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Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice.

Noblanc A, Peltier M, Damon-Soubeyrand C, Kerchkove N, Chabory E, Vernet P, Saez F, Cadet R, Janny L, Pons-Rejraji H, Conrad M, Drevet JR, Kocer A - PLoS ONE (2012)

Bottom Line: We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2)O(2)-scavenger activity especially in the cauda epididymis.Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity.Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation.

View Article: PubMed Central - PubMed

Affiliation: Genetics Reproduction & Development laboratory, CNRS UMR 6293 - INSERM U1103 - Clermont Université, Aubière, France.

ABSTRACT
We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2)O(2)-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.

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Cauda-retrieved spermatozoa of DKO animals suffer oxidative damage.A: Left panel: Typical picture of fragmented (arrowhead) or non-fragmented (arrow) sperm nucleus as shown by the modified Sperm Chromatin Dispersion Assay. Scale bar = 5 µm. Right panel: Histograms show the proportion of WT and DKO cauda collected spermatozoa with a fragmented DNA in animals aged 4 or 8 months. B: Left panel: Typical immunodetection of the nuclear adduct 8-oxodG in cauda epididymidis-retrieved spermatozoa preparations from DKO male mice aged 8 months. Scale bar = 5 µm. Right panel: Histograms show the percentage of 8-oxodG positive spermatozoa in cauda epididymidis-retrieved spermatozoa preparations, respectively from WT, positive control (WT spermatozoa treated with H2O2) and DKO male mice aged 4 and 8 months. (Mean+/− SEM; n = 5; *: p≤0.05; **: p≤0.01; ***: p≤0.001). C: Histograms show global H2O2-scavenging activity using H2O2 as a substrate in cauda epididymidis tissue extracts from WT or DKO animals aged 4 or 8 months. (Mean +/− SEM; n = 5; *: p≤0.05; **: p≤0.01).
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pone-0038565-g007: Cauda-retrieved spermatozoa of DKO animals suffer oxidative damage.A: Left panel: Typical picture of fragmented (arrowhead) or non-fragmented (arrow) sperm nucleus as shown by the modified Sperm Chromatin Dispersion Assay. Scale bar = 5 µm. Right panel: Histograms show the proportion of WT and DKO cauda collected spermatozoa with a fragmented DNA in animals aged 4 or 8 months. B: Left panel: Typical immunodetection of the nuclear adduct 8-oxodG in cauda epididymidis-retrieved spermatozoa preparations from DKO male mice aged 8 months. Scale bar = 5 µm. Right panel: Histograms show the percentage of 8-oxodG positive spermatozoa in cauda epididymidis-retrieved spermatozoa preparations, respectively from WT, positive control (WT spermatozoa treated with H2O2) and DKO male mice aged 4 and 8 months. (Mean+/− SEM; n = 5; *: p≤0.05; **: p≤0.01; ***: p≤0.001). C: Histograms show global H2O2-scavenging activity using H2O2 as a substrate in cauda epididymidis tissue extracts from WT or DKO animals aged 4 or 8 months. (Mean +/− SEM; n = 5; *: p≤0.05; **: p≤0.01).

Mentions: Based on a modified method of the classical sperm chromatin dispersion assay (SCDA, [28]) Figure 7A illustrates that cauda spermatozoa of double mutant animals presented a higher level of DNA fragmentation than WT spermatozoa at the same ages. Figure 7B also shows that for double mutant animals aged 4 months the level of sperm DNA oxidative damage was well controled by the epididymis antioxidant response since cauda spermatozoa were as reactive to an 8 oxo-dG antibody as WT spermatozoa. However, the situation was rather different with spermatozoa collected from older double mutant animals (8 months) where a high proportion of spermatozoa revealed oxidized guanine residues typical of DNA oxidative alterations. Figure 7C shows that this weaker protection of cauda collected spermatozoa in older animals was probably due to a general physiological decrease in the cauda epididymidis antioxidant response since we show that at this age (8 months), global H2O2/LOOH-recycling activity was reduced by 2.5-fold both in WT and double mutant cauda epididymidis protein extracts compared to the situation in animals aged 4 months.


Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice.

Noblanc A, Peltier M, Damon-Soubeyrand C, Kerchkove N, Chabory E, Vernet P, Saez F, Cadet R, Janny L, Pons-Rejraji H, Conrad M, Drevet JR, Kocer A - PLoS ONE (2012)

Cauda-retrieved spermatozoa of DKO animals suffer oxidative damage.A: Left panel: Typical picture of fragmented (arrowhead) or non-fragmented (arrow) sperm nucleus as shown by the modified Sperm Chromatin Dispersion Assay. Scale bar = 5 µm. Right panel: Histograms show the proportion of WT and DKO cauda collected spermatozoa with a fragmented DNA in animals aged 4 or 8 months. B: Left panel: Typical immunodetection of the nuclear adduct 8-oxodG in cauda epididymidis-retrieved spermatozoa preparations from DKO male mice aged 8 months. Scale bar = 5 µm. Right panel: Histograms show the percentage of 8-oxodG positive spermatozoa in cauda epididymidis-retrieved spermatozoa preparations, respectively from WT, positive control (WT spermatozoa treated with H2O2) and DKO male mice aged 4 and 8 months. (Mean+/− SEM; n = 5; *: p≤0.05; **: p≤0.01; ***: p≤0.001). C: Histograms show global H2O2-scavenging activity using H2O2 as a substrate in cauda epididymidis tissue extracts from WT or DKO animals aged 4 or 8 months. (Mean +/− SEM; n = 5; *: p≤0.05; **: p≤0.01).
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Related In: Results  -  Collection

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pone-0038565-g007: Cauda-retrieved spermatozoa of DKO animals suffer oxidative damage.A: Left panel: Typical picture of fragmented (arrowhead) or non-fragmented (arrow) sperm nucleus as shown by the modified Sperm Chromatin Dispersion Assay. Scale bar = 5 µm. Right panel: Histograms show the proportion of WT and DKO cauda collected spermatozoa with a fragmented DNA in animals aged 4 or 8 months. B: Left panel: Typical immunodetection of the nuclear adduct 8-oxodG in cauda epididymidis-retrieved spermatozoa preparations from DKO male mice aged 8 months. Scale bar = 5 µm. Right panel: Histograms show the percentage of 8-oxodG positive spermatozoa in cauda epididymidis-retrieved spermatozoa preparations, respectively from WT, positive control (WT spermatozoa treated with H2O2) and DKO male mice aged 4 and 8 months. (Mean+/− SEM; n = 5; *: p≤0.05; **: p≤0.01; ***: p≤0.001). C: Histograms show global H2O2-scavenging activity using H2O2 as a substrate in cauda epididymidis tissue extracts from WT or DKO animals aged 4 or 8 months. (Mean +/− SEM; n = 5; *: p≤0.05; **: p≤0.01).
Mentions: Based on a modified method of the classical sperm chromatin dispersion assay (SCDA, [28]) Figure 7A illustrates that cauda spermatozoa of double mutant animals presented a higher level of DNA fragmentation than WT spermatozoa at the same ages. Figure 7B also shows that for double mutant animals aged 4 months the level of sperm DNA oxidative damage was well controled by the epididymis antioxidant response since cauda spermatozoa were as reactive to an 8 oxo-dG antibody as WT spermatozoa. However, the situation was rather different with spermatozoa collected from older double mutant animals (8 months) where a high proportion of spermatozoa revealed oxidized guanine residues typical of DNA oxidative alterations. Figure 7C shows that this weaker protection of cauda collected spermatozoa in older animals was probably due to a general physiological decrease in the cauda epididymidis antioxidant response since we show that at this age (8 months), global H2O2/LOOH-recycling activity was reduced by 2.5-fold both in WT and double mutant cauda epididymidis protein extracts compared to the situation in animals aged 4 months.

Bottom Line: We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2)O(2)-scavenger activity especially in the cauda epididymis.Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity.Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation.

View Article: PubMed Central - PubMed

Affiliation: Genetics Reproduction & Development laboratory, CNRS UMR 6293 - INSERM U1103 - Clermont Université, Aubière, France.

ABSTRACT
We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2)O(2)-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.

Show MeSH
Related in: MedlinePlus