Epigenetics, cryptorchidism, and infertility

Background Cryptorchid boys with defective mini-puberty and impaired differentiation of Ad spermatogonia (high infertility risk) have altered expression of several genes encoding histone methyltransferases compared to patients with intact differentiation of gonocytes into Ad spermatogonia (low infertility risk). Results High infertility risk cryptorchid boys display hypogonadotropic hypogonadism, which, together with the diminished expression of histone deacetylases and increased expression of HDAC8 decrotonylase, indicates altered histone marks and, thus, a perturbed histone code. Curative GnRHa treatment induces normalization of histone methyltransferase, chromatin remodeling, and histone deacetylase gene expression. As a result, histone changes induce differentiation of Ad spermatogonia from their precursors and, thus, fertility. In this short report, we describe key functions of histone lysine methyltransferases, chromatin remodeling proteins, and long-noncoding RNAs, and discuss their potential roles in processes leading to infertility. Conclusion Our findings suggest that epigenetic mechanisms are critical to better understanding the root causes underlying male infertility related to cryptorchidism and its possible transgenerational transmission.


Introduction
Two major goals in the field of male reproductive biology are to elucidate the molecular mechanisms that underlie cryptorchidism and to develop an effective treatment to prevent infertility.Male-specific epigenetic information involves global re-organization of and localized changes in the chromatin structure during different stages of the male germ cell differentiation [1].Multiple studies have demonstrated that histone lysine methyltransferases regulate gene transcription, thereby influencing cell proliferation, cell differentiation, cell migration, and tissue invasion [1].In humans, inherited alterations in nuclear maturity contributing to subfertility have been found in spermatozoids of adult males who had grandfathers diagnosed with cryptorchidism [2].This may indicate an epigenetic mode of transmission underlying the disorder.
Using the model for artificially induced cryptorchidism, Nishio et al. found that Kdm5a (lysine demethylase 5a) expression is significantly higher in undescended testes than in descended testes.Kdm5a over-expression led to increased expression of Esr2, Neurog3, Pou5f1, Ret, and Thy1.Nishio et al. concluded that Kdm5a is likely involved in the transformation of gonocytes into spermatogonial stem cells by transcriptionally regulating specific genes via H3K4 histone modification [3].
Cryptorchidism could also be caused by prenatal exposure to external disruptors of normal embryogenesis.Any effect observed on male reproductive functions is probably due to altered epigenetic modifications following disruption of DNA methyltransferases and histone marks in the neonatal and/or adult testis [4][5][6][7].
In this short report, we present research that extends our previously published work, describe the key functions of histone lysine methyltransferases and chromatin remodeling, and summarize their role in infertility.

Patients and methods
The patients, biopsy samples, histological analyses, and RNA sequencing protocol were described in detail in the previous study [8].The high infertility risk (HIR) group was defined by the presence of Ad spermatogonia (< 0.005 Ad spermatogonia per tubular cross section), whereas the low infertility risk (LIR) group had a normal distribution of Ad spermatogonia [8].Here, we interpreted the gene expression patterns observed in different prepubertal testicular cell types using our own RNA profiling data, and single-cell RNA sequencing data for adult testis (Table 1) provided by the Human Protein Atlas (www.prote inatl as.org) [9].We analyzed the HIR and LIR groups and the HIR group before and after GnRHa treatment.Analyzed testes were not from boys with syndromic or familial cryptorchidism.

Results
Different methyltransferase and chromatin remodeling genes were found to be preferentially expressed in the prepubertal testis.Relevant genes were grouped into classes based on expression in Leydig and Sertoli cells (class 1), in all testicular cells (class 2), and in germ cells (class 3; Table 1).

Chromatin regulators show distinct testicular expression patterns Class 1
KDM6A and TET1 are two demethylase genes that are mostly expressed in Leydig/Sertoli cells in HIR testes (Table 1).KDM6A plays a critical role in the differentiation of embryonic stem cells [10].TET1 is predominately expressed in Leydig cells and their precursors and is highly methylated in the pluripotent state (Table 1) [11].Its expression results in reduced cell proliferation [12].

Class 2
Eighteen genes encoding modifiers of histone marks were primarily expressed in all three testicular cell types (Leydig, Sertoli, and germ cells, Fig. 1).Except for the three histone deacetylase genes, all 15 genes had increased expression in the HIR group (Table 1).ARID4A and ARID5B function as transcriptional coactivators for androgen receptor and play an integral role in androgen receptor signaling pathways [13].Methyltransferase ATRX is an ATP-dependent chromatin remodeling factor with high homology to SWI/SNF.The protein is important for genome stability, DNA damage repair, and heterochromatin formation, and functions as a transcriptional repressor [14].The histone acetyltransferase EPC1 is a component of the NuA4 histone acetyltransferase complex and can act as both a transcriptional activator and repressor [15].Catalytically active methyl transferase DNMT3A is particularly active during germ cell development [16].
A putative regulatory component of the chromatin remodeling INO80D complex is involved in transcriptional regulation, DNA replication, and cell cycle control [17].INO80D and KDM4A expression, and thus likely signaling, was enhanced in the HIR group (Table 1).Histone demethylase KDM4A participates in transcriptional repression and plays a central role in histone code modification [18].KMT2E protein associates with chromatin regions downstream of transcriptional start sites of actively expressed genes and regulates DNA repair and apoptosis [18].The chromatin remodeler PBRM1 acts as a negative regulator of cell proliferation, whereas the arginine methyltransferase PRMT2 is involved in histone methylation, regulation of androgen receptor signaling, and the regulation of transcription [19].The histone lysine methyltransferase SETD7 is involved in DNA repair [20].
SMARCA genes belong to the SWI1/SNF1 family and are responsible for chromatin remodeling and DNA repair [21].Chromatin regulators SMARCA1 and SMARCA2 act as negative regulators of chromatin remodelers by forming inactive complexes (Table 1).TSPYL4 is thought to possess chromatin and histone binding activity (see www. genec ards.org for references) and is involved in cholesterol metabolism [22].
Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysine residues in histones and other proteins, often in association with transcriptional repression.We found that testicular HDAC2 mRNA is down-regulated in the HIR group (Table 1).Expression of the decrotonylase gene HDAC8 was increased in HIR samples (Table 1).HDAC8 protein mediates decrotonylation of histones, inducing global transcriptional regression.It acts independently, without forming any co-complexes to exert this activity [23].

Class 3
Six genes had increased gene expression predominately in germ cells (spermatogonia and spermatocytes, Table 1).Chromatin remodeler ARID2 is involved in transcriptional activation and repression of its target genes by chromatin remodeling, which is defined as an alteration of the DNA-nucleosome topology (see.genecards.org for reference).Methyltransferase ASH1L is required for efficient expression and H3K4 methylation of HOXA10 [24].Members of the BAZ2B gene family encode proteins that are integral components of chromatin remodeling complexes (see.genecards.org for reference) (Table 1).Histone transcriptional repressor SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression [25].SETD2 is a histone methyltransferase and represents the main enzyme generating H3K36me3, a specific mark associated with transcriptional activity that plays an essential role in the maintenance of a heterochromatic state by recruiting DNA methyltransferase DNMT3A [26].TRDMT1 is an arginine methyltransferase, one of a group of enzymes that catalyze the transfer of methyl groups from S-adenosylmethionine to the arginine residues on histones and other proteins.This gene participates in DNA damage repair [27].

Molecular changes following GnRHa treatment
Genes that are involved in transcriptional repression or DNA damage repair and/or negative regulation of chromatin exhibit lower expression levels after GnRHa treatment (Table 2 and 3).We found no significant differences in gene expression between the LIR group and hormone-treated HIR group (Table 2 and 3).Importantly, a previous study found no significant differences in gene expression between their LIR group and a control group [28].Thus, lower gene expression may be interpreted as a normalization of signaling induced by testosterone stimulation during GnRHa treatment.

Long noncoding RNA and chromatin
Long non-coding RNAs (lncRNAs) are critical for modulating chromatin during development [29].One lncRNA downregulated in the HIR group was TINCR, which produces a spliced long noncoding RNA to bind SEDT7, ARID5B, KDM5A, and LINC00222.TINCR is a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression [30].Another lncRNA, HOX antisense intergenic RNA (HOTAIR), coordinates with chromatin-modifying enzymes, regulates gene silencing, and is transcriptionally induced by estradiol [31].We found that LINC00261 stimulates the expression of HOTAIR and HOTTIP together with FOXA1 [8].HOT-TIP and HOTAIR expression was downregulated in the

Discussion
Post-translational modification of histone proteins and their interpretation by specific binding proteins, the socalled histone code, represents a fundamental regulatory mechanism that has an impact on most chromatin-templated processes, with far-reaching consequences for cell fate decisions and both normal and pathological development [32].This epigenetic phenomenon is likely altered in HIR samples.The observed increase in gene signaling in boys with HIR may be interpreted as compensation for disturbed acetylation.GnRHa treatment induced LH and testosterone secretion, which normalizes the expression of the most methyl transferases and chromatin remodeler mRNA levels (Table 1).This is different from the results obtained with experimentally induced cryptorchidism.
In artificially induced cryptorchidism, Kdm5a overexpression led to increased stimulation of five testicular development genes [3].In contrast, in the HIR group, developmental genes NEUROG3, POU5F1, and RET (-2.1 log2FC; 0.0001 FDR) were downregulated and ESR2 and THY1 were not increased compared to the LIR group [28,33].In addition, no differences were found in the gene expression between the LIR group and a control group [28,33].Therefore, the differences between the HIR group and control group would be expected to be identical to the differences in gene expression between the LIR and HIR groups.Furthermore, we could not confirm overexpression of KDM5A.
Of particular interest is that, in contrast to artificial cryptorchidism, GnRHa treatment led to normalization of KDM5A mRNA levels from 31.7 to 20.0 RPKM (-0.66 log2FC; 0.008 FDR).Thus, transformation of gonocytes into spermatogonial stem cells in humans is not a result of overexpression of Kdm5a, but involves an intact hypothalamus-pituitary-gonadal axis [28,33].We have described three different expression patterns of methyltransferase, deacetylase, and acetylase proteins and chromatin remodelers in prepubertal testes.It seems that chromatin formation in testicular cells requires different sets of these enzymes in different cell types.

Estrogen effect
17-β-estradiol and its receptors are key regulators of gene transcription by binding to estrogen-responsive elements in the genome.Its receptors are important regulators of passive and active DNA demethylation [34].Furthermore, estrogen receptor bound to estradiol recruits histone acetyl transferases (HATs), altering the balance of HATs and HDACs.The HDACs are confronted with greater amounts of acetylated histone substrates, requiring a longer time to deacetylate the acetylated histones [35,36].We observed increased methyltransferase gene expression and decreased deacetylase gene expression in HIR samples, indicating impaired acetylation.In pregnant women, viral infections trigger an immune response that leads to an increased concentration of 17-β-estradiol in the syncytiotrophoblast.Elevated estradiol in syncytiotrophoblasts from women who have given birth to cryptorchid boys are indicative of increased estradiol levels in the fetus [37].Thus, hypogonadotropic hypogonadism and cryptorchidism have been hypothesized to be the result of elevated fetal estradiol levels caused by viral infection during pregnancy [37].In a prospective study, we showed that the placentas of cryptorchid newborns had significantly higher levels of estradiol compared with control placentas of boys with bilateral descended testes [38].Furthermore, analyzed testes were from idiopathic, non-syndromic, and non-familial cryptorchid boys who had Leydig cell atrophy, retarded tubular development implicating Sertoli cell maldevelopment, and decreased number of germ cells [38].Testicular histology resembled that observed by Niestal et al., which was described as estrogen-induced pathological changes [39].A negative effect of estrogen was found in the fractions of spermatozoa from infertile men with shortened ano-genital distance.This fraction is likely to contain transposable elements harboring an estrogen receptor response element and their sperm shows substantial hypomethylation in estrogenic Alu sequences [40].In summary, our observations are consistent with a postulated role of estrogen in modulating the expression of enzymes that modify histones, impacting the histone code.
LncRNAs have emerged as a critical layer of epigenetic regulation in which different lncRNAs are associated with distinctive epigenetic states but share a common mechanism; they physically associate with chromatin-modifying and chromatin-remodeling complexes and guide them to specific genomic loci that are crucial for proper cellular function [41].A good example is HOTAIR, an lncRNA that coordinates with chromatin-modifying enzymes, regulates gene silencing, and is transcriptionally induced by estradiol [42].HOTAIR expression is negatively regulated by estrogen, positively regulated by FOXA1, and inversely correlated with estrogen receptor expression [43].
Almost all estrogen receptor-chromatin interactions and gene expression changes are dependent on the presence of FOXA1.As such, FOXA1 is a major determinant of estrogen-estrogen receptor activity [43].Fendrr regulates Foxa1 and other genes via a Polycomb-dependent epigenetic mechanism [44].FENDRR expression increased substantially following curative GnRHa treatment (2.05 log2FC; FDR 7.21E-05).

Possible transgenerational effect of estrogen
Transgenerational epigenetic inheritance in humans has been challenged and dismissed because of difficulties ruling out the possibility that epimutation induction depends on genetic variants [45].By generating DNA methylation-edited mice, Takahashi et al. showed that acquired methylation of CGIs can be transmitted to offspring through the parental germ line in subsequent generations of mice [45].Moreover, they found that the CGIs with heritable DNA methylation can be demethylated in primordial germ cells.This suggests that DNA methylation memory, elicited by as yet unidentified factors, is transmitted to the next generation in mammals instead of inheritance of epigenetic information [45].Given the commonalities in biological systems between humans and mice, Takahashi et al. 's findings may support the hypothesis that transgenerational inheritance of CGI methylation can occur in humans and, thus, could contribute to heritable susceptibility to cancer and obesity [45], as well as cryptorchidism and infertility.Reports suggest that prenatal exposure to endocrine disruptors may induce transgenerational effects on male reproductive functions, probably due to altered epigenetic modification following disruption of DNMTs and histone marks in the neonatal and/or adult testis [5,6].In humans, alterations of nuclear maturity able to contribute to the subfertility have been found in the spermatozoids of adult males whose grandfathers had cryptorchidism [2].Given that histones transfer genetic material to the next generation after being transformed into protamines, abrogated histone code in HIR samples due to hypogonadotropic hypogonadism may contribute to the observed transgenerational effect in cryptorchid men.Persistent effects over several generations occur due to changes in the level of expression of master regulator genes, such as the key pluripotency gene POU5F1, which could contribute to propagating the epigenetic effects [46].Pou5f1 could directly alter the expression of up to 400 genes, which in turn would modulate many downstream target genes, ultimately affecting the global transcriptional network [46,47].The observed POU5F1 master regulator gene downregulation in HIR samples may contribute to propagating epigenetic effects [28].

Can orchidopexy alone rescue fertility in high infertility risk group?
Current treatment recommendations are early orchidopexy without hormonal treatment with the expectation that successful surgery will be sufficient to protect from infertility [48][49][50].However, early and successful orchidopexy fails to induce transformation of gonocytes into Ad spermatogonia in cryptorchid boys with HIR [51].Failure to develop Ad spermatogonia results in infertility despite successful orchidopexy [51].The incidence of HIR estimated with semi-thin sections of testicular biopsies ranges from 50 to 70% [51,52].Therefore, hormonal therapy provides a better chance of obtaining adequate sperm quality in adulthood [53][54][55].

Limitations of the study
A critical issue, especially when working with human samples, is the number of cases that are included in a given analysis.First, the number of replicates affects the statistical confidence level.Second, human tissue samples exhibit intrinsic variability that needs to be controlled for.In this exploratory RNA profiling study, we included seven patients taken sequentially from a large ongoing study based on randomized patient samples.Their inclusion in the cohorts to be treated or to remain untreated was completely unbiased by any parameter other than undescended testes, which were surgically corrected.This sample size, though small, is sufficient for an initial transcriptome study as we present here.Furthermore, the current study lacks validation experiments for RNA profiling data.However, we previously validated the transcriptome data via qPCR [28].
In conclusion, we found impaired chromatin remodeling due to diminished expression of histone deacetylase and increased expression of methyltransferase and HDAC8 decrotonylase in HIR testes.Assuming that lncR-NAs can cooperate with chromatin-modifying enzymes to promote epigenetic regulation of genes, GnRHa treatment may act as a surrogate for mini-puberty by triggering the differentiation of Ad spermatogonia via lncRNA-mediated epigenetic effects.Our observations indicate that Linc00261, FENDRR, HOTAIR, and FOXA1 participate in the alternate pathway for curative GnRHa treatment to rescue impaired fertility.It is unlikely that the described epigenetic changes could be corrected by early orchidopexy.In this regard, appropriate guidance needs to be adopted for cryptorchidism treatment.

Fig. 1
Fig. 1 Class 2 epigenetic factors.Genes are grouped together according to their broad activities in the indicated epigenetic control mechanisms

Table 2
Gene expression profiles in testicular cells before and after GnRHa treatment RNA-Seq data are indicated for biopsies from high/low infertility risk patients before (-) and after ( +) GnRH treatment.RNA levels (reads per kilobase and million, RPKM), log2 fold changes (log2FC), and false discovery rates (FDRs) are given n.s.not significant, Lc Leydig cell, Sc Sertoli cell, Sptg spermatogonium, Sptc spermatocyte, Sptd spermatid

Table 3
Gene expression profiles following GnRHa treatment in HIR samples, compared to LIR RNA-Seq data are indicated for biopsies from high/low infertility risk (HIR/LIR) patients and samples with/without GnRH treatment.RNA levels (reads per kilobase and million, RPKM), log2 fold-changes (log2FC), and false discovery rates (FDRs) are given n.s.not significant, Lc Leydig cell, Sc Sertoli cell, Sptg spermatogonium, Sptc spermatocyte, Sptd spermatid