Iatrogenic genetic damage of spermatozoa

Abstract

Different factors can affect sperm morphology and physiology that negatively influence men fertility. Many studies on humans and animals suggest that both radiation and chemotherapy alter sperm chromatin thus promoting significant damage on sperm DNA and, decreasing the level of protamination, thus altering DNA compaction. Spermatozoa from cancer survivors are affected by chemotherapy even years after the end of the treatment. We are exposed to different toxicants present in the environment (products of air pollution, pesticides, plasticizers, etc), which their impact on men reproduction has not been yet established.

This chapter aims to update our knowledge on how sperm chromatin structure is modified by external agents and to describe the different strategies available to better study this complex structure in the infertile men.

INTRODUCTION

Sperm chromatin structure

The ultimate goal of any given spermatozoon is to deliver the paternal genetic information into a mature oocyte during fertilization. In order to accomplish this essential task for any species, a round cell ‘spermatogonia’ should go through several divisions and transformations, in the testis, to become a fully formed spermatozoon [1]. Then, the spermatozoa leave the testis to start a journey through the epididymis, a long single and highly convoluted tube, to complete their maturation acquiring some elements needed for fertilization and the ability to move [2]. Finally, they must reside in the female reproductive tract, specifically the oviduct, to achieve fertilizing ability and be able to recognize the oocyte, undergo the acrosome reaction needed to go through the zona pellucida that surrounds the oocyte. This process is called ‘capacitation’ and involves a series of biochemical and morphological changes that prepare the spermatozoon for the fertilization [3–5].

In order to accomplish this long journey through the testicular, epididymal and female tract environments, the sperm chromatin is transformed into a complex structure, with association of DNA with basic proteins called protamines and other elements, forming a toroid structure; this transformation aims to avoid potential damage to the genomic material (Fig. 8.1). During spermatogenesis, histones are replaced by protamines allowing a tighter compaction of the sperm DNA compared to somatic cells [6–8]. Histones are replaced first by transitional protein [9] and then by protamines 1 and 2 (P1 and P2) in human spermatids [6–8] during the spermiogenesis [1]. In vitro studies suggested that the hyperacetylation, an epigenetic modification of histones, allows the replacement of histones by protamines [10–12]. A cycle of phosphorylation-dephosphorylation occurs in protamines before binding to DNA and during nucleosome maturation [13, 14]. Protamines have a high number of positively charged residues, thus allowing the formation of a highly condensed complex with the sperm DNA that has strong negative charge [15–19].

Fig. 8.1. Organization of sperm chromatin structure.

The intimate interaction between the DNA strands and protamine and the formation of disulfide bridges (-SS-) among protamines and the incorporation of zinc (Zn²⁺) make the structure tightly compacted.

Despite this massive protein exchange, promoting that 85–95% of sperm DNA is associated with protamines, 5%–15% remains associated to histones [20–22]. Several histone isoforms (e.g. H2A, H2B, H3 and H4) and isoform variants are present in human spermatozoa, being histone H 2B the predominant isoform [23]; the increased levels of histones and/or histones variants are associated with abnormal DNA compaction and DNA damage in astenozoospermic infertile men [24]. Similarly, the change in P1/P2 ratio due to an increase of protamine 2 together with increased levels of the pre-P2 are associated with sperm DNA fragmentation in infertile men [25, 26] and low P1/P2 ratio has been associated with low pregnancy rates [27].

The stabilization of sperm chromatin is accomplished, in part, by addition of zinc (Zn²⁺) to the sperm nucleus at the time of the beginning of nuclear compaction [1, 28, 29]. This micronutrient is important for fertility as Zn²⁺ deficiency promotes arrest at spermiogenesis, decrease in germ cell proliferation, impairment of sperm motility in different species including humans [30, 31]. Zn²⁺ contributes to stabilize sperm chromatin by binding to free thiol (-SH) groups and forming Zn²⁺ bridges among protamines [21, 29, 32, 33]. The stabilization of sperm chromatin is completed by the formation of disulfide (-SS-) bridges among protamines during epididymal maturation [34–36]; in normal human spermatozoa, less than 1.5% of cysteines are found as reactive -SH [37]. An increased [38] or decreased [39–41] levels of free -SH has been observed in infertile men, indicating that under- or over-oxidation of -SH are associated with abnormal sperm function. This alteration of thiol oxidation can be attributed to abnormal epididymal maturation due to an improver oxidation of –SH groups in most of sperm proteins including protamines. It is still poorly understood which are the players and the intrinsic mechanisms to promote sperm chromatin condensation during spermiogenesis. A candidate to perform such task could be the nuclear isoform of gluthatione peroxidase (nGPX4) which is necessary for the protamine thiol cross-linking during sperm maturation [42]. Mice lacking nuclear isoform of glutathione peroxidase 4 (nGPX4) have spermatozoa with increased DNA decondensation, however, these animals are viable and fertile [43]. More research is necessary to reveal other players in the sperm DNA condensation mechanisms to understand better how the sperm chromatin is shaped during sperm maturation.

Alterations of the sperm chromatin structure

The sperm chromatin can be altered and therefore is susceptible to damage at different stages of the sperm production and maturation. Starting in the testis, it could be affected by apoptosis during spermatocytogenesis or during the chromatin remodeling during spermiogenesis [1]. Approximately 50% of germ cells that enter into meiosis become apoptotic and are removed by Sertoli cells. Sometimes, this process is not as efficient as required and some defective germ cells continue developing and can be found in the ejaculate [44, 45]. It is then possible that spermatozoa carrying apoptotic markers such as Fas, caspase activities, p53 and annexin-V can found in semen [46–50]. Most of these spermatozoa have normal morphology and good motility, being impossible to discriminate between affected and the healthy sperm cells. Many strategies are under study to overcome this problem but will not be discussed further in this chapter.

As mentioned above, the sperm chromatin remodeling during spermiogenesis is another stage where this structure is susceptible to damage. The replacement of histone by protamines requires nuclease activity that creates DNA nicks to provide relief of torsional tension in order to achieve the chromatin arrangement during the histone replacement in spermatids from many species including humans [51–53]. A deregulation of this process may promote abnormal chromatin packaging or DNA fragmentation that can be detected in the ejaculated spermatozoa. After spermiation, spermatozoa enter into the epididymis in order to achieve the ability to move and to acquire fertilizing ability [2, 3]. During the journey through the epididymis, that varies among species but in humans is 5–6 days, spermatozoa can be damaged and showed significantly DNA fragmentation [54, 55]. This is the result of reactive oxygen species (ROS) action or ROS-modified metabolites, for instance it is known that hydrogen peroxide produce DNA fragmentation in human spermatozoa [56] and that the radical hydroxyl can attack DNA bases promoting oxidation and the increase of 8-oxoguanosine (8-oxoG) among other DNA metabolites. Another consequence of ROS attack is the production of abasic sites, which destabilize the double helix and can result in strand breaks [57].

Drugs and other chemicals can affect human sperm chromatin [58–61]. Common lesions found associated with agents are the presence of inter strand cross-linking and chemical adducts. These are highly toxic DNA lesions that prevent translation and replication by inhibiting DNA strand separation [62, 63]. This property is convenient when the goal is to eliminate cancerous cells; however, chemotherapy severely affects germ cells [64, 65]. Animals’ studies suggest that spermatogonia [66, 67] and Sertoli cells [68, 69] are deeply affected by chemotherapeutic agents.

DNA methylation and histone modifications (e.g. phosphorylation, methylation and acethylation) are epigenetics changes that occur during spermatogenesis in the spermatozoon; they are important to assure the development of the future embryo. Thus, any changes in the programming epigenetic modifications during spermatogenesis by different causes (e.g.: drugs, diseases, etc.) may promote men infertility [70, 71].

Evaluation of sperm chromatin structure

Different assays have been developed to determine sperm DNA or other components of the sperm chromatin; interestingly, each of them measures a certain type of damage, thus having limited used to characterize the entire sperm chromatin structure. This is important to mention because only a multi-assay approach will allow a proper characterization of this complex structure; however, this type of analysis is limited by the amount of spermatozoa in a given sample [40].

Assays to determine sperm DNA damage

Acridine orange/Sperm chromatin structure assay (SCSA^®)

The acridine orange (AO) is a metachormatic probe that binds to single or double strands of DNA giving a red or green fluorescence, respectively, when it is excited at 470 to 490nm [72]. This property is useful to identify spermatozoa with denaturated (single stranded) DNA. Sperm DNA can then be analyzed by fluorescence microscope or flow cytometry [73, 74].

This assay determines the susceptibility of sperm DNA to acid denaturation (pH 1.20); the low pH induced the opening of the sperm DNA strands at the sites of DNA breaks. Then, sperm are incubated with AO and the red and green fluorescence is acquired using a flow cytometer [75, 76].

The SCSA is one of the most well tested assays to study sperm chromatin structure and has been used by different laboratories worldwide. Three main sperm populations can be distinguished after analysis of the acquired data: 1) sperm with no DNA damage, 2) sperm with moderate or high DNA damage and 3) sperm with high AO DNA stainability. Based on these sperm populations, data is expressed as mean DNA fragmentation index (DFI), standard deviation of DFI, percentage of DFI (%DFI, corresponding to the percentage of cells outside the main population) and as percentage of spermatozoa with high green fluorescence or high DNA stainability (%HDS), as an indication of sperm DNA compaction [72]. The DFI and HDS are the most used SCSA parameters to characterize sperm chromatin but the mean DFI and SD DFI are also powerful parameters to determine the presence of sperm DNA damage. For instance, significant sperm DNA fragmentation can be found with the DFI, mean and SD DFI parameters in semen samples from infertile men, whereas the mean DFI indicated significant DNA damage in samples from Hodgkin’s lymphoma patients when DFI value similar to that of healthy controls [40].

Comet assay

The single-cell gel electrophoresis assay or comet assay is a common tool in male reproductive toxicology to study DNA fragmentation in spermatozoa in humans and animal models [40, 77–80]. This assay is based on the electrophoretic migration of DNA fragments from the core of chromatin after the sperm suspension is treated with a buffer with neutral or alkaline pH. These fragments are originated from single and double strand breaks of sperm DNA. Then, the slides are staining with a DNA labeling dye (propidium iodide, SBYR green, etc) and individual pictures of spermatozoa are taken using a fluorescence microscopy. The extension of the sperm DNA damage can be determined using a software that provides the following parameters: percentages of the DNA in the head or the tail of the comet, the tail length and the tail extent moment [40]. It is a very reliable assay to determine sperm DNA damage in humans, particularly in severe olizoospermic samples from infertile [40] or cancer survivors [59].

TUNEL assay

Another way to detect single- and double-strand DNA breaks is the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay [81]. The 3′ OH openings of single and double strands breaks are labelled by the addition of FITC-labeled deoxyuridine triphosphate nucleotides in a reaction catalized by the deoxynucleotidyl transferase [82, 83]. Labelled spermatozoa are then analyzed by fluorescence microscopy or flow cytometry [40, 84–86]. Both the microscope- or flow cytometry-based methods were extensively used to determine sperm DNA damage in human spermatozoa [40, 84–88].

Sperm DNA oxidation determination

The oxidation of sperm DNA generates the formation of 8-hydroxy-2′ deoxyguanosine (8-OHdG) promoting single and double strand DNA breaks; this damage is associated with oxidative stress, which is a major component on the pathophysiology of men infertility [89–95]. Therefore, the determination of the 8-OHdG levels in spermatozoa should be an important part of the semiology in infertile men. Immunocytochemistry- and flow cytometry-based techniques using antibodies or binding proteins that recognize 8-OHdG were used to determine sperm DNA oxidation in spermatozoa from different species including humans [96–98]. Recently, Cambi et al, developed a flow cytometry-based method using an anti-8-OHdG moiety antibody that overcomes the problem of false negatives that can give the method using binding proteins [99]. In this study, they found negative correlations between levels of 8OH-G and sperm parameters in a cohort of 94 infertile men, thus demonstrating the clinical relevance of using the detection of 8-OHdG in combination with the standard semen analysis for diagnosis of men infertility.

Sperm chromatin dispersion test

The sperm chromatin dispersion (SCD) assay was developed to determine sperm DNA damage for diagnosis of men infertility [100]. This is a microscope-based assay that detects sperm DNA fragmentation in samples that were previously acid-denatured. The treated sperm are then stained with a probe that binds to DNA (e.g. DAPI) and pictures are taken to quantify the dispersion area of the DNA using image analyzer software [100, 101]. The SDC detected significant more sperm DNA fragmentation in infertile men with varicocele than in fertile controls, thus promising its use in the clinic. It has also the potential advantage to be combined with other assays that could detect other abnormalities in the sperm chromatin (e.g., aneuplody, DNA oxidation, DNA methylation, etc) [102]; although this strategy is very appealing, more work has to be done to confirm whether these combinatory techniques are possible to study sperm chromatin structure in humans.

Assays to evaluate sperm DNA compaction

Monobromobimame (mBBr) thiol groups labeling

As mentioned above, sperm DNA is tightly compacted due to specific interactions among components of the sperm chromatin. The -SH groups present in the protamines are important elements to maintain the chromatin closely compacted in the sperm nucleus [34, 103]. The amount of –SH groups can be determined by labeling them with the fluorescent probe mBBr [104]. It is important to mention that –SH are not only present in the protamines within the sperm nucleus but also in the tail, thus it is essential to separate the head from the tail by sonication before labeling the spermatozoa with mBBr [40]. The intensity of the mBBr-labeling in the sperm head is then determined by flow cytometry [40, 105]. Parallel samples previously treated with DTT are necessary to determine the percentage of free –SH (total fluorescence – fluorescence of DTT sample). A high fluorescence intensity value corresponds to a high percentage of free –SH present in the sample and therefore an indication of less sperm DNA compaction.

Chormomycin A3 (CMA3) labeling

CMA3 is a fluorochrome that specifically binds to guanosine-cytosine-rich sequences where the protamines are prone to bind; it was observed that CMA3 competes with sperm protamines for binding to the minor groove of DNA [106, 107]. Thus, an increased CMA3 labeling is an indication of lower protamination in spermatozoa and lower DNA compaction [40, 108–112]. The CMA3 labeling can be determined by either microscopy or flow cytometry and, although some studies associate the increased CMA3 labeling with high percentage of abnormal spermatozoa [113, 114], others reported the presence of high levels of CMA3 labeling in morphologically normal spermatozoa [40, 109]. This discrepancy could be based on the method used to determine CMA3 labeling (e.g. microscopy or flow cytometry) or the way to determine teratozoospermia (e.g. strict criteria).

Aniline blue and toluidine blue assays

The aniline blue (AB) and the toluidine blue (TB) assays are based on the property of these dyes to bind to different components of the sperm chromatin and can be performed with light microscopy, thus with minimum costs at a clinical settings. The Aniline blue (AB) assay detects histones, which are rich in lysine, and binds to them at low pH [115]. AB assay showed significant correlation between high content of histone, abnormal sperm chromatin and men infertility. However, the correlation of AB results with other sperm parameters is inconsistent and controversial [116].

Toluidine blue (TB) is a basic planar nuclear dye useful to stain sperm chromatin. It binds to DNA phosphate residues of sperm DNA in nuclei with loosely packed chromatin and/or impaired DNA, providing a metachromatic shift due to dimerization of the dye molecules from light blue to purple–violet color [117–120]. Recently, it has been suggested that TB assay can be a complementary tool for the semen analysis to diagnose men infertility [121].

Both AB and TB are simple an inexpensive assays that can be performed with a light microscope and using smears previously prepared and stored. The main disadvantage is that, as a microscope-based assay, the number of spermatozoa to be counted is limited compared to assays based on flow cytometry.

High DNA stainability (HDS, SCSA^®)

Another parameter that can be obtained with the AO/SCS^® is the percentage of spermatozoa with high DNA stainability (HDS) [72, 122]. This population of spermatozoa can be visualized in the top of the scatter plot after the flow cytometry acquisition and those showing a higher AO green intensity due to the increased accessibility of the dye for the sperm nucleus (Fig. 1.2). This population does not have increased DNA damage and is consider immature due to the high amounts of histones remaining in sperm cells [72].

The assays described above were used by several laboratories to study human sperm chromatin with the goal to associate their outcome with the regular semen analysis [72, 123–128]. Different results were obtained in these studies and there is still controversy on whether, which is the best approach to evaluate human sperm chromatin. A multi-assay approach appears to be useful to better characterize the sperm chromatin structure in a given semen sample than the use of an individual test [40, 60].

Sperm chromatin and cancer treatments

There is growing evidence that support the detrimental effects that chemo- or radio-therapy produce in male reproduction and, particularly, in sperm chromatin structure [40, 58–61, 129–131]. The oncology field has been advanced with the design of chemotherapeutic agents and new drug combinations that target cancerous cells with minimal toxic effects on normal cells; although there is a high survival rates over 5-years (80–96% of cases) in young adults for some malignancies such as testicular cancer and Hodgkin’s lymphoma [132–135], cancer survivors must suffer another burden which is the possibility of facing infertility [64, 134, 136, 137]. Moreover, depending of the type of cancer and treatment, childhood cancer survivors may have severe oligozoospermia to azoospermia in adult life [138].

As mentioned above, chemotherapeutic agents promote a variety of damage to sperm chromatin; depending on the cancer treatment, differential changes in components of the sperm chromatin structure can be documented. This damage is translated to impairment of DNA integrity and compaction [40, 59, 60]. It is important to mention that there is individual variability among patients in terms on how fast the chromatin integrity will be restored. For instance, patients with advanced testicular cancer or Hodgkin’s lymphoma showed high sperm DNA damage, determined by the alkaline comet assay, over a period of two years after the end of chemotherapy [59]. Concurrently, levels of HDS (SCSA^®) were higher in cancer patients compared to those from healthy donors, suggesting that poor DNA compaction was still present in sperm from cancer patients during this period of time [60]. It is possible that insufficient Zn²⁺ bridges were formed to stabilize the sperm chromatin [28] and thus, making this structure still susceptible to acid denaturation in spermatozoa from cancer patients. However, the level of protamination (as measured by CMA3 labeling) and of free -SH in sperm from cancer patients were similar to those from sperm from healthy donors at 18 months after chemotherapy [60]. Overall, these data indicate that some components of the sperm chromatin are repaired over time after chemotherapy; however, there is still significant sperm DNA damage and low compaction even in normozoospermic samples from cancer survivors. These findings stress the need to use a complementary approach to evaluate sperm chromatin quality; only one assay may not be sufficient to determine whether spermatozoa from cancer survivors have good chromatin quality [40, 60].

Sperm chromatin and environmental toxicants

Rising evidence supports the hypothesis that the exposure to environmental toxicants including pesticides and air pollution (products from motor combustion and waste incineration) is a cause of low sperm chromatin quality [139, 140]. It is worrisome the non-occupational exposure to pesticides or their metabolites present in the environment such as 3-phenoxybenzoic acid (3-PBA), the pyrethroid metabolite with the highest detected rate in the general population [141–144]. High levels of sperm DNA fragmentation were associated with high levels of urinary 3-PBA in infertile men [145]. Similar associations were found also with other metabolites of the pesticide chlorpyrifos present in urine of infertile men [146].

Animal studies suggest that pesticides affect sperm chromatin structure promoting a variety of alterations; diazinon, an organophosphorous pesticide (OP), promotes increased DFI (sperm DNA fragmentation) and of CMA3 labeling (low protamination) values along with phopshorylation of protamines in spermatozoa recovered from treated males mice after 8 days of the end of the treatment [147]. This study suggests that late spermatids are affected by diazinon resulting in alteration of sperm chromatin including increased DNA decondensation, low protamination and DNA damage. Similar toxic effects were observed in humans; spermatozoa treated with different OPs showed increased levels of DNA fragmentation [148]. It is noteworthy that the toxicity of OPs is not equal among them and, in some cases; the oxon metabolite is 10 times more toxic than the original compound [148].

Air pollutants are associated with low sperm chromatin quality at a level that can be associated with men infertility [139]. In this study, men exposed to high levels of air pollution have normal semen analysis. This evidence, along with other studies in different cohort of men (i.e. infertile men, cancer patients) showing low or no correlation between sperm chromatin assays and semen analysis [40, 122, 126–128, 139], supports the need for a complementary analysis of the sperm chromatin quality to better characterize the health of human spermatozoa.

Toxicants that are closely looking, nowadays, as potential detrimental compounds for male reproduction are plasticizers and bisphenols, compounds used in the plastic industry. Since the discovery of the properties of these compounds, humans are more exposed to those compounds that can leak from the plastic containers into the food, water and other liquids. Urinary levels of different phthalates (plasticizers) metabolites suggest that exposure to these compounds is much higher and common that previously suspected [149] and they might be associated with sperm abnormalities including poor chromatin quality [150]. Although other studies suggest low or no association between phthalates metabolites and impairment of sperm quality in humans, there is a need for further research with appropriate epidemiological studies [151] to rule out the possibility that these compounds are responsible for alterations of sperm chromatin quality and other sperm parameters that can explain cases of men infertility.

CONCLUSIONS AND FUTURE DIRECTIONS

Based on basic and clinical studies, it is now evident that human sperm chromatin can be altered by different agents or conditions some of them still difficult to control. This damaged chromatin may impact negatively on the development of the embryo promoting miscarriages. Therefore, it is necessary the development of tools to better analyze the sperm chromatin quality to assure that the paternal genome is not altered, especially at the time of selecting sperm for artificial reproductive technologies (ARTs). Although there are many tests to evaluate individual aspects of the sperm chromatin, there is still a limitation to characterize this structure in a more global approach. The combination of two or more assays will allow overcoming this difficulty. More research is needed to improve the tools that we have today to evaluate this vital element of the spermatozoon. Moreover, large-scale epidemiological studies are necessary to help to understand the extent of the exposure to drugs and environmental toxicants. The combination of the knowledge harvested by basic and clinical research and the data generated by the epidemiological studies will serve to design better strategies to detect and possibly isolate those sperm samples carrying significant sperm chromatin damage in order to obtain a semen sample safe to be use in ARTs.

Fig. 8.2.

SCSA scatterplot.