L. E. Bennetts

University of Newcastle, Newcastle, New South Wales, Australia

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Publications (6)15.25 Total impact

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    ABSTRACT: A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (beta-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17beta-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male infertility is warranted.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 06/2008; 641(1-2):1-11. · 3.90 Impact Factor
  • Liga E Bennetts, R John Aitken
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    ABSTRACT: Alkaline gel electrophoresis, pulsed field gel electrophoresis, and quantitative PCR analyses (QPCR) of the nuclear (nDNA) and mitochondrial (mtDNA) genomes were used to assess DNA integrity in the spermatozoa of three species exposed to oxidative stress. In human and murine spermatozoa, the mtDNA was significantly more susceptible to H2O2-mediated damage than nDNA. In both eutherian species, exposure to 250 microM H2O2 induced around 0.6 lesions/10 kb of mtDNA. The mtDNA of human spermatozoa was particularly vulnerable to oxidative stress; 0.25, 1, and 5 mM H2O2 inducing DNA damage equivalent to 0.62, 1.34, and 1.42 lesions/10 kb, respectively. Such results emphasize the diagnostic significance of mtDNA as a biomarker of oxidative stress in the male germ line. In contrast, no damage could be detected by QPCR in the nDNA of either eutherian species, on exposure to H2O2 at doses as high as 5 mM. However, electrophoretic analysis indicated that severe oxidative stress could induce detectable nDNA fragmentation in human, but not murine spermatozoa. The mtDNA of tammar wallaby spermatozoa was relatively resistant to oxidative stress, only exhibiting damage (0.6 lesions/10 kb DNA) on exposure to 5 mM H2O2. By contrast, the nDNA of wallaby spermatozoa was significantly more susceptible to this oxidant than the other species. Such vulnerability is consistent with the lack of disulfide cross-linking in marsupial sperm chromatin and suggests that chromatin condensation during epididymal maturation may be important in establishing the resistance of these cells to the genotoxic effects of reactive oxygen species.
    Molecular Reproduction and Development 06/2005; 71(1):77-87. · 2.81 Impact Factor
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    ABSTRACT: Concern has arisen over human exposures to radio frequency electromagnetic radiation (RFEMR), including a recent report indicating that regular mobile phone use can negatively impact upon human semen quality. These effects would be particularly serious if the biological effects of RFEMR included the induction of DNA damage in male germ cells. In this study, mice were exposed to 900 MHz RFEMR at a specific absorption rate of approximately 90 mW/kg inside a waveguide for 7 days at 12 h per day. Following exposure, DNA damage to caudal epididymal spermatozoa was assessed by quantitative PCR (QPCR) as well as alkaline and pulsed-field gel electrophoresis. The treated mice were overtly normal and all assessment criteria, including sperm number, morphology and vitality were not significantly affected. Gel electrophoresis revealed no gross evidence of increased single- or double-DNA strand breakage in spermatozoa taken from treated animals. However, a detailed analysis of DNA integrity using QPCR revealed statistically significant damage to both the mitochondrial genome (p < 0.05) and the nuclear beta-globin locus (p < 0.01). This study suggests that while RFEMR does not have a dramatic impact on male germ cell development, a significant genotoxic effect on epididymal spermatozoa is evident and deserves further investigation.
    International Journal of Andrology 06/2005; 28(3):171-9. · 3.37 Impact Factor
  • L. E. Bennetts, R. J. Aitken
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    ABSTRACT: Defects in the male genome produced as a consequence of oxidative insult have been associated with decreased fertility levels, an elevated incidence of childhood cancer and dominant genetic disease in the offspring (1). The objective of this study was to determine the relative susceptibility of sperm DNA of different mammalian species to oxidative injury. We applied a highly sensitive quantitative PCR assay (QPCR) to measure gene-specific DNA damage in nuclear and mitochondrial compartments of spermatozoa treated with H2O2. Human, murine and tammar wallaby (Macropus eugenii) spermatozoa were treated with H2O2 (0–5 mM) over a 1 h period. After DNA purification, DNA damage was assessed in a nuclear and a mitochondrial fragment of DNA by quantitative polymerase chain reaction assay (QPCR). DNA damage was detected as a decrease amplification of the target sequences. In murine and human spermatozoa, mitochondrial DNA exhibited greater sensitivity to oxidative damage than nuclear DNA. Doses ranging from 0.25–5 mM H2O2 induced DNA damage of up to 0.65 lesions/10 kb in the mouse, and 1.42 lesions /10 kb in the human. No significant effect on DNA damage was observed over this dose range in the nuclear DNA fragments investigated in these species. In contrast, tammar wallaby spermatozoa were susceptible to DNA damage at the 5 mM H2O2 dose in both nuclear (0.51 lesions/10 kb) and mitochondrial (0.55 lesions/10 kb) genomes. This study is the first to compare DNA damage in specific DNA sequences in spermatozoa of different mammalian species. Nuclear DNA of the metatherian species, the tammar wallaby, was more susceptible to oxidative damage than that of the eutherian species. A major difference between metatherian and eutherian spermatozoa is that, in general, the former possess protamines that are not stabilised by disulfide cross-linkage. These findings therefore suggest that sperm chromatin packaging affects the susceptibility of sperm DNA to oxidative damage. (1) Sawyer and Aitken (2000) Reprod. Med. Rev. 8, 107–126.
    Reproduction Fertility and Development 08/2004; 16(9):215-215. · 2.58 Impact Factor
  • Liga Bennetts, Minjie Lin, R John Aitken
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    ABSTRACT: Despite considerable advances in our understanding of the molecular mechanisms regulating eutherian sperm function, there is a paucity of such knowledge for the Metatheria. In eutherian spermatozoa, the attainment of functional competence is associated with a redox-regulated, cAMP-mediated tyrosine phosphorylation cascade, activated during capacitation. In this report we investigate whether tammar wallaby (Macropus eugenii) spermatozoa possess a similar signal transduction pathway. Western blot analysis of phosphotyrosine expression in caudal and ejaculated populations of tammar spermatozoa revealed that elevation of intracellular cAMP levels, but not exposure to oxidants or NADPH, induced a dramatic increase in the overall level of tyrosine phosphorylation. Washed, ejaculated spermatozoa exhibited more pronounced increases in tyrosine phosphorylation than unwashed sperm populations. Localisation of tyrosine phosphorylation by immunocytochemistry showed that phosphotyrosine residues were principally located along the tammar sperm flagellum, and occasionally at a small region of the sperm head, adjacent to the acrosome. Associated with the tyrosine phosphorylation of tammar spermatozoa, was a change in sperm head conformation to a T-shaped orientation, further implying the importance of these pathways to normal tammar sperm function. Redox activity, as detected by lucigenin-dependent chemiluminescence, was stimulated by NADPH in caudal sperm preparations but not ejaculated spermatozoa. However, neither sperm population responded to treatment with NADPH with changes in intracellular cAMP or tyrosine phosphorylation. In conclusion, tammar spermatozoa possess the same cAMP-mediated, tyrosine phosphorylation-dependent signal transduction cascade that has been associated with capacitation in eutherian spermatozoa. However in Metatherian spermatozoa we could find no evidence that this pathway was redox regulated.
    Journal of Experimental Zoology Part A Comparative Experimental Biology 03/2004; 301(2):118-30.
  • L. E. Bennetts, D. E. Sawyer, R. J. Aitken
    Reproduction Fertility and Development 08/2003; 15(9):5-5. · 2.58 Impact Factor