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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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Evaluation of spermatozoa integrity by flow cytometry.A: Protamine association with sperm chromatin determined by chromomycin A3 (CMA3) staining. Histograms show the proportion of CMA3 incorporated in caput and cauda sperm of WT versus DKO animals aged 4 months. B: Disulfide bonds/free thiol quantification by monobromobimane (mBrB) staining. Histograms showing the incorporation of mBrB in caput and cauda sperm of WT versus DKO animals aged 4 months. (Mean +/− SEM; n = 6; *: p≤0.05; **: p≤0.01).
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pone-0038565-g005: Evaluation of spermatozoa integrity by flow cytometry.A: Protamine association with sperm chromatin determined by chromomycin A3 (CMA3) staining. Histograms show the proportion of CMA3 incorporated in caput and cauda sperm of WT versus DKO animals aged 4 months. B: Disulfide bonds/free thiol quantification by monobromobimane (mBrB) staining. Histograms showing the incorporation of mBrB in caput and cauda sperm of WT versus DKO animals aged 4 months. (Mean +/− SEM; n = 6; *: p≤0.05; **: p≤0.01).

Mentions: Since we recorded no differences in the spermatozoa phenotype of the double mutant animals at 4 and 8 months, all further experiments were carried out on spermatozoa from animals aged 4 months. Because snGPx4 is located in the sperm nucleus during spermiogenesis, it may be argued that its absence could somehow impair the process of histone replacement by protamines, eventually leading to defective sperm nuclear protamination in the double mutant. We used flow cytometry and chromomycin A3 as a probe to evaluate indirectly the protamine content of WT and double mutant spermatozoa. Figure 5A first shows that sperm nuclear compaction was increased during spermatozoa epididymal journey from the caput to the cauda irrespective of the genetic background. This Figure also indicates that there was no significant difference in the percentage of recorded fluorescence due to chromomycin A3 staining both in caput and cauda double mutant spermatozoa when compared to WT spermatozoa. Defective testicular protamination is therefore unlikely to explain sperm nuclear decondensation in the double mutants.


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)

Evaluation of spermatozoa integrity by flow cytometry.A: Protamine association with sperm chromatin determined by chromomycin A3 (CMA3) staining. Histograms show the proportion of CMA3 incorporated in caput and cauda sperm of WT versus DKO animals aged 4 months. B: Disulfide bonds/free thiol quantification by monobromobimane (mBrB) staining. Histograms showing the incorporation of mBrB in caput and cauda sperm of WT versus DKO animals aged 4 months. (Mean +/− SEM; n = 6; *: p≤0.05; **: p≤0.01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375294&req=5

pone-0038565-g005: Evaluation of spermatozoa integrity by flow cytometry.A: Protamine association with sperm chromatin determined by chromomycin A3 (CMA3) staining. Histograms show the proportion of CMA3 incorporated in caput and cauda sperm of WT versus DKO animals aged 4 months. B: Disulfide bonds/free thiol quantification by monobromobimane (mBrB) staining. Histograms showing the incorporation of mBrB in caput and cauda sperm of WT versus DKO animals aged 4 months. (Mean +/− SEM; n = 6; *: p≤0.05; **: p≤0.01).
Mentions: Since we recorded no differences in the spermatozoa phenotype of the double mutant animals at 4 and 8 months, all further experiments were carried out on spermatozoa from animals aged 4 months. Because snGPx4 is located in the sperm nucleus during spermiogenesis, it may be argued that its absence could somehow impair the process of histone replacement by protamines, eventually leading to defective sperm nuclear protamination in the double mutant. We used flow cytometry and chromomycin A3 as a probe to evaluate indirectly the protamine content of WT and double mutant spermatozoa. Figure 5A first shows that sperm nuclear compaction was increased during spermatozoa epididymal journey from the caput to the cauda irrespective of the genetic background. This Figure also indicates that there was no significant difference in the percentage of recorded fluorescence due to chromomycin A3 staining both in caput and cauda double mutant spermatozoa when compared to WT spermatozoa. Defective testicular protamination is therefore unlikely to explain sperm nuclear decondensation in the double mutants.

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