Oxidative Stress DNA Damage and Protamines

The molecular structure of the human protamine-DNA complex is still poorly understood [48-51]. Most of the models proposed to date are a mere approximation of the actual structure. There is general consensus that the sperm chromatin responds differentially to stressing factors. The reason for this differential response still belongs to the field of speculation. One of the explanations for this differential sensitivity could be related to the fact that the protamines facilitate an efficient packing of the DNA molecule. Two main types of protamine families have been described in mammalian species. The P1 family has been reported in all species of vertebrates studied to date [52-54], while the family of P2 proteins is only present in primates and most of the rodents. The sperm of stallions, lagomorphs and proboscides have also been found to contain processed protamine P2 [52, 53, 55-57]. But in the case of bull and boar sperm, two species that present the lowest rate to iatrogenic sperm DNA damage, the gene for P2 is present, but it appears to be dysfunctional or that produces an aberrant protein [56]. Among the putative functions ascribed to protamines [57, 58] are those related to imprinting of the paternal genome during spermatogenesis and the control of transcription factors to allow its reprogramming by the oocyte. However, one of the functions that should be underlined is its primary role in configuring a compact and hydrodynamic nucleus which plays a crucial role in protecting the paternal genome during migration through the male and female reproductive tract. The quasi-paracrystalline packaging of the sperm chromatin renders the DNA molecule virtually inaccessible to nucleases or mutagens that may be present in the internal or in the external media where the sperm have to endure for variable periods of time. Variations in the levels of protamination by an unbalanced P1/P2 ratio may result in male infertility. In infertile humans, where these aspects have been more deeply analyzed, these unbalance differentially impacts on the integrity of the DNA and in the reproductive outcome of these couples [59, 60]. It has been recently reported that the P1/P2 ratio in human sperm correlates with the levels of sperm DNA fragmentation and also with the rate of sperm DNA damage after ejaculation. Statistical differences were found between fertile controls and patients with different types of pathologies [61]. An altered P1/P2 ratio is associated with an increased amount of histones and intermediate proteins thus supporting the role of a balance between both fractions in order to produce a stable chromatin. Incomplete protamination makes the sperm chromatin more vulnerable to attack by endogenous and exogenous agents, such as mutagenic compounds and disulphide bond reductors [ 62] or oxygen radicals [63, 64], Therefore, it appears that the protamine ratio determines the ability of the DNA molecule to reach the oocyte in an intact manner. This situation is not present in those species lacking P2 in the sperm chromatin. Thus, mammalian species such as boar or bull sperm, which only show one type of protamine for the assembling of the sperm chromatin, appear to elude the negative effects of a P1/P2 unbalance.

Additionally, we should bare in mind the role of -SS- disulphide bonds, as well as the number of arginine-lysine residues present in the protamines, that play a decisive role in the highly efficient organization of the sperm chromatin. P1 protamines of eutherian mammals are characterized by the presence of a variable number (6-9) of cysteine (Cys) residues per molecule. During spermiogenesis, SH groups are oxidized to -SS- to form a three-dimensional network of disulphide bridges between and within protamine molecules in the sperm chromatin, conferring the sperm chromatin high stability [65, 66]. In fact, once the sperm chromatin has been tightly packed with these Cys residues, an effective sperm chromatin disorganization is only possible by a reduction in the extent of -SS- cross-linking using, for example, reducing agents such as dithiothreitol or beta-mercaptoethanol [62, 67] and it has been suggested that the differential resistance of the sperm chro-matin to relaxation is linked to the protective effect of the Cys residues to produce intra- or inter-protamine cross-linking [68] , The model proposed by Vilfan et al., applies to most of the mammalian species known because is mainly based in the simultaneous implication of one of the two Cys at position 5-6 and 38-39 in intermolecular cross-linking, while the other participate in a intermolecular connection (see Figure 2a in [68]). Moreover, a specific sperm nuclear glutathione peroxidase (snGPx) with properties similar to that of phospholipid hydroperoxide glutathione peroxidase (PHGPx) and identified as a 34-kDa selenoenzyme, acts as a protamine thiol peroxidase and is directly involved in the stabilization of the condensed chro-matin by specific cross-linked protamine disulphide bridges [69]. With regard to the possible disulphide bonding occurring in P1, variations in the number and position varies from one species to another. However, the number of Cys residues by itself does not offer a full convincing explanation for the differential rates of sperm iatro-genic DNA damage. For example, boar containing ten Cys residues shows a low rate of sperm DNA fragmentation, while human presenting six Cys shows a relatively high rate. However, boar and bull sperm, both exhibiting a low rate of sperm DNA damage, reveal different number of Cys residues. Moreover, identical number and distribution of Cys residues among different species (stallion, ram and bull) result in large differences in the rate of sperm DNA damage.

In a recent study, the chromatin resistance to the effect of oxidative stress has been compared using sperm from Homo sapiens, Mus musculus and Sus domesticus and three metatherian species (Vombatus ursinus, Phascolarctos cinereus, Macropus giganteus). In this experiment, semen samples were exposed to increasing concentrations of hydrogen peroxide. The main result was that sperm DNA of marsupial species was significantly more sensitive to oxidative stress than spermatozoa of eutherian species [69]. It is known that protamines of eutherian species are cysteine-rich and are extensively cross-linked by disulphide bonds during epididymal transit, whereas the protamines of most marsupial species lack cysteine residues. Therefore, this observed differential susceptibility is consistent with the lack of disulphide cross-linking in marsupial sperm chromatin and suggests that the oxidation of thiols to disulphides for chromatin condensation during epididymal transit in eutherian mammals is likely to be important in order to provide stability and protect spermatozoa against the genotoxic effects of hostile environments. In fact, some studies have revealed a higher vulnerability of marsupial sperm when exposed to different treatments [70-72],

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