Benjamin J. Curry

Redox Regulation of Human Sperm Function: From the Physiological Control of Sperm Capacitation to the Etiology of Infertility and DNA Damage in the Germ Line

Defective sperm function is the largest single defined cause of human infertility and one of the major reasons we are witnessing an exponential increase in the uptake of assisted conception therapy in the developed world. A major characteristic of defective human spermatozoa is the presence of large amounts of DNA damage, which is, in turn, associated with reduced fertility, increased rates of miscarriage, and an enhanced risk of disease in the offspring. This DNA damage is largely oxidative and is closely associated with defects in spermiogenesis. To explain the origins of this DNA damage, we postulate that spermiogenesis is disrupted by oxidative stress, leading to the creation of defective gametes with poorly remodeled chromatin that are particularly susceptible to free radical attack. To compound the problem, these defective cells have a tendency to undergo an unusual truncated form of apoptosis associated with high amounts of superoxide generation by the sperm mitochondria. This leads to significant oxidative DNA damage that eventually culminates in the DNA fragmentation we see in infertile patients. In light of the significance of oxidative stress in the etiology of defective sperm function, a variety of antioxidant therapies are now being assessed for their therapeutic potential.

Polymerase chain reaction detection of melanoma cells in the circulation: relation to clinical stage, surgical treatment, and recurrence from melanoma.

PURPOSE The detection of melanoma cells in the circulation by polymerase chain reaction (PCR) assays has been shown by several investigators to correlate with the stage of the disease and possibly with prognosis. PATIENTS AND METHODS We performed prospective studies on 276 patients with primary melanoma and regional lymph node (LN) metastases to assess the predictive value of PCR detection of tyrosinase and melanoma antigen recognized by T cells-1 (MART-1) in the blood for recurrence of melanoma. RESULTS PCR tests for gp 100, Muc-18, and p97 reacted with RNA in blood from healthy subjects and were considered unsuitable for patient monitoring. The tests were most frequently positive in the first 3 months after surgery. There were 47 recurrences in 123 patients who had been followed up for 18 months. Assays within 3 months of surgery predicted recurrence from melanoma in 66% of the latter (tests for tyrosinase alone detected 51% and MART-1 alone 21% of the patients). Hence, 34% of recurrences were not predicted by tests in the early postoperative period. This did not appear to be because of marker-negative melanoma because summation of tests over the first year identified 89% of those with recurrent disease. CONCLUSION Positive tests were recorded in 35% of patients who remained disease free, but it is too early to assess whether these represent false-positive results. The false-negative results raise the question of whether the assays will provide a reliable basis for selection of patients for adjuvant therapy.

MART-1 Is Expressed Less Frequently on Circulating Melanoma Cells in Patients Who Develop Distant Compared With Locoregional Metastases

PURPOSE Polymerase chain reaction (PCR) with tyrosinase and with MART-1 permits detection of small numbers of circulating melanoma cells (CMCs) in patients who have undergone surgical resection of localized disease. In a previous study, we showed that PCR with MART-1 had sensitivity and specificity similar to those of PCR with tyrosinase in terms of detection of CMCs but that PCR with MART-1 seemed to identify a different but overlapping subgroup of patients. In the current study, we examined the utility and prognostic significance of PCR with tyrosinase and with MART-1. PATIENTS AND METHODS We analyzed the prognostic significance of the patterns of expression of tyrosinase and MART-1 in 186 patients followed sequentially before and after surgical removal of American Joint Committee on Cancer stage I, II, or III melanoma. RESULTS PCR with tyrosinase and with MART-1 in the first 3 months after surgery identified 68.5% of 73 patients who developed recurrence in the 2-year period after surgery. Approximately 35% of patients with positive tests remained disease-free at 2-year follow-up. We found that patients with disseminated recurrence had a significantly lower incidence of MART-1-positive CMCs (16%) than of tyrosinase-positive CMCs (63%). Patients with locoregional metastases had CMCs that expressed tyrosinase and MART-1 at similar rates. These differences in expression of the markers in patients with disseminated recurrence were also associated with a much lower disease-free survival, in those who had CMCs that were positive for tyrosinase but negative for MART-1. The reverse applied in those with locoregional disease. CONCLUSION These findings suggest that PCR with MART-1 and with tyrosinase identifies subgroups of patients who develop disseminated or locally recurrent metastases. We hypothesize that immune responses against MART-1 may reduce the establishment of disseminated metastases.

Prolactin Exerts a Prosurvival Effect on Human Spermatozoa via Mechanisms that Involve the Stimulation of Akt Phosphorylation and Suppression of Caspase Activation and Capacitation

The purpose of this study was to examine the impact of prolactin (PRL) on human sperm function, in light of a recent proteomic analysis indicating that these cells express the PRL receptor (PRLR). Immunocytochemical analyses confirmed the presence of PRLR in human spermatozoa and localized this receptor to the postacrosomal region of the sperm head as well as the neck, midpiece, and principal piece of the sperm tail. Nested PCR analysis indicated that these cells possess four splice variants of the PRLR: the long form and three short isoforms, one of which is reported for the first time. A combination of Western blot analyses and immunocytochemistry demonstrated that PRL inhibited sperm capacitation in a dose-dependent manner, suppressing SRC kinase activation and phosphotyrosine expression, two hallmarks of this process. The suppression of sperm capacitation was accompanied by a powerful prosurvival effect, supporting the prolonged motility of these cells and preventing the formation of spontaneous DNA strand breaks via mechanisms that involved the concomitant suppression of caspase activation. Western blot analyses indicated that the prosurvival effect of PRL on human spermatozoa involved the stimulation of Akt phosphorylation, whereas inhibitors of phosphatidylinositol-3-OH kinase and Akt negated this effect, as did the direct induction of sperm capacitation with cAMP analogues. We conclude that PRL is a prosurvival factor for human spermatozoa that prevents these cells from defaulting to an intrinsic apoptotic pathway associated with cell senescence. These findings have implications for preservation of sperm integrity in vivo and in vitro.

Identification of Cytochrome P450-Reductase as the Enzyme Responsible for NADPH-Dependent Lucigenin and Tetrazolium Salt Reduction in Rat Epididymal Sperm Preparations

Abstract Lucigenin-dependent chemiluminescence and WST-1 reduction can be detected following addition of NADPH to many cell types, including rat epididymal sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other probes—such as MCLA and luminol—that are capable of detecting reactive oxygen metabolites do not produce a chemiluminescent signal in this model system. Our aim was to purify and identify the enzyme catalyzing the NADPH-dependent lucigenin and WST-1 reduction from rat epididymal spermatozoa preparations. Here, we show the identity of this enzyme as cytochrome P450-reductase. In support of this, a homogenous preparation of this protein was capable of reducing lucigenin and WST-1 in the presence of NADPH. Moreover, COS-7 cells overexpressing cytochrome P450-reductase displayed a 3-fold increase in the aforementioned activity compared with mock-transfected cells. Immunolocalization studies and biochemical analysis suggest that the majority of the NADPH-lucigenin activity is localized to the epithelial cells present within the epididymis. These results emphasize the importance of the direct NADPH-dependent reduction of superoxide-sensitive probes by cytochrome P450-reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

Phosphorylation and consequent stimulation of the tyrosine kinase c-Abl by PKA in mouse spermatozoa: its implications during capacitation

Upon ejaculation, spermatozoa undergo a series of post-translational modifications in a process known as capacitation in order to prepare for fertilization. In the absence of capacitation, fertilization cannot occur. Spermatozoa are unusual in that one of the hallmarks of capacitation is a global up-regulation in phosphotyrosine expression, which is known to be mediated upstream by PKA. Little is known about the signaling events downstream of PKA apart from the involvement of SRC, as a key mediator of PKA-induced tyrosine phosphorylation in the sperm tail. Here we describe the presence of c-Abl in mouse spermatozoa. In vitro analysis confirmed that PKA can up-regulate c-Abl kinase activity. In vivo, this tyrosine kinase was found to associate, and become threonine phosphorylated by PKA in the sperm flagellum. By treating spermatozoa with hemolysin we could demonstrate that a significant proportion of the tyrosine phosphorylation associated with capacitation could be suppressed by the c-Abl inhibitor, Gleevac. This is the first report of c-Abl being up-regulated by PKA for any cell type. We present a model, whereby these kinases may operate together with SRC to ensure optimal levels of tyrosine phosphorylation in the sperm flagellum during the attainment of a capacitated state.

Identification of Cytochrome-b5 Reductase as the Enzyme Responsible for NADH-Dependent Lucigenin Chemiluminescence in Human Spermatozoa

Abstract Lucigenin-dependent chemiluminescence together with 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) reduction can be detected following addition of NADH to many cell types, including human sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other oxygen detecting metabolite probes, such as MCLA and luminol, do not produce a chemiluminescent signal in this model system. The enzyme responsible for NADH-dependent lucigenin chemiluminescence was purified and identified as cytochrome-b5 reductase. In support of this concept, COS-7 cells overexpressing cytochrome-b5 reductase displayed at least a 3-fold increase in the previously mentioned activity compared with mock-transfected cells. Fractions containing cytochrome-b5 reductase were capable of inducing both lucigenin-dependent chemiluminescence and WST-1 reduction. Oxygen radicals clearly did not mediate the cytochrome b5-mediated activation of these probes in vitro since neither luminol nor MCLA gave a chemiluminescence response in the presence of the enzyme and the cofactor NADH. These results emphasize the importance of the direct NADH-dependent reduction of these putative superoxide-sensitive probes by cytochrome-b5 reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

Investigation of the stallion sperm proteome by mass spectrometry

Stallion spermatozoa continue to present scientific and clinical challenges with regard to the biological mechanisms responsible for their survival and function. In particular, deeper understanding of sperm energy metabolism, defence against oxidative damage and cell-cell interactions should improve fertility assessment and the application of advanced reproductive technologies in the equine species. In this study, we used highly sensitive LC-MS/MS technology and sequence database analysis to identify and characterise the proteome of Percoll-isolated ejaculated equine spermatozoa, with the aim of furthering our understanding of this cells complex biological machinery. We were able to identify 9883 peptides comprising 1030 proteins, which were subsequently attributed to 975 gene products. Gene ontology analysis for molecular and cellular processes revealed new information about the metabolism, antioxidant defences and receptors of stallion spermatozoa. Mitochondrial proteins and those involved in catabolic processes constituted dominant categories. Several enzymes specific to β-oxidation of fatty acids were identified, and further experiments were carried out to ascertain their functional significance. Inhibition of carnitine palmitoyl transferase 1, a rate-limiting enzyme of β-oxidation, reduced motility parameters, indicating that β-oxidation contributes to maintenance of motility in stallion spermatozoa.

Utility of Tests for Circulating Melanoma Cells in Identifying Patients Who Develop Recurrent Melanoma

Prospective studies were carried out on 186 patients with AJCC stage I (13), II (76) and III (97 patients) melanoma before and after surgical removal of their tumour. The goal was to determine if PCR tests on blood samples for MART-1 and tyrosinase were predictive of recurrence of melanoma in a 2-year follow-up period. (PCR assays for MUC-18, p97 and gp100 were positive in blood samples from normal subjects and excluded from the study.) PCR tests for MART-1 and tyrosinase were most commonly positive in the first 3 months following surgical removal of melanoma, and three tests over this period gave maximum sensitivity in the identification of patients who subsequently developed recurrences. Positive tests for the first time in the second year of follow-up had similar predictive power. The tests identified 68.5% of patients who developed recurrences in the 2-year follow-up period. Assays for MART-1 were mainly positive in patients with locoregional recurrences, whereas tyrosinase was detected in blood samples from patients with both locoregional and disseminated recurrences. (Positivity rate for tyrosinase in 48 patients with disseminated melanoma was 60.4% compared to 14.6% for MART-1.) Tests for MART-1 and tyrosinase were strongly predictive of disease-free survival (DFS) and were more powerful predictors of DFS than lymph node status or thickness of the primary melanoma. (Hazard ratios by Cox analysis were 2.97 in patients with disseminated recurrences and 2.93 in those with locoregional recurrences.) These results indicate that PCR tests for MART-1 and tyrosinase are powerful prognostic indicators, but their practical utility for selecting patients for adjuvant therapy is limited by the high false-negative rate of approximately 30%. Intermittent shedding of melanoma cells into the circulation would appear to be the most likely explanation for the latter.

Human spermatozoa possess an IL4I1 L-amino acid oxidase with a potential role in sperm function

Reactive oxygen species (ROS) are known to play an important role in the regulation of human sperm function. In this study, we demonstrate for the first time that human spermatozoa possess interleukin-induced gene 1 (IL4I1), an l-amino acid oxidase (LAAO) which is capable of generating ROS on exposure to aromatic amino acids in the presence of oxygen. The preferred substrates were found to be phenylalanine and tryptophan while the enzyme was located in the acrosomal region and midpiece of these cells. In contrast to equine and bovine spermatozoa, enzyme activity was lost as soon as the spermatozoa became non-viable. On a cell-to-cell basis human spermatozoa were also shown to generate lower levels of hydrogen peroxide than their equine counterparts on exposure to phenylalanine. Stimulation of LAAO activity resulted in the induction of several hallmarks of capacitation including tyrosine phosphorylation of the sperm flagellum and concomitant activation of phospho-SRC expression. In addition, stimulation of LAAO resulted in an increase in the levels of acrosomal exocytosis in both the presence and absence of progesterone stimulation, via mechanisms that could be significantly reversed by the presence of catalase. As is often the case with free radical-mediated phenomena, prolonged exposure of human spermatozoa to phenylalanine resulted in the stimulation of apoptosis as indicated by significant increases in mitochondrial superoxide generation and the activation of intracellular caspases. These results confirm the existence of an LAAO in human spermatozoa with a potential role in driving the redox regulation of sperm capacitation and acrosomal exocytosis.