scholarly journals Species-Specific Differences in Sperm Chromatin Decondensation Between Eutherian Mammals Underlie Distinct Lysis Requirements

2021 ◽  
Vol 9 ◽  
Author(s):  
Jordi Ribas-Maynou ◽  
Estela Garcia-Bonavila ◽  
Carlos O. Hidalgo ◽  
Jaime Catalán ◽  
Jordi Miró ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Condensation ◽  
Proteinase K ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Dna Breaks ◽  
Chromatin Decondensation ◽  
The Third ◽  
Core Length ◽  
Sperm Dna

Sperm present a highly particular DNA condensation that is acquired during their differentiation. Protamines are key elements for DNA condensation. However, whereas the presence of protamine 1 (P1) is conserved across mammalian species, that of protamine 2 (P2) has evolved differentially, existing only few species that use both protamines for sperm DNA condensation. In addition, altered P1/P2 ratios and alterations in the expression of P1 have previously been associated to infertility and DNA damage disorders. On the other hand, different methods evaluating DNA integrity, such as Sperm Chromatin Dispersion (SCD) and Comet tests, need a previous complete DNA decondensation to properly assess DNA breaks. Related with this, the present study aims to analyze the resilience of sperm DNA to decodensation in different eutherian mammals. Sperm samples from humans, horses, cattle, pigs and donkeys were used. Samples were embedded in low melting point agarose and treated with lysis solutions to induce DNA decondensation and formation of sperm haloes. The treatment consisted of three steps: (1) incubation in SDS + DTT for 30 min; (2) incubation in DTT + NaCl for 30 min; and (3) incubation in DTT + NaCl with or without proteinase K for a variable time of 0, 30, or 180 min. How incubation with the third lysis solution (with or without proteinase K) for 0, 30, and 180 min affected DNA decondensation was tested through analyzing core and halo diameters in 50 sperm per sample. Halo/core length ratio was used as an indicator of complete chromatin decondensation. While incubation time with the third lysis solution had no impact on halo/core length ratios in species having P1 and P2 (human, equine and donkey), DNA decondensation of pig and cattle sperm, which only present P1, significantly (P < 0.05) increased following incubation with the third lysis solution for 180 min. In addition, the inclusion of proteinase K was found to accelerate DNA decondensation. In conclusion, longer incubations in lysis solution including proteinase K lead to higher DNA decondensation in porcine and bovine sperm. This suggests that tests intended to analyze DNA damage, such as halo or Comet assays, require complete chromatin deprotamination to achieve high sensitivity in the detection of DNA breaks.

scholarly journals Complete Chromatin Decondensation of Pig Sperm Is Required to Analyze Sperm DNA Breaks With the Comet Assay

2021 ◽  
Vol 9 ◽  
Author(s):  
Jordi Ribas-Maynou ◽  
Ariadna Delgado-Bermúdez ◽  
Estela Garcia-Bonavila ◽  
Elisabeth Pinart ◽  
Marc Yeste ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Comet Assay ◽  
Dnase I ◽  
Proteinase K ◽  
Farm Animals ◽  
Sperm Chromatin ◽  
Dna Breaks ◽  
Chromatin Decondensation ◽  
Sperm Dna

Sperm quality is usually evaluated prior to artificial insemination in farm animals. In addition to conventional semen analysis, other biomarkers, such as mitochondrial activity, integrity and lipid disorder of plasma membrane, generation of reactive oxygen species (ROS) and sperm DNA integrity, have been found to be related to fertility rates in different species. While mounting evidence indicates that the Comet assay is a sensitive method for the detection of DNA breaks, complete sperm chromatin decondensation is required in order to properly analyze the presence of single- and double-strand DNA breaks. In this sense, a previous study showed that longer lysis treatment with proteinase K is needed to achieve complete chromatin decondensation. The current work sought to determine which specific lysis treatment leads to complete chromatin decondensation in pig sperm, as this is needed for the measurement of DNA damage in this species. With this purpose, incubation with a lysis solution containing proteinase K for 0, 30, and 180 min was added to the conventional protocol. The impact of the DNA damage induced by hydrogen peroxide (H2O2; 0.01 and 0.1%) and DNAse I (1U and 4U) was also evaluated. Complete chromatin decondensation was only achieved when a long additional lysis treatment (180 min) was included. Furthermore, olive tail moment (OTM) and percentage of tail DNA (TD) indicated that a higher amount of DNA breaks was detected when hydrogen peroxide and DNAse I treatments were applied (P < 0.05). The comparison of treated and control sperm allowed defining the thresholds for OTM; these thresholds revealed that the percentage of sperm with fragmented DNA determined by the alkaline Comet does not depend on chromatin decondensation (P > 0.05). In conclusion, complete chromatin decondensation prior to alkaline and neutral Comet assays is needed to analyze DNA breaks in pig sperm.

scholarly journals Are semen quality parameters sufficient for biomonitoring spermatozoa DNA integrity and oxidatively damaged DNA

Biomonitoring ◽  
2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Hueiwang Anna Jeng ◽  
Ruei-Nian Li ◽  
Wen-Yi Lin
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Semen Quality ◽  
Quality Parameters ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Phase System ◽  
Sperm Dna Fragmentation ◽  
Sperm Dna

Abstract:The present study aimed to investigate the relationship between semen quality parameters and DNA integrity, and determine whether semen quality parameters could serve as a reliable biomarker for monitoring sperm DNA damage. Conventional semen parameters from a total of 202 male human subjects were analyzed. DNA fragmentation and 8-oxo-7,8-dihydro-2′- deoxyguanosine (8-oxoGuo) were used to assess sperm DNA integrity. DNA fragmentation was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and sperm chromatin structure assay (SCSA), while 8-oxodGuo was quantified by the liquid chromatography/tandem mass spectrometry (LC-MS/MS) coupled with an on-line solid phase system. The levels of 8-oxodGuo levels in sperm were related to the percentages of DNA fragmentation measured by both the TUNEL and SCSA (r = 0.22, p = 0.048; r = 0.12, p = 0.039). Sperm vitality, motility and morphology from all of the participants exhibited a weak correlation with the levels of 8-oxodGuo and the percentages of DNA fragmentation. Semen quality parameters may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Semen quality parameters may be insufficient to monitor sperm DNA fragmentation and oxidative damage. DNA damage in sperm is recommended to be included in routine measurements.

scholarly journals Effect of Alcohol Consumption on the Sperm DNA Integrity: A Systematic Review

2017 ◽  
Vol 9 (13) ◽  
pp. 136
Author(s):  
Farah Hanan Fathihah Jaafar ◽  
Khairul Osman ◽  
Jaya Kumar ◽  
Siti Fatimah Ibrahim
Keyword(s):  
Dna Damage ◽  
Alcohol Consumption ◽  
Alcohol Withdrawal ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Damage ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Keywords Alcohol

There is no solid conclusion on the conventional sperm parameters in association with alcohol consumption, evaluation of sperm DNA integrity thus become a more reliable parameter. Hereby, this literature search was performed to summarize alcohol consumption on the sperm DNA integrity. A computerized database search was done through MEDLINE via Ovid (since 1946 until August 2017) and Cochrane was used. The following set of keywords: ‘alcohol consumption OR alcohol intake OR alcohol diet OR drinking alcohol OR ethanol diet’ AND ‘sperm DNA OR sperm chromatin OR sperm genome OR sperm histone OR sperm protamine’ were utilised. 24 articles were retrieved where only five studies conform to the inclusion criteria All studies demonstrated a negative effect of alcohol consumption on sperm DNA integrity, regardless of various range of alcohol doses and duration of alcohol consumption. Out of five studies reviewed, four studies were using a different approach to measure the sperm DNA damage. Hereby, this review identified a need to use a single approach of DNA damage test by having various method of alcohol administration and/or vice versa so that the extension of sperm DNA damage to alcohol consumption will have a better conclusion. On the same note, a few studies have reported the reversibility on conventional semen parameters, none has been done on the sperm DNA damage upon alcohol withdrawal. Therefore, the role of alcohol withdrawal on the reversibility of sperm DNA damage needs to be as well investigated further.

scholarly journals Sperm DNA fragmentation induced by DNAse I and hydrogen peroxide: an in vitro comparative study among different mammalian species

Reproduction ◽  
2010 ◽  
Vol 140 (3) ◽  
pp. 445-452 ◽  
Author(s):  
Paola Villani ◽  
Patrizia Eleuteri ◽  
Maria Giuseppa Grollino ◽  
Michele Rescia ◽  
Pierluigi Altavista ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Comet Assay ◽  
Chromatin Structure ◽  
Dnase I ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Dna Breaks ◽  
Sperm Dna

Sperm DNA damage may have adverse effects on reproductive outcome. Sperm DNA breaks can be detected by several tests, which evaluate DNA integrity from different and complementary perspectives and offer a new class of biomarkers of the male reproductive function and of its possible impairment after environmental exposure. The remodeling of sperm chromatin produces an extremely condensed nuclear structure protecting the nuclear genome from adverse environments. This nuclear remodeling is species specific, and differences in chromatin structure may lead to a dissimilar DNA susceptibility to mutagens among species. In this study, the capacity of the comet assay in its two variants (alkaline and neutral) to detect DNA/chromatin integrity has been evaluated in human, mouse, and bull sperm. The hypothesis that chromatin packaging might influence the amount of induced and detectable DNA damage was tested by treating sperm in vitro with DNAse I, whose activity is strictly dependent upon its DNA accessibility. Furthermore, hydrogen peroxide (H2O2) was used to assess whether spermatozoa of the three species showed a different sensitivity to oxidative stress. DNAse I-induced damage was also assessed by the sperm chromatin structure assay and the TUNEL assay, and the performances of these two assays were compared and correlated with the comet assay results. Results showed a different sensitivity to DNAse I treatment among the species with human sperm resulting the most susceptible. On the contrary, no major differences among species were observed after H2O2 treatment. Furthermore, the three tests show a good correlation in revealing sperm with DNA strand breaks.

scholarly journals Impaired protamination and sperm DNA damage in a Nellore bull with high percentages of morphological sperm defects in comparison to normospermic bulls

2015 ◽  
Vol 67 (2) ◽  
pp. 417-423 ◽  
Author(s):  
J.T. Carreira ◽  
J.T. Trevizan ◽  
B.H. Kipper ◽  
S.H.V. Perri ◽  
I.R. Carvalho ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Cell Damage ◽  
Sperm Concentration ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Dna Breaks ◽  
Mitochondrial Potential ◽  
Sperm Dna Damage ◽  
Sperm Dna

The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%), and were selected for complementary tests. Damage of acrosomal (76.9±8.9%) and plasma membranes (75.7±9.3%) as well as sperm DNA strand breaks (13.8±9.5%) and protamination deficiency (3.7±0.6%) were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively) (P<0.05). Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology) may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.

scholarly journals Single and Double Strand Sperm DNA Damage: Different Reproductive Effects on Male Fertility

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 105 ◽  
Author(s):  
Jordi Ribas-Maynou ◽  
Jordi Benet
Keyword(s):  
Dna Damage ◽  
Male Fertility ◽  
Sperm Chromatin ◽  
Dna Breaks ◽  
Sperm Dna Damage ◽  
Reproductive Effects ◽  
Double Strand Dna Breaks ◽  
Sperm Dna ◽  
Break Points ◽  
Double Strand Dna

Reproductive diseases have become a growing worldwide problem and male factor plays an important role in the reproductive diagnosis, prognosis and design of assisted reproductive treatments. Sperm cell holds the mission of carrying the paternal genetic complement to the oocyte in order to contribute to an euploid zygote with proper DNA integrity. Sperm DNA fragmentation had been used for decades as a male fertility test, however, its usefulness have arisen multiple debates, especially around Intracytoplasmic Sperm Injection (ICSI) treatments. In the recent years, it has been described that different types of sperm DNA breaks (single and double strand DNA breaks) cause different clinical reproductive effects. On one hand, single-strand DNA breaks are present extensively as a multiple break points in all regions of the genome, are related to oxidative stress and cause a lack of clinical pregnancy or an increase of the conception time. On the other hand, double-strand DNA breaks are mainly localized and attached to the sperm nuclear matrix as a very few break points, are possibly related to a lack of DNA repair in meiosis and cause a higher risk of miscarriage, low embryo quality and higher risk of implantation failure in ICSI cycles. The present work also reviews different studies that may contribute in the understanding of sperm chromatin as well as treatments to prevent sperm DNA damage.

scholarly journals A cost for high levels of sperm competition in rodents: increased sperm DNA fragmentation

2016 ◽  
Vol 283 (1826) ◽  
pp. 20152708 ◽  
Author(s):  
Javier delBarco-Trillo ◽  
Olga García-Álvarez ◽  
Ana Josefa Soler ◽  
Maximiliano Tourmente ◽  
José Julián Garde ◽  
...  
Keyword(s):  
Dna Damage ◽  
Sperm Competition ◽  
Dna Fragmentation ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Sperm Dna Damage ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Physical And Chemical

Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage.

P–051 Differential resilience of sperm from different mammals to DNA decondensation

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
J Ribas-Maynou ◽  
E Garcia-Bonavila ◽  
M Llavanera ◽  
J Miró ◽  
S Bonet ◽  
...  
Keyword(s):  
Human Sperm ◽  
Dna Condensation ◽  
Proteinase K ◽  
Dna Integrity ◽  
Sperm Cells ◽  
Core Diameter ◽  
Bovine Sperm ◽  
Sperm Dna ◽  
Protamine 1 ◽  
Protamine 2

Abstract Study question Does sperm from different species with different protamine 1/protamine 2 ratios have different resilience to sperm decondensation? Summary answer Sperm cells from species whose DNA is condensed with both protamine 1 and protamine 2 require less time in deprotamination steps. What is known already Sperm cells present a highly particular DNA condensation that is acquired during sperm differentiation, where most part of histones are replaced by protamines. Protamines are key elements for DNA condensation and, while protamine 1 is more conserved among species, protamine 2 has evolved differentially, existing only a few species that retain the mature protein in their sperm DNA. Changes in protamine expression rates have been described to be associated to head sperm size and shape. In addition, reduced amounts of protamine 2 are related to male infertility in species in which this protein is present. Study design, size, duration Cryopreserved sperm samples were treated with lysis solutions to induce DNA decondensation and formation of sperm haloes. In these treatments, the effect of different incubation times with proteinase K added to the lysis solution upon DNA decondensation was tested by analyzing core diameter, halo diameter and the Halo/core ratio in at least 50 sperm per sample. Participants/materials, setting, methods Species included in the study were Human, Equine, Donkey, Porcine and Bovine. Sperm samples from five different individuals for each species were included in the study. DNA decondensation included three lysis steps: first, a SDS + DTT incubation for 30 minutes; second, a DTT + NaCl treatment for 30 minutes; and third, a DTT + NaCl + Proteinase K treatment with a variable time of 0, 30 or 180 minutes. Main results and the role of chance The halo/core diameter, used as a representation of the degree of DNA decondensation, for 0 minutes, 30 minutes and 180 minutes of proteinase K incubation were: 4.68±0.51, 4.32±0.51 and 4.77±0.64, respectively for human sperm; 4.15±0.41, 4.57±0.53 and 4.68±0.63, respectively for Equine sperm; 4.40±0.64, 4.00±0.37 and 4.17±0.19, respectively for donkey sperm; 1.77±0.2, 3.05±0.14 and 4.13±0.39, respectively for porcine sperm; and 2.40±0.40, 3.36±0.22 and 4.19±0.38, respectively for bovine sperm. Differences of halo/core ratio in different times were only observed in porcine and bovine sperm, where increasing degrees of DNA decondensation were found (p &lt; 0.05). Therefore, these results show that while longer incubations in lysis solutions with proteinase K lead to higher DNA decondensation in porcine and bovine, they do not induce higher decondensation in human, equine and donkey. This evidence, coupled to the fact that porcine and bovine sperm present null or very low protamine 2 content, suggests that its presence might confer higher DNA decondensation susceptibility. Limitations, reasons for caution Only sperm cells with normal sperm haloes were analyzed in the present study. As multiple studies show, haloes exhibited by sperm cells with DNA damage display higher diameter, that is why they were strictly excluded in this study with the aim to elucidate the average DNA decondensation. Wider implications of the findings: Sperm DNA might have different degrees of DNA condensation, which can be associated to a higher difficulty of DNA decondensation, thus having implications in the sensitivity tests that assess sperm DNA integrity. Trial registration number Not applicable.

scholarly journals Can the SCD test and terminal uridine nick-end labeling by flow cytometry technique (TUNEL/FCM) be used interchangeably to measure sperm DNA damage in routine laboratory practice?

2019 ◽  
Vol 29 (1) ◽  
Author(s):  
Cécile Grèze ◽  
Aline Guttmann ◽  
Hanae Pons-Rejraji ◽  
Marie-Paule Vasson ◽  
Jacqueline Lornage ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dna Damage ◽  
Gradient Centrifugation ◽  
Intraclass Correlation ◽  
Systematic Difference ◽  
Test Reliability ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Damage ◽  
Sperm Dna

Abstract Background Numerous tests have been proposed to evaluate sperm DNA integrity. To assess the sperm chromatin dispersion (SCD) test in an andrology laboratory, twenty-five men attending Clermont-Ferrand (France) University Hospital’s Center for Reproductive Medicine were recruited. Sperm DNA damage was measured in the same semen samples using the SCD test and the Terminal Uridine Nick-end Labeling by flow cytometry technique (TUNEL/FCM) after density gradient centrifugation. Results SCD test reliability between readings, readers or slides was clearly established with very high agreement between measurements (Intraclass correlation coefficient (ICC) at 0.97, 0.95 and 0.98 respectively). Despite very good agreement between the SCD test and TUNEL/FCM (ICC at 0.94), the SCD test tended to slightly but significantly underestimate DNA damage compared with TUNEL (p = 0.0127). This systematic difference between the two techniques was − 3.39 ± 1.45% (mean ± SE). Conclusions Andrology laboratories using the SCD test to measure sperm DNA damage need to know that it appears to give slightly underestimated measurements compared to TUNEL/FCM. However, this systematic underestimation is very small in amplitude. Both techniques give almost perfectly congruent results. Our study underlines the importance for each laboratory to validate its method to assess sperm DNA damage before implementing it in routine andrology lab practice.

The assessment of sperm DNA fragmentation in the saltwater crocodile (Crocodylus porosus)

2017 ◽  
Vol 29 (3) ◽  
pp. 630 ◽  
Author(s):  
S. D. Johnston ◽  
C. López-Fernández ◽  
F. Arroyo ◽  
J. L. Fernández ◽  
J. Gosálvez
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Fragmented Dna ◽  
Sperm Dna ◽  
Saltwater Crocodile ◽  
Crocodylus Porosus ◽  
Dispersion Test ◽  
Sperm Nuclei

Herein we report a method of assessing DNA fragmentation in the saltwater crocodile using the sperm chromatin dispersion test (SCDt) after including frozen–thawed spermatozoa in a microgel (Halomax; Halotech DNA, Madrid, Spain). Following controlled protein depletion, which included a reducing agent, sperm nuclei with fragmented DNA showed a homogeneous and larger halo of chromatin dispersion with a corresponding reduced nucleoid core compared with sperm with non-fragmented DNA. The presence of DNA damage was confirmed directly by incorporation of modified nucleotides using in situ nick translation (ISNT) and indirectly by studying the correlation of the SCDt with the results of DNA damage visualisation using a two-tailed comet assay (r = 0.90; P = 0.037). Results of the SCDt immediately following thawing and after 5 h incubation at 37°C in order to induce a range of DNA damage revealed individual crocodile differences in both the baseline level of DNA damage and DNA longevity.

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