Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function.

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.

Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa.

Human spermatozoa are characterized by poor functionality and abundant DNA damage that collude to generate the high incidences of male infertility and miscarriage seen in our species. Although apoptosis has been suggested as a possible cause of poor sperm quality, the ability of these cells to enter an apoptotic state and the factors that might trigger such an event are unresolved. In the present study we provide evidence that the commitment of these cells to apoptosis is negatively regulated by PI3K (phosphoinositide 3-kinase)/AKT. If PI3K activity is inhibited, then spermatozoa default to an apoptotic cascade characterized by rapid motility loss, mitochondrial reactive oxygen species generation, caspase activation in the cytosol, annexin V binding to the cell surface, cytoplasmic vacuolization and oxidative DNA damage. However, the specialized physical architecture of spermatozoa subsequently prevents endonucleases activated during this process from penetrating the sperm nucleus and cleaving the DNA. As a result, DNA fragmentation does not occur as a direct result of apoptosis in spermatozoa as it does in somatic cells, even though oxidative DNA adducts can clearly be detected. We propose that this unusual truncated apoptotic cascade prepares spermatozoa for silent phagocytosis within the female tract and prevents DNA-damaged spermatozoa from participating in fertilization.

DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress.

DNA damage in human spermatozoa has been associated with a range of adverse clinical outcomes, including infertility, abortion, and disease in the offspring. We have advanced a two-step hypothesis to explain this damage involving impaired chromatin remodeling during spermiogenesis followed by a free radical attack to induce DNA strand breakage. The objective of the present study was to test this hypothesis by determining whether impaired chromatin protamination is correlated with oxidative base damage and DNA fragmentation in human spermatozoa. DNA fragmentation, chromatin protamination, mitochondrial membrane potential, and formation of the oxidative base adduct, 8-hydroxy-2'-deoxyguanosine (8OHdG), were monitored by flow cytometry/fluorescence microscopy. Impairment of DNA protamination during late spermatogenesis was highly correlated (P < 0.001) with DNA damage in human spermatozoa. The disruption of chromatin remodeling also was associated with a significant elevation in the levels of 8OHdG (P < 0.001), and the latter was itself highly correlated with DNA fragmentation (P < 0.001). The significance of oxidative stress in 8OHdG formation was demonstrated experimentally using H2O2/Fe2+ and by the correlation observed between this base adduct and superoxide generation (P < 0.001). That 8OHdG formation was inversely associated with mitochondrial membrane potential (P < 0.001) suggested a possible role for these organelles in the creation of oxidative stress. These results clearly highlight the importance of oxidative stress in the induction of sperm DNA damage and carry significant implications for the clinical management of this condition.

Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa.

CONTEXT: Male infertility has been linked with the excessive generation of reactive oxygen species (ROS) by defective spermatozoa. However, the subcellular origins of this activity are unclear. OBJECTIVE: The objective of this study was to determine the importance of sperm mitochondria in creating the oxidative stress associated with defective sperm function. METHOD: Intracellular measurement of mitochondrial ROS generation and lipid peroxidation was performed using the fluorescent probes MitoSOX red and BODIPY C(11) in conjunction with flow cytometry. Effects on sperm movement were measured by computer-assisted sperm analysis. RESULTS: Disruption of mitochondrial electron transport flow in human spermatozoa resulted in generation of ROS from complex I (rotenone sensitive) or III (myxothiazol, antimycin A sensitive) via mechanisms that were independent of mitochondrial membrane potential. Activation of ROS generation at complex III led to the rapid release of hydrogen peroxide into the extracellular space, but no detectable peroxidative damage. Conversely, the induction of ROS on the matrix side of the inner mitochondrial membrane at complex I resulted in peroxidative damage to the midpiece and a loss of sperm movement that could be prevented by the concomitant presence of alpha-tocopherol. Defective human spermatozoa spontaneously generated mitochondrial ROS in a manner that was negatively correlated with motility. Simultaneous measurement of general cellular ROS generation with dihydroethidium indicated that 68% of the variability in such measurements could be explained by differences in mitochondrial ROS production. CONCLUSION: We conclude that the sperm mitochondria make a significant contribution to the oxidative stress experienced by defective human spermatozoa.

Proteomic changes in mammalian spermatozoa during epididymal maturation.

Epididymal maturation is associated with the activation of a cAMP-induced tyrosine phosphorylation cascade, which is ultimately associated with the expression of capacitation-dependent sperm functions, such as hyperactivated movement and acrosomal exocytosis. As spermatozoa progress through the epididymis they first acquire the capacity to phosphorylate tyrosine on targets on the principal piece, followed by the midpiece. By the time these cells have reached the cauda epididymidis they can phosphorylate the entire tail from neck to endpiece. This particular pattern of phosphorylation is associated with the ontogeny of fully functional spermatozoa that are capable of fertilizing the oocyte. Proteomic analyses indicate that this change is associated with the phosphorylation of several mitochondrial proteins, creation of a mitochondrial membrane potential and activation of mitochondrial free radical generation. At least in rodent species, activation of sperm mitochondria appears to be a particularly important part of epididymal maturation.

Cis-unsaturated fatty acids stimulate reactive oxygen species generation and lipid peroxidation in human spermatozoa.

CONTEXT: Defective sperm function is the largest defined cause of human infertility; however, the etiology of this condition is poorly understood. Although oxidative stress is acknowledged as a key contributor to this pathology, there are also data indicating that defective human spermatozoa contain abnormally high amounts of cis-unsaturated fatty acids. This study investigated whether a causative relationship exists between these two attributes of impaired semen quality. OBJECTIVE: The objective of this study was to determine whether polyunsaturated fatty acids can induce oxidative stress in human spermatozoa. METHOD: Dihydroethidium and SYTOX Green were used in conjunction with flow cytometry and HPLC to investigate reactive oxygen species (ROS) generation by human spermatozoa after fatty acid exposure. RESULTS: Arachidonic acid (AA) induced a time- and dose-dependent increase in ROS generation by human spermatozoa that led to the promotion of peroxidative damage and a loss of sperm motility. This effect could not be blocked with inhibitors of the cyclooxygenase or lipoxygenase pathways of AA metabolism, rotenone, protein kinase C antagonists, or known inhibitors of plasma membrane redox systems. However, ROS generation could be triggered with other cis-unsaturated fatty acids including linoleic and docosahexaenoic acids. Saturated fatty acids, methyl esters of unsaturated fatty acids, or other amphiphiles were all ineffective. However in a cell-free system, AA could trigger a redox signal via mechanisms that were profoundly disrupted by diphenylene iodonium, a flavoprotein inhibitor. CONCLUSIONS: The presence of excess unsaturated fatty acids in defective human spermatozoa may precipitate the oxidative stress encountered in male infertility.

Definitive evidence for the nonmitochondrial production of superoxide anion by human spermatozoa.

CONTEXT: Oxidative stress in the male germ line has been associated with poor fertility, impaired embryonic development, miscarriage, and childhood disease. Such stress is known to be associated with the peroxidation of unsaturated fatty acids in the sperm plasma membrane and oxidative DNA damage to both the nuclear and mitochondrial genomes. However, the source of the free radicals responsible for such damage is still unresolved. OBJECTIVE: The objective of this study was to chemically validate the use of dihydroethidium (DHE) as a probe for detecting the generation of superoxide anion by human spermatozoa and to examine the relationship between this activity and defective sperm function. METHOD: DHE and SYTOX green were used in conjunction with flow cytometry and HPLC to investigate superoxide generation by human spermatozoa. Cause and effect relationships were established using menadione to artificially drive superoxide production by these cells. RESULTS: HPLC, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and spectrofluorometry were used to demonstrate that human spermatozoa generate the superoxide-specific product, 2-hydroxyethidium, from DHE. Spontaneous superoxide production by human spermatozoa was found to originate from a nonmitochondrial source and was inversely correlated with sperm motility. A causative relationship between superoxide generation and sperm function was demonstrated when the pharmacological stimulation of this activity with menadione was shown to result in both severe motility loss and DNA damage. CONCLUSIONS: These studies validate a methodology for investigating the origins of oxidative stress in the male germ line and demonstrate, for the first time, the significance of superoxide generation by human spermatozoa in the etiology of this condition.

Apoptosis and DNA damage in human spermatozoa.

DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis, resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apoptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.

The role of cysteine-rich secretory proteins in male fertility.

The cysteine-rich secretory proteins (CRISPs) are a subgroup of the CRISP, antigen 5 and Pr-1 (CAP) protein superfamily, and are found only in vertebrates. They show a strong expression bias to the mammalian male reproductive tract and the venom of poisonous reptiles. Within the male reproductive tract CRISPs have been implicated in many aspects of male germ cell biology spanning haploid germ cell development, epididymal maturation, capacitation, motility and the actual processes of fertilization. At a structural level, CRISPs are composed of two domains, a CAP domain, which has been implicated in cell-cell adhesion, and a CRISP domain, which has been shown to regulate several classes of ion channels across multiple species. Herein, we will review the current literature on the role of CRISPs in male fertility, and by inference to related non-mammalian protein, infer potential biochemical functions.

Stimulation of mitochondrial reactive oxygen species production by unesterified, unsaturated fatty acids in defective human spermatozoa.

Male infertility is a relatively common condition affecting 1 in 20 men of reproductive age. The etiology of this condition is thought to involve the excessive generation of reactive oxygen species by human spermatozoa; however, the cause of this aberrant activity is unknown. In this study we demonstrate that defective human sperm populations are characterized by high cellular contents of both esterified and unesterified fatty acids and a decrease in the proportion of the total fatty acid pool made up by docosahexaenoic acid. The free unsaturated fatty acid content of these cells was positively correlated with the induction of mitochondrial superoxide generation (P<0.001). This relationship was causal and mediated by the range of unesterified, unsaturated fatty acids that are present in human spermatozoa. Thus direct exposure of these cells to free unsaturated fatty acids stimulated mitochondrial superoxide generation and precipitated a loss of motility and an increase in oxidative DNA damage, two key attributes of male infertility. We conclude that defective human spermatozoa are characterized by an abnormally high content of fatty acids that, in their unesterified, unsaturated form, promote ROS generation by sperm mitochondria, creating a state of oxidative stress and a concomitant loss of functional competence.