Spermiogenesis and DNA Repair: A Possible Etiology of Human Infertility and Genetic Disorders

This paper reviews the possible origin of sperm DNA fragmentation and focuses on the nuclear events associated with spermiogenesis as a potential source of genetic instability and reduced fertilizing potential of the mature male gamete. Recent findings suggest a programmed DNA fragmentation and DNA damage response during the chromatin remodeling steps in spermatids. We also discuss the spermatid DNA repair mechanisms and the possible involvement of condensing proteins, such as transition proteins and protamines, in the process, as this DNA fragmentation is normally not found in late spermatids. We propose that alterations in the chromatin remodeling steps or DNA repair in elongating spermatids may lead to persistent DNA breaks. This vulnerable step of spermiogenesis may provide a clue to the etiology of sperm DNA fragmentation associated with infertility in humans. This vulnerability is further emphasized given the haploid character of spermatids that must resolve programmed double-stranded breaks by an error-prone DNA repair mechanism. Therefore, spermiogenesis has probably been overlooked as an important source of genetic instability.     

Spermiogenesis and DNA repair: a possible etiology of human infertility and genetic disorders

This paper reviews the possible origin of sperm DNA fragmentation and focuses on the nuclear events associated with spermiogenesis as a potential source of genetic instability and reduced fertilizing potential of the mature male gamete. Recent findings suggest a programmed DNA fragmentation and DNA damage response during the chromatin remodeling steps in spermatids. We also discuss the spermatid DNA repair mechanisms and the possible involvement of condensing proteins, such as transition proteins and protamines, in the process, as this DNA fragmentation is normally not found in late spermatids. We propose that alterations in the chromatin remodeling steps or DNA repair in elongating spermatids may lead to persistent DNA breaks. This vulnerable step of spermiogenesis may provide a clue to the etiology of sperm DNA fragmentation associated with infertility in humans. This vulnerability is further emphasized given the haploid character of spermatids that must resolve programmed double-stranded breaks by an error-prone DNA repair mechanism. Therefore, spermiogenesis has probably been overlooked as an important source of genetic instability.     

精子DNA完整性与精液常规参数相关性分析

目的探讨精子DNA完整性与精液参数间的相关性,探讨精子DNA完整性检测在评估男性生育力中的应用价值。方法对206例男性不育患者行精液常规分析,并采用精子染色质扩散法(sperm chromatin dispersion,SCD)检测精子DNA完整性情况,计算精子DNA碎片指数(DNA fragmentation index,DFI),对DFI与各项精液常规参数(浓度、活力和形态等)的相关性进行分析。结果将DFI值按DFI≤10%、10%DFI≤30%和DFI30%分为3组,发现3组间禁欲时间、前向运动精子、不动精子、头/颈部缺陷精子和存活率等差异均有统计学意义(P0.05)。双变量相关分析表明精子DFI与不动精子、头/颈部缺陷精子、精子畸形指数(sperm deformity index,SDI)和畸形精子指数(teratozoospermia index,TZI)存在正相关,而与前向运动精子、正常形态精子、精子浓度和存活率存在负相关。结论精子DNA损伤可能会影响精子形态学异常和活力下降,在分析精液常规的同时,检测精子DNA完整性并将二者结合能够更好的评估患者的生育力。     

极低频电磁场对小鼠睾丸细胞DNA以及精子染色质结构的影响

目的研究50Hz电磁场暴露对小鼠睾丸细胞DNA以及精子染色质结构的影响。方法将昆明种雄性小鼠暴露于0.2mT或6.4mT、50Hz电磁场中,持续4周,分别采用单细胞凝胶电泳法和精子染色质结构检测法分析睾丸细胞DNA断裂情况以及精子染色质结构。结果经电磁场暴露后,0.2、6.4mT组睾丸细胞彗星的相对尾长、彗尾相对DNA含量分别为17.86%±14.60%、2.32%±4.26%及17.88%±13.71%、2.35%±3.87%,与对照组(13.06%±12.38%、1.52%±3.25%)相比,均有明显上升,差异有统计学意义(P<0.05)。0.2、6.4mT组发生DNA迁移的细胞百分率分别为37.83%、39.38%,对照组为25.64%。流式细胞术对精子染色质结构检测的结果表明,S.D.αT(αT分布标准偏差)、XαT(αT均值)没有发生明显变化,但COMPαT(主群以外的精子占总精子的百分数)与对照组相比,有上升的趋势。结论极低频电磁场可引发睾丸细胞DNA链断裂增加,可能引起精子核染色质浓缩异常。     

精子DNA损伤的研究进展

随着男性不育症发病率的升高,精液常规检测已不足以精确评估男性生育力。近年来,精子DNA损伤检测作为一项评估精子质量及男性生育力更精准的指标,逐步成为生殖医学领域的研究热点。精子DNA损伤的机制包括精子发生异常、氧化应激损伤、精子凋亡异常等。目前,精子染色质结构分析法(SCSA)已经成为检测精子DNA完整性的金标准。精子DNA损伤可能与男性不育、辅助生殖结局及后代生长、发育相关。笔者拟就精子DNA损伤的机制、检测方法、对男性生育力的影响及其与辅助生殖技术的关系进行综述,旨在为男性不育症的临床诊断提供依据。     

Loss of livestock breeding efficiency due to uncompensable sperm nuclear defects

An important goal of modern analyses of semen is to elucidate the molecular traits of mammalian sperm chromatin structural abnormalities, defined here as 'uncompensable', that lead to abnormalities in fertility, pronuclear formation, early embryo quality and pregnancy outcome. Sperm with uncompensable nuclear abnormalities are able to fertilize oocytes both in vivo and in vitro; however, due to the uncompensable trait(s), the embryo development may be abnormal. Uncompensable nuclear traits can be experimentally induced in bull sperm by a mild thermal insult to the testis. Sperm nuclear morphology abnormalities seen in ejaculates 11-days post stress are likely related to molecular changes in chromatin observed 3-days post stress by the flow cytometric sperm chromatin structure assay (SCSA). The SCSA measures the susceptibility of sperm nuclear DNA to denaturation in situ. This susceptibility has been correlated with the presence of DNA strand breaks that may be derived in part by oxidative stress and possibly by a unique, abortive apoptotic mechanism. The extent of DNA denaturation is not significantly related to the level of disulfide bonding between the chromatin protamines. The use of human sperm with uncompensable nuclear traits for artificial reproductive techniques is also discussed. The goal of this research is to remove from semen doses those sperm with uncompensable nuclear traits and thereby increase male fertility potential.     

THE GENETICS OF MALE INFERTILITY: A FIELD OF STUDY WHOSE TIME IS NOW

Idiopathic male infertility is often associated with genetic and epigenetic abnormalities. Such abnormalities include chromosome translocations and aneuploidies, Y chromosome microdeletions, and mutations of the CFTR gene. The unraveling of the human genome and ongoing animal transgenic studies have identified numerous other genes likely to be associated with male infertility. Initial reports from human studies have identified several candidate genes, including the protamine genes, SPO11, EIF5A2, USP26, ACT, and others. In addition to gene mutations and polymorphisms, damage to the chromatin resulting in single and double strand DNA breaks affects fertility. Recent studies are highlighting the role of such abnormalities in male infertility, and point to protamine defects as one cause of DNA damage. Epigenetic abnormalities also are being investigated, including the role of residual sperm mRNA in embryogenesis, and the effects of abnormal spermatogenesis on gene imprinting. These studies are pointing to complex etiologies and clinical ramifications in many infertile men.     

Ambient air pollution exposure and damage to male gametes: human studies and in situ 'sentinel' animal experiments

Globally there is concern that adverse reproductive outcomes and fertility impairment in humans may be caused by exposure to environmental contaminants. Air pollution in particular has been linked to DNA damage, abnormal sperm morphology, and reduced sperm performance in men. Experimental studies using model species (mice and rats) exposed in situ provide evidence that ambient air pollution can cause damage to the respiratory system and other tissues or organs. This can take the form of DNA damage and other genetic changes throughout the body, including induced mutations, DNA strand breaks, and altered methylation patterns in male germ cells. Human and animal studies together provide strong evidence that air pollution, especially airborne particulate matter, at commonly occurring ambient levels is genotoxic to male germ cells. The mechanistic link between air pollution exposure and induced genetic changes in male germ cells is currently unclear. 'Sentinel' animal experiments explicitly examining air pollution affects on sperm quality in laboratory rodents have not been conducted and would provide a critical link to observations in humans. The importance of air pollution compared to other factors affecting fertility and reproductive outcomes in humans is not clear and warrants further investigation.     

SPERM MORPHOLOGY AND CHROMATIN CONDENSATION BEFORE AND AFTER SEMEN PROCESSING

Semen analysis constitutes the most important investigation of male infertility. However, the true anomalies present in defective sperm cells have been only partially characterized. The integrity of the sperm chromatin may play the most important role, particularly in ICSI, where most of the natural selection mechanisms are bypassed. This study was carried out to characterize sperm morphology (strict criteria), to evaluate chromatin condensation and sperm count in native semen as well as after semen preparation by the swim-up technique, and to eventually evaluate any correlation between these parameters. Semen from 90 men was analyzed for the above parameters in both the fresh and processed semen. Whereas the sperm count decreased after sperm preparation by the swim-up technique in comparison to the value in the fresh semen (p&lt;.001), there was an increase in the percentage of morphologically normal (p&lt;.001) and chromatin-condensed sperm (p=.99). However, there was no correlation between sperm morphology, chromatin condensation, and sperm count either in the fresh or in the processed semen samples. These results suggest that sperm morphology, sperm count, and chromatin condensation are independent parameters that should be evaluated separately in the assessment of male fertility in an assisted reproduction program.     

精子染色质完整性检测与辅助生殖技术

随着男科学研究的深入,精子评估已由传统的精液常规分析向细胞、分子水平深入发展。精子染色质完整性是反映男性生育力的新指标,其影响到辅助生殖技术(ART)中的受精率、胚胎质量和妊娠率等,与ART结局密切相关。检测精子染色质完整性,采取适当措施获得染色质无损伤或损伤较小的精子,对改善ART结局十分必要。精子染色质结构分析是目前检测精子染色质完整性最常用的方法之一。现就精子染色质损伤的机制与检测方法、其与ART结局的关系以及ART中染色质损伤的防治做文献综述。     

Ambient air pollution exposure and damage to male gametes: human studies and in situ ‘sentinel’ animal experiments

Globally there is concern that adverse reproductive outcomes and fertility impairment in humans may be caused by exposure to environmental contaminants. Air pollution in particular has been linked to DNA damage, abnormal sperm morphology, and reduced sperm performance in men. Experimental studies using model species (mice and rats) exposed in situ provide evidence that ambient air pollution can cause damage to the respiratory system and other tissues or organs. This can take the form of DNA damage and other genetic changes throughout the body, including induced mutations, DNA strand breaks, and altered methylation patterns in male germ cells. Human and animal studies together provide strong evidence that air pollution, especially airborne particulate matter, at commonly occurring ambient levels is genotoxic to male germ cells. The mechanistic link between air pollution exposure and induced genetic changes in male germ cells is currently unclear. ‘Sentinel’ animal experiments explicitly examining air pollution affects on sperm quality in laboratory rodents have not been conducted and would provide a critical link to observations in humans. The importance of air pollution compared to other factors affecting fertility and reproductive outcomes in humans is not clear and warrants further investigation.     

Cyproterone acetate affects protamine gene expression in the testis of adult male rat

The temporal effects of oral administration of cyproterone acetate (CPA), a progestational androgen receptor blocker, were studied on the fertility of adult male rat sires, at a dose of 20 mg kg-1 day-1 after 15 days of gavage. The treatment reduced the fertility and weights of accessory sex glands, without altering the serum levels of luteinizing hormone, follicle-stimulating hormone (FSH) and testosterone (T). Sperm counts were significantly reduced after treatment. Several changes were evident in caput epididymal sperm chromatin in treated rats. The in vitro decondensation rates of sperm chromatin and total fluorescent acridine orange (AO) dye uptake were enhanced. The fluorescent AO dye uptake by the double- and single-stranded sperm chromatin increased. The uptake of thiol-specific monobromobimane fluorescent dye by sperm chromatin was significantly reduced. Sperm of treated rats exhibited hypoprotamination. Protamine levels in the testis were significantly reduced after treatment. Androgen-binding protein (ABP) expression was significantly reduced in testis after treatment. A slight but significant increase was observed in cyclic AMP immunoexpression in testis after treatment. The expression and levels of transition proteins 1 (TP1) and 2 (TP2) as well as cyclic AMP response element modulator protein-tau were maintained at control levels in the testis of treated rats. The present study reports that androgen receptor occupation by CPA preferentially reduces the levels of spermatidal protamine in testis and spermatozoa involved in nuclear chromatin condensation. It is inferred that ABP could be mediating the effects of T in modulating the sequential expression of TPs and protamines during nuclear chromatin condensation. It is likely that indirect effects of T involve its aromatization in spermatids.     

Teratozoospermia with amorphous sperm head associate with abnormal chromatin condensation in a Chinese family

Male infertility affects approximately 7% of the male population. In about 40% of affected patients, the etiology remains unknown. Here, we report the cases of two infertile brothers who have a uniquely prevalent sperm phenotype with completely amorphous sperm heads. To investigate the mechanisms of familial teratozoospermia with amorphous sperm heads, chromatin condensation was assessed by aniline blue staining, western blot, sperm chromatin structure assay and atomic force microscopy in both the two brothers and 40 control fertile donors. Our results showed an abnormal condensation of chromatin with amorphous headed sperm. We suggest that abnormal chromatin condensation which was induced by disturbances in the process of histone–protamine replacement may be a possible cause of familial teratozoospermia with amorphous head, and the elasticity of sperm nuclei could be a new index to assess sperm quality. Additionally, for the first time, the current study provided a new biomechanics strategy for evaluating pathological sperm contributes to our understanding of teratozoospermia.Abbreviations: SCSA: sperm chromatin structure assay; AFM: atomic force microscopy; ICSI: intracytoplasmic sperm injection; HDS: high DNA stainability; DFI: DNA fragmentation index; PBS: phosphate-buffered saline; DTT: dithiothreitol; FITC: fluorescein isothiocyanate; DAPI: 4?,6-diamidino-2-pheneylindole; SSC: standard saline citrate     

The relationship among sperm global DNA methylation,telomere length,and DNA fragmentation in varicocele: a cross-sectional study of 20 cases

Varicocele pathophysiology is related to increased oxidative stress, which might result in loss sperm DNA integrity as well as in genomic instability. Sperm telomere shortening and loss of global DNA methylation are the main features of genomic instability, leading to cell senescence and death, whereas sperm DNA fragmentation (SDF) characterizes the loss of chromatin integrity. We hypothesize that sperm genomic stability and DNA integrity is reduced in infertile men with moderate and large-sized varicoceles, thus being candidate markers of sperm quality in varicocele-related infertility. Here, we assessed the sperm global DNA methylation, telomere length, and SDF in men with and without clinically palpable varicoceles. While the rates of SDF and telomere length were not statistically different between varicocele patients and controls, global sperm DNA methylation seems to be lower in men with varicocele (49.7% ± 20.7%) than controls (64.7% ± 17.1%). A negative correlation between SDF and sperm motility and a positive correlation between sperm morphology and telomere length were observed. Our results suggest that varicocele may result in genomic instability, in particular, global DNA hypomethylation. However, a large sample size may confirm these findings. The understanding of the molecular mechanisms involved in the pathophysiology of varicocele-related infertility may help to better select candidates for varicocele repair.     

精子DNA完整性检测的临床应用

男性生育力受到许多因素的影响,其中精子DNA损伤一直是生殖医学领域研究的热点之一。精子线粒体、核DNA损伤会导致受精失败或影响受精后原核的形成、胚胎着床及子代的健康。目前,定量检测精子中异常DNA数量的方法有多种,虽然尚不理想,但各有其临床应用价值。精子DNA损伤分析比传统精液常规分析参数更稳定、更敏感。胞浆内单精子注射(ICSI)前先对精子DNA进行评估十分必要。精子DNA损伤程度是新的评价精液质量和预测生育能力的指标。     

Vitamin C in Cultured Human (HeLa) Cells: Lack of Effect on DNA Protection and Repair

Aims: Dietary antioxidants, including vitamin C, may be in part responsible for the cancer-preventive effects of fruits and vegetables. Human intervention trials with clinical endpoints have failed to confirm their protective effects, and mechanistic studies have given inconsistent results. Our aim was to investigate antioxidant/ pro-oxidant effects of vitamin C at the cellular level. Experimental approach: We have used the comet assay to investigate effects of vitamin C on DNA damage, antioxidant status, and DNA repair, in HeLa (human tumor) cells, and HPLC to measure uptake of vitamin C into cells. Results: Even at concentrations in the medium as high as 200 μM, vitamin C did not increase the background level of strand breaks or of oxidized purines in nuclear DNA. Vitamin C is taken up by HeLa cells and accumulates to mM levels. Preincubation of cells with vitamin C did not render them resistant to strand breakage induced by H₂O₂ or to purine oxidation by photosensitizer plus light. Vitamin C had no effect on the rate of repair of strand breaks or oxidized bases by HeLa cells. However, vitamin C at a concentration of less than 1 μM, or extract from cells preincubated for 6 h with vitamin C, was able to induce damage (strand breaks) in lysed, histone-depleted nuclei (nucleoids). Conclusion: In these cultured human cells, vitamin C displays neither antioxidant nor pro-oxidant properties; nor does it affect DNA strand break or base excision repair.     

Analysis of sperm nuclear protein gene polymorphisms and DNA integrity in infertile men

Sperm nuclear proteins, the protamines (PRM) and transition nuclear proteins (TNP) play a crucial role in sperm nuclear condensation. The compact packaging of sperm DNA by protamines maintains sperm genome integrity, which is prerequisite for normal sperm function. However the effect of nucleotide variations in PRM and TNP genes on sperm DNA integrity and male fertility is not clear. This case-control study was planned to analyze PRM and TNP gene nucleotide variations and sperm DNA integrity in 100 oligozoospermic infertile men and 100 fertile controls. Protamine and TNP genes were amplified by polymerase chain reaction and sequenced. Flow cytometry-sperm chromatin structure assay (FC-SCSA) was applied to measure the DNA fragmentation index (DFI) in sperm. Semen analysis was performed as per WHO [1999] guidelines with slight modification. In total, 7 nucleotide variations including two novel changes, a non-synonymous mutation in the exon-2 of PRM2 gene (c.443C?>?A) and a novel insertion of T (c.396_397InsT) at the 3′ UTR region of TNP1 were detected. None of the nucleotide changes were observed with increased risk frequency in the oligozoospermic infertile men compared to the controls. Though overall DFI was significantly (p?<?0.0001) higher in infertile men compared to controls (36.31?±?7.25 vs. 26.49?±?2.78) irrespective of nucleotide changes, no such difference was observed between 100 infertile men or pooled population of 200 with and without mutations. However it was observed that two cases with novel nucleotide changes PRM2 c.443C?>?A and TNP1 c.396_397InsT had higher DFI value of 34.82% and 43.85%, respectively. In conclusion, our pilot study for the first time in the Indian population revealed two rare novel mutations in sperm nuclear protein genes that are perhaps associated with higher sperm DNA fragmentation.     

泌尿生殖道支原体属感染男性不育患者精子DNA完整性分析及应用

目的探讨泌尿生殖道支原体属感染男性不育患者精子DNA完整性的改变及其临床意义。方法对79例泌尿生殖道支原体属感染男性不育患者(患者组)和16例健康生育男性(对照组)同时进行精子染色质扩散试验(SCD)、与精液常规参数检测;在患者组随机选取32例进行抗菌药物治疗,用SCD试验检测治疗前后的精子DNA损伤情怳。结果患者组的精子DNA碎片化指数(DFI)为(35.7±14.7)%,对照组精子DFI为(11.2±5.9)%,2组差异有统计学意义(P<0.01);32例患者治疗前的精子DFI为(35.3±14.2)%,抗菌药物治疗后的精子DFI为(19.6±9.7)%,2组差异有统计学意义(P<0.01);患者治疗前后的精子密度、活动率比较,差异无统计学意义;患者治疗后的精子畸形率明显减少,差异有统计学意义(P<0.05)。结论泌尿生殖道支原体属感染男性不育患者的精子DNA损伤,明显高于健康生育男性,相应抗菌药物治疗可以降低感染造成的精子DNA损伤,精子DNA完整性分析在支原体感染不育男性诊疗中,比精液常规参数检测更有价值。     

Function of the Sperm Nuclear Matrix

Mammalian spermatozoa contain some of the most highly compact chromatin. This is due to the DNA binding proteins, the protamines, which replace most of the histones during spermiogenesis. This chromatin, however, shares some features with somatic cell chromatin. One of these is the organization of DNA into loop domains attached at their bases to a proteinaceous nuclear matrix. Several groups have shown that the sites at which DNA associates with the sperm nuclear matrix contain chromatin structures that are linked with specific functions. Recent data also suggest that the sperm nuclear matrix plays essential roles in the paternal pronucleus of the newly fertilized oocyte, suggesting that the sperm cell provides more information to the new embryo than solely the genetic material it delivers. Here, we will review these data which together give insight into the functional significance and requirements of sperm nuclear structure.     

Analysis of sperm nuclear protein gene polymorphisms and DNA integrity in infertile men

Sperm nuclear proteins, the protamines (PRM) and transition nuclear proteins (TNP) play a crucial role in sperm nuclear condensation. The compact packaging of sperm DNA by protamines maintains sperm genome integrity, which is prerequisite for normal sperm function. However the effect of nucleotide variations in PRM and TNP genes on sperm DNA integrity and male fertility is not clear. This case-control study was planned to analyze PRM and TNP gene nucleotide variations and sperm DNA integrity in 100 oligozoospermic infertile men and 100 fertile controls. Protamine and TNP genes were amplified by polymerase chain reaction and sequenced. Flow cytometry-sperm chromatin structure assay (FC-SCSA) was applied to measure the DNA fragmentation index (DFI) in sperm. Semen analysis was performed as per WHO [1999] guidelines with slight modification. In total, 7 nucleotide variations including two novel changes, a non-synonymous mutation in the exon-2 of PRM2 gene (c.443C?>?A) and a novel insertion of T (c.396_397InsT) at the 3' UTR region of TNP1 were detected. None of the nucleotide changes were observed with increased risk frequency in the oligozoospermic infertile men compared to the controls. Though overall DFI was significantly (p??A and TNP1 c.396_397InsT had higher DFI value of 34.82% and 43.85%, respectively. In conclusion, our pilot study for the first time in the Indian population revealed two rare novel mutations in sperm nuclear protein genes that are perhaps associated with higher sperm DNA fragmentation.