxidation of the methyl group of 5mC is followed by the formation of 5-hydroxymethylcytosine (5hmC). The latter leads to oxidation of intermediates, 5-formyl cytosine (5fC) and 5-carboxyl cytosine (5caC).6,16 Both 5fC and 5caC are recognized and cleaved by thymine DNA glycosylase, and the abasic sites formed afterward are repaired by base excision repair.6 Thus, methylated cytosines are replaced by unmethylated cytosines at the end of this process. Each of the TET enzymes has been shown to be expressed depending on the stage during spermatogenesis in the testis.17 However, TET enzymes have also been suggested to contribute in passive demethylation, a replication-dependent process, and have a specific preference for intermediates in demethylation.16 This preference likely leads to different genomic levels of the intermediates in demethylation, mostly because 5hmC is more stable in the genome.5,16 Indeed, in recent years, 5hmC has gained importance, especially in methylation studies. Immunofluorescence analyzes of testicular samples of NOA patients revealed that the values of 5mC and 5hmC were different from each other in spermatogenic cells. However, the level of 5hmC was found to be less compared to 5mC.18

Methylation of single genes

DNA methylation errors in various genes have been reported to be associated with NOA.7,8,19,20,21,22 Among these genes, the well-known example is methylenetetrahydrofolate reductase (MTHFR) gene, which is involved in folate metabolism and has been prominent in NOA.23,24,25 MTHFR is a folate-metabolizing enzyme that catalyzes the generation of intermediates utilized in purine biosynthesis.24,25 Seminal plasma folate levels of azoospermic patients are low compared to normozoospermic men. This finding suggests changes in expression levels of the MTHFR gene play a role in folate metabolism.25 Methylation-specific polymerase chain reaction (MSP) analysis of the promoter of the MTHFR gene showed that testicular biopsies had a higher number of methylated alleles in NOA compared to obstructive azoospermic (OA) patients. However, this difference in methylation level of the MTHFR gene was not found in blood samples.23 However, another MSP study has shown no MTHFR promoter hypermethylation in 35 idiopathic NOA patients.24 These studies suggest that, despite the importance of folate in spermatogenesis, more comprehensive and new technology studies are needed to investigate the possible effects on the methylation profiles of genes that express key molecules in certain phenotypic subtypes of NOA.

Discoidin domain receptor 1 (DDR1) is one of the receptor tyrosine kinases and is involved in essential pathways such as proliferation, differentiation, and cell migration.26 Fibroblast cultures from testicular tissues of idiopathic patients with NOA showed abnormal hypermethylation of one CpG in particular in the promoter of DDR1 compared to fertile controls.26 Researchers later found alterations in the expression of the DDR1 gene in the testicular tissues of all patients with NOA participating in the study.26 On the other hand, testicular tissues of patients with spermatogenic defects, including SCOS samples, were analyzed and some genes responsible for gamete formation and PIWI-interacting RNA (piRNA) pathways (spermatogenesis-related 16 [SPATA16], PIWI-like protein 1 [PIWIL1], PIWI-like protein 2 [PIWIL1], MutS homolog 4 [MSH4], insulin-like peptide 3 [INSL3], cyclic nucleotide-gated channel subunit alpha 1 [CNGA1], Fanconi anemia protein G [FANCG], and HIST1H1T) were found to be hypermethylated in patients.27 Recent studies also showed that the promoter region of the SPATA16 gene, which is involved in the formation of acrosomes, was methylated in azoospermic men, and also there was a correlation between promoter methylation and the degree of spermatogenic disorder.22 In a study, the highest hypermethylation of SPATA16 promoter in testicular tissue samples was determined in SCOS, followed by MA and HS.22

In addition, the genes involved in piRNA pathways, methylation changes in the maelstrom spermatogenic transposon silencer (MAEL) gene, which expresses a protein known to have function in suppressing transposable elements, have also been reported in NOA.7,28 In NOA patients with HS, including idiopathic cases, it was found that CpGs in the promoter regions of boule homolog, RNA-binding protein (BOLL), DEAD-box helicase 4 (DDX4), HORMA domain containing 1 (HORMAD1), and MAEL genes showed increased methylation and decreased messenger RNA (mRNA) expressions in testis.29 Mouse studies showed that Mael was expressed in spermatocytes and elongated spermatids, leading to piRNA levels decrease and silencing of long interspersed element 1 (LINE-1) impairment in the testes of Mael null animals.28 In another study involving 26 patients with NOA and HS, a specific site known to affect the expression of the MAEL promoter was found to be hypermethylated in the testes of patients with HS compared to controls with normal spermatogenesis.7 However, LINE-1 transcripts were also observed to increase in cell culture models obtained by enriching the germ cells of HS patients.7 In addition, deficiency in the expression of SRY-box transcription factor 30 (SOX30) from the SOX family, which is known to be necessary for male fertility, has been reported to reduce the expressions of genes that provide hyperactivation and motility of sperm.20 Testicular specimens from 58 patients with NOA were compared with those of patients with OA, and more than 5800 genome-wide differentially methylated regions (DMRs) were analyzed.30 This study showed that 25 CpGs in SOX30 promoter were hypermethylated in patients with NOA and SOX30 methylation was strongly associated with testicular impairment in NOA.30 Sox30 null mice showed complete arrest at the beginning of spermiogenesis, without spermatid or sperm production.19 A recent study revealed that germ cell differentiation in the testes of Sox30 null male mice was halted at the zygotene stage of meiosis, and reexpression of Sox30 also reversed testicular damage.21 Another study, in which the gene expressions and DNA methylations of testicular samples of patients with NOA and OA were analyzed together, found that zinc finger CCHC-type containing 13 (ZCCHC13) gene showed a high negative correlation between methylation and expression.8 Immunohistochemistry results revealed that the level of ZCCHC13 protein decreased in NOA samples compared to OA. Using a mouse cell line, the researchers also reported that ZCCHC13 expression was higher in germ cells compared to Sertoli cells, and demethylation in the ZCCHC13 promoter increased its expression level.8 Subsequent studies have shown that ZCCHC13 acts as a cell cycle regulatory signaling molecule in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) pathways.31 As a noninvasive method, it has been reported that the level of cell-free DNA promoter methylation may be associated with idiopathic NOA.32 Testis-specific promoter methylations were correlated in both the seminal cell-free DNA and in testicular biopsy samples from patients with NOA and SCOS, MA, and HS phenotypes.32 However, methylation variations were detected among patients with NOA, which may be probably due to epigenetic differences during gametogenesis.32 Among testis-specific genes, especially cyclin A1 (CCNA1) and doublesex and mab-3 related transcription factor 1 (DMRT1), methylation patterns in cell-free DNA samples were different in HS compared to those in other NOA groups.32

Global and genome-wide DNA methylation

Until now, limited studies have performed global DNA methylation analyses from tissues of men diagnosed with NOA (Table 1). Although genome-wide methylation studies are important to identify candidate genes responsible for the DNA methylation changes observed in NOA, the presence of somatic DNA is a condition that should be taken into account, especially in the epigenetic evaluation of testicular tissue.27 High-throughput studies in determining the DNA methylation profile allow screening of both gene bodies and intergenic regions.8,33 Thus, genes that could help to explain spermatogenic failure in patients and DNA methylation changes in the noncoding region might be determined.34

Table 1.

Global DNA methylome studies with nonobstructive azoospermia patients

Method	Patients and control	Origin	Number of genes/CpGs found to be affected	Main results for NOA	Reference
Whole-genome bisulfite sequencing, RNA-Seq	NOA (MA) and OA	Testis	Within upstream and downstream regions along with gene body regions, the methylation levels of different functional regions were different	Hypermethylated in DAZL; differential methylation in NR5A1, Topaz1, DND1, and transposable elements	34
Whole-genome bisulfite sequencing, methylation microarray	NOA and OA	Testis	5832 DMRs (2189 genes) were detected, 1391 hypermethylated and 798 hypomethylated genes	Hypermethylated genes were mainly located on chromosome 3, 18, and 5, twenty-five CpGs in SOX30 promoter were hypermethylated	30
DNA methylation bead microarray with >450 000 CpG sites, deep sequencing	Azoospermic men and normozoospermic fertile controls	Blood	Identified 1680 DMCs between infertile cases and fertile controls, 329 annotated DMCs which referred to 245 genes	Overall hypomethylation in GSTM1, GSTM5, HCG4P6, HLAC, and RNF39, hypermethylation in HLA-DQA1, HLA-DRB5, HLA-DRB6, RPTOR, SMAD3, ZFYVE28, and mixed methylation in the infertile group for C6orf10, and HLA-DRB1	33
DNA methylation bead microarray with >450 000 CpG sites	NOA and OA	Testis	10 600 hypomethylated CpG sites in 4253 genes and 20 097 hypermethylated CpG sites in 7889 genes	Hypermethylated loci included the CDK gene family, including CDK4, CDK5, CDK6, CDK18, CDK19, and CDK20; hypomethylation of CECR2, GRAP2, CCL23, SLA2, and ZCCHC13	8
Whole-genome methylation, methylation microarray, qRT-PCR, pyrosequencing for validation	Mild, moderate, and severe HS and OA with normal spermatogenesis	Testis	255 gene promoters were hypermethylated	ANKRD36, BOLL, CRISP2, DAZL, DDX3Y, DDX4, DMRTC2, HIST1H1T, HORMAD1, MAEL, RAN, RPS21, SOHLH2, and TCP11-hypermethylated	29
Cloning-based bisulfite sequencing, microarray	Azoospermic men and men with proven fertility	Testis	Differentially methylated CpG sites (approximately 20 000), 20 genes with >30% difference in DNA methylation within the promoter region of men with NOA and fertile controls	Hypermethylated six genes (MRI1, DCAF12L1, TMEM95, CECR2, DDR1, and NPHS2)	26
DNA methylation bead microarray with >27 578 CpG sites	NOA (HS, MA, and SCO) and OA	Testis	NOA and OA groups differed significantly in terms of their respective DNA methylation profiles at over 9000 CpG sites	78 hypomethylated CpG sites (68 hypomethylated genes) and 134 hypermethylated CpG sites (126 hypermethylated genes); 39 of the testicular function-related 47 genes were hypermethylated in NOA	170

CpG: cytosine-phosphate-guanine; DMRs: differentially methylated regions; DMCs: differentially methylated CpG sites; MA: maturation arrest; HS: hypospermatogenesis; NOA: nonobstructive azoospermia; OA: obstructive azoospermia; SCO: Sertoli-cell only; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; DAZL: deleted in azoospermia-like; NR5A1: steroidogenic factor-1; Topaz1: testis and ovary-specific PAZ domain gene 1; DND1: dead end 1; SOX30: SRY-box transcription factor 30; GSTM1: glutathione S-transferase M1; RNF39: RING finger protein 39; HLA: human leukocyte antigen; SMAD3: small mother against decapentaplegic family member 3; ZFYVE28: zinc finger FYVE-type containing 28; C6orf10: chromosome 6 open reading frame 10; CDK: cyclin-dependent kinase; CECR2: cat eye syndrome chromosome region candidate 2; CCL23: CC chemokine ligand 23; SLA2: SRC-like adaptor 2; ZCCHC13: zinc-finger CCHC-type containing 13; ANKRD36: ankyrin repeat domain 36; CRISP2: cysteine-rich secretory protein 2; DDX3Y: DEAD-box helicase 3 Y-linked; DMRTC2: doublesex and mab-3-related transcription factors like family C2; HIST1H1T: histone H1t; HORMAD1: HORMA domain-containing protein 1; MAEL: maelstrom; RPS21: ribosomal protein S21; SOHLH2: spermatogenesis- and oogenesis-specific basic helix-loop-helix 2; TCP11: T-complex protein 11; DCAF12L1: DDB1- and CUL4-associated factor 12-like protein 1; DDR1: discoidin domain receptor 1

Methylation of imprinted genes

Although many studies investigate the relationship between spermatogenic errors and imprinted genes, most of these studies were based on the methylation of imprinting genes analyzed in ejaculated spermatozoa. On the other hand, testicular spermatozoa may be used in assisted reproductive technology (ART) for azoospermic patients to have children, and imprinting diseases have been reported to be seen up to three times more in babies born with ARTs.35,36 Azoospermia may also be detected in male patients with Beckwith–Wiedemann syndrome, which affects mainly the maternally expressed transcript (H19)insulin-like growth factor 2 (IGF2) genes and the chromosome 11p15 containing the site controlling these genes.36

In a study including azoospermic and anejaculation patients, CTCF-binding site 6, which regulates the expression of H19 and IGF2 in testicular tissues of patients with NOA and hypospermatogenesis, was significantly reduced compared to other patient groups. It was also shown that patients with NOA had no methylation in the H19 gene.35 Similarly, testicular sperm analyses also showed that the level of H19 methylation in samples of patients with NOA was lower compared to proven fertile men.37 However, no difference was observed in the hypomethylation of paternally expressed gene 1 (PEG1)/mesoderm-specific transcript homolog (MEST) gene in both studies in NOA patients. In addition, a study found differences in DMRs of both H19 and MEST genes in primary spermatocytes of NOA patients with MA other than testicular sperm and in H19 DMRs in elongated spermatids.9 These results and previous studies draw attention to the complete lack of methylation in H19, especially from the primary spermatocytes to the testicular spermatozoa of patients with NOA.

m⁶A methylation (N⁶-methyladenosine) in NOA

Epigenetic changes that are effective in regulation of gene expression can also occur in RNA, DNA, and histones. The best described and the most common mRNA modification is N6-methyladenosine.38 While methylation of adenosine occurs by a methyltransferase complex (on both RNA and DNA) including methyltransferase 3 (METTL3) and METTL14, demethylation is catalyzed by fat mass and obesity-associated protein (FTO) alpha-ketoglutarate-dependent dioxygenase (ALKBH9) and ALKBH5.39,40 m⁶A methylation occurs in the evolutionarily highly conserved motif.38 Adenosine methylation may alter the structure and stabilization of mRNA, thereby regulating protein binding to sites close to the modification.41,42

Studies have shown that m⁶A methylation may be an important epigenetic regulator of spermatogenesis.40,43,44 Mouse models have shown that tissue with the highest expression of Alkbh5 was testis.40,44 Alkbh5 null mice reached adulthood, but their testicles were smaller than normal, and histological examination showed abnormal seminiferous tubules in structure and size.40,44 Mutant mice had a lower secondary spermatocyte and spermatid count, which was considered a spermatogenic arrest.40 As a result, those mice have been shown few mature spermatozoa with increased apoptosis.44 However, transcriptome and single-cell RNA sequencing analyzes performed in Alkbh5 null mice showed that the expressions of many genes, especially spermatogenic differentiation and apoptosis, were changed by demethylation of m⁶A.40,44 In a recent study, the level of global m⁶A RNA methylation in testicular tissues of idiopathic NOA patients was stated to decrease.43 Researchers also determined that methylation was the lowest in the SCOS group, followed by MA and HS, proportional to the degree of spermatogenic damage.43

CHROMATIN REMODELING AND HISTONE MODIFICATIONS

The genomic DNA of somatic cells is packaged into chromatin in arrays of nucleosomes. Nucleosomes contain nucleosome cores comprised of DNA coiled around a histone (H) octamer (two copies of core histones H2A, H2B, H3, and H4) and linker histone protein (H1).45,46,47 Sperm cells, however, have a unique genomic organization via chromatin remodeling.48,49 The chromatin remodeling is a crucial epigenetic process in male gametogenesis and required for sperm nuclear condensation, sperm maturation, and fertility.49,50,51 Defects in chromatin condensation have been observed in elongated spermatids of men with NOA who referred to testicular sperm extraction (TESE)-intracytoplasmic sperm injection (ICSI).52 Although testicular spermatozoa have less condensed chromatin than ejaculated spermatozoa, the differences in the chromatin condensation rate do not affect ICSI outcome.53

The chromatin remodeling process occurs in the final stage of spermatogenesis, and about 85% of histones are gradually replaced by smaller protamines in human spermatozoa.54,55 This multistep reorganization process is facilitated by several specific factors and mechanisms, including histone variants and modifications as well as transient DNA strand breaks.56,57,58 Histone variants are noncanonical histones distinct from canonical ones in primary amino acid sequences and function in the regulation of chromatin structure. H1T, H1T2, HILS1, TH2A, H2AL1, H2AL2, TH2B, H3.1T, H3.3, H3T, and H3.5 are involved in testis-specific histone variants in mammals.57,59,60,61 H1t is merely expressed in mouse pachytene spermatocytes, whereas H1T protein persists in round to elongated spermatids.62 The chromatin condensation capability of testicular H1T is lower than somatic subtypes, indicating the role of that in establishing an open chromatin structure and ultimately facilitating histone-to-protamine transition.63,64 However, H1t is not essential for normal spermatogenesis and fertility, as the function of H1T may be implemented by other H1 variants.65 Furthermore, certain genetic variations in the coding and regulatory regions of HIST1H1T gene (coding HT1 protein) have not been associated with NOA.66 However, the c190C>T (Arg64Cys) single-nucleotide polymorphism in the H3T gene was found to be correlated with SCOS as a potential reason for NOA.67 H3T is expressed in differentiating spermatogonia to elongating spermatids but lost in spermatozoa. It has vital functions in the formation of open chromatin structure and spermatogonial differentiation. Homozygous mutation in the H3t gene causes azoospermia in male mice.68 H3.5 is exclusively expressed in seminiferous tubules in the human testis,69 and it shows differential expression patterns in spermatogonia or primary spermatocytes.70 NOA men had lower expression levels of H3.5 in their testis than men with OA. The quantity of H3.5-positive germ cells also showed variation in hypospermatogenesis and early/late maturation arrest in the testes of NOA men.70

The assembly of histone variants in chromatin structure proceeds with the interactions of transition proteins (TPs) and protamines. This unique event is also supported by posttranslational modifications (PTMs).58 PTMs of proteins are chemical modifications of specific amino acids in the protein structure through a proteolytic cleavage or an attachment of a modifying group to one or more amino acid residues.71 Various PTMs of histones have been characterized in distinct stages of mouse spermatogenesis and mature mouse and human spermatozoa. Acetylation, methylation, ubiquitination, crotonylation, and oxidation are involved in these PTMs (Figure 2). H1, H2A, and H2B were found to show little conservation in terms of PTMs between mouse and human sperm, even though PTMs of H3 and H4 were highly conserved between them. Mass spectrometry analysis has illustrated that acetylation of H4 protein at lysine 16 was the most abundant form of single PTMs in human sperm. Furthermore, di-, tri-, and monomethylation of H4K20 were included in the prevailing modifications of H4 tail domain.57 H4 acetylation controls chromatin condensation and functional interactions of nonhistone proteins with chromatin fiber.72 Hyperacetylation of H4 was primarily detected in elongating spermatids, indicating its role in the histone-to-protamine replacement.73 Defective H4-hyperacetylation caused spermatogenesis abnormalities. H4 hyperacetylation or H4K12ac, including spermatogonia level, diminished in NOA men with mixed atrophy compared to men with complete spermatogenesis.74 However, for normal spermiogenesis process, not only acetylation but also deacetylation is required in a balance, which accomplish by several enzymes and/or proteins including bromodomain testis-specific protein (BRDT), sirtuin 1 (SIRT1), and zinc finger MYND-type containing 15 (ZMYND15).75 Sirtuins are deacetylases involved in the catalyzation of acetyl group removal from modified protein substrates in a nicotinamide adenine dinucleotide (NAD⁺)-dependent manner.76 Testicular expression of SIRT1 is not limited to a single type of germ cell, which is expressed in multiple germ cell types. Male mice with a specific deletion of Sirt1 in their germ cells displayed a disruption in the chromatin condensation due to a lack of H4 hyperacetylation (H4K5ac, H4K8ac, and H4K12ac) during gametogenesis, culminating in reduced fertility.77 Zmynd15 codes a transcriptional repressor, MYND-containing zinc-binding protein, which takes part in the spermatogenesis. The function of this transcriptional factor relies on histone deacetylase. Knockout mutation in the Zmynd15 gene caused untimely expression of certain genes (such as protamine 1 [Prm1] and transition protein 1 [Tnp1]), resulting in azoospermia and male infertility in mice.78 Moreover, the testicular expression levels of ZMYND15 and its target genes (TNP1, PRM1, and spermatid maturation 1 [SPEM1]) have decreased in men with NOA. The ZMYND15 expression level has also been proposed as a predictor value for sperm retrieval in these men.79

Figure 2.

(a) Histone acetylation and deacetylation. (b) Methylation, phosphorylation, and ubiquitination of histones. HAT: histone acetyltransferase; HDAC: histone deacetylases.

Acetylation can act synergistically with other histone modifications in chromatin remodeling (Figure 2). Ubiquitination is one of them in which one or more ubiquitin proteins are covalently attached to specific lysine residues in a three-step enzymatic cascade. Ubiquitin-activating, conjugating, and ligating enzymes (E1, E2, and E3, respectively) are involved in this cascade.80,81 Ubiquitination of H2A, H2B, H3, and TH3 have been detected in pachytene spermatocytes and elongating spermatids. Ubiquitinated histones have facilitated chromatin remodeling via nucleosome destabilization, which was also seen in the DNA damage response (DDR) under the controlling of really interesting new gene (RING) E3 ligase, RING finger protein 8 (RNF8).82,83,84,85 RNF8 is involved in the adding of ubiquitin protein to H2A and H2AX during the DDR followed by the recruitment of DDR factors.84

In addition, RNF8-dependent histone ubiquitination stimulates nucleosome eviction by regulating the H4K16ac. RNF8-deficient testes of male mice show abnormalities in nucleosome removal due to the reduction in the H2A and H2B ubiquitination and H4K16ac level, ultimately culminating in infertility. Since RNF8-dependent ubiquitination promotes the H4K16 acetylation via regulating the association of male absent on the first (MOF) with chromatin. The MOF is involved in the acetylation of H4K16.80 In the RNF8-dependent ubiquitination process, PIWI protein may also have a regulatory role. PIWI proteins with a function in maintaining genomic integrity and the repression of transposable elements are exclusively expressed in the germline of animals. The mouse genome encodes three Piwi paralogs (Piwi-like protein 1 [Miwi], Piwi-like protein 2 [Mili], and Piwi-like protein 4 [Miwi2]), all of which are essential for male fertility. Human genome codes human Piwi (HIWI), human PIWIL (HILI) , HILI2, and PIWIL3 are expressed in the testis.86 MIWI and HIWI proteins have a destruction (D) box domain that is evolutionarily conserved in mice and humans. This mutation causes the stabilization of MIWI proteins, thus sequestering RNF8 in the cytoplasm of late spermatids and preventing histone ubiquitination. Azoospermic patients showed HIWI ubiquitination-deficient D box mutations.86 However, variants of the RNF8 gene have not been associated with NOA in the Chinese Han population.87

Chromatin remodeling is characterized by multiple histone methylation, acetylation, and ubiquitination. Histones have undergone some methylation changes during mammalian spermatogenesis, and specific enzymes, including methylases and demethylases, have dynamically regulated these changes. Mono-, di-, and trimethylation of H3K4, di- and trimethylation of H3K79, and trimethylation of H3K27 are some of them.88,89,90 JmjC-domain-containing histone demethylase 1A (JMJD1A), JMJD2A, and K-specific demethylase 3A (KDM3A) enzymes possess histone demethylase activity that removes methyl groups from H3 proteins.91 JMJD1A (also known as JHDM2A), which functions to remove one and two methyl groups on H3K9, is mainly expressed in pachytene and secondary spermatocytes. Jmjd1a null mice exhibited severe oligozoospermia and infertility due to extensive germ cell apoptosis, preventing spermatid elongation. Since deficient expression of Jmjd1a has caused an increase in H3K9me1 and H3K9me2 levels in pachytene spermatocytes and elongating spermatids while causing a reduction in histone acetylation. These modification alterations of histones have been indicated to decrease the expression of certain chromatin-packaging genes involved in the chromatin condensation in spermatids.92 JMJD1A (JHDM2A) has directly targeted Tnp1 and Prm1 genes in round spermatids and its deficiency resulted in chromatin condensation defects.93 Histone demethylases JMJD1A and JMJD1B are also required for male germline maintenance by regulating H3K9 methylation after birth. Removal of methyl groups from lysine residues in H3 (H3K9me2) by JMJD1A and JMJD1B has controlled prospermatogonia to spermatogonia differentiation in mouse germline. Deficiency of JMJD1A/JMJD1B has been associated with complete loss of male germ cells after puberty.94 Testicular expression of JMJD1A has decreased in patients with spermatid maturation arrest SMA and SCOS. The incorporation of JMJD1A on promoter regions of TNP1, TNP2, PRM1, and PRM2 genes has been detected by chromatin immunoprecipitation and found to be lower in round SMA and SCOS patients when compared to that of OA men.95 Furthermore, the expressions of JMJD1A and KDM3A/PRM1 have been correlated with the success of sperm retrieval in NOA men.96,97

Apart from acetylation, ubiquitination, and methylation, several PTMs have a role in chromatin condensation during spermatogenesis. Phosphorylation, poly(ADP)-ribosylation, butyrylation, and crotonylation are involved in them.98,99,100,101 Disruption of the PARslation and crotonylation has resulted in subfertility in mice.102,103

During the histone-to-protamine transition, prior to protamine incorporation in nuclei of spermatids, intermediate transition proteins (TPs) compose the majority (90%) of chromatin basic proteins.104 There are two major types of TPs in rodents, TP1 and TP2, which are encoded by Tnp1 and Tnp2, respectively.105 The exact role(s) of TPs has not been clarified yet. However, it has been implicated that TP1 causes chromatin decondensation through the decreasing melting temperature of DNA while TP2 has reverse function.104,106,107 TP1 may also induce single-strand break repair in mammalian cell culture.108 Male mice with mutations in either Tnp1 or Tnp2 had some subtle sperm abnormalities even though they were fertile, proposing that TP1 and TP2 have partially complemented each other.109,110,111 However, male mice carrying mutations in both Tnp1 and Tnp2 genes (Tnp1^−/− and Tnp2^−/−) were infertile. TPs have enabled proper chromatin condensation via repairing single-strand breaks, preventing sperm defects, and ensuring genomic integrity.112 In humans, TNP1 expression level was similar in spermatids of men with NOA with that of OA men, and it was not related to pregnancy rate following TESE-ICSI.113

Transition proteins are exchanged by protamines in late spermatids. There are two types of protamine in human sperm: protamine 1 (P1) and the family of protamine 2 (P2) proteins (P2–4), which were encode by PRM1 and PRM2 genes, respectively. P2 is firstly transcribed as a precursor (pre-P2) followed by the proteolytic process of it to form P2,114 as pre-P2 is not complex with DNA. Pre-P2 is processed precisely in the subacrosomal domain (DAPI-intense doughnut- and cap-like structure) of human sperm concomitantly by a decrease in the nucleosome and certain histones, indicating the chromatin remodeling initiation during spermiogenesis.115 NOA men have exhibited alterations in the chromatin remodeling initiation.116 The testicular expression of PRM1/PRM2 demonstrated alterations in NOA men,117 which could be a predictor for ICSI outcome in these infertile patients.118 PRM1 expression of testicular spermatid cells was also related to pregnancy rate and post-ICSI embryonic development but not to PRM2 expression.119

NONCODING RNAS (NCRNA)

ncRNAs are categorized into two main classes, which are sncRNA and lncRNA. sncRNA and lncRNAs differ from each other according to their length; sncRNAs are shorter than 200 nucleotides, while lncRNAs are longer than 200 nucleotides. ncRNAs have a regulator role in reproduction, and the association of certain sncRNAs with reproduction has been intensively studied in different pathologies (Figures 3 and 4). microRNA (miRNA) and piRNA are involved in these sncRNAs.24,120

Figure 3.

ncRNAs and translation regulation via miRNAs and lncRNAs. ncRNA: noncoding RNA; miRNA: microRNA; lncRNA: long noncoding RNA; mRNA: messenger RNA.

Figure 4.

Epigenetic mechanisms playing a role in infertility. TNP1: transition protein 1; miRNA: microRNA; lncRNA: long noncoding RNA; piRNA: P-element-induced wimpy testis (PIWI)-interacting RNA; tsRNA: transfer RNA (tRNA)-derived small RNA; circRNA: circular RNA; DNMT: DNA methyltransferase.

SNCRNA

miRNAs

miRNAs are one type of the small RNA, that take part in the cleavage or translational repression of target mRNA transcripts. miRNA biogenesis is strictly controlled by specific enzymes, Drosha and Dicer, and the dysregulation of miRNAs, as well as the biogenesis, is associated with several diseases, including male infertility.121,122,123,124 Recent studies have indicated that testis, semen, and seminal plasma of NOA men exhibit differential miRNA profiles.125,126,127,128

Testicular miRNA

NOA is characterized by abnormalities in spermatogenesis, resulting in the lack of spermatozoa in the ejaculate.129 miRNAs play a role in spermatogenesis.130 Testes of NOA men have illustrated altered miRNA profiles compared to normal controls, 154 of which were downregulated and approximately 20 miRNAs upregulated. Differentially expressed miRNAs, including miR-302a, miR-491-3p, miR-520d-3p, miR-372, and miR-371-5p, target genes with high expression levels in infertile testes such as sex-determining region Y-box (SOX9) and tissue inhibitor of metalloproteinase 3 (TIMP3).131 Similarly, Zhang et al.132 have found that the testicular expression of 129 miRNAs was different in NOA men and these miRNAs were related to spermatogenesis, cell cycle, mitotic prometaphase, and sexual reproduction. They have proposed that the combination of miR-10b-3p and miR-34b-5p could be a biomarker for azoospermia. Infertile men with impaired spermatogenesis and/or various testicular pathologies have been identified by differential miRNA expression profiles in their testicular tissue, although sharing a small number of deregulated miRNAs.133,134 Patients with SCOS, mixed atrophy, and germ cell arrest have altered testicular expression of 197 miRNAs, 68 miRNAs, and 46 miRNAs, respectively. Putative genes of differentially expressed and validated miRNAs (hsa-miR-34b, hsa-miR-34b, hsa-miR-34c-5p, hsa-miR-449a, and hsa-miR-449b) were found to be functional in apoptosis, cell proliferation, and differentiation, implicating the role of these miRNAs in spermatogenesis.121 As testicular tissue miRNAs expression compared to HS, HS with azoospermic factor c (AZFc) deletion, SCOS and MA with normal testicular tissue, all groups have exhibited altered expression of miRNAs except the HS group. Their integrated mRNAs have been involved in the tumor necrosis factor-related apoptosis-inducing ligand signaling pathway, regulating apoptosis.134 Moreover, global analysis of testicular miRNAs of men with NOA with SCOS has recently revealed that miRNAs are crucial regulators of spermatogenesis,126 and the success of sperm retrieval by microdissection TESE (micro-TESE) has been related to testicular miRNAs in patients with NOA. One hundred and eighty miRNAs showed distinct expression profiles between successful and unsuccessful micro-TESE groups, and 86 miRNAs were only expressed in the successful micro-TESE group.135

The expression of miRNAs is not only varied in testicular tissue with several pathologies but also can change even in different developmental stages of germ cells during spermatogenesis in NOA.136 RNA deep sequencing revealed that miRNA profiles of spermatogonia, pachytene spermatocytes, and round spermatids differed between patients with OA and NOA. While the expression of 76 miRNAs was only detected in spermatogonia of OA men, the expression of 91 miRNAs was determined in that of men with NOA. Approximately 400 miRNAs demonstrated differential expression patterns in pachytene spermatocytes between men with OA and NOA. Ninety-four miRNAs and 61 miRNAs were found particularly in round spermatids of OA and NOA patients, respectively. hsa-miR-99b-5p, hsa-miR-140-3p, hsa-miR-424-5p, hsa-miR-221-3p, hsa-miR-100-5p, and hsa-Let-7e-5p were involved in differentially expressed miRNAs, targeting certain genes such as fibroblast growth factor receptor 3 (FGFR3), SMAD family member 3 (SMAD3), and RAR-related orphan receptor A (RORA).136 The cellular miRNA content of developed germ cells also relies on the efficacy of the spermatogenic process.137

mRNA targets of differentially expressed miRNAs have been characterized by integrated analysis of miRNA and mRNA in NOA. Differentially expressed 4712 genes and 93 miRNAs have been detected between NOA and OA patients. The integrated analysis identified the interaction of 193 miRNAs with 2163 genes, including hsa-miR-199a-5p/spermatid flower-like structure protein (SPERT), hsa-miR-376c/polypeptide N-acetylgalactosaminyltransferase-like 5 (GALNTL5), and hsa-let-7c/immunity-related GTPase cinema (IRGC). The identified miRNA/mRNA pairs have been mainly involved in development of secondary sexual characteristics, cell cycle, meiosis, and male gametogenesis.138 Idiopathic infertile patients with NOA have also exhibited altered expression of miRNAs and miRNAs levels as well as the expression of lncRNAs. miR-509 and miR-27b have suggested to be integrated by nine lncRNAs, including LNC00661, LNC00301, LNC00654, and LNC00238 in NOA patients.139

Seminal fluidal miRNAs

Infertile men with NOA and asthenozoospermia displayed differential miRNA expression patterns in their seminal plasma as compared to that of fertile ones. Expressions of miR-34c-5p, miR-122, miR-146b-5p, miR-181a, miR-374b, miR-509-5p, and miR-513a-5p were downregulated in NOA men whereas upregulated in asthenozoospermic men.140 miR-141, miR-429, and miR-7-1-3p have been suggested as biomarkers for idiopathic NOA, expressions of which increased in seminal plasma of these patients according to fertile controls.141 The seminal plasma expression levels of miR-509-5p, miR-122-5p, miR-34b-3p, and miR-34c-5p decreased in patients with OA, NOA, and Klinefelter syndrome. Alterations in the expression profile have been suggested to result from defective spermatogenesis and germ cell damage in NOA and Klinefelter syndrome patients.125 Testicular histopathology types have also had an effect on seminal fluidal miRNA expression profile in NOA. In patients with SCOS and spermatogenesis arrest, 210 miRNAs and 122 miRNAs were differentially expressed compared with fertile men, respectively. Target genes of these miRNAs were involved in the Notch signaling pathway, an effector of Hes5, and were higher in the seminal plasma of men with NOA.142 Furthermore, seminal hsa-miR-3687, hsa-miR-3195, hsa-miR-202-3p, hsa-miR-3656, hsa-miR-3653, hsa-miR-1225-3p, hsa-miR-6752-5p, hsa-miR-4651, hsa-miR-1181, and hsa-miR-513c-5p have been recently found to be a predictor for the existence of spermatogonia in NOA men.128

piRNAs

piRNAs are a novel type of sncRNAs with a length of 21–35 nucleotides and were discovered initially in germline cells of Drosophila melanogaster.143 Biogenesis of piRNAs starts from pre-pre-piRNA by binding to PIWI proteins in germ cells and is completed with the production of mature piRNAs during mammalian spermatogenesis. Fetal piRNAs are enriched in transposon sequences and bind PIWIL2 and PIWIL2 proteins in humans. These piRNAs play an essential role in silencing transposons and, therefore, protect the integrity of the spermatozoal genome.144 Additionally, pachytene piRNAs are also involved in differentiation and maturation of spermatozoa via binding PIWIL1.145 Recent studies showed that the abnormalities in the piRNA biosynthesis pathway lead to failure in spermatogenesis and infertility in men.146 Indeed, whole-exome sequencing of protein poly(A)-specific RNase-like domain containing 1 (PNLDC1) gene characterized four point mutations in 924 men with NOA. The histopathological evaluation of the testicular tissues of these patients showed errors in meiosis and spermatogenic arrest with round spermatids.147

Transfer RNA (tRNA)-derived small RNA (tsRNA)

tsRNAs are also novel sncRNAs rich in body fluid148 and are characterized based on biogenesis, functions, nucleotide compositions, and length.149 tsRNAs are involved in various physiological processes and pathological conditions and found to be enriched in seminal plasma, playing a crucial role in sperm maturation. A recent study indicated their potential use as biomarkers in the prediction of the presence of sperm in patients with NOA undergoing micro-TESE.150 Expression levels of exosomal tRF-Gly-GCC-002 and tRF-Glu-CTC-005 were also suggested as a promising predictive biomarker candidate in sperm retrieval through micro-TESE.151

LNCRNAS AND LNCRNAS-MEDICATED COMPETING ENDOGENOUS RNA (CERNA)

lncRNAs regulate gene expression by interacting with DNA, RNA, and proteins at transcriptional, posttranscriptional, and translational levels, respectively.152 Spermatogenesis is regulated by multiple genes;153 therefore, it is inevitable that lncRNAs have a regulator function in this unique process. It has also been confirmed by recent studies.154,155,156,157 lncRNAs HOX antisense intergenic RNA (HOTAIR) and AK015322 play a role in the maintaining mouse spermatogonial stem cell population.154,156 Inhibition of HOTAIR has resulted in the suppression of proliferation as well as the promoting of spermatogonia apoptosis via ceRNA mechanism. ceRNA mechanism is one of the mechanisms modulating gene expression by lncRNAs.156 lncRNAs may function as a miRNA sponge to competitively regulate the expression of target genes.158 Moreover, expressions of lncRNAs and mRNAs have correlated with each other in spermatogonial stem cells, type A spermatogonia, pachytene spermatocytes, and round spermatid. Sequential expression of these ncRNAs showed that their roles occur in spermatogenesis in a coordinated manner.159 Testes of NOA men are identified by differential expression patterns of lncRNAs. Nearly 190 lncRNAs showed differential expression and most of them (174 lncRNAs) were downregulated in testes with NOA. LINC00884, long noncoding RNA LEMD1 antisense RNA 1 (LEMD1-AS1), zinc finger NFX1-type containing 1 (ZNFX1) antisense RNA 1 (ZFAS1), CSNK1G2-AS1, LINC00467, SPATA42, and ZNF295-AS1 were involved in these lncRNAs. One thousand two hundred and nine interaction pairs of miRNAs, lncRNAs, and mRNAs were determined by ceRNA regulatory network analysis. There was a positive correlation between lncRNAs and mRNAs, while a negative correlation was found between miRNAs and mRNAs, implicating that lncRNAs have an effect on spermatogenesis through ceRNA mechanism.158 Disruptions in the interaction between miRNA-mRNA and miRNA-lncRNA have been associated with idiopathic NOA. There are interactions between miR-509-5p/polo-like kinase 1 (PLK1) and miR-27b-3p/cysteine-rich secretory protein 2 (CRISP2) in men. PLK1 plays a role in meiotic prophase, and CRISP2 has a function in the sperm motility and fertilization. Single-nucleotide polymorphisms in the PLK1 and CRISP2 genes affect pairwise interaction between miR-509-5p and PLK1 and between miR-27b-3p and CRISP2, respectively, resulting in idiopathic NOA due to dysregulation in the mRNAs–miRNAs–lncRNAs interactions.139 LOC100505685, SPATA42, CCDC37-DT, GABRG3-AS1, LOC440934, LOC101929088, LOC101929088, LINC00343, and LINC00301 are extracellular vesicles lncRNAs detected in seminal plasma of infertile men with NOA . These lncRNAs have been recently proposed to be a biomarker for the presence of sperm cells in NOA testes.160

CIRCULAR RNAS (CIRCRNAS)

circRNA is one of the classes of endogenous RNAs that is involved in the suppressing of miRNA function, promoting target gene splicing, and sequestering RNA-binding proteins and regulating their interactions.161 In NOA, circRNAs displayed differential expression profiles in testes. In total, the expression of 894 circRNAs has altered in testicular tissue of men with NOA as compared to controls, most of which were upregulated in these patients. hsa_circRNA_0023313 was one of the upregulated circRNAs, taking part in the ubiquitination process and chromatin binding.162 Bo et al.163 have also reported the role of circRNAs in the pathogenesis of NOA using microarray analysis. They have detected 82 circRNAs with altered expression in NOA testis, including hsa_circRNA_101130, hsa_circRNA_402130, hsa_circRNA_103075, hsa_circRNA_072697, hsa_circRNA_030050, hsa_circRNA_406267, hsa_circRNA_403874, hsa_circRNA_100812, and hsa_circRNA_406168. CircRNA-miRNA-mRNA interaction and enrichment analysis revealed that hsa_circRNA_402130 and hsa_circRNA_072697 have related to many transcription factors and biological processes involving in NOA pathogenesis.163 Men with NOA have been characterized by high level of hsa_circ_0049356 in their blood samples, whereas characterized by low level of hsa_circ_0049356 in their seminal plasma as compared to healthy controls. The analysis of hsa_circ_0049356-miRNA-mRNA interaction indicated that circ_0049356 has targeted mRNAs participating in the actin cytoskeleton rearrangement of germ cells during spermatogenesis.164 Moreover, in a recent study, approximately 37 900 differentially expressed circRNAs characterized in testes with NOA, and targeting mRNAs of these circRNAs determined by ceRNA network have participated in axoneme assembly, microtubule-based processes, and cell proliferation.165 The link between microtubular dysfunction and male infertility has been known.166 Testicular or seminal plasma circRNAs, circ_0008045 (circ_monoglyceride lipase), hsa_circ_0000116, hsa_circ_0000277, hsa_circ_0060394, and hsa_circ_0007773, have been indicated to be a predictor for the successful sperm retrieval by micro-TESE in NOA men.167,168,169

Male factor infertility and decrease in sperm count have become a vital medical and social problem for couples in developing and underdeveloped countries. NOA is the most severe and complex form of infertility regarding the success of treatment. Although various factors are well defined in the disruption of spermatogenesis, not all factors due to the heterogeneity of the disorder have been determined yet. Therefore, understanding the molecular epigenetic mechanisms underlying subforms NOA becomes essential in the upregulation of spermatogenesis.

AUTHOR CONTRIBUTIONS

SG conceived and designed the review and revised the manuscript. AMM and NH contributed to the writing of the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.