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Differential gene susceptibility to sperm DNA damage: analysis of developmental key genes in trout.

González-Rojo S, Fernández-Díez C, Guerra SM, Robles V, Herraez MP - PLoS ONE (2014)

Bottom Line: Sperm chromatin in mammals is packaged in different blocks associated to protamines (PDNA), histones (HDNA), or nuclear matrix proteins.The absence of histones in the sperm nuclei was confirmed by Western blot.Location of 8-hydroxyguanosine (8-OHdG) was analyzed by immunocytochemistry and confocal microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of León, León, Spain.

ABSTRACT
Sperm chromatin in mammals is packaged in different blocks associated to protamines (PDNA), histones (HDNA), or nuclear matrix proteins. Differential packaging has been related to early or late transcription and also to differential susceptibility to genotoxic damage. Genes located in the more accessible HDNA could be more susceptible to injuries than those located in PDNA, being potential biomarkers of paternal DNA damage. Fish sperm chromatin organization is much diversified, some species lacking protamines and some others totally depleted of histones. Analyzing genotoxic damage in a species homogeneously compacted with some sperm nuclear basic protein type, could help in deciphering the clues of differential susceptibility to damage. In the present study we analyzed in rainbow trout the differential susceptibility of nine genes to UV irradiation and H2O2 treatment. The absence of histones in the sperm nuclei was confirmed by Western blot. The chromatin fractionation in sensitive and resistant regions to PvuII (presumably HDNA-like and PDNA-like, respectively) revealed that the nine genes locate in the same resistant region. The number of lesions promoted was quantified using a qPCR approach. Location of 8-hydroxyguanosine (8-OHdG) was analyzed by immunocytochemistry and confocal microscopy. UV irradiation promoted similar number of lesions in all the analyzed genes and a homogenous distribution of 8-OHdG within the nuclei. 8-OHdG was located in the peripheral area of the nucleus after H2O2 treatment, which promoted a significantly higher number of lesions in developmental-related genes (8.76-10.95 lesions/10 kb) than in rDNA genes (1.05-1.67 lesions/10 kb). We showed for the first time, that differential susceptibility to damage is dependent on the genotoxic mechanism and relies on positional differences between genes. Sensitive genes were also analyzed in cryopreserved sperm showing a lower number of lesions than the previous treatments and a predominant peripheral distribution of oxidative damage (8-OHdG).

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Number of DNA lesions per 10 kb in nine nuclear genes.UV irradiation (400 µW/cm2, 10 min) and H2O2 treatment (250 mM, 20 min) induces different levels of DNA damage in early or late transcribed genes. DNA damage was calculated by the 2-ΔΔCt method and transformed into a DNA lesions rate. Data are expressed as media ± SEM (n = 7). Asterisks show significant differences among genes for the same treatment (P<0.05).
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pone-0114161-g004: Number of DNA lesions per 10 kb in nine nuclear genes.UV irradiation (400 µW/cm2, 10 min) and H2O2 treatment (250 mM, 20 min) induces different levels of DNA damage in early or late transcribed genes. DNA damage was calculated by the 2-ΔΔCt method and transformed into a DNA lesions rate. Data are expressed as media ± SEM (n = 7). Asterisks show significant differences among genes for the same treatment (P<0.05).

Mentions: However, oxidative stress promoted by H2O2 caused a significantly lower number of lesions in rDNA genes 18S and 28S (1.67±0.8 and 1.05±0.9, respectively), than in the rest of the analyzed genes (ranging from 8.76±0.7 for HoxA3a-1 to 10.95±0.9 for HoxD4ai, expressed as lesions per 10 kb ± SEM) (Fig. 4).


Differential gene susceptibility to sperm DNA damage: analysis of developmental key genes in trout.

González-Rojo S, Fernández-Díez C, Guerra SM, Robles V, Herraez MP - PLoS ONE (2014)

Number of DNA lesions per 10 kb in nine nuclear genes.UV irradiation (400 µW/cm2, 10 min) and H2O2 treatment (250 mM, 20 min) induces different levels of DNA damage in early or late transcribed genes. DNA damage was calculated by the 2-ΔΔCt method and transformed into a DNA lesions rate. Data are expressed as media ± SEM (n = 7). Asterisks show significant differences among genes for the same treatment (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257556&req=5

pone-0114161-g004: Number of DNA lesions per 10 kb in nine nuclear genes.UV irradiation (400 µW/cm2, 10 min) and H2O2 treatment (250 mM, 20 min) induces different levels of DNA damage in early or late transcribed genes. DNA damage was calculated by the 2-ΔΔCt method and transformed into a DNA lesions rate. Data are expressed as media ± SEM (n = 7). Asterisks show significant differences among genes for the same treatment (P<0.05).
Mentions: However, oxidative stress promoted by H2O2 caused a significantly lower number of lesions in rDNA genes 18S and 28S (1.67±0.8 and 1.05±0.9, respectively), than in the rest of the analyzed genes (ranging from 8.76±0.7 for HoxA3a-1 to 10.95±0.9 for HoxD4ai, expressed as lesions per 10 kb ± SEM) (Fig. 4).

Bottom Line: Sperm chromatin in mammals is packaged in different blocks associated to protamines (PDNA), histones (HDNA), or nuclear matrix proteins.The absence of histones in the sperm nuclei was confirmed by Western blot.Location of 8-hydroxyguanosine (8-OHdG) was analyzed by immunocytochemistry and confocal microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of León, León, Spain.

ABSTRACT
Sperm chromatin in mammals is packaged in different blocks associated to protamines (PDNA), histones (HDNA), or nuclear matrix proteins. Differential packaging has been related to early or late transcription and also to differential susceptibility to genotoxic damage. Genes located in the more accessible HDNA could be more susceptible to injuries than those located in PDNA, being potential biomarkers of paternal DNA damage. Fish sperm chromatin organization is much diversified, some species lacking protamines and some others totally depleted of histones. Analyzing genotoxic damage in a species homogeneously compacted with some sperm nuclear basic protein type, could help in deciphering the clues of differential susceptibility to damage. In the present study we analyzed in rainbow trout the differential susceptibility of nine genes to UV irradiation and H2O2 treatment. The absence of histones in the sperm nuclei was confirmed by Western blot. The chromatin fractionation in sensitive and resistant regions to PvuII (presumably HDNA-like and PDNA-like, respectively) revealed that the nine genes locate in the same resistant region. The number of lesions promoted was quantified using a qPCR approach. Location of 8-hydroxyguanosine (8-OHdG) was analyzed by immunocytochemistry and confocal microscopy. UV irradiation promoted similar number of lesions in all the analyzed genes and a homogenous distribution of 8-OHdG within the nuclei. 8-OHdG was located in the peripheral area of the nucleus after H2O2 treatment, which promoted a significantly higher number of lesions in developmental-related genes (8.76-10.95 lesions/10 kb) than in rDNA genes (1.05-1.67 lesions/10 kb). We showed for the first time, that differential susceptibility to damage is dependent on the genotoxic mechanism and relies on positional differences between genes. Sensitive genes were also analyzed in cryopreserved sperm showing a lower number of lesions than the previous treatments and a predominant peripheral distribution of oxidative damage (8-OHdG).

Show MeSH
Related in: MedlinePlus