Free radical-induced sperm DNA damage may take place during the process of spermatogenesis, during sperm transit through the epididymis [1, 2] and in vitro during sperm processing [ 3, 4]. When the fine balance between oxygen radical (reactive oxygen species [ROS]) production and antioxidant defences is tilted towards net ROS presence in the intracellular and extracellular medium, oxidative stress develops. This can trigger apoptotic mechanisms and cause direct damage of critical cell structures such as the plasma and acrosomal membranes and the chro-matin. Exceedingly high levels of ROS may also cause necrosis [5, 6]. Oxygen radicals, including free radicals and peroxides, are particularly deleterious to the cell through direct, indirect or synergistic mechanisms. Alternatively, ROS, through oxi-doreduction reactions, may be converted into more reactive radical species capable of causing more extensive cell damage [7]. Although the short-term effects of excessive ROS production are mainly expressed as loss of overall cell viability, once the DNA molecule is affected, these effects may be long-lasting, since they can be inherited [8]. If sperm DNA is damaged, given the lack of DNA repair mechanisms in the male germ line during sperm maturation, there is the risk of transmitting DNA mutations to the oocyte after fertilization [9] [ Although some of these mutations could be repaired at the pronuclear stage embryo, if the transmitted DNA damage affects constitutive genes or DNA motifs related with gene regulation of epigenetic control, embryo viability could be greatly compromised.

Oxygen radicals are among the most damaging molecules capable of modifying cell components. These radicals include the superoxide anion (O2-0, hydrogen peroxide (H[O2), the hydroxyl radical (OH-0, peroxynitrite, organic hydroperoxides (ROOH), alkoxy and peroxy radicals and hypochlorous acid (HOCl). Those that are liposoluble, such as H2O2, HOCl or ROOH, may alter membrane stability and, in the particular case of sperm motility. Free radicals such as O2-\ may cross-react to produce Fe2+ using proteins as a target and generate H2O2 or be the precursors for the metal-catalyzed OH~* formation. Although the biological consequences of many DNA base modifications as products of oxidative stress are known, attack of only OH free radicals, is particularly effective in modifying, for example, the C4-5 double bond of pyrimidine and the OH radical of purines. This generates a spectrum of oxidative stable conformations such as 8-OHdG, 8-OHdA, formamidopyrimidines thymine glycol, uracil glycol, urea residue, 5-OHdU, 5-OHdC, hydantoin (Fig. 11.1), which may cause unrepaired DNA lesions. Thus, for example, thymine glycol is able to block DNA replication and subsequently is potentially lethal to cells. The case of unrepaired 8-oxo-dG mismatching with dA is a well-known situation which increases G to T transition mutations with the subsequent consequences for gene expression if affecting structural genes. Although little is known about the potential negative effects of nitric oxide-derived oxidative processes, they have been shown to decrease sperm motility [10], but also to increase it [11]. Chemical studies suggest that nitric oxide may produce single-stranded DNA breaks (SSBs) and abasic sites (AP sites [12, 13]), predominately in regions with guanine residues, producing base modifications such as 8-Oxo-dG and 8-Oxo-nitro-G [14].

Fig. 11.1 Possible nucleotide modifications caused by attack of OH radicals. First raw, pyrimidine modifications: 5-hydroxy-dU, 5-hydroxy-dC, thymine glycol, uracil glycol. Second raw, purine modifications: 8-Oxo-dA, 8-Oxo-dG, Fapy-dA (N4-(2-deoxy-a,p-D-erytro-pentofuranosyl)-4,6-diamino-5-formamidopyrimidine) and Fapy-dG (N6-(2-deoxy-a,p-D-erytro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine)

Fig. 11.1 Possible nucleotide modifications caused by attack of OH radicals. First raw, pyrimidine modifications: 5-hydroxy-dU, 5-hydroxy-dC, thymine glycol, uracil glycol. Second raw, purine modifications: 8-Oxo-dA, 8-Oxo-dG, Fapy-dA (N4-(2-deoxy-a,p-D-erytro-pentofuranosyl)-4,6-diamino-5-formamidopyrimidine) and Fapy-dG (N6-(2-deoxy-a,p-D-erytro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine)

11.1.1 Oxygen Radical-Induced DNA Damage: Candidate DNA Sequences to Be Affected

The large variability of functional and non-functional DNA sequences in the genome is determined by a combination of A, T, C and G nucleotides. However, the mutation sensitivity of different DNA motifs made up of combinations of these four base pairs differs at different genome domains. For example, when human peripheral blood leukocytes are exposed to X-ray radiation for the analysis of the initial level of DNA breakage, using FISH and targeting for different satellite DNA sequences, such as alphoid, satellite 1, 5-bp classical satellite or telomeric DNA sequences (TEL-DNA), differences in sensitivity are observed. Irradiation of nucleoids obtained after protein removal show that alkaline unwinding solutions generate about half the amount of signal when the DNA breaks are present in the 5-bp classical DNA satellites than when the same number of breaks are present in the whole genome. Furthermore, the signal is slightly stronger when the breaks are within the alphoids or the satellite 1 sequence. Therefore, chromatin containing the 5-bp classical satellite arrangement proved to be more sensitive to breakage than the overall genome, whereas DNA in the chromatin corresponding to alphoids or satellite 1 show sensitivity similar to that of the whole genome. Interestingly, TEL-DNA sequences appear to be maximally labelled, even in unirradiated cells [15].

Similarly to the DNA damage induced by exposure to X-ray radiation, oxygen radical-induced damage may result in random DNA damage leading to whole DNA damage during the process of spermatogenesis affecting structural genes (introns or exons) or DNA sequences not directly involved in RNA production but regulating epigenetic functions. Particularly, the telomeres (TEL-DNA) seem to be highly susceptible to DNA damage [16]. Oxygen radical-induced DNA damage may not only lead to single strand DNA breaks, with the subsequent production of apurinic or apirimidinic sites (AP-sites), but also predispose to apoptosis. The particular DNA damage experimented by the telomeres of the germ line is largely unknown, but should not be overlooked, since the transmission and size maintenance of the TEL-DNA for each species is tissue specific. Large variations in the copy number of TEL-DNA sequences among male and female gametes are not expected, since this would compromise the continuity of the species. In fact, in some insects it has been shown that the largest TEL-DNA sequences are harboured in germ line tissues although a certain level of heterozygosity between homologous chromosomes has been reported. This is interesting because during meiosis, synapsis of the bivalents takes place through the telomeres and the presence of heterozygosity may disrupt bivalent synapsis. Moreover, large differences in the size of TEL-DNA between homologous chromosomes may lead to asynchrony in the replication timing assigned to these particular regions. This could be critical during the first stages of embryo development. Studies performed in fibroblasts have shown that oxygen radicals can induce telomere damage leading to defects in synapsis [17]. The highly mutagenic capacity of TEL-DNA is also found when the interstitial telomere-like DNA sequence arrays of Chinese hamster Don cells are tested for the effects of certain damaging agents. In this regard, it has been reported that around 40% of the exchanges involved a telomere-like block of DNA sequences within the rearrangement site [ 16] . Interestingly, this effect was independent of the DNA-damaging agent. This chromosomal behaviour suggests a general recombination capacity of interstitial telomere-like DNA sequence repeats that does not seem to be related to the initial mechanism of DNA damage, although its consequences for cell arrest, senescence or apoptotic triggering have been largely demonstrated [18]. Nitric oxide-induced DNA damage is particularly effective in inducing intragenomic heterogeneous damage since, for example, a higher density of DNA damage has been found in the telomeric DNA sequence repeats region, since they are particularly enriched in guanine residues [19]. That is precisely why it has been suggested that chronic exposure to nitric oxide in vivo may lead to premature ageing and neoplastic development. In fact, it has been shown that sperm subpopulations selected by swim-up are not homogeneous for telomere length [20] . Telomerase activity is low in mature oocytes and in cleavage stage embryos, but high in blastocysts. After fertilization, the presence of critically shortened telomeres, coming from either the sperm or the oocyte, may contribute to abnormal cleavage and development, as shown in knock-out mice. However, telomere lengthening is remarkable during the early cleavage cycles through a recombination-based mechanism [21],

It is also worth mentioning that ejaculated spermatozoa are not homogeneous in terms of telomere size [22] and that the routine sperm selection techniques used in ART, such as density gradient centrifugation and swim-up, unintentionally allow for the selection of spermatozoa with the largest TEL-DNA repeats. The question is why and where this heterogeneity for these particular chromosome domains is produced? Are they the product of DNA replicative arrested processes before meiosis or are they "allowed mutations" of telomere shortening produced during spermato-genesis and spermiogenesis?

Oxygen radical-induced telomere damage and synaptic anomalies may also occur in germ cells leading to chromosomal defects in ejaculated spermatozoa. After fertilization, these synaptic anomalies could result in defects in chromosomal segregation during embryo division, leading to embryo aneuploidy and to a reduction in pregnancy rate and an increase in miscarriage.

Get Pregnant - Cure Infertility Naturally

Get Pregnant - Cure Infertility Naturally

Far too many people struggle to fall pregnant and conceive a child naturally. This book looks at the reasons for infertility and how using a natural, holistic approach can greatly improve your chances of conceiving a child of your own without surgery and without drugs!

Get My Free Ebook

Post a comment