1,406 research outputs found
Mechanisms of environmental reproductive toxicity: Sperm DNA damage
No full textThe spermatogenetic process in the human produces a heterogeneous cell population showing different degrees of maturation, variable morphological features and fertilizing capacity. There is a large body of evidence that some of the ejaculated spermatozoa possess a variety of abnormalities at the nuclear, cytoskeletal, and organelle levels and that these anomalies impact fertility. Damage of sperm DNA or its chromatin structure can occur at any step of whole spermatogenesis and could have endogenous (i. e. pathological altered germ cell maturation) or exogenous origin (i. e. exposure to xenobiotic substances). Three main theories have been proposed to explain DNA anomalies in the ejaculated human spermatozoa (Sakkas and Alavarez, 2010). The first theory supports that DNA damage in mature spermatozoa is associated with poor chromatin packaging or abnormal packing due to underprotamination and/or alteration of Topisomerase II activity which results in the presence of endogenous nicks in DNA (Manicardi et al., 1995). The second theory proposes that sperm DNA damage could be related to a process called “abortive apoptosis” whereby sperm cells are earmarked for apoptosis during spermatogenesis but survive to be present in the ejaculate (Sakkas et al., 2002). A major factor affecting sperm is also their vulnerability to oxidative stress, because they are deficient in both antioxidant and DNA-repair systems and rich in targets for oxidative attack (Aitken et al., 1998). The correct DNA packaging and stability appears to be fundamental for the protection against both endogenous and exogenous DNA damage. Transmission of damaged DNA to the offspring, particularly at levels that exceed the DNA repair capacity of the oocyte, has been shown to have serious consequences in animal models and is also being more and more implicated in the human. Normal sexual development, differentiation and function in the human has been shown to be altered by the presence of synthetic chemicals in the environment acting as endocrine disruptors or mutation agents. Moreover, exposure to physical agents or chemicals, including therapeutic drugs and environmental toxicants, either individually or together, can affect the integrity of sperm chromatin (Stronati et al., 2006). The observation that ejaculated human spermatozoa possess DNA damage raises numerous problems relating to why and how these spermatozoa arise in the ejaculate of some men and what consequences they have if they succeed in their genetic project. Understanding the mechanisms responsible will improve our knowledge about certain causes of male infertility
Chromosomal localization of a highly repeated EcoRI DNA fragment in Megoura viciae (Homoptera, Aphididae) by nick translation and fluorescence in situ hybridization.
No full textTo investigate the genome of the aphid Megoura viciae at molecular level, we have studied total DNA by agarose gel electrophoresis after cleavage with different restriction endonucleases. EcoRI digestion produced a highly repeated DNA fragment, about 600 pb long. The contribution of this EcoRI element to the total genome of M. viciae was estimated at about 6% by means of densitometric scanning of agarose gel photographs. The chromosomal localization of this fragment, investigated by fluorescent in situ hybridization (FISH), constantly showed one large and two narrower fluorescent bands located on the X chromosome, all corresponding to C-positive heterochromatic areas. These results are in full accordance with the data obtained by in situ nick translation experiments carried out after EcoRI digestion, and clearly demonstrate that a substantial amount of M. viciae heterochromatin consists of EcoRI fragments which are mainly located on the X chromosome. Using the EcoRI restriction fragment as a molecular probe may be a practical tool for the investigation of taxonomic and evolutionary relationships in this group of insects
Basic and clinical aspects of Sperm Chromomycin A3 assay.
No full textSemen quality is conventionally determined according to the number, motility, and morphology ofspermatozoa in an ejaculate. In turn, it is generally accepted that an association exists between these semenparameters and fertilizing ability. With the advent of in vitro fertilization (IVF) and related techniquessuch as intracytoplasmic sperm injection (ICSI), it has become increasingly apparent that the number,motility, and morphology of spermatozoa are not always indicative of a male’s fertility status. Methodsexploring sperm DNA stability and integrity have been applied during the last decade to evaluate fertilitydisorders and to increase the predictive value of sperm analysis for procreation in vivo and in vitro. It hasbeen shown that infertile men have an increased sperm histone–protamine ratio than fertile counterparts.This alteration of histone–protamine ratio, also called abnormal packing, increases susceptibility of spermDNA to external stresses due to poorer chromatin compaction. Recent studies have also underlined the linkbetween protamine deficiency and sperm DNA damage that resulted in poor fertilizing capacity
Possible consequences of performing intracytoplasmic sperm injection (ICSI) with sperm possessing nuclear DNA damage
No full textA number of studies have confirmed that sperm with damaged nuclear DNA are present in human ejaculate. It appears that these sperm are more likely to occur in men with low concentrations of sperm, or poor sperm motility or morphology. In assisted reproductive techniques, in particular intracytoplasmic sperm injection (ICSI), there is a higher statistical chance that sperm possessing damaged DNA will be selected and used to fertilize oocytes. In light of this observation, the question of whether human sperm with damaged DNA can impair fertilization and embryo development is examined
EFFECT OF DEOXYRIBONUCLEIC-ACID PROTAMINATION ON FLUOROCHROME STAINING AND IN-SITU NICK-TRANSLATION OF MURINE AND HUMAN MATURE SPERMATOZOA
No full textA major event in enhancing sperm chromatin stability is the replacement of the histones by protamines during spermiogenesis. In this study, we present results indicating that chromomycin A3 (CMA3) Can be used to show protamine deficiency in sperm chromatin. Fixed chromatin of mature mouse spermatozoa showed high fluorescence after treatment with ethidium bromide (EB), but was completely unstained after treatment with CMA3. The same chromatin was found to be highly resistant to in situ nick-translation. In contrast, a substantial fraction of human spermatozoa were positive for CMA3. The accessibility of CMA3 to the DNA of human sperm was eliminated if the slides were previously treated with protamine in situ. This treatment did not affect the accessibility of EB to the chromatin. Individual human sperm samples revealed a substantial frequency of spermatozoa with endogenous nicks, which was found to be the same as the frequency of spermatozoa responding positively to CMA3 staining. Treatment of preparations with protamines prevented the identification of the endogenous nicks. These data as a whole suggest that CMA3 could represent a useful tool for the detection of protamine deficiency in sperm chromatin. Furthermore, confirmation of experiments relating sensitivity to nick translation and positivity to CMA3 may allow an indirect in situ visualization of nicked and partially denatured DNA, which could correlate with certain forms of male factor infertility
Sperm decondensation during fertilization in the mouse: presence of DNaseI hypersensitive sites in situ and a putative role for topoisomerase II.
No full textIn this study our aim was to characterise the presence and the role of DNA alterations during sperm decondensation in the mouse. To visualise the changes during decondensation we investigated for the presence of DNase I hypersensitive sites in situ and for a putative role for topoisomerase II by examining the effect of teniposide, a topoisomerase II inhibitor, during fertilisation. In situ nick translation without the previous addition of DNase I failed to reveal the presence of endogenous nicks in decondensing sperm and pronuclei whereas preincubation of fixed oocytes with DNase I indicated that decondensing sperm were sensitive to this enzyme. Addition of 100 mu M teniposide did not completely inhibit pronuclei formation but its addition to the fertilisation medium did lead to the presence of endogenous DNA nicks in decondensing sperm. These observations suggest that DNase I hypersensitivity during sperm decondensation is related to the dramatic conformational changes that the chromatin undergoes during the decondensation process, in which topoisomerase II may be implicated
Cytological and electrophoretic analysis of DNA methylation in the holocentric chromosomes of Megoura viciae (Homoptera, Aphididae).
No full textChromosomal and purified DNA methylation patterns were determined in the holocentric chromosomes of Megoura viciae by treatment with MspI and HpaII. Both enzymes produced a clear C-like banding pattern but widely digested one telomere of the X chromosome, which appeared as heterochromatic after C-banding treatment and brightly fluorescent after chromomycin A(3) staining. Quantitative microfluorometric evaluations of DNA extraction performed on cytological preparations showed that both isoschizomers resulted in the same DNA extraction (about 30%). Contrary to what was found by in situ endonuclease treatment, the electrophoretic patterns of purified and digested DNA showed that digestion with MspI was slightly more extensive than that with HpaII in a zone of fragments ranging from 23 to 9 kb. This result indicates that aphid chromatin is not wholly unmethylated. The discrepancy between electrophoretic and cytological data has been explained by taking into consideration that DNA fragments with high molecular weights could be cleaved in situ by the enzymes but not extracted from the chromatin
Cytogenetic analysis on the holocentric chromosomes of the cabbage aphid Brevicoryne brassicae.
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