SPANX蛋白的研究进展及其与男性生育力的相关性

男性不育症病因十分复杂,遗传、环境、内分泌等许多因素都会导致男性不育。而现今临床上多依据精液常规分析对男性不育做出诊断和治疗,但仅依赖精液常规参数存在一定局限性。探寻男性生育力的潜在生物标志分子是当前男性不育的迫切需求。精子X染色体核结合精子蛋白(The sperm protein associated with the nucleus on the X chromosome,SPANX)是在精子中表达的一类小分子蛋白,SPANX蛋白家族基因定位在X染色体上,它随精子的成熟而迁徙,参与精子结构的形成,在精子成熟的不同时期,蛋白定位和蛋白表达均存在差异。在精液参数正常的不育男性和自发弱精症的男性中,SPANX表达下调;同时在活性氧自由基(reactive oxygen species,ROS)阴性的精子中,SPANXC表达降低,在DNA碎片率低的精子中,SPANX表达增高;这些表明SPANX与男性生育力存在一定的相关性,但其与生育力的影响极其相关机制还需要进一步的研究。     

DNA双链断裂检测技术研究进展 *

细胞DNA的完整性受到不同因素的影响,可分为内源性及外源性因素,这些因素均可引起不同程度的DNA损伤。其中,DNA双链断裂是最严重的一种DNA损伤,若未能进行及时的修复,则会引起一系列的损伤反应。严重的DNA双链断裂甚至可以造成细胞凋亡、肿瘤的发生等严重后果。因此,快速并准确地检测细胞DNA双链断裂程度能帮助评估DNA的完整性、内外环境的遗传毒性效应、临床诊断和放化疗监测。DNA双链断裂检测技术近年来发展迅速,目前可基于物理或化学方法、免疫荧光法和高通量测序技术检测DNA双链断裂程度。对这些检测方法的最新研究进展、应用以及其优缺点进行介绍,旨在为后续DNA双链断裂程度检测的研究和临床提供参考。     

DNA双链断裂检测技术研究进展

细胞DNA的完整性受到不同因素的影响,可分为内源性及外源性因素,这些因素均可引起不同程度的DNA损伤。其中,DNA双链断裂是最严重的一种DNA损伤,若未能进行及时的修复,则会引起一系列的损伤反应。严重的DNA双链断裂甚至可以造成细胞凋亡、肿瘤的发生等严重后果。因此,快速并准确地检测细胞DNA双链断裂程度能帮助评估DNA的完整性、内外环境的遗传毒性效应、临床诊断和放化疗监测。DNA双链断裂检测技术近年来发展迅速,目前可基于物理或化学方法、免疫荧光法和高通量测序技术检测DNA双链断裂程度。对这些检测方法的最新研究进展、应用以及其优缺点进行介绍,旨在为后续DNA双链断裂程度检测的研究和临床提供参考。     

流式细胞术在哺乳动物精液质量检测中的应用

精液检测的首要目标就是快速准确地确定精子的生育力。同时具备多种特性和功能完整的精子才能受精,因而只有同时客观地检测多个指标,才能更好地反映精子的生育力。精子检测的传统方法费时费力,检测精子数量少,指标单一,而且易受操作者的主观影响,不能准确地反映精子功能。流式细胞术(FCM)为精子功能研究提供了一种快速、客观、多指标、大通量的检测手段;利用FCM检测精子的质膜完整性、顶体状态、染色质结构、线粒体功能以及细胞凋亡等,可以得知精子功能的相关情况。随着新的荧光探针、染色方法的不断开发和改进,FCM为精液质量检测提供了一种新的检测平台,应用前景极其广阔。     

精子的运动特性

精子的运动特性与生育力有密切关系。本文从超微结构水平和分子生物学基础出发,阐述精子运动机制的微管滑动学说;介绍了精子在液体介质中的运动形式、检测精子活动力的主要技术、影响精子活动力的因素、从异常精液中选择正常精子的技术,以及精子活动力在雄性和雌性生殖道中的演变过程。     

猪精子冷冻损伤研究进展

综述了猪冻存精子顶体、质膜和线粒体的形态学变化及其对精子受精能力的影响,同时分析了冻融精子中所发生的蛋白质、DNA等生物大分子的变化及其可能对冻存精子质量及受精能力的影响,指出冷冻保存过程对精子蛋白质、DNA等生物大分子质和量的影响是精子冷冻损伤的实质,应用先进的蛋白组学进行猪精子冷冻损伤机理研究,有助于深刻揭示冷冻损伤的分子机理,为推动猪精液冷冻保存技术研究取得突破性进展提供理论依据。     

正常人及不育者精子核DNA的定量研究

用流式细胞术,Feulgen显微分光光度法,荧光显微分光光度法测定正常人精子核DNA相对含量:结果表明,上述方法所测得的精子核DNA相对含量稳定,变化范围小。同时用Feulgen显微分光光度法测定不育者精子核DNA相对含量。显示不育者精子核DNA相对含量高于正常人,提示精子核DNA核蛋白复合物异常可能是某些男性不育症的原因。此项研究为诊断男性不育症提供了新方法,在男性学精子核分子研究方面提供了依据。     

精子因素对精子载体法制备转基因山羊的影响

精子具有主动结合、转运、整合外源DNA的能力,并在受精时导入卵母细胞,获得转基因动物.精子介导基因转移(sperm-mediated gene transfer,SMGT)是目前获得转基因动物简单而高效的方法之一.精子因素是影响SMGT方法生产转基因动物的重要方面.本论文结合我们的研究针对转染用山羊(Capra hircus)精液的来源、精子质膜完整性、精液品质及发育阶段等精子因素影响精子结合外源DNA和SMGT方法生产转基因山羊的效率进行了论述,并从这些影响因素入手,提出了筛选精子供体、保持精液品质、调控质膜等措施,提高精子转染外源DNA能力和生产转基因动物的效率.     

特发性少精子症和无精子症与Pygo2基因蛋白编码区SNPs的相关性

男性不育常伴随精子数量减少。Pygo2基因在染色质重塑的伸长精细胞中表达, 其功能受损会导致精子形成阻滞和精子生成减少而引发不育。文章旨在检测引起人特发性少精子症和无精子症的Pygo2基因突变。从77例正常生育力男性和195例特发性少精子症和无精子症患者静脉血提取DNA, 采用聚合酶链式反应-测序方法对Pygo2基因3个蛋白质编码区进行测序对比, 非同义单核苷酸多态性(Single nucleotide polymorphisms, SNPs)位点分别用SIFT、Polyphen-2和 Mutation Taster软件进行诱发蛋白质结构和表型改变的检测和分析。结果表明, 195例患者中, 178例(30例轻度或中度少精子症, 57例重度少精子症和91例无精子症)基因序列分析报告完好, 无精子症中3例患者分别在2个位点(rs61758740, rs141722381)发生了非同义突变SNPs, 重度少精子症中1例患者在位点rs61758741发生了非同义突变, 3个突变位点在SNPs基因数据库都已有报道, 轻度或中度少精子症患者以及正常生育力男性中不存在SNPs。rs61758740可使PYGO2蛋白第141位蛋氨酸(M)变为异亮氨酸(I), rs61758741使PYGO2蛋白第261位碱性赖氨酸(K)变为酸性谷氨酸(E), rs141722381使PYGO2蛋白第240位亲水侧链天冬酰胺(N)变为疏水侧链异亮氨酸(I)。软件分析表明, 在所发现的3个SNP非同义突变位点中, rs141722381引起的单个氨基酸改变会导致PYGO2蛋白空间结构破坏和诱发相关疾病。因此, Pygo2基因蛋白质编码序列区SNPs可能是特发性少精子症和无精子症的诱发因素之一, 导致男性不育。     

哺乳动物卵母细胞的DNA损伤与修复研究进展

DNA损伤是影响配子发生和胚胎发育的关键因素之一。卵母细胞容易被各种内外源因素(如活性氧、辐射、化疗药物等)诱发DNA损伤。目前研究发现,对于各类DNA损伤,各发育阶段的卵母细胞能够做出相应的DNA损伤反应,通过复杂的机制对DNA进行修复或者启动细胞凋亡。相比于进入生长阶段的卵母细胞,原始卵泡卵母细胞更容易被DNA损伤诱导凋亡。DNA损伤不易诱导卵母细胞减数分裂成熟进程停滞,然而携带DNA损伤的卵母细胞的发育能力明显下降。在临床上,衰老、放疗和化疗是导致女性卵母细胞DNA损伤、卵巢储备降低和不孕的常见原因。为此,人们尝试了能够减轻卵母细胞DNA损伤和增强DNA修复能力的多种方法,试图保护卵母细胞。本文对哺乳动物的各发育阶段卵母细胞的DNA损伤与修复的相关研究进行了梳理和总结,并讨论了其潜在的临床价值,以期为生育力保护提供新的策略。     

The use of the acridine orange test and the TUNEL assay to assess the integrity of freeze-dried bovine spermatozoa DNA

The ability to detect nuclear damage is an important tool for the development of sperm preservation methods. We used the acridine orange test (AOT) and the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) assay to assess the DNA status of sperm cells preserved with different lyophilization media. The AOT did not detect any differences between different lyophilization media. However, differences in DNA integrity were observed among treatments with the TUNEL assay, suggesting that TUNEL is a more sensitive method to evaluate sperm DNA. The use of TCM 199 and 10% FCS as a lyophilization medium resulted in 14% of the cells with DNA fragmentation in TUNEL test. The AOT indicated only 4% of the cells with chromatin damage, with this same treatment, with no significant differences when compared to the other treatments. The degree of DNA fragmentation was negatively related to fertilizing potential, as sperm DNA damage was inversely correlated with pro-nucleus formation. The TUNEL assay was found to be an efficient method to detect DNA damage in sperm, and it could be used as a tool to predict male fertility.     

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health.     

Spermatozoal sensitive biomarkers to defective protaminosis and fragmented DNA

Human sperm DNA damage may have adverse effects on reproductive outcome. Infertile men possess substantially more spermatozoa with damaged DNA compared to fertile donors. Although the extent of this abnormality is closely related to sperm function, the underlying etiology of ensuing male infertility is still largely controversial. Both intra-testicular and post-testicular events have been postulated and different mechanisms have been proposed to explain the presence of damaged DNA in human spermatozoa. Three among them, i.e. abnormal chromatin packaging, oxidative stress and apoptosis, are the most studied and discussed in the present review. Furthermore, results from numerous investigations are presented, including our own findings on these pathological conditions, as well as the techniques applied for their evaluation. The crucial points of each methodology on the successful detection of DNA damage and their validity on the appraisal of infertile patients are also discussed. Along with the conventional parameters examined in the standard semen analysis, evaluation of damaged sperm DNA seems to complement the investigation of factors affecting male fertility and may prove an efficient diagnostic tool in the prediction of pregnancy outcome.     

Alterations of sperm DNA integrity during cryopreservation procedure and in vitro incubation of bull semen

An association between sperm DNA integrity and fertility was recently shown for frozen–thawed Norwegian Red (NRF) bull semen diluted in skimmed milk egg yolk (SMEY). In general the fertility of NRF cattle is high, however, in comparison with NRF semen in SMEY, NRF semen diluted in Tris EY based extenders has shown reduced fertility. The aim of the present study was to do a split-sample comparison of sperm DNA integrity of NRF bull semen (n = 20) in SMEY and Triladyl^® (Tris EY based) during routine cryopreservation procedure and during in vitro incubation of frozen–thawed semen in modified synthetic oviduct fluid (mSOF). In contrast to the high fertility of NRF cattle, Holstein cattle are experiencing a marked decline in fertility. Therefore, the present study also aimed to compare sperm DNA integrity of NRF (n = 20) and Holstein (n = 20) semen diluted in Triladyl^® during in vitro incubation. The sperm DNA integrity was measured by susceptibility to in situ acid induced denaturation by the Sperm chromatin structure assay (SCSA). Compared to initial values of frozen neat semen, an increase in DNA damage was observed after dilution and cooling (5 °C) and after freezing–thawing of NRF semen in SMEY, but only after freezing–thawing for NRF semen diluted in Triladyl^®. Sperm DNA damage of NRF semen increased during in vitro incubation in mSOF; the increase in percentage of spermatozoa with DNA damage was more prominent in SMEY than in Triladyl^®, while the degree of damage was higher in Triladyl^®, throughout the incubation period. However, while the correlation between DNA damage and sperm survival was negative in SMEY throughout the incubation period, a positive correlation was observed in Triladyl^® after 9 h of incubation, indicating a higher presence of DNA damage in the live sperm population. In comparison with Holstein spermatozoa, the sperm DNA integrity of NRF semen reflected a better ability to withstand alterations induced during in vitro incubation in mSOF. In conclusion, sperm DNA integrity of NRF bull semen was altered during the cryopreservation procedure and in vitro incubation in mSOF. Dilution in Triladyl^® maintained bull sperm DNA integrity better than dilution in SMEY. Furthermore, alterations in Holstein sperm DNA integrity was more pronounced during in vitro incubation in mSOF compared to NRF bull spermatozoa.     

Sperm aneuploidy and DNA fragmentation in unexplained recurrent pregnancy loss: a multicenter case-control study

Background

Recurrent pregnancy loss (RPL) is defined as the loss of at least three pregnancies in the first trimester. Although the most common cause is embryo aneuploidy, and despite female checkup and couple karyotyping, in about 50% of cases RPL remain unexplained. Male implication has little been investigated and results are discordant. In this context, we conducted a multi-center prospective case-control study to investigate male gamete implication in unexplained RPL.

Methods

A total of 33 cases and 27 controls were included from three university hospitals. We investigated environmental and family factors with a detailed questionnaire and andrological examination, sperm characteristics, sperm DNA/chromatin status using the sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and sperm aneuploidy using fluorescence in situ hybridization (FISH). The Mann-Whitney test and the Wilcoxon or Fisher exact tests were used. A non-parametric Spearman correlation was performed in order to analyze the relationship between various sperm parameters and FISH and sperm DNA fragmentation results.

Results

We found significant differences between cases and controls in time to conceive, body mass index (BMI), family history of infertility and living environment. In cases, total sperm motility and the percentage of morphologically normal spermatozoa were significantly decreased. No difference was found between cases and controls in sperm DNA fragmentation or chromatin integrity. In cases, spermatozoa with aneuploidy, hyperhaploidy and chromosome 18 disomy were significantly increased.

Conclusions

This prospective case-control study is one of the largest to examine environmental factors, sperm characteristics, sperm DNA fragmentation and chromatin, and chromosome anomalies in spermatozoa in relation to unexplained recurrent pregnancy loss. The originality of our study lies in the comprehensive andrological examination and search for risk factors and fertility history. Further studies are needed to confirm the links between unexplained RPL and a male family history of infertility or miscarriages. The increased sperm aneuploidy observed in unexplained RPL supports a male etiology. These data pave the way for further studies to demonstrate the value of preimplantation genetic screening in men with increased sperm aneuploidy whose partners experience unexplained RPL.

     

Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test

The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm.     

Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia

Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.     

Bulky DNA adducts in human sperm: relationship with fertility,semen quality,smoking, and environmental factors

The integrity of DNA of spermatogenic cells can be affected by endogenous and exogenous genotoxic factors. Resulting DNA damage in spermatozoa may significantly contribute to impaired fertility. Here, the 32P-postlabeling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 males, either healthy donors or patients with an impaired fertility. When all donors were analyzed, the levels of bulky DNA adducts was 1.2-fold higher in smokers than in non-smokers, but the difference was not statistically significant (P=0.054). However, a statistically significant difference existed between current smokers and never smokers among the healthy individuals (1.7-fold increase, P=0.008). No correlation between alcohol or coffee consumption and sperm DNA adducts was found. The levels of DNA adducts in sperm seemed to be unaffected by environmental and occupational factors. On the other hand, groups of healthy persons and patients with male-factor infertility differed significantly with respect to the level of bulky DNA adducts (P=0.012). A significant negative correlation between DNA adducts and sperm concentration or sperm motility existed among patients with an impaired fertility (n=93; P<0.029, r(S)=-0.225). These results suggest that DNA adducts in sperm cells can be applied as potential biomarkers in studies of human infertility.     

Genetic factors of male infertility. The role of complex examination in the case of spermatogenesis disturbances

Data about some genetic factors of male infertility are presented, and methods, which may be used for its diagnosing, are studied. Among genetic factors the following are distinguished: changes in the level of genes (mutations), chromosomes (chromosomal aberrations), and total DNA (chromatin dispersion and DNA fragmentation). As well as standard cytogenetic methods of investigation there are a number of molecular-cytogenetic methods like FISH (fluorescent in situ hybridization), TUNEL (Terminal uridine deoxynucleotidyl transferase dUTP nick end labeling), SCSA (sperm chromatin structural assay), SCGE (single cell gel electrophoresis), and SCD (sperm chromatin dispersion). The thorough study of the sperm of infertile men on several levels of organization let us assess the informativity of each method separately and together, and also develop an optimal algorithm of diagnostics with the aim to choose further tactics in the treatment of male infertility.