Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells

Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issuesThis review was supported by the project BFU2010-1673

DNA damage and repair in human reproductive cells

The fundamental underlying paradigm of sexual reproduction is the production of male and female gametes of sufficient genetic difference and quality that, following syngamy, they result in embryos with genomic potential to allow for future adaptive change and the ability to respond to selective pressure. The fusion of dissimilar gametes resulting in the formation of a normal and viable embryo is known as anisogamy, and is concomitant with precise structural, physiological, and molecular control of gamete function for species survival. However, along the reproductive life cycle of all organisms, both male and female gametes can be exposed to an array of “stressors” that may adversely affect the composition and biological integrity of their proteins, lipids and nucleic acids, that may consequently compromise their capacity to produce normal embryos. The aim of this review is to highlight gamete genome organization, differences in the chronology of gamete production between the male and female, the inherent DNA protective mechanisms in these reproductive cells, the aetiology of DNA damage in germ cells, and the remarkable DNA repair mechanisms, pre- and post-syngamy, that function to maintain genome integrit

Association of polymorphisms in genes coding for antioxidant enzymes and human male infertility

This is the peer reviewed version of the following article: Annals of Human Genetics 83.1 (2019): 63-72, which has been published in final form at https://doi.org/10.1111/ahg.12286. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsPurpose: Although oxidative stress is thought to be an important cause of male infertility, primarily due to DNA and cell membrane damage, little is known about the genetic causes underlying suboptimal function of the seminal enzymatic antioxidant system. The aim of this study was to investigate the relationship of four potentially functional polymorphisms associated with oxidative stress pathway genes (superoxide dismutase—SOD2 lle58Thr and SOD2 rs4880, catalase—CAT C-262T, glutathione peroxidase 1—GPX1 Pro200Leu) and two null variants of the glutathione S transferase (GSTT and GSTM) genes and infertility risk. Methods: A case control study was conducted on 313 infertile patients and 80 fertile donors. Each ejaculate was subjected to a seminal analysis that included the classical parameters seminal volume, sperm concentration, sperm motility, and sperm morphology, as well as sperm DNA fragmentation (patients only). Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and PCR multiplex methods were carried out for genotyping. Results: Statistically significant differences were found between fertile donors and infertile patients for SNP CAT C-262T; the CC genotype was related with a twofold increased risk of infertility (odds ratio [OR] = 2.262; 95% confidence interval [CI] = 1.369–3.733; P = 0.001), whereas the CT genotype was associated with a protective effect (OR = 0.401; 95% CI = 0.241–0.667; P = 0.001). Surprisingly, the SOD2 Ile58ssThr SNP was not represented in the sample population, so its frequency in the current population frequenting fertility clinics in Madrid may be very low. Conclusions: Our results suggest that the CAT SNP C-262T is potentially associated with an increased risk of male infertilityThe Ministry of Economy, Industry and Competitiveness of Spain supported this stud

Evaluación de la calidad seminal en pilotos de vuelos internacionales transoceánicos

Motivación: El factor masculino en reproducción está presente en la mitad de los casos de infertilidad, un problema de salud pública en auge en las ultimas décadas. Muchos estudios han demostrado que la calidad seminal se ha visto afectada por los contaminantes ambientales y los cambios en el estilo de vida, que pueden inducir procesos de fragmentación del DNA espermático o inducir estrés oxidativo(1). El objetivo de este trabajo consiste en evaluar la calidad seminal de un grupo concreto, los pilotos de vuelos transoceánicos que, por su profesión, pueden suponer un grupo de riesgo de problemas de fertilidad debido a sus condiciones laborales. Los cambios constantes de presión, temperatura y estación, pueden ocasionar daños a nivel celular con afectación del DNA, que implique procesos de fragmentación masiva y, consecuentemente, una disminución o pérdida de la fertilidad con el subsecuente fracaso en el embarazo. Este trabajo también pretende poner de manifiesto la necesidad de un seminograma avanzado, mas allá de los parámetros básicos analizados convencionalmente.Métodos:Se llevó a cabo un seminograma estándar para evaluar concentración, volumen, motilidad, morfología y vitalidad, con ayuda de sistemas informáticos (SCA) para los ensayos de concentración y motilidad, microscopía tanto de campo claro como de fluorescencia, así como el kit Vitaltest para el ensayo de vitalidad.Para el análisis de fragmentación de DNA se realizaron 2 ensayos: el SCD (Sperm Chromatin Dispersion) para evaluar la fragmentación total (2) y el ensayo COMET, el cual identifica específicamente las roturas de cadena doble(3). Ambos se realizaron mediante la utilización de kits comerciales (Halotech DNA) y microscopía de fluorescencia para su recuento.Asimismo se realizó un ensayo para medir el estrés oxidativo, también mediante el uso de un kit comercial para un análisis colorimétrico (Halotech DNA).Conclusiones: A pesar de no tener aún los resultados necesarios, hay muchos estudios que demuestran que la calidad seminal, sobre todo respecto al DNA, se ve afectada por múltiples factores, ya sean ambientales, genéticos o referentes al estilo de vida, por lo que cabe esperar que este grupo concreto se vea afectado negativamente debido a sus condiciones laborales. Cada vez van surgiendo mas casos en los que el DNA espermático está tan dañado, que el ovocito es incapaz de reparar ese daño, con lo que se justifica el uso de un seminograma avanzado que estudie el material genético

A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis

BACKGROUND: Antibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. We have adapted a kit for rapid determination of bacterial DNA fragmentation, to assess cell wall integrity. RESULTS: Cells incubated with the antibiotic were embedded in an agarose microgel on a slide, incubated in an adapted lysis buffer, stained with a DNA fluorochrome, SYBR Gold and observed under fluorescence microscopy. The lysis affects the cells differentially, depending on the integrity of the wall. If the bacterium is susceptible to the antibiotic, the weakened cell wall is affected by the lysing solution so the nucleoid of DNA contained inside the bacterium is released and spread. Alternatively, if the bacterium is resistant to the antibiotic, it is practically unaffected by the lysis solution and does not liberate the nucleoid, retaining its normal morphological appearance. In an initial approach, the procedure accurately discriminates susceptible, intermediate and resistant strains of Escherichia coli to amoxicillin/clavulanic acid. When the bacteria came from an exponentially growing liquid culture, the effect on the cell wall of the beta-lactam was evident much earlier that when they came from an agar plate. A dose-response experiment with an E. coli strain susceptible to ampicillin demonstrated a weak effect before the MIC dose. The cell wall damage was not homogenous among the different cells, but the level of damage increased as dose increased with a predominant degree of effect for each dose. A microgranular-fibrilar extracellular background was evident in gram-negative susceptible strains after beta-lactam treatment. This material was digested by DNase I, hybridised with a specific whole genome probe, and so recognized as DNA fragments released by the bacteria. Finally, 46 clinical strains from eight gram-negative and four gram-positive species were evaluated blind for susceptibility or resistance to one of four different beta-lactams and vancomycin, confirming the applicability of the methodology. CONCLUSION: The technique to assess cell wall integrity appears to be a rapid and simple procedure to identify resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis

Urinary concentrations of bisphenol A, parabens and benzophenone-type ultra violet light filters in relation to sperm DNA fragmentation in young men: A chemical mixtures approach

People are daily exposed to multiple endocrine disruptor compounds (EDCs) that may interfere with different molecular and cellular processes, promoting a potential estrogenic, androgenic, or anti-androgenic state. However, most epidemiological studies attempting to establish relationships between EDCs exposure and health effects are still considering individual compounds. A few studies have shown associations between exposure to individual non-persistent EDCs and sperm DNA fragmentation (SDF) in different male populations. Thus, the aim of this study was to investigate associations between combined exposure to non-persistent EDCs and SDF index in young men. A cross-sectional study was conducted with 158 healthy university students from Southeaster Spain. The participants provided spot urine and semen samples on the same day. The concentrations of urinary bisphenol A (BPA), benzophenones [2,4-dihydroxybenzophenone (BP-1); 2,2′,4,4′-tetrahydroxybenzophenone (BP-2), 2-hydroxy-4-methoxybenzophenone (BP-3), 2,2′-dihydroxy-4-methoxybenzophenone (BP-8), 4-hydroxybenzophenone (4OHBP)], and parabens (methylparaben, ethylparaben, propylparaben, butylparaben) were measured by dispersive liquid-liquid microextraction and ultrahigh-performance liquid chromatography with tandem mass spectrometry detection. SDF was analysed using a Sperm Chromatin Dispersion test. Statistical analyses were carried out using Bayesian Kernel Machine Regression models to evaluate associations between combined exposure to these compounds and SDF index while adjusting by relevant covariates. The increase in urinary concentration of 4OHBP was found to be the most important contributor to the negative association between urinary EDCs concentrations and SDF index, being of -5.5 % [95 % CI: -10.7, -0.3] for those in percentile 50, and -5.4 % [95 % CI: -10.8, -0.1] for those in percentile 75. No significant associations were observed between other EDCs and SDF index. Our findings show that urinary 4OHBP levels may be associated with a decrease in the SDF index. Nonetheless, the effects we observed were likely to be small and of uncertain clinical significance. Further research is needed to replicate our findings in other male populations.Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia [08808/PI/08, 19443/PI/14]Consejería de Innovación, Junta de Andalucía [P09-CTS-5488]Ministerio de Economía, Industria y Competitividad, Instituto de Salud Carlos III (Acción Estratégica en Salud, AES) [PI10/00985, PI13/01237, PI13/ 02406

Use of quinolones in bull semen extenders to reduce sperm deoxyribonucleic acid damage

Cryopreserved sperm samples from Holstein bulls (n = 20) were examined for bacterial presence and Sperm DNA Fragmentation (SDF) dynamics. SDF was assessed after thawing (T0) and at 4, 24 and 48 h of incubation (37°C) and the rate of SDF (r-SDF), as an estimator of the DNA degradation over time, was calculated. Two groups of bulls were identified based on the presence or absence of bacteria: One group (n = 10) had a readily detectable bacterial presence, while the other group (n = 10) had an undetectable bacterial presence. Differences in the SDF at T0 were not observed between these two groups. However, statistically different results were found after 24 h of incubation at 37°C (Kaplan-Meier estimator; Log- Rank Matel-Cox, p0.05) were not detected between the control and the quinolone treated sample for those samples without bacteria. However, differences (p<0.000) in SDF were observed for quinolone treated samples that previously presented bacteria. Incubation of sealed straws showed that bacterial contamination occurred prior to cryopreservation. These results call attention to three points: (1) sperm samples were in contact with bacteria before cryopreservation; (2) the r-SDF can be directly correlated with bacterial presence but this effect remains cryptic after thawing and (3) the r-SDF can be reduced by treating the semen samples with an adequate antibiotic such as quinolones, a finding not previously reported in the scientific literature, but important in terms of reproductionThis work was supported by the Ministry of Education and Science, Spain (Grant BFU2010-16738/BFI

Expression of the HPV18/E6 oncoprotein induces DNA damage

Abstract This study investigated possible variations in DNA damage in HeLa cells with silenced expression of the HPV/E6oncogene compared with HeLa cells with normal expression of the E6oncogene using the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) technique and a whole human genome DNA probe. The variable levels of DNA breaks present were measured quantitatively using image analysis after whole-genome DNA hybridization. HeLa cells with silenced expression of the HPV18/E6 oncogene showed a significant decrease in DNA damage compared with parental cells with normal expression of the E6oncogene. These results were confirmed by alkaline comet assay. In conclusion, we demonstrated a decrease in DNA damage in HeLa clones associated with low expression of the HPV/E6 oncogene

Fast assessment of resistance to carbapenems and ciprofloxacin of clinical strains of Acinetobacter baumannii

Infections caused by multidrug-resistant Acinetobacter baumannii constitute a major life-threatening problem worldwide, and early adequate antibiotic therapy is decisive for success. For these reasons, rapid detection of antibiotic susceptibility in this pathogen is a clinical challenge. Two variants of the Micromax kit were evaluated for a rapid detection in situ of susceptibility or resistance to meropenem or ciprofloxacin, separately, in 322 clinical isolates. Release of the nucleoid is the criterion of susceptibility to the beta-lactams (carbapenems), whereas diffusion of DNA fragments emerging from the nucleoid characterizes the quinolone activity. All the susceptible and resistant strains were correctly categorized in 100 min according to the MIC results and CLSI criteria. Thus, our technology is a promising tool for rapid identification of carbapenem and quinolone resistance of A. baumannii strains in hospital settingsThis work has been supported by grants from the European Community, FP 7, ID: 278232 (MagicBullet), Xunta de Galicia 10CSA916020PR, and by REIPI, Spanish Network for Research in Infectious Diseases (Instituto de Salud Carlos III, RD06/0008/0025) and the Fondo de Investigaciones Sanitarias (PS09/00687) to G.B

Male meiosis in Crustacea:synapsis, recombination, epigenetics and fertility in Daphnia magna

We present the first detailed cytological study of male meiosis in Daphnia (Crustacea: Branchiopoda: Cladocera)—an aquatic microcrustacean with a cyclical parthenogenetic life cycle. Using immunostaining of the testes in Daphnia magna for baseline knowledge, we characterized the different stages of meiotic division and spermiogenesis in relation to the distribution of proteins involved in synapsis, early recombination events and sister chromatid cohesion. We also studied post-translational histone modifications in male spermatocytes, in relation to the dynamic chromatin progression of meiosis. Finally, we applied a DNA fragmentation test to measure sperm quality of D. magna, with respect to levels of inbreeding. As a proxy for fertility, this technique may be used to assess the reproductive health of a sentinel species of aquatic ecosystems. Daphnia proves to be a model species for comparative studies of meiosis that is poised to improve our understanding of the cytological basis of sexual and asexual reproduction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00412-015-0558-1) contains supplementary material, which is available to authorized users