Round Spermatid Maturation Arrest Research Articles

Epigenetic Dysregulation of BRDT Gene in Testis Tissues of Infertile Men: Case-Control Study.

Objective Bromodomain testis associated (BRDT), a testis-specific member of the Bromo- and Extra-Terrminal domain (BET) protein family, is involved in spermatogenesis and, more specifically, chromatin remodeling. In the post-meiotic spermatogenic cells, BRDT protein binds to the hyperacetylated histones and facilitates their replacement with transition proteins (TPs), particularly protamines, which are essential for chromatin condensation. The current research was conducted to assess the expression and epigenetic profile of BRDT in the testis tissues of infertile men. Materials and Methods In this case-control study, three groups were included: positive control group: obstructive azoospermia (OA, n=10), round spermatid maturation arrest group (SMA, n=10) and negative control group: sertoli cell- only syndrome (SCOS, n=10). Using quantitative real-time polymerase chain reaction (PCR), the expression profile of BRDT was generated. Also, ChIP-real time PCR was used to measure the following histone marks: H3K9ac, H3K9me3, H3K4me3, H3K27me3 on the promoter region of BRDT. ResultsOur data indicated that BRDT expression decreased in the SMA group in comparison with the positive control group and this finding is in line with the ChIP results obtained in this group.ConclusionBased on these data, we postulate that BRDT gene has a vital role in the spermatogenesis and its decreased expression due to an aberrant epigenetic signaling might be associated with male infertility.

Dynamic Expression and Chromatin Incorporation of ACT and CREM Transcription Factors in Testis Tissues of Infertile Men.

ObjectiveActivator of CREM in the testis (ACT) is a tissue specific transcription factor which activates cAMP responsive element modulator (CREM), a key transcription factor in differentiation of round spermatids into mature spermatozoa. They bind to CRE region in the promoters of transition protein genes (TNP1, TNP2) and protamine genes (PRM1 and PRM2), which are essential for sperm chromatin compaction, and regulates their transcription. This study was conducted to consider the expression of ACT and CREM and their regulatory roles on the expression of PRM1, PRM2, TNP1 and TNP2 genes in testis tissues of infertile men.Materials and MethodsIn this case-control study, testicular biopsies were collected from 40 infertile men and classified into three groups: obstructive azoospermia (OA, n=10, positive control), round spermatid maturation arrest (SMA, n=20), Sertoli cell-only syndrome (SCOS, n=10, negative control group). Using quantitative real-time polymerase chain reaction (PCR), the expression profile of ACT, CREM, TNP1, TNP2, PRM1 and PRM2 genes were assessed in testicular samples and incorporation of ACT and CREM proteins on the promoters of PRM1, PRM2, TNP1 and TNP2 genes were also evaluated by ChIP-real time PCR. ResultsOur results demonstrated significant decrease in the expression levels of ACT, CREM and in their incorporations on their target genes in SMA group in comparison to control groups (P<0.05).ConclusionThese data confirm that there is low expression and incorporation of ACT and CREM and of their target genes in infertilities which are associated with post-meiotic arrest.

Role of peroxisome proliferator-activated receptor gamma (PPARγ) in the regulation of fatty acid metabolism related gene expressions in testis of men with impaired spermatogenesis

Although male infertility is a multifactorial syndrome in which genetic factors are responsible for up to 15 % of cases, there are few studies of genes involved in lipid metabolism and male infertility. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor in testis tissue. PPARγ binds to DNA and regulates the genes for fatty acid (FA) metabolism. Thus, it has a key role in male reproduction. The current study assessed the expressions of fatty acid desaturase 2 (FADS2), elongation of very‐long‐chain fatty acids‐like 2 (ELOVL2), stearoyl-CoA desaturase-1 (SCD), and lipoprotein lipase (LPL) and incorporation of PPARγ in the promoter regions of these genes in testicular tissue biopsies from 30 infertile males who underwent testicular sperm extraction. The samples were classified into three groups: obstructive azoospermia (OA), which was the positive control (n = 10); round spermatid maturation arrest (SMA, n = 10); and Sertoli cell-only syndrome (SCOS, n = 10). There were significantly lower relative mRNA expression levels of the FADS2, ELOVL2, SCD, and LPL genes in the SCOS (P < 0.01) and SMA (P < 0.01) groups compared to the OA control group. We observed a significant decrease in chromatin incorporation of PPARγ on the promoter regions of the candidate FA metabolism genes (P < 0.05). For the first time, the present study results show that PPARγ is a strong mediator for regulation of FA metabolism in human testis tissue and we confirmed its critical role in normal spermatogenesis.

Protamine 1/Protamine 2 mRNA ratio in nonobstructive azoospermic patients.

Altered protamine 1 (PRM1)/ protamine 2 (PRM2) mRNA ratio in testicular biopsy samples correlates with sperm quality and its fertilising ability. This study is planned to assess PRM1/ PRM2 mRNA ratio in subgroups of azoospermia to suggest a more reliable and accurate marker for assessing sperm quality in nonobstructive azoospermia (NOA). A cross-sectional study was done on testicular biopsy samples, taken from 106 azoospermic patients. Samples were histologically classified into subgroups: 36 obstructive azoospermia (OA), and two groups of NOA: 41 round spermatid maturation arrest (SMA) and 29 Sertoli cell-only syndrome (SCOS). OA samples showed histologically normal spermatogenesis and serve as a positive control. mRNA expression of jumonji domain-containing 1A (JMJD1A), PRM1, PRM2 and transition nuclear proteins (TNP1, TNP2) genes was determined, by RT-qPCR. Significantly lower expression of JMJD1A (p<.001), PRM1 (p=.0265) and PRM2 (p=.0032) has been seen in the SCOS group of NOA. We found significant (p<.001) increase in PRM1/PRM2 mRNA ratio in testicular biopsy samples of SCOS group of NOA patients and significant negative correlation of PRM1/PRM2 mRNA ratio with JMJD1A. Hence, PRM1/PRM2 mRNA ratio may represent a more reliable and accurate marker to assess sperm quality in NOA in addition to standard semen parameters.

A homozygous FANCM frameshift pathogenic variant causes male infertility

PurposeFanconi anemia (FA) genes play important roles in spermatogenesis. In mice, disruption of Fancm impairs male fertility and testicular integrity, but whether FANCM pathogenic variants (PV) similarly affect fertility in men is unknown. Here we characterize a Pakistani family having three infertile brothers, two manifesting oligoasthenospermia and one exhibiting azoospermia, born to first-cousin parents. A homozygous PV in FANCM (c.1946_1958del, p.P648Lfs*16) was found cosegregating with male infertility. Our objective is to validate that FANCM p.P648Lfs*16 is the PV causing infertility in this family. MethodsExome and Sanger sequencing were used for PV screening. DNA interstrand crosslink (ICL) sensitivity was assessed in lymphocytes from patients. A mouse model carrying a PV nearly equivalent to that in the patients (FancmΔC/ΔC) was generated, followed by functional analysis in spermatogenesis. ResultsThe loss-of-function FANCM PV increased ICL sensitivity in lymphocytes of patients and FancmΔC/ΔC spermatogonia. Adult FancmΔC/ΔC mice showed spermatogenic failure, with germ cell loss in 50.2% of testicular tubules and round-spermatid maturation arrest in 43.5% of tubules. In addition, neither bone marrow failure nor cancer/tumor was detected in all the patients or adult FancmΔC/ΔC mice. ConclusionThese findings revealed male infertility to be a novel phenotype of human patients with a biallelic FANCM PV.

Deficient expression of JMJD1A histone demethylase in patients with round spermatid maturation arrest

JMJD1A (jumonji domain-containing 1A), a known histone H3K9 demethylase, has been identified as a critical epigenetic regulator in male germ cells, activating the sperm chromatin-packaging genes encoding protamines (PRM) and transition proteins (TNP) required for spermatid elongation and condensation. This research investigated the expression pattern of JMJD1A protein in testicular biopsies of 79 infertile men who had undergone testicular sperm extraction. Samples were classified into obstructive azoospermia (OA, n = 26), round spermatid maturation arrest (SMA, n = 29) and Sertoli cell only syndrome (SCOS, n = 24). Experiments including the detection of mRNA and protein expressions of JMJD1A revealed a severe decrease of JMJD1A/JMJD1A in samples with SMA and SCOS compared with samples with OA (P < 0.005, Kruskal−Wallis test). Additional experiments, including incorporation of JMJD1A on the promoter regions of TNP and PRM genes, and the expression of these genes, showed a significant decrease in the SMA and SCOS versus the OA testes (P < 0.005, Kruskal-Wallis test). These findings show the low expression of JMJD1A/JMJD1A, as well as its low incorporation into chromatin in testes with round spermatid maturation arrest, suggesting that a deficient expression of JMJD1A/JMJD1A might be reflecting and/or contributing to round spermatid maturation arrest.

EXPRESSION PROFILING OF CREM GENE IN TESTIS WITH NORMAL AND IMPAIRED SPERMATOGENESIS IN EGYPTIAN MALES

cAMP responsive element modulator (CREM) is one of the most important transcription factors expressed in male testes which connect the extra cellular signals to gene regulation. CREM expression switches from a repressor to an activator in testes during spermatogenesis. Any alteration in this switch leads to different testicular patterns. Methods: Total RNA was extracted from Eighty eight testicular biopsy and analyzed for CREM expression profiling. Normal spermatogenesis showed expression of CREM2and gamma repressor, the same finding was detected in Hypospermatogenesis group but with another longer repressor belong to alpha repressor type, 95% of Round spermatid maturation arrest group showed two repressor patterns, and 5% showed two activator transcripts of CREM2 activator. Sertoli cells only group didn’t show any activator transcripts of CREM. In conclusion studying the mechanisms of interaction between CREM repressors and activators in the normal and impaired spermatogenesis may lead to better understanding of genetic control of the spermatogenesis and consequently to better treatment options.

Aberrant mRNA expression of chromatin remodelling factors in round spermatid maturation arrest compared with normal human spermatogenesis

During human spermiogenesis, chromatin condensation is associated with replacement of histones by protamines. This exchange is supported by acetylated histones and chromatin remodelling factors. Ten chromatin remodelling factor protein families are known. This study aims to analyse whether a different chromatin remodelling factor expression pattern exists between normal spermatogenesis and round spermatid maturation arrest as potential reason for impaired spermatogenesis and idiopathic male infertility. Laser capture microdissection was used to excise seminiferous tubules from testicular biopsies with normal spermatogenesis and round spermatid maturation arrest. RNA was isolated, first strand cDNA synthesis and pre-amplification were performed using Epigenetic Chromatin Remodelling Factors PCR arrays with 84 genes. Applying hierarchical cluster analysis, three gene expression clusters with six subgroups were identified. The expression pattern ranges from a few high expressed genes in round spermatid maturation arrest to a multitude of genes (74) which are more highly expressed in normal spermatogenesis than in maturation arrest. A total of 22 genes showed a significant difference between normal spermatogenesis and round spermatid maturation arrest (1 gene was up-regulated and 21 genes were down-regulated in the developmental arrest). The significantly different expression of chromatin remodelling factors between normal spermatogenesis and round spermatid maturation arrest may lead to impaired epigenetic information and aberrant transcription during sperm development representing one possible reason for developmental arrest of round spermatids.

Chronic exposure to environmentally-relevant, low-doses of cadmium (Cd) in a rat model mimics effects on spermatogenesis observed in infertile men with varicoceles

Infertile men with varicoceles typically exhibit reduced sperm count, reduced motility, round spermatid maturation arrest and altered Leydig cell function. Testicular Cd is elevated in 60% of biopsies of varicocele testes. Our goal was to determine, using an animal model, whether elevated Cd contributes to abnormal semen parameters and, if so, to identify underlying mechanisms with the aim of developing medical therapies. Rats were exposed to low level Cd in drinking water from puberty to maturity (PN weeks 5–21). We have previously reported that sperm motility is decreased in this model system. In this study, we looked for changes in sperm number, loss of testicular germ cells and changes in RNA expression profiles compared to controls. Male Wistar Hanover rats were fed a nutritionally complete solid diet without Cd and given deionized drinking water containing one of four levels of Cd (0, 5, 50 or 100 mg/L; corresponding to 0–13 mg/kg/d Cd) in the form of Cd Cl2 for 1,4, 8 or 16 weeks. At each time, epididymal sperm and testes were collected. Sperm count was assessed using a hemocytmeter, testicular Cd levels by atomic absorption, testicular testosterone (T) by RIA, histology by H&E staining, apoptosis by DAPI staining, and gene expression using Affymetrix GeneChip Rat Genome 230 2.0 microarrays. Cd accumulated in testes in response to time (ANOVA, P<0.001) and dose (ANOVA, P<0.001). T fell with Cd exposure (ANOVA, P<0.014–P<0.003), as did epididymal sperm count (ANOVA, P<0.007) while apoptosis increased (t-test, P<0.01). Testes sections showed selective loss of postmeiotic stages with increasing Cd. Cd exposures were associated with increased nitric oxide synthase mRNA levels (iNOS, eNOS), NOS inhibitor mRNA down-regulation (e.g., Prmt3), a decrease in mRNAs functioning in late stages of sperm maturation (TP1, TP2, protamines), disregulation of apoptosis factors (caspase 8 decreases; FasL, p53 and Bid increase), and up-regulation of genes involved in steroid hormone biosynthesis (e.g., HSD17β3). Cd alone induces many aspects of human varicocele-related infertility in this rat model: altered apoptosis, increased oxidative stress enzymes, endocrine disruption. Cd induces these changes by affecting levels of testicular mRNAs. This argues that Cd is a significant co-morbidity factor and suggests the need for the systematic study of specific supplements, e.g., antioxidants and antiestrogens.

Epigenetics in Male Germ Cells

Epigenetics in Male Germ Cells

Crem Activator Isoforms in Normal and Impaired Human Spermatogenesis Analyzed by Real Time RT-PCR

cAMP responsive element modulator (CREM) activator isoforms are involved in mammalian spermatogenesis and spermiogenesis. CREM proteins are highly expressed in postmeiotic germ cells of rodents and primates. Homozygous CREM inactivated mice exhibit round spermatid maturation arrest. The lack of CREM expression at both the mRNA and protein levels is associated with spermatid maturation arrest in infertile patients. Using real-time RT-PCR, we have examined the levels of CREM activator isoform mRNAs: CREMtheta1, CREMtheta2 and CREMt2 + Ex-gamma in gametogenic and interstitial cell fractions from normal human testis, in homogenized tissue samples from spermatogenic arrest and from testicular tumors. We have shown for the first time the presence of CREM activator isoform containing exon gamma (CREMtau2 + Exgamma) in normal human spermatogenesis. Among the three CREM isoforms, CREMtheta1 was expressed in its highest level in the male gonads. In comparison, CREMtheta2 mRNA was significantly less suggesting that the P3 promoter is much more active in human testis than the P4 promoter. Minimal-nill levels of mRNA for either of the CREM activator isoforms were detected in lymphocytes or in gonadal tissues from patients with SCOS (Sertoli Cell Only Syndrome). This data underlines the significance of CREMtheta1 isoform in the regulation of transcription during post-meiotic germ cell differentiation.

DNMT1 and HDAC1 gene expression in impaired spermatogenesis and testicular cancer

DNA methylation catalyzed by DNA methyltransferases (DNMTs) and histone deacetylation catalyzed by histone deacetylases (HDACs) play an important role for the regulation of gene expression during carcinogenesis and spermatogenesis. We therefore studied the cell-specific expression of DNMT1 and HDAC1 for the first time in human testicular cancer and impaired human spermatogenesis. During normal spermatogenesis, DNMT1 and HDAC1 were colocalized in nuclei of spermatogonia. While HDAC1 was additionally present in nuclei of Sertoli cells, DNMT1 was restricted to germ cells exhibiting a different expression pattern of mRNA (in pachytene spermatocytes and round spermatids) and protein (in round spermatids). Interestingly, in infertile patients revealing round spermatid maturation arrest, round spermatids lack DNMT1 protein, while pachytene spermatocytes became immunopositive for DNMT1. In contrast, no changes in the expression pattern could be observed for HDAC1. This holds true also in testicular tumors, where HDAC1 has been demonstrated in embryonal carcinoma, seminoma and teratoma. Interestingly, DNMT1 was not expressed in seminoma, but upregulated in embryonal carcinoma.

Expression of activator of CREM in the testis (ACT) during normal and impaired spermatogenesis: correlation with CREM expression.

The cAMP-responsive element modulator (CREM) is involved in regulating gene expression in haploid spermatids. Transcriptional activity of the CREM protein is thought to be regulated by activator of CREM in the testis (ACT). Applying RT-PCR and in situ hybridization, cell-specific gene expression of ACT was demonstrated in man, cynomolgus monkey and mouse. During normal spermatogenesis, RT-PCR revealed a strong signal in all three species. We sequenced monkey ACT cDNA and demonstrated that the putative amino acid sequence is highly conserved between these species. In situ hybridization demonstrated ACT mRNA in mid and late pachytene spermatocytes and in round spermatids. Among four infertile men with round spermatid maturation arrest (RSMA), only one patient revealed a strong signal for ACT, while three patients displayed a weak signal for both RT-PCR and in situ hybridization, although germ cells normally expressing ACT were present in these patients. In addition, CREM knockout mice known to be infertile due to RSMA also exhibited only a weak amplification product for ACT cDNA. ACT mRNA was barely detectable in some round spermatids, but was completely absent in pachytene spermatocytes. Database search revealed two and one CRE within the putative human and mouse ACT promoters respectively. Our findings indicate a conserved function of ACT during the evolution of mammalian spermatogenesis and suggest a role for CREM in ACT transcriptional regulation.

Different CREM-isoform gene expression between equine and human normal and impaired spermatogenesis

Histone-to-protamine exchange causes chromatin condensation ceasing gene expression in elongating spermatids. Gene expression of protamines is regulated by the transcription factor cAMP-responsive element modulator (CREM). Altered CREM expression results in male infertility, as shown by CREM-knock-out mice being sterile due to round spermatid maturation arrest and patients exhibiting round spermatid maturation arrest revealing a lack or substantial reduction of both CREM-mRNA and CREM-protein. Similar defects in histone-to-protamine exchange have been suggested in infertile stallions exhibiting enlarged sperm heads. The CREM-gene consists of 14 exons. Alternative exon splicing results in the production of both activator and repressor proteins. To further clarify the role of different CREM-isoforms for male infertility, the expression pattern of various CREM-isoforms during equine and human normal and impaired spermatogenesis was investigated by RT-PCR. Stallions with normal spermatogenesis expressed six activators and three repressors. In men three activators and seven different repressors were detected. In one stallion and patients with impaired spermatogenesis, only repressors were found. It is concluded that (i) stallion and man reveal a different CREM expression pattern, (ii) the expression of CREM activators is a prerequisite for normal spermatogenesis, and (iii) the lack of CREM activator expression results in male infertility.

Expression of hyperacetylated histone H4 during normal and impaired human spermatogenesis.

Histone-to-protamine exchange in haploid spermatids is preceded by hyperacetylation of core histones resulting in decreased DNA-histone interaction. During normal spermatogenesis, immunohistochemistry with a polyclonal antihyperacetylated histone H4 antibody displayed a strong signal in nuclei of elongating spermatids and, in addition, spermatogonia. Quantitative analysis revealed 98.2 +/- 1.1% of immunopositive spermatids. The percentage of positive spermatids was significantly reduced in infertile men exhibiting at least qualitatively normal spermatogenesis (scores 10-8, 93.1 +/- 6.6%) and impaired spermatogenesis (scores 7-1, 74.9 +/- 23.4%). In seminiferous tubules showing spermatogenic arrest at the level of round spermatids, only 59.5 +/- 16.5% of spermatids were immunopositive for hyperacetylated histone H4. These data demonstrate that the decrease of histone acetylation in spermatids associated with impaired spermatogenesis corresponds with the well known reduction of protamine expression in these cells and confirms the essential role of histone hyperacetylation for correct histone-to-protamine exchange. In seminiferous tubules exhibiting round spermatid maturation arrest, there was an additional signal in nuclei of spermatocytes, suggesting that premature hyperacetylation of histone H4 may result in precocious histone-to-protamine exchange followed by infertility. This is in accordance with data from transgenic mice, where it has been demonstrated that premature expression of protamine-1 results in precocious chromatin condensation followed by sterility.

Transplantation of wild-type spermatogonia leads to complete spermatogenesis in testes of cyclic 3',5'-adenosine monophosphate response element modulator-deficient mice.

The cAMP response element modulator (CREM) gene encodes transcription factors that are highly expressed in spermatids. A deficiency of the CREM gene leads to male infertility in mice due to round spermatid maturation arrest. However, CREM is also expressed in testicular Sertoli cells. We investigated whether CREM deficiency affects the germ line alone or whether the testicular environment is also dependent on CREM function. We examined the restoration of donor-derived spermatogenesis in CREM-deficient testes after transfer of wild-type spermatogonia (16 animals) and after transplantation of germ cells from CREM-deficient or heterozygous donors into spermatogenic tubules of wild-type hosts (16 and 12 animals). Six wk after endogenous spermatogenesis had been depleted by busulphan treatment, spermatogonia were transferred via the rete testis. Production of donor-derived germ cells in the deficient recipients was confirmed by testicular histology and polymerase chain reaction (PCR) analysis of testis fragments 7 and 13 wk after germ cell transfer. Sperm with donor genotype, as detected by PCR, also were flushed from the epididymis. Germ cell transfer using heterozygous donors was also successful. CREM-deficient germ cells largely failed to colonize wild-type recipient testes. According to these findings, germ cell differentiation is dependent on CREM function. The testicular environment of CREM-deficient mice is not essentially affected and is able to support complete spermatogenesis in the presence of wild-type germ cells.

The CREM system in human spermatogenesis.

Spermatogenesis is a multi-step process, regulated by endocrine, paracrine and autocrine signals, through which immature germ cells differentiate into spermatozoa. The cAMP-dependent transcription factor CREM directs in the testis the expression of genes related to the structuring of mature spermatozoa. CREM gene expression originates different isoforms, which in turn can be divided into activators or repressors of gene expression. Whereas only CREM repressors are expressed in pre-meiotic germ cells in mice, a switch to the expression of the CREM activator tau occurs in post-meiotic germ cells. In addition, mice lacking CREM gene are infertile and present an arrest of germ cell maturation at the stage of round spermatid. We have demonstrated that CREM gene is expressed also in human germ cells and that a switch from the expression of repressors to activators is present in normospermic men. In particular, CREM tau mRNA is located in the cytoplasm of round spermatids. Conversely, in patients showing a testicular pattern of round spermatid maturation arrest only CREM repressors are expressed. Other reports have confirmed our data, supporting the hypothesis that CREM plays a role also in human spermatogenesis and that the absence of "the CREM switch" can be associated to spermatogenic arrest.

Testicular cAMP responsive element modulator (CREM) protein is expressed in round spermatids but is absent or reduced in men with round spermatid maturation arrest.

Mice lacking the functional cAMP responsive element modulator (CREM) gene, a component of cAMP-mediated signal transduction, exhibit a specific arrest of round spermatid development although follicle stimulating hormone (FSH) and androgen secretion are not impaired. We studied testicular expression of CREM protein by immunocytochemistry in four patients with complete spermatogenesis (obstructive azoospermia), in 20 infertile patients with round spermatid maturation arrest (n = 10) or mixed atrophy (n = 10) and in six prostate cancer patients undergoing orchidectomy. Concentrations of testosterone were below normal in three patients. Concentrations of luteinizing hormone (LH) were lowered in two patients and elevated in one patient. FSH concentrations were above normal in ten patients. During normal spermatogenesis, CREM was expressed in nuclei of round spermatids in stages I-III of spermatogenesis but not in elongating spermatids. Western blot analysis of testes from prostate cancer patients indicated a major CREM band of approximately 35 kDa. Among patients with predominant round spermatid maturation arrest, CREM expression was significantly reduced (P < 0.05) or undetectable as revealed by quantitative image analysis. CREM-negative spermatids failed to progress beyond stage III of spermatogenesis. Our observations suggest a role for CREM in human spermatid development and raise the possibility that altered CREM expression could be associated with spermatid maturation defects in some cases of idiopathic male infertility.