Mark A. Baker

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.

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.

Oxidative stress in the male germ line and its role in the aetiology of male infertility and genetic disease

The human male is characterized by extremely poor semen quality as reflected in the number, morphology and motility of the spermatozoa and a high incidence of nuclear and mitochondrial DNA damage. As a consequence of these factors, defective sperm function is thought to be a major contributor to the aetiology of human infertility, as well as childhood diseases including dominant genetic mutations such as achondroplasia and cancer. Factors associated with the origin of poor semen quality include: (i) a lack of selection pressure for high fecundity genes in developed countries, (ii) an evolutionary lineage associated with the deterioration of several male fertility genes in humans and their close ancestors, (iii) genetic factors including, but not limited to, Y-chromosome deletions (iv) paternal age and (v) environmental factors. A model is proposed whereby factors such as ageing or environmental toxicants initiate DNA strand breakage in the spermatozoa of affected males, eventually leading to a mutation in the embryo. This hypothesis stresses the importance of discovering the identity of those environmental factors that are capable of damaging DNA integrity in the male germ line. Such information could make an important contribution to understanding of the origins of both male infertility and a variety of pathological conditions that affect humans, including cancer and dominant genetic disease.

Oxidative stress and male reproductive health

One of the major causes of defective sperm function is oxidative stress, which not only disrupts the integrity of sperm DNA but also limits the fertilizing potential of these cells as a result of collateral damage to proteins and lipids in the sperm plasma membrane. The origins of such oxidative stress appear to involve the sperm mitochondria, which have a tendency to generate high levels of superoxide anion as a prelude to entering the intrinsic apoptotic cascade. Unfortunately, these cells have very little capacity to respond to such an attack because they only possess the first enzyme in the base excision repair (BER) pathway, 8-oxoguanine glycosylase 1 (OGG1). The latter successfully creates an abasic site, but the spermatozoa cannot process the oxidative lesion further because they lack the downstream proteins (APE1, XRCC1) needed to complete the repair process. It is the responsibility of the oocyte to continue the BER pathway prior to initiation of S-phase of the first mitotic division. If a mistake is made by the oocyte at this stage of development, a mutation will be created that will be represented in every cell in the body. Such mechanisms may explain the increase in childhood cancers and other diseases observed in the offspring of males who have suffered oxidative stress in their germ line as a consequence of age, environmental or lifestyle factors. The high prevalence of oxidative DNA damage in the spermatozoa of male infertility patients may have implications for the health of children conceived in vitro and serves as a driver for current research into the origins of free radical generation in the germ line.

XPS investigation of preferential sputtering of S from MoS2 and determination of MoSx stoichiometry from Mo and S peak positions

Abstract The preferential sputtering of S from bulk MoS2 standard samples exposed to 3 keV Ar+ ion bombardment has been studied by XPS. The MoSx stoichiometry decreases from MoS2 to MoS1.12 with a concomitant reduction in the Mo 3d5/2 binding energy from 229.25 to 228.35 eV. The altered layer extends to a depth of 3.8 nm and is proposed to consist of a single amorphous MoSx phase in which Mo has a varying number of nearest neighbour S atoms. Using peak positions alone it is possible to determine the MoSx stoichiometry to an accuracy of x±0.1 from a plot of MoSx stoichiometry against (Mo 3d5/2–S 2p3/2) binding energy. The results are of strong current interest for coating analysis applications as MoS2 is a compound capable of providing low friction properties when incorporated into hard coatings.

Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility

Human spermatozoa generate low levels of reactive oxygen species in order to stimulate key events, such as tyrosine phosphorylation, associated with sperm capacitation. However, if the generation of these potentially pernicious oxygen metabolites becomes elevated for any reason, spermatozoa possess a limited capacity to protect themselves from oxidative stress. As a consequence, exposure of human spermatozoa to intrinsically- or extrinsically- generated reactive oxygen intermediates can result in a state of oxidative stress characterized by peroxidative damage to the sperm plasma membrane and DNA damage to the mitochondrial and nuclear genomes. Oxidative stress in the male germ line is associated with poor fertilization rates, impaired embryonic development, high levels of abortion and increased morbidity in the offspring, including childhood cancer. In this review, we consider the possible origins of oxidative damage to human spermatozoa and reflect on the important contribution such stress might make to the origins of genetic disease in our species.

Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa

Fertilization of the mammalian oocyte depends on the ability of spermatozoa to undergo a process known as capacitation as they ascend the female reproductive tract. A fundamental feature of this process is a marked increase in tyrosine phosphorylation by an unusual protein kinase A (PKA)-mediated pathway. To date, the identity of the intermediate PKA-activated tyrosine kinase driving capacitation is still unresolved. In this study, we have identified SRC as a candidate intermediate kinase centrally involved in the control of sperm capacitation. Consistent with this conclusion, the SRC kinase inhibitor SU6656 was shown to suppress both tyrosine phosphorylation and hyperactivation in murine spermatozoa. Moreover, SRC co-immunoprecipitated with PKA and this interaction was found to lead to an activating phosphorylation of SRC at position Y416. We have also used difference-in-2D-gel-electrophoresis (DIGE) in combination with mass spectrometry to identify a number of SRC substrates that become phosphorylated during capacitation including enolase, HSP90 and tubulin. Our data further suggest that the activation of SRC during capacitation is negatively controlled by C-terminal SRC kinase. The latter was localized to the acrosome and flagellum of murine spermatozoa by immunocytochemistry, whereas capacitation was associated with an inactivating serine phosphosphorylation of this inhibitory kinase.

Identification of gene products present in Triton X-100 soluble and insoluble fractions of human spermatozoa lysates using LC-MS/MS analysis

A comprehensive analysis of the proteins found in human spermatozoa is essential for understanding the events leading up to, and including, fertilization and development. Proteomics offers a platform for investigating this process, provided that the dynamic range is relatively low. In this report, spermatozoa from a number of human sperm ejaculates were isolated in a pure state using discontinuous Percoll gradient centrifugation. Triton X‐100 soluble and insoluble proteins were recovered and separated by SDS‐PAGE. The separation lanes were dissected into 96 fractions and analyzed individually by LC‐MSn. A comprehensive protocol, involving LC‐MS/MS analysis eventually down to the ninth most intense peak found in the MS‐survey scan, was performed. Analysis of purified human sperm populations resulted in the identification of 1056 gene products, of which approximately 8% have not previously been characterized. The data were supported by the large number of proteins represented by expressed sequence tags in the testis. Bioinformatic analysis demonstrated that 437 of the gene products were involved in various metabolic pathways including glycolysis and oxidative phosphorylation. The inventory of proteins present in the human sperm proteome includes a number of notable discoveries including the first description of a nicotinamide adenine dinucleotide phosphate oxidase, dual‐oxidase 2, finally laying to rest any doubts about the presence of such enzymes in spermatozoa. Furthermore, a number of different classes of receptor have also been detected in these cells and are potential regulators of sperm function. This list includes at least six seven‐pass transmembrane receptors, six tyrosine kinase receptors, a tyrosine phosphatase receptor, glutamate‐gated ion channel receptors, transient receptor potential cation channels, and a non‐genomic progesterone receptor. This is the first published list of identified proteins in human spermatozoa using LC‐MS/MS analysis.

The importance of redox regulated pathways in sperm cell biology

Redox regulated events are fundamental to our understanding of many cellular pathways and pathological processes. On the one hand, production of reactive oxygen species by mammalian spermatozoa has been associated with a loss of cell function and DNA integrity as a consequence of oxidative stress. These cells are exquisitely sensitive to such damage as a consequence of their relative lack of cytosolic antioxidant enzymes and relative abundance of polyunsaturated fatty acids. Given this susceptibility, it is surprising to discover that spermatozoa are intensely redox active cells and professional generators of reactive oxygen species. The latter are physiologically important to the spermatozoa in regulating every aspect of sperm function examined, including their movement characteristics, capacitation, sperm-zona interaction, the acrosome reaction and sperm-oocyte fusion. The molecular basis of this redox drive is still poorly understood in terms of the source of the reactive oxygen species and the mechanisms by which these reactive metabolites enhance sperm function. Recent advances include the discovery of NOX5 in the male germ line and elucidation of the role of reactive oxygen species in controlling a unique signal transduction cascade associated with sperm capacitation. Given the central importance of redox chemistry in the control of sperm function further research in this area may uncover valuable targets for contraceptive intervention.

The mouse sperm proteome characterized via IPG strip prefractionation and LC-MS/MS identification

Proteomic profiling of the mouse spermatozoon has generated a unique and valuable inventory of candidates that can be mined for potential contraceptive targets and to further our understanding of the PTMs that regulate the functionality of this highly specialized cell. Here we report the identification of 858 proteins derived from mouse spermatozoa, 23 of which demonstrated testis only expression. The list contained many proteins that are known constituents of murine spermatozoa including Izumo, Spaca 1, 3, and 5, Spam 1, Zonadhesin, Spesp1, Smcp, Spata 6, 18, and 19, Zp3r, Zpbp 1 and 2, Spa17, Spag 6, 16, and 17, CatSper4, Acr, Cylc2, Odf1 and 2, Acrbp, and Acrv1. Certain protein families were highly represented in the proteome. For example, of the 42 gene products classified as proteases, 26 belonged to the 26S‐proteasome. Of the many chaperones identified in this proteome, eight proteins with a TCP‐1 domain were found, as were seven Rab guanosine triphosphatases. Finally, our list yielded three putative seven‐transmembrane proteins, two of which have no known tissue distribution, an extragenomic progesterone receptor and three unique testis‐specific kinases all of which may have some potential in the future regulation of male fertility.