R. J. Aitken

Oxidative stress, sperm survival and fertility control.

The human spermatozoon is highly susceptible to oxidative stress. This process induces peroxidative damage in the sperm plasma membrane and DNA fragmentation in both the nuclear and mitochondrial genomes. Such stress may arise from a variety of sources including a lack of antioxidant protection, the presence of redox cycling xenobiotics, infiltrating leukocytes and excess reactive oxygen species production by the spermatozoa. Whenever the levels of oxidative stress in the male germ line are high, the peroxidation of unsaturated fatty acids in the sperm plasma membrane ensures that normal fertilization cannot occur. However, at lower levels of oxidative stress, spermatozoa may retain their capacity for fertilization while carrying significant levels of oxidative damage in their DNA. Epidemiological evidence suggests that subsequent aberrant repair of such damage in the zygote may result in the creation of mutations associated with pre-term pregnancy loss and a variety of pathologies in the offspring, including childhood cancer. Thus, while the induction of oxidative stress in spermatozoa is causally involved in the aetiology of male infertility, the prospects of using such a strategy for male contraception is fraught with potential problems, should the suppression of fertility be incomplete and DNA-damaged spermatozoa gain access to the oocyte.

ANTIOXIDANT STRATEGIES IN THE EPIDIDYMIS

Spermatozoa are very specialized cells, dedicated to fertilization of the oocyte. The attainment of this biological role is partly due to the fusogenic properties of the sperm plasma membrane, which is particularly rich in polyunsaturated fatty acids (PUFA). This predominance of PUFA renders spermatozoa highly susceptible to lipid peroxidation due to attacks from reactive oxygen species (ROS). These attacks ultimately lead to the impairment of sperm function through oxidative stress. Despite such disruptive effects, it should be also emphasized that these molecules also play an important positive, physiological role in the regulation of sperm physiology through their participation in apoptosis and the signal transduction cascades that control sperm maturation and capacitation. In this article, the different sources of ROS are examined and then the antioxidant strategies that protect these cells during epididymal transit are reviewed. While the major focus is on the involvement of glutathione peroxidase in this process, consideration will also be given to a range of additional antioxidant enzymes (catalase, indolamine dioxygenase and superoxide dismutase) that have evolved to protect spermatozoa during this extremely vulnerable phase in their life history. Besides the classical enzymatic roles of these enzymes in recycling ROS, additional features are discussed in the light of contraceptive development.

Oxidative stress and male reproductive biology

Spermatozoa were the first cell type in which the cellular generation of reactive oxygen was demonstrated. This activity has now been confirmed in spermatozoa from all mammalian species examined including the rat, mouse, rabbit, horse, bull and human being. Under physiological circumstances, cellular redox activity is thought to drive the cAMP-mediated, tyrosine phosphorylation events associated with sperm capacitation. In addition to this biological role, human spermatozoa also appear to suffer from oxidative stress, with impacts on the normality of their function and the integrity of their nuclear and mitochondrial DNA. Recent studies have helped to clarify the molecular basis for the intense redox activity observed in defective human spermatozoa, the nature of the subcellular structures responsible for this activity and possible mechanisms by which oxidative stress impacts on these cells. Given the importance of oxidative damage in the male germ line to the origins of male infertility, early pregnancy loss and childhood disease, this area of sperm biochemistry deserves attention from all those interested in improved methods for the diagnosis, management and prevention of male-mediated reproductive failure.

Founders' Lecture. Human spermatozoa: fruits of creation, seeds of doubt.

Deoxyribonucleic acid damage in the male germline is associated with defective fertilisation, impaired embryonic development, reduced implantation, abortion and childhood disease. Oxidative stress and the retention of excess residual cytoplasm by the spermatozoa are frequently associated with the induction of such damage. The redox cycling of xenobiotics by oxido-reductases in the germline, the patients age, the incidence of genital tract infections and Sertoli cell dysfunction are all possible contributors to DNA damage in germ cells. Collateral peroxidation of unsaturated fatty acids in the sperm plasma membrane generally ensures that spermatozoa experiencing severe oxidative DNA damage cannot participate in the process of fertilisation. The adaptive termination of pregnancy through the selective vulnerability of genes involved in placentation may also help prevent the vertical transmission of damaged DNA. However, the ultimate safeguard against this form of damage will be to understand the biochemical basis of oxidative stress in human spermatozoa, so that the underlying causative mechanisms can be addressed in a logical manner.

PROTEOMIC INSIGHTS INTO SPERMATOZOA: CRITIQUES, COMMENTS AND CONCERNS

Understanding the cellular mechanisms that regulate mammalian sperm function is strategically important for both the management of male infertility and the development of novel approaches to male contraception. The spermatozoon is a transcriptionally and translationally suppressed cell that is released from the testes in a functionally inert state. Functional activation occurs in the epididymis and female tract via mechanisms that are entirely dependent on post-translational modifications. Proteomics is, therefore, the ideal technology to investigate this cell type. Herein, we comment on the proteomic analyses that have been applied to mammalian spermatozoa, including some concerns relating to data interpretation. Three comprehensive liquid chromatography–mass spectrometry lists of human, mouse and rat spermatozoa are then compared, insights into the molecular regulation of sperm function discussed and future directions speculated upon.

Cellular mechanisms regulating sperm–zona pellucida interaction

For mammalian spermatozoa to exhibit the ability to bind the zona pellucida (ZP) they must undergo three distinct phases of maturation, namely, spermatogenesis (testis), epididymal maturation (epididymis) and capacitation (female reproductive tract). An impressive array of spermatozoa surface remodeling events accompany these phases of maturation and appear critical for recognition and adhesion of the outer vestments of the oocyte, a structure known as the ZP. It is becoming increasingly apparent that species-specific zona adhesion is not mediated by a single receptor. Instead, compelling evidence now points toward models implicating a multiplicity of receptor-ligand interactions. This notion is in keeping with emerging research that has shown that there is a dynamic aggregation of proteins believed to be important in sperm-ZP recognition to the regions of sperm that mediate this binding event. Such remodeling may in turn facilitate the assembly of a multimeric zona recognition complex (MZRC). Though formation of MZRCs raises questions regarding the nature of the block to polyspermy, formation and assembly of such a structure would no doubt explain the strenuous maturation process that sperm endure on their sojourn to functional maturity.

On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid

Oxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5‐diaminofluorescein diacetate and 2′,7′‐dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2‐hydroxyestradiol, menadione, 4‐hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol‐peroxidase and, to a lesser extent, 2′,7′‐dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function.

The Simmet Lecture: New Horizons on an Old Landscape – Oxidative Stress, DNA Damage and Apoptosis in the Male Germ Line

Our ability to diagnose and treat male infertility is gradually improving in concert with advances in our understanding of the molecular mechanisms underpinning defective sperm function. In this context, one of the factors to emerge as a major causative agent in male infertility is oxidative stress. Spermatozoa are particularly susceptible to such stress because they are exceptionally rich in vulnerable substrates such as polyunsaturated fatty acids, proteins and DNA. The lack of sperm cytoplasm also provides these cells with little capacity to protect themselves from oxidative attack or to effect any repair, should damage occur. Similarly, sperm chromatin is in a quasi-crystalline state and has very little capacity to respond to any DNA damage induced by oxidative attack. When the latter does occur, it appears to be initiated by reactive oxygen species (ROS) generated by the sperm mitochondria. These free radicals attack the lipids present in the sperm mitochondria generating electrophilic aldehydes, which bind to components of the mitochondrial electron transport chain stimulating yet more ROS production. The oxidative stress created via this self-propagating mechanism initiates an apoptotic cascade as a result of which the spermatozoa loose their capacity for fertilization and suffer damage to their DNA. Phosphatidylserine externalization is a late event in sperm apoptosis and may facilitate the silent phagocytosis of moribund cells in the female reproductive tract, that is, the phagocytosis of senescent spermatozoa without the accompanying generation of an inflammatory response. Encouragingly, the involvement of oxidative stress in the aetiology of male infertility has opened up new opportunities for therapeutic interventions involving the judicious administration of nucleophiles and other forms of antioxidants.

Seminal fluid analysis and sperm function testing

The diagnosis of male infertility is a rapidly developing field of investigation. The traditional descriptive approach to semen analysis is still at the heart of the diagnostic workup of male patients and important advances have been made in the standardization of the procedures used to construct the conventional semen profile, as embodied in the WHO handbook. Significant improvements have also been made in the techniques used to assess the quality of sperm motility. While this was once an entirely subjective exercise, the introduction of CASA systems to take objective measurements of the trajectories of human spermatozoa has revolutionized this form of analysis. The speed and accuracy of such systems has permitted detailed analyses of the relationships between sperm movement and sperm function, which has greatly enhanced the diagnostic power of this form of descriptive analysis. Notwithstanding the importance of the descriptive approach to semen analysis, it has also been recognized that to achieve an accurate diagnosis of male infertility such criteria should be supplemented with assays designed to reveal the functional competence of the spermatozoa. Bioassays have therefore been developed to measure such functions as the penetration of cervical mucus, sperm-zona interaction, the acrosome reaction, and sperm-oocyte fusion. All of these assays have been shown to generate information predictive of the fertilizing potential of human spermatozoa in vivo and in vitro. Despite their diagnostic value, the time, expense, and expertise required to run such functional assays has meant that they have not been widely used by infertility specialists. These functional assays are of value, however, in fundamental studies designed to elucidate the biochemical basis of defective sperm function. The first fruits of this research effort are now beginning to appear in the identification of a number of cytoplasmic markers for defective sperm function and the realization that lipid peroxidation plays a key role in the etiology of male infertility. In the wake of these fundamental studies will flow a new generation of biochemical tests for the diagnosis of defective sperm function and, it is hoped, the development of rational therapies with which to treat this condition.

Potential importance of transition metals in the induction of DNA damage by sperm preparation media

STUDY QUESTION What are the mechanisms by which the preparation of spermatozoa on discontinuous density gradients leads to an increase in oxidative DNA damage? SUMMARY ANSWER The colloidal silicon solutions that are commonly used to prepare human spermatozoa for assisted reproduction technology (ART) purposes contain metals in concentrations that promote free radical-mediated DNA damage. WHAT IS KNOWN ALREADY Sporadic reports have already appeared indicating that the use of colloidal silicon-based discontinuous density gradients for sperm preparation is occasionally associated with the induction of oxidative DNA damage. The cause of this damage is however unknown. STUDY DESIGN, SIZE, DURATION This study comprised a series of experiments designed to: (i) confirm the induction of oxidative DNA damage in spermatozoa prepared on commercially available colloidal silicon gradients, (ii) compare the levels of damage observed with alterative sperm preparation techniques including an electrophoretic approach and (iii) determine the cause of the oxidative DNA damage and develop strategies for its prevention. The semen samples employed for this analysis involved a cohort of >50 unselected donors and at least three independent samples were used for each component of the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS The setting was a University biomedical science laboratory. The major techniques employed were: (i) flow cytometry to study reactive oxygen species generation, lipid peroxidation and DNA damage, (ii) computer-aided sperm analysis to measure sperm movement and (iii) inductively coupled mass spectrometry to determine the elemental composition of sperm preparation media. MAIN RESULTS AND THE ROLE OF CHANCE Oxidative DNA damage is induced in spermatozoa prepared on PureSperm(®) discontinuous colloidal silicon gradients (P < 0.001 versus repeated centrifugation) because this medium contains metals, particularly Fe, Al and Cu, which are known to promote free radical generation in the immediate vicinity of DNA. This damage can be significantly accentuated by reducing agents, such as ascorbate (P < 0.001) and inhibited by selective chelation (P < 0.001). This problem is not confined to PureSperm(®); analysis of additional commercial sperm preparation media revealed that metal contamination is a relatively constant feature of such products. LIMITATIONS, REASONS FOR CAUTION While the presence of metals, particularly transition metals, may exacerbate the levels of oxidative DNA damage seen in human spermatozoa, the significance of such damage has not yet been tested in suitably powered clinical trials. WIDER IMPLICATIONS OF THE FINDINGS The results explain why the preparation of spermatozoa on discontinuous colloidal silicon gradients can result in oxidative DNA damage. The results are of immediate relevance to the development of safe, effective protocols for the preparation of spermatozoa for ART purposes. STUDY FUNDING/COMPETING INTERESTS The study was funded by the Australian Health and Medical Research Council. One of the authors (R.J.A.) has had a consultantship with a biotechnology company, NuSep, interested in the development of electrophoretic methods of sperm preparation. He has no current financial interest in this area. None of the other authors have a conflict of interest to declare.