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Paternal exposure to cyclophosphamide affects the progression of sperm chromatin decondensation and activates a DNA damage response in the prepronuclear rat zygote.

ABSTRACT: Spermatozoon decondensation in the zygote leads to the initiation of chromatin remodeling during which protamines are removed and replaced with maternal histones. We hypothesize that damage to male germ cells induced by paternal exposure to cyclophosphamide may alter the timing of spermatozoal decondensation and the pattern of chromatin remodeling in the prepronuclear rat zygote. A specific order of sperm decondensation was observed, starting at the posterior end, proceeding to the ventral sides, followed by the tip, and finally the midbody region of the sperm head nucleus; subgroups of partially decondensed type a sperm nuclei were defined as types a1, a2, a3, and a4. Based on their frequencies relative to controls, paternal exposure to cyclophosphamide accelerated the timing of spermatozoal decondensation. Two distinct patterns of chromatin remodeling were observed for totally decondensed (type b) and recondensing (type c) sperm nuclei: H4K12ac showed a homogenous staining, whereas H3S10ph displayed a ring-like staining around the sperm nucleus; the distribution of these posttranslationally modified histones was not affected by cyclophosphamide exposure. In contrast, paternal cyclophosphamide treatment increased the number of gammaH2AX foci found in decondensing sperm nuclei. Small foci were significantly increased in type a2 and a3 nuclei, whereas a significant increase in the numbers of large foci was found in type b and c nuclei. This increase in gammaH2AX foci in the decondensing male genome suggests that damage recognition and repair pathways are initiated in prepronuclear rat zygotes. Thus, exposure of male rats to chronic low doses of cyclophosphamide accelerates spermatozoal decondensation and leads to the activation of gammaH2AX recognition of DNA damage in the male genome of the prepronuclear zygote.

SUBMITTER: Grenier L

PROVIDER: S-EPMC6322429 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Paternal exposure to cyclophosphamide affects the progression of sperm chromatin decondensation and activates a DNA damage response in the prepronuclear rat zygote.

Grenier Lisanne L Robaire Bernard B Hales Barbara F BF

Biology of reproduction 20100414 2

Spermatozoon decondensation in the zygote leads to the initiation of chromatin remodeling during which protamines are removed and replaced with maternal histones. We hypothesize that damage to male germ cells induced by paternal exposure to cyclophosphamide may alter the timing of spermatozoal decondensation and the pattern of chromatin remodeling in the prepronuclear rat zygote. A specific order of sperm decondensation was observed, starting at the posterior end, proceeding to the ventral sides ...[more]

PMID: 20393171

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Project description: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.

Dataset Information

Paternal exposure to cyclophosphamide affects the progression of sperm chromatin decondensation and activates a DNA damage response in the prepronuclear rat zygote.

Publications

Paternal exposure to cyclophosphamide affects the progression of sperm chromatin decondensation and activates a DNA damage response in the prepronuclear rat zygote.

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