Low 25-hydroxyvitamin D levels are more prevalent in Canadians of South Asian than European ancestry inhabiting the National Capital Region of Canada.

The US Institute of Medicine defined serum 25-hydroxyvitamin D (25OHD) cut point values of 30 nmol/L and 40 nmol/L were used to assess the vitamin D status of South Asian and European Canadians of self-identified ancestry living in the National Capital Region of Canada. Serum 25OHD values were measured in the spring and fall of 2012 to represent status during the winter and summer months, respectively. A total of 1238 measurements were obtained from 669 participants (49% South Asian ancestry): some participants were measured only once (spring or fall). Median 25OHD values were significantly higher in participants of European ancestry: 70.8 nmol/L (68.1, 73.5; 95% CI) versus South Asian ancestry: 42.7 nmol/L (40.5, 45.0; P<0.001). Spring vs. fall differences were small for each ethnic group and significant only for those of European ancestry (2.9, CI: 1.0-4.9 nmol/L; P = 0.01). Among participants of South Asian ancestry, 27.3% (fall) and 29.1% (spring) of females had values <40 nmol/L while the percentages for males were considerably higher (36.5% and 44.2%, respectively). The corresponding values for participants of European ancestry were ≤10%, showing that the South Asian participants were less likely to achieve the 25OHD concentrations established by the IOM for optimum bone health. Investigation of the factors related to serum 25OHD levels showed that supplement intake and ethnic background were associated with the biggest differences. Skin color was not a major factor, suggesting that genetic factors are responsible for the observed differences between participants of different ethnic backgrounds.

Cardio-Metabolic Disease Risks and Their Associations with Circulating 25-Hydroxyvitamin D and Omega-3 Levels in South Asian and White Canadians.

OBJECTIVES: This study compared cardio-metabolic disease risk factors and their associations with serum vitamin D and omega-3 status in South Asian (SAC) and White Canadians (WC) living in Canada's capital region. METHODS: Fasting blood samples were taken from 235 SAC and 279 WC aged 20 to 79 years in Ottawa, and 22 risk factors were measured. RESULTS: SAC men and women had significantly higher fasting glucose, insulin, homeostasis model assessment for insulin resistance (HOMA-IR), apolipoprotein B (ApoB), ratios of total (TC) to HDL cholesterol (HDLC) and ApoB to ApoA1, leptin, E-selectin, P-selectin, ICAM-1 and omega-3 (p < 0.05), but lower HDLC, ApoA1, vitamin D levels than WC (p < 0.05). SAC women had higher CRP and VEGF than WC women. Adequate (50-74.9 nmol/L) or optimal (≥ 75 nmol/L) levels of 25(OH)D were associated with lower BMI, glucose, insulin, HOMA-IR, TG, TC, low density lipoprotein cholesterol (LDLC), ApoB/ApoA1 ratio, CRP, leptin, and higher HDLC, ApoA1, omega-3 index, L-selectin levels in WC, but not in SAC. Intermediate (>4%-<8%) or high (≥ 8%) levels of omega-3 indices were related to lower E-selectin, P-selectin, ICAM-1 and higher HDLC, 25(OH)D levels in WC, but not in SAC. The BMIs of ≤ 25 kg/m2 were related to lower LDLC, ApoB, VEGF, creatinine and higher 25(OH)D in WC, but not in SAC. CONCLUSIONS: The associations of vitamin D, omega-3 status, BMI and risk factors were more profound in the WC than SAC. Compared to WC, vitamin D status and omega-3 index may not be good predictive risk factors for the prevalence of CVD and diabetes in SAC.

Exposure to a northern contaminant mixture (NCM) alters hepatic energy and lipid metabolism exacerbating hepatic steatosis in obese JCR rats.

Non-alcoholic fatty liver disease (NAFLD), defined by the American Liver Society as the buildup of extra fat in liver cells that is not caused by alcohol, is the most common liver disease in North America. Obesity and type 2 diabetes are viewed as the major causes of NAFLD. Environmental contaminants have also been implicated in the development of NAFLD. Northern populations are exposed to a myriad of persistent organic pollutants including polychlorinated biphenyls, organochlorine pesticides, flame retardants, and toxic metals, while also affected by higher rates of obesity and alcohol abuse compared to the rest of Canada. In this study, we examined the impact of a mixture of 22 contaminants detected in Inuit blood on the development and progression of NAFLD in obese JCR rats with or without co-exposure to 10% ethanol. Hepatosteatosis was found in obese rat liver, which was worsened by exposure to 10% ethanol. NCM treatment increased the number of macrovesicular lipid droplets, total lipid contents, portion of mono- and polyunsaturated fatty acids in the liver. This was complemented by an increase in hepatic total cholesterol and cholesterol ester levels which was associated with changes in the expression of genes and proteins involved in lipid metabolism and transport. In addition, NCM treatment increased cytochrome P450 2E1 protein expression and decreased ubiquinone pool, and mitochondrial ATP synthase subunit ATP5A and Complex IV activity. Despite the changes in mitochondrial physiology, hepatic ATP levels were maintained high in NCM-treated versus control rats. This was due to a decrease in ATP utilization and an increase in creatine kinase activity. Collectively, our results suggest that NCM treatment decreases hepatic cholesterol export, possibly also increases cholesterol uptake from circulation, and promotes lipid accumulation and alters ATP homeostasis which exacerbates the existing hepatic steatosis in genetically obese JCR rats with or without co-exposure to ethanol.

EPA and DHA status of South Asian and white Canadians living in the National Capital Region of Canada.

To minimize the risk of cardiovascular disease (CVD), most dietary guidelines have recommended consuming 500 mg/day of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) or two servings of oily fish/week. The sum of percent EPA and DHA in red blood cell (RBC) total fatty acids-termed the omega-3 index-has been proposed as a biomarker for assessing the risk of death from CVD. The omega-3 indices of ≤4, >4 to <8 and ≥8 % have been proposed to be associated with high, intermediate and low CVD risks, respectively. In this study, we determined the EPA + DHA intake level and the omega-3 index of South Asian Canadians (SAC; n = 308) and white Canadians (WC; n = 341) age 20-79 years living in the National Capital Region of Canada. The mean EPA + DHA intake levels were 569 ± 571 mg/day for SAC and 684 ± 865 mg/day for WC and 46 % of SAC and 43 % of WC met the recommended EPA + DHA intake level of 500 mg/day. The mean omega-3 indices were 6.6 and 5.9 % for SAC and WC respectively. The suggested cardio-protective target level for the omega-3 index of ≥8 % was observed only in 19.8 % of SAC and in 9.4 % of WC subjects. The majority of the participants (74.4 % of SAC and 82.7 % of WC) were in the >4 to <8 % range. These results suggest that although study participants' dietary intake of EPA + DHA is adequate, this intake was not sufficient to provide an omega-3 index that is considered cardio-protective.

Mandatory trans fat labeling regulations and nationwide product reformulations to reduce trans fatty acid content in foods contributed to lowered concentrations of trans fat in Canadian women's breast milk samples collected in 2009-2011.

BACKGROUND: Recent efforts in Canada to reduce industrial trans fatty acids (TFAs) in foods include mandated inclusion of TFA content on food labels and recommendations by Health Canada that encourage the food industry to voluntarily limit TFA content in all vegetable oils and soft margarines and in all other prepackaged foods to <2% and <5% of total fat, respectively. OBJECTIVE: To assess the impact of these efforts, we measured the concentration of TFAs in human breast milk samples. DESIGN: The TFA content in 639 breast milk samples collected in 2009, 2010, and 2011 from breastfeeding mothers in 10 major cities across Canada was analyzed by gas chromatography. RESULTS: The mean (±SD) TFA contents were 2.7 ± 0.9% (n = 153, range: 1.4-7.2%), 2.2 ± 0.7% (n = 309, range: 1.0-6.8%), and 1.9 ± 0.5% (n = 177, range: 0.9-3.4%) of total milk fat for samples collected in 2009, 2010, and 2011, respectively. These values are considerably lower than the value of 7.2 ± 3.0% (range: 0.1-17.2%) found previously for Canadian human milk in 1992. On the basis of a linear correlation between the percentage of TFAs in the diet and human milk fat established by Craig-Schmidt et al, and assuming that 30% of energy of a lactating mother's diet is derived from fat, we estimated from the TFA human milk fat data that TFA intake of Canadian breastfeeding mothers was 0.9%, 0.5%, and 0.3% of total energy in 2009, 2010, and 2011, respectively. These estimated values are lower than the WHO's maximum recommended intake of 1% of total energy for a healthy diet. CONCLUSIONS: The results suggest that the trans fat labeling regulations introduced in 2003 and recommendations by Health Canada in 2007 instructing the food manufacturers and restaurants to limit TFAs in foods have resulted in significant reductions in TFAs in the diets of Canadian breastfeeding mothers and their breast milk.

High-dose supplemental selenite to male Syrian hamsters fed hypercholesterolaemic diets alters Ldlr, Abcg8 and Npc1l1 mRNA expression and lowers plasma cholesterol concentrations.

The aim of the present study was to elucidate possible cholesterol-lowering mechanism(s) of high-dose supplemental Se in the form of selenite, a known hypocholesterolaemic agent. Male Syrian hamsters (four groups, ten per group) were fed semi-purified diets for 4 weeks containing 0.1 % cholesterol and 15 % saturated fat with selenite corresponding to varying levels of Se: (1) Se 0.15 parts per million (ppm), control diet; (2) Se 0.85 ppm; (3) Se 1.7 ppm; (4) Se 3.4 ppm. Lipids were measured in the bile, faeces, liver and plasma. The mRNA expression of several known regulators of cholesterol homeostasis (ATP-binding cassette transporters g5 (Abcg5) and g8 (Abcg8), 7-hydroxylase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor (LdLr) and Nieman-Pick C1-like 1 protein (Npc1l1)) were measured in the liver and/or jejunum. Oxysterols including 24-(S)-hydroxycholesterol, 25-hydroxycholesterol and 27-hydroxycholesterol (27-OHC) were measured in the liver. Significantly lower total plasma cholesterol concentrations were observed in hamsters consuming the low (0.85 ppm) and high (3.4 ppm) Se doses. The two highest doses of Se resulted in decreased plasma LDL-cholesterol concentrations and increased mRNA levels of hepatic Abcg8, Ldlr and jejunal Ldlr. Higher hepatic 27-OHC and TAG concentrations and lower levels of jejunal Npc1l1 mRNA expression were noted in the 1.7 and 3.4 ppm Se-treated hamsters. Overall, Se-induced tissue changes in mRNA expression including increased hepatic Abcg8 and Ldlr, increased jejunal Ldlr and decreased jejunal Npc1l1, provide further elucidation regarding the hypocholesterolaemic mechanisms of action of Se in the form of selenite.

Antioxidant Supplements Improve Profiles of Hepatic Oxysterols and Plasma Lipids in Butter-fed Hamsters.

Hypercholesterolemic diets are associated with oxidative stress that may contribute to hypercholesterolemia by adversely affecting enzymatically-generated oxysterols involved in cholesterol homeostasis. An experiment was conducted to examine whether the cholesterol-lowering effects of the antioxidants selenium and α-tocopherol were related to hepatic oxysterol concentrations. Four groups of male Syrian hamsters (n = 7-8) were fed high cholesterol and saturated fat (0.46% cholesterol, 14.3% fat) hypercholesterolemic semi-purified diets: 1) Control; 2) Control + α-tocopherol (67 IU all-racemic-α-tocopheryl-acetate/kg diet); 3) Control + selenium (3.4 mg selenate/kg diet); and 4) Control + α-tocopherol + selenium. Antioxidant supplementation was associated with lowered plasma cholesterol concentrations, decreased tissue lipid peroxidation and higher hepatic oxysterol concentrations. A second experiment examined the effect of graded selenium doses (0.15, 0.85, 1.7 and 3.4 mg selenate/kg diet) on mRNA expression of the oxysterol-generating enzyme, hepatic 27-hydroxylase (CYP27A1, EC 1.14.13.15), in hamsters (n = 8-9) fed the hypercholesterolemic diets. Supplementation of selenium at 3.4 mg selenate/kg diet was not associated with increased hepatic 27-hydroxylase mRNA. In conclusion, the cholesterol lowering effects of selenium and α-tocopherol were associated with increased hepatic enzymatically generated oxysterol concentrations, which appears to be mediated via improved antioxidant status rather than increased enzymatic production.

Trans fatty acids: current contents in Canadian foods and estimated intake levels for the Canadian population.

Research conducted in the mid-1990s indicated that the levels of trans fats in Canadian diets were among the highest in the world. The consumption of trans fats raises blood levels of low-density lipoprotein (LDL)-cholesterol, while reducing levels of high-density lipoprotein (HDL)-cholesterol. In June 2007, Health Canada called on the food industry to voluntarily reduce levels of trans fats in vegetable oils and soft (tub)-margarines to < 2% of total fat, and in all other foods, to < 5%. Industry must show satisfactory progress by June 2009, or Health Canada might have to introduce legislation to ensure that recommended limits are achieved. Since 2005, Health Canada has been performing a national assessment of prepackaged and restaurant foods that likely contain trans fats. From 2005 to 2009, 1120 samples were analyzed, of which 852 or approximately 76% met the recommended trans fat limits. As a result of reformulation, most of the products had decreased trans + saturated fat content. The estimated average intake of trans fatty acids (TFA) in Canada significantly dropped from the high value of 8.4 g/day in the mid-1990s to 3.4 g/day (or 1.4% food energy) in 2008. However, this TFA intake of 1.4% of energy is still above the World Health Organization recommended limit of TFA intake of < 1% of energy, which suggests that the Canadian food industry needs to put more effort into reducing the TFA content in its products, especially in tub-margarines, donuts, and bakery products.

Protein kinase C is an important signaling mediator associated with motility of intact sea urchin spermatozoa.

Numerous kinases and phosphatases are most likely implicated in sperm motility initiation and maintenance. Data on these signaling molecules were mostly obtained from studies conducted on in vitro demembranated-reactivated sperm models but are not necessarily representative of the in vivo situation. We therefore investigated the effect of a variety of cell-permeable chemicals, mostly kinase inhibitors, on the motility initiation and maintenance of intact sea urchin spermatozoa. Among the 20 substances tested, the protein kinase C (PKC) inhibitor chelerythrine was the most potent, arresting motility at concentrations starting from 1.5-2 mumol l(-1). Motility was also inhibited by two other PKC inhibitors as well as staurosporine. Furthermore, these inhibitors prevented the motility-associated increase in phosphorylation of at least four PKC substrates. These phospho-PKC target proteins, as assessed with an antibody specific to phosphorylated motifs of PKC substrates, were found to be associated with the flagellum, either in the Triton X-100 soluble portion or the axoneme (Triton X-100 insoluble). A phosphorylated PKC-like enzyme was also detected by immunoblotting in the flagellum, as well as a significant 50 kDa PKC cleavage product. Taken together, the data strongly indicate for the first time that, in vivo, which means on intact spermatozoa, PKC is a key signaling mediator associated with the maintenance of sea urchin sperm motility.

Positive role of reactive oxygen species in mammalian sperm capacitation: triggering and modulation of phosphorylation events.

The role of reactive oxygen species (ROS) as signal transduction elements in physiological phenomena is a recent concept that changes the paradigm of these active species as harmful molecules that promote deleterious effects and even cell death. Capacitation is a term used to define a complex and not well-characterized process that allows spermatozoa to complete their preparation to fertilize oocytes. Spermatozoa from many species incubated under specific conditions have the ability to produce small amounts of ROS without harming cell function and rather promoting signal transduction pathways associated with capacitation. This review summarizes the findings regarding the role of ROS during mammalian sperm capacitation, specifically as physiological mediators that trigger phosphorylation events. The role of ROS as regulators of protein tyrosine phosphorylation has been known for a decade but other novel phosphorylations, such as those of PKA substrates, of MEK-like proteins, and of proteins with the threonine-glutamine-tyrosine motif, were recently evidenced. Here we stress the involvement of PKA and the ERK pathway as two signal mechanisms acting independently that contribute to the modulation of protein tyrosine phosphorylation required for spermatozoa to achieve capacitation. Moreover, integration of all these data reinforces the concept that although some phosphorylation events are independent of the others, cross talk is also needed among the various pathways involved.

Reactive oxygen species modulate independent protein phosphorylation pathways during human sperm capacitation.

Spermatozoa must undergo capacitation to acquire fertilizing ability. Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide H2O2, and nitric oxide (NO*), are involved in this process. We investigated the roles and interactions of ROS, the ERK cascade, and the phosphoinositide 3-kinase (PI3K)/Akt axis during human sperm capacitation. Two different agents, fetal cord serum ultrafiltrate and bovine serum albumin, similarly promoted capacitation and the associated phosphorylation of protein tyrosine residues (P-Tyr), threonine-glutamine-tyrosine (P-Thr-Glu-Tyr-P) motif, and MEK-like proteins (P-MEK-like proteins). Components of the ERK pathway modulated these phosphorylation events. ROS increased P-MEK-like proteins and NO* induced P-Thr-Glu-Tyr-P, possibly by acting on or downstream of Ras. The PI3K/Akt axis participated in capacitation and phosphorylation of Tyr and Thr-Glu-Tyr but not MEK-like proteins. H2O2 and NO* induced P-Tyr even in the presence of ERK pathway inhibitors, indicating that ROS also act downstream of this pathway. These new results indicate that ROS act on different transduction elements during sperm capacitation and regulate phosphorylation events that occur in parallel pathways that eventually lead to late phosphorylation of Tyr. These new data reinforce the concept that a complex network of differentially modulated pathways is needed for spermatozoa to become capacitated.

Mice lacking the UBC4-testis gene have a delay in postnatal testis development but normal spermatogenesis and fertility.

Activation of ubiquitination occurs during spermatogenesis and is dependent on the induction of isoforms of the UBC4 family of ubiquitin-conjugating enzymes. The UBC4-testis isoform is testis specific, is induced in round spermatids, and demonstrates biochemical functions distinct from a ubiquitously expressed isoform UBC4-1. To explore further the function of UBC4-testis, mice bearing inactivation of this gene were produced. Homozygous (-/-) mice showed normal body growth and fertility. Although testis weight and morphology were normal in testes from adult mice, examination of young mice during the first wave of spermatogenesis revealed that testes were approximately 10% smaller in weight at 40 and 45 days of age but had become normal at 65 days of age. Overall protein content, levels of ubiquitinated proteins, and ubiquitin-conjugating activity did not differ between wild-type and homozygous (-/-) mice. Spermatid number, as well as the motility of spermatozoa isolated from the epididymis, was also normal in homozygous (-/-) mice. To determine whether the germ cells lacking UBC4-testis might be more sensitive to stress, testes from wild-type and knockout mice were exposed to heat stress by implantation in the abdominal cavity. Testes from both strains of mice showed similar rates of degeneration in response to heat. The lack of an obvious phenotype did not appear to be due to induction of other UBC4 isoforms, as shown by two-dimensional gel immunoblotting. Our data indicate that UBC4-testis plays a role in early maturation of the testis and suggest that the many UBC4 isoforms have mixed redundant and specific functions.

Reactive oxygen species and protein kinases modulate the level of phospho-MEK-like proteins during human sperm capacitation.

Capacitation is an essential process by which spermatozoa acquire fertilizing ability. Reactive oxygen species (ROS), protein kinase A (PKA), protein kinase C (PKC), protein tyrosine kinases (PTKs), and the extracellular signal-regulated protein kinase (ERK or mitogen-activated protein kinase [MAPK]) pathway regulate sperm capacitation. Our aim was to evaluate the phosphorylation of MEK (MAPK kinase or MAP2K) or MEK-like proteins in human sperm capacitation and its modulation by ROS and kinases. Immunoblotting using an anti-phospho-MEK antibody indicated that the phosphorylation of three protein bands (55, 94, and 115 kDa) increased in spermatozoa treated with fetal cord serum ultrafiltrate (FCSu), BSA, or isobutylmethylxanthine plus dibutyryl cAMP as capacitating agents. These phospho-MEK-like proteins are localized along the sperm flagellum. The MEK-inhibitors PD98059 and U126 prevented this phosphorylation, suggesting that these proteins are MEK-like proteins. The ROS scavengers prevented, and the addition of H(2)O(2) or spermine-NONOate (nitric oxide donor) triggered, the increase of phospho-MEK-like proteins. The capacitation-related increases in phospho-MEK-like proteins induced by FCSu, H(2)O(2), and spermine-NONOate were similarly modulated by PKA, PKC, and PTK, suggesting ROS as mediators in this phenomenon. These results indicate that phospho-MEK-like proteins are modulated by ROS and kinases and probably represent an intermediary step between the early events and the late tyrosine phosphorylation associated with capacitation.

Two anti-radial spoke monoclonal antibodies inhibit Chlamydomonas axonemal motility by different mechanisms.

In the 9 + 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules. They protrude toward the central pair microtubule complex with which they have transient but regular interactions for the normal flagellar motility to occur. Flagella of Chlamydomonas mutants deficient in entire radial spokes or spoke heads are paralyzed. In this study the importance of two radial spoke proteins in the flagellar movement is exemplified by the potent inhibitory action of two monoclonal antibodies on the axonemal motility of demembranated-reactivated Chlamydomonas models. We show that one of these proteins is localized on the stalk of the radial spokes, whereas the other is a component of the head of the same structure and most likely correspond to radial spoke protein 2 and 1, respectively. Fine motility analysis by videomicrography further indicates that these two anti-radial spoke protein antibodies at low concentration affect motility of demembranated-reactivated Chlamydomonas by changing the flagellar waveform without modifying axonemal beat frequency. They also modify wave amplitude differently during motility inhibition. This brings more direct evidence for the involvement of both radial spoke stalk and head in the fine tuning of the waveform during flagellar motility.

Implication of cAMP during porcine sperm capacitation and protein tyrosine phosphorylation.

Second messengers are involved in sperm fertilizing potential, as both motility and the acrosome reaction are influenced by cAMP. Moreover, the activity of cyclic nucleotides is implicated in the appearance of tyrosine phosphorylated sperm proteins, which is associated with capacitation in the mammalian spermatozoa. Nevertheless, the involvement of the cAMP/protein kinase A (PK-A) pathway during pig sperm capacitation may be different from that observed in other mammals. The objective of the present study was to clarify the cAMP/PK-A pathway during the capacitation of porcine spermatozoa and to evaluate this impact on the p32 sperm tyrosine phosphoprotein appearance. The presence of p32 was assessed after incubating fresh pig sperm with IBMX/db-cAMP, H-89, a PK-A inhibitor or bistyrphostin, a tyrosine kinase inhibitor, in capacitating (CM) or non-capacitating conditions (NCM) by immunoblotting SDS-extracted and separated sperm proteins using an anti-phosphotyrosine antibody. When pig spermatozoa were incubated in CM supplemented with H-89 (50 microM) or bistyrphostin (1.2 microM), capacitation decreased significantly (P < 0.001). The p32 sperm tyrosine phosphoprotein, previously shown to be associated with capacitation of porcine sperm though not necessarily an end point of this phenomenon, was not modulated by IBMX/db-cAMP (100 microM/1 mM), H-89 (50 microM) nor bistyrphostin (1.2 microM). Our results indicate, therefore, that pig sperm are regulated somewhat differently than as described for other mammals, because although the cAMP/PK-A and tyrosine kinase pathways are involved in capacitation, they do not influence the appearance of p32.

Inhibitors of phosphoinositide 3-kinase, LY294002 and wortmannin, affect sperm capacitation and associated phosphorylation of proteins differently: Ca2+-dependent divergences.

Sperm capacitation is regulated by multiple pathways that also control sperm motility and tyrosine (Tyr) phosphorylation of several sperm proteins. Among the reported pathways, phosphoinositide 3-kinase (PI3K) signaling and its role in modulating sperm postejaculatory changes and motility remain elusive. It was shown that wortmannin, a selective inhibitor of PI3K, prevents human sperm acrosome reaction. Using LY294002 (2-(4-morphlinyl)-8-phenyl-4H-1-benzopyran-4-one), another chemically different inhibitor of PI3K, it was suggested that this enzyme inhibits human sperm motility. In this study, we used the 2 known inhibitors of PI3K to investigate their effect on sperm capacitation and associated protein phosphorylation events. Our data show that sperm incubated with LY294002 undergo capacitation and increased Tyr phosphorylation of specific sperm proteins in a manner similar to that promoted by the capacitation inducer fetal cord serum ultrafiltrate (FCSu), as well as double phosphorylation of the threonine (Thr)-glutamine (Glu)-Tyr motif. Under similar conditions, wortmannin did not affect these sperm functions on its own, although it did prevent the effect induced by FCSu. Consistently, wortmannin decreased the phospho (P)-Tyr content of sperm proteins and prevented the phosphorylation of their Thr-Glu-Tyr motif. We also show by means of immunoblotting and cell fractionation experiments the presence of PI3K and its downstream effector Akt (protein kinase B) at the membrane level, as well as sperm heads and flagella. Our data show that human spermatozoa contain a consensus motif usually phosphorylated by Akt and that its P-serine (Ser)/Thr content is increased by both LY294002 and FCSu, while it is decreased by wortmannin. In addition, the 2 inhibitors differently affected the intracellular calcium concentration, [Ca(2+)](i). While LY294002 increased [Ca(2+)](i), wortmannin did not affect its content and did not prevent the LY294002 effect. Thus, we propose that the LY294002-promoted increase in [Ca(2+)](i) operates independently of PI3K. In conclusion, we suggest that special care be taken when using LY294002 to investigate the role that PI3K plays in a cellular phenomenon.

Redox control of changes in protein sulfhydryl levels during human sperm capacitation.

Capacitation, the series of transformations that spermatozoa undergo to become fertile, is regulated by reactive oxygen species (ROS) and associated with an increase in the sulfhydryl content of Triton-soluble proteins. Our aims were to determine the fate of sulfhydryl groups in Triton-soluble proteins from capacitating human spermatozoa using two-dimensional (2D) gel electrophoresis, to evaluate the role of ROS in the changes observed, and to correlate the time course of the changes with that of the sperm generation of O(2)(*)(-). Triton-soluble proteins of control and capacitating human spermatozoa were labeled with 3-(N-maleimidylpropionyl) biocytin, separated by 2D gel electrophoresis, and probed with horseradish peroxidase-conjugated streptavidin. The sulfhydryl content of 10 out of the 14 proteins studied (pI: 4-7) was modified by the induction of capacitation, and the increases (by 200-400%, five proteins) and decreases (by 45-95%, five proteins) were prevented by superoxide dismutase and/or catalase. The alterations in protein sulfhydryl content occurred within 5-15 min but were reversed within 30-120 min. Three capacitation inducers triggered similar modifications. Therefore, human sperm capacitation is associated with rapid and reversible changes in protein sulfhydryl groups that appear to be redox regulated. The number of proteins affected, the types, and the kinetics of changes emphasize the complexity of sperm capacitation.

Total suspended particulate manganese in ambient air in Montreal 1981-2000.

Our research group previously established the time variation of atmospheric Mn in Montreal for the period 1981-1992. The results indicated stable Mn concentrations between 1981 and 1990 followed by a decrease of almost 50% attributed to the closing of a ferromanganese plant located approximately 25 km from Montreal. The aim of this study is to assess the atmospheric concentrations of Mn in Montreal between 1993 and 2000, to compare these data to those for the period 1981-1992, and to evaluate the presence of any trend. Three sampling stations belonging to the Montreal Urban Community (MUC) air quality surveillance network were selected. Filters from 1993 to 2000 were obtained from the MUC archives. The first sample of each month was selected and chemical analysis was performed by neutron activation analysis. The mean annual atmospheric Mn concentrations in Montreal from 1993 to 2000 were found to be very stable. The mean Mn concentration calculated for this time period was 0.01 microg/m(3) for station 99 (characterized by a low traffic density), which was significantly different (P<0.05) from the mean of 0.03 microg/m(3) for stations 13 and 68 which had higher traffic densities. Even though Mn represented a small percentage of the TSP (varying between 0.02% and 0.14%), the comparison between Mn and TSP is interesting since the stable temporal profile of Mn since 1993 contrasts with the continuously decreasing atmospheric TSP concentrations. This observation suggests that the combustion of MMT used in gasoline could be contributing to maintaining stable atmospheric Mn levels.

Nitric oxide is a signaling molecule in spermatozoa.

Low and controlled concentrations of nitric oxide play an important role in sperm physiology. Nitric oxide is produced by spermatozoa and acts as an intracellular signaling molecule in the processes of capacitation and acrosome reaction. It has been documented that during capacitation, nitric oxide interacts with the cAMP-protein kinase A pathway and also is involved in tyrosine nitration of sperm proteins. On the other hand, during the acrosome reaction, two different pathways have been postulated for nitric oxide to exert its effects. During the progesterone-induced acrosome reaction, nitric oxide stimulates a heme-containing enzyme, named cyclooxygenase with a subsequent increase in prostaglandin E(2). Furthermore, the acrosome reaction inducing effect of NO-releasing compounds occurs via an increase in cGMP levels and protein kinase G activation. Taken together, these data support a role for nitric oxide in sperm function. This review focuses on providing new evidence for the physiological role of nitric oxide (NO) on sperm function. We will first present a brief description on nitric oxide chemistry and on the events leading to sperm fertilizing ability followed by the observations obtained on the participation of NO on fertilization.

Nitric oxide regulates the phosphorylation of the threonine-glutamine-tyrosine motif in proteins of human spermatozoa during capacitation.

Reactive oxygen species (superoxide anion, hydrogen peroxide, and nitric oxide) are involved in human sperm capacitation and associated tyrosine (Tyr) phosphorylation through a cAMP- and protein kinase A-mediated pathway. Recently, we evidenced the double phosphorylation of the threonine-glutamine-Tyr motif (P-Thr-Glu-Tyr-P) in human sperm proteins of 80 and 105 kDa during capacitation. The objective of the present study was to investigate the role of reactive oxygen species in the regulation of this process and to immunolocalize the P-Thr-Glu-Tyr-P motif in human spermatozoa. Superoxide dismutase and catalase did not prevent, and exogenous addition of superoxide anion or hydrogen peroxide did not trigger, the increase in P-Thr-Glu-Tyr-P related to sperm capacitation. However, l-NAME (a competitive inhibitor of l-arginine for nitric oxide synthase) prevented, and a nitric oxide donor promoted, the increase in P-Thr-Glu-Tyr-P related to sperm capacitation. In addition, l-arginine reversed the inhibitory effect of l-NAME on capacitation and the associated increase of P-Thr-Glu-Tyr-P. Therefore, the regulation of P-Thr-Glu-Tyr-P is specific to nitric oxide and not to superoxide anion or hydrogen peroxide. The nitric oxide-mediated increase of P-Thr-Glu-Tyr-P involved protein Tyr kinase, MEK or MEK-like kinase, and protein kinase C but not protein kinase A. The P-Thr-Glu-Tyr-P motif was immunolocalized to the principal piece region of spermatozoa. In conclusion, nitric oxide regulates the level of P-Thr-Glu-Tyr-P in sperm proteins of 80 and 105 kDa during capacitation. These data evidence, to our knowledge for the first time, a specific role for nitric oxide in signal transduction events leading to sperm capacitation.