Genomic integrity in the male germ line: evidence in support of the disposable soma hypothesis

in Reproduction
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The Big Blue λSelect-cII selection system has been employed along with whole-exome sequencing to examine the susceptibility of the male germ line to mutation in two challenging situations (i) exposure to a chemotherapeutic regime including bleomycin, etoposide and cis-platinum (BEP) and (ii) the ageing process. A 3-week exposure to BEP induced complete azoospermia associated with a loss of developing germ cells and extensive vacuolization of Sertoli cell cytoplasm. Following cessation of treatment, spermatozoa first appeared in the caput epididymis after 6 weeks and by 12 weeks motile spermatozoa could be recovered from the cauda, although the count (P < 0.001) and motility (P < 0.01) of these cells were significantly reduced and superoxide generation was significantly elevated (P < 0.001). Despite this increase in free radical generation, no evidence of chromatin instability was detected in these spermatozoa. Furthermore, embryos obtained from females mated at this 12-week time point showed no evidence of an increased mutational load. Similarly, progressive ageing of Big Blue mice had no impact on the quality of the spermatozoa, fertility or mutation frequency in the offspring despite a significant increase in the mutational load carried by somatic tissues such as the liver (P < 0.05). We conclude that the male germ line is highly resistant to mutation in keeping with the disposable soma hypothesis, which posits that genetic integrity in the germ cells will be maintained at the expense of the soma, in light of the former’s sentinel position in safeguarding the stability of the genome.

 

    Society for Reproduction and Fertility

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    Histological profile of the testes in BEP-exposed mice during the recovery phase in relation to corresponding controls. Wk represents the numbers of weeks following a 3-week BEP exposure. Images to the left are the controls, images to the right are BEP-exposed animals. Arrows indicate seminiferous tubule sections exhibiting a severe disruption of spermatogenesis in association with extensive vacuolization of the Sertoli cell cytoplasm.

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    Analysis of BEP-exposed mice during the recovery phase. (A) Percentage of testicular cross sections not revealing evidence of severely disrupted spermatogenesis, (B) testes weights, (C) mean litter size and (D) number of embryonic resorptions at 14 dpc. Open bars are control treatments and closed bars are chemotherapy-treated animals. Probability values inserted into the top right-hand corner of each panel indicates the overall significance due to treatment according to ANOVA. Asterisks at the head of the columns indicate the significance of the difference at each time point. *P < 0.05; **P < 0.01; ***P < 0.001. At least three independent replicates per group.

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    Sperm quality following exposure to BEP. (A) Number of spermatozoa recovered from the caput epididymis, (B) number of spermatozoa recovered from the cauda epididymis, (C) percentage of motile cells (D) percentage of progressively motile cells, (E) percentage of viable spermatozoa exhibiting high levels of mitochondrial ROS generation by flow cytometry according to the probe MitoSox Red (MSR). (F) Percentage of viable spermatozoa exhibiting a high level of total cellular ROS generation according to the probe dihydroethidium (DHE). Open bars are control treatments and closed bars are chemotherapy treated animals. Probability values inserted into the top right-hand corner of each panel indicates the overall significance due to treatment according to ANOVA. Asterisks at the head of the columns indicate the significance of the difference at each time point. *P < 0.05; **P < 0.01; ***P < 0.001. At least three independent replicates per group.

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    Levels of DNA damage in the spermatozoa of mice recovering from BEP exposure as measured by SCSA. Results of SCSA analysis for (A) caput epididymal spermatozoa and (B) cauda epididymal spermatozoa. Open bars are control treatments and closed bars are chemotherapy-treated animals. At least three independent replicates per group.

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    Impact of age on male reproduction in the Big Blue mouse. (A) Number of de novo mutations in the whole-exome of embryos of pairs of young mice (1352 ± 20 mutations) and older mice (1357 ± 9 mutations), showed no statistical differences (Mann–Whitney U test, P > 0.1, n = 4). (B) Mutation frequency in the whole-exome of embryos was not statistically different (Mann–Whitney U test, P > 0.1, n = 4) between the offspring of young (2.98 × 10−7 ± 4 × 10−9 mutation/bp) and aged mice (2.99 × 10−7 ± 2 × 10−9 mutations/bp). Age-dependent changes in (C) testes weight; (D) sperm motility; (E) DNA damage as measured by the SCSA assay; (F) flow cytometry analysis of percentage of cell expressing abnormally high levels of 8OHdG formation; (G) mitochondrial superoxide generation in caput and caudal epididymal spermatozoa; (H) mitochondrial membrane potential in caput and caudal epididymal spermatozoa as measured by JC-1. Probability values inserted into the top right-hand corner of each panel indicates the overall significance of differences due to the source of the spermatozoa (caput or cauda epididymis). Asterisks at the head of the columns indicate the significance of the difference at each time point. *P < 0.05; **P < 0.01; ***P < 0.001. At least three independent replicates per group.

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