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Expression of the HAHB4 sunflower transcription factor confers drought tolerance to wheat event IND-ØØ412-7 (HB4® wheat). After confirming the compositional equivalence of event IND-ØØ412-7 with conventional wheat, its nutritional similarity to its non-genetically modified (GM) counterpart was analyzed by performing a 42-day broiler feeding study. Isoenergetic diets containing 40% flour from wheat event IND-ØØ412-7, its non-GM counterpart Cadenza, and a commercial variety were included in the study. Broilers' performance was analyzed by measuring feed intake, weight gain, feed conversion, and time to reach 2.8 kgs. The yield was evaluated by carcass weight, breast meat, and abdominal fat. No differences were found between wheat event IND-ØØ412-7 and the non-GM counterpart. A few significant differences were found with the commercial variety which were associated with the genetic background, different from the other two materials. These results support the nutritional equivalence of event IND-ØØ412-7 with conventional wheat.

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Soybean (Glycine max L.) is the world's largest source of protein feed and the second largest source of vegetable oil. Water restriction is the main limiting factor to achieve maximum soybean yields. Therefore, development of varieties that maintain yield under environmental stresses is a major objective of soybean breeding programs. The HaHB4 (Helianthus annuus homeobox 4) gene from sunflower encodes for a transcription factor involved in the plant´s tolerance to environmental stress. The introduction of HaHB4 in soybean led to the development of event IND-ØØ41Ø-5 (HB4® soybean), which displayed higher yield in environments having low productivity potential, compared with the parental control variety. Compositional analyses of soybean event IND-ØØ41Ø-5 were conducted both in Argentina and the United Sates. A total of 44 components were analyzed in grain and 9 components in forage. Based on the results of these studies it was concluded that soybean event IND-ØØ41Ø-5 was compositionally equivalent to its non-transgenic parental control.

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Wheat is the most widely grown cereal grain, occupying a significant portion of the total cultivated land. As drought is the major environmental stressor affecting crop production, yield maintenance under water deficit conditions appears as a highly desirable phenotype for crop improvement. The HaHB4 (Helianthus annuus homeobox 4) gene from sunflower encodes for a transcription factor involved in tolerance to environmental stress. The introduction of HaHB4 in wheat led to the development of event IND-ØØ412-7 (HB4® wheat), which displayed higher yield in production environments of low productivity potential. Compositional analysis of IND-ØØ412-7 wheat, including 41 nutrients and 2 anti-nutrients for grain and 10 nutrients in forage, was performed. Results of these studies indicated that IND-ØØ412-7 is compositionally equivalent to non-transgenic wheat.

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After leaving the testis, sperm undergo two sequential maturational processes before acquiring fertilizing capacity: sperm maturation in the male epididymis, and sperm capacitation in the female reproductive tract. During their transit through the epididymis, sperm experience several maturational changes; the acquisition of motility is one of them. The molecular basis of the regulation of this process is still not fully understood. Sperm are both transcriptionally and translationally silent, therefore post-translational modifications are essential to regulate their function. The post-translational modification by the addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) can act as a counterpart of phosphorylation in different cellular processes. Therefore, our work was aimed to characterize the O-GlcNAcylation system in the male reproductive tract and the occurrence of this phenomenon during sperm maturation. Our results indicate that O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAcylation, is present in the testis, epididymis and immature caput sperm. Its presence is significantly reduced in mature cauda sperm. Consistently, caput sperm display high levels of O-GlcNAcylation when compared to mature cauda sperm, where it is mostly absent. Our results indicate that the modulation of O-GlcNAcylation takes place during sperm maturation and suggest a role for this post-translational modification in this process.

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Mammalian seminal plasma contains membranous vesicles (exosomes), with a high content of cholesterol and sphingomyelin and a complex protein composition. Their physiological role is uncertain because sperm stabilization and activation effects have been reported. To analyze a putative modulatory role for semen exosomes on sperm activity in the boar, the effects of these vesicles on several sperm functional parameters were examined. Additionally, boar exosome proteins were sequenced and their incorporation into sperm was explored. Boar sperm were incubated under conditions that induce capacitation, manifested as increased tyrosine phosphorylation, cholesterol loss and greater fluidity in apical membranes, and the ability to undergo the lysophosphatidylcholine-induced acrosome reaction. After establishing this cluster of capacitation-dependent functional parameters, the effect produced by exosomes when present during or after sperm capacitation was analyzed. Exosomes inhibited the capacitation-dependent cholesterol efflux and fluidity increase in apical membranes, and the disappearance of a 14-kD phosphorylated polypeptide. In contrast, the acrosome reaction (spontaneous and lysophosphatidylcholine-induced) was not affected, and sperm binding to the oocyte zona pellucida was reduced only when vesicles were present during gamete coincubation. Liposomes with a lipid composition similar to that present in exosomes mimicked these effects, except the one on zona pellucida binding. Interaction between exosomes and sperm was confirmed by transfer of aminopeptidase activity. In addition, the major exosome protein, identified as actin, appeared to associate with sperm after coincubation. Exosome composition had a predominance for structural proteins (actin, plastin, ezrin, and condensin), enzymes, and several porcine seminal plasma-specific polypeptides (e.g., spermadhesins). Transfer of proteins from exosome to sperm and their ability to block cholesterol efflux supports a direct interaction between these vesicles and sperm, whereas inhibition of some capacitation-dependent features suggests a stabilizing function for exosomes in boar semen.

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Calcium (Ca(2+)) is an absolute requirement for a decisive sperm function event: the acrosome reaction (AR). Physiologically, sperm capacitation is a prerequisite for this specialized exocytosis and both events are intimately related. In an effort to separate capacitation from AR, we have been using a modified sperm incubation medium where Ca(2+) is replaced by Strontium (Sr(2+)). The aim of this report is to analyze with more detail the difference between sperm incubated with Ca(2+) or Sr(2+) in several events. We found that sperm undergo the capacitation-related changes in the chlortetracycline (CTC) pattern and tyrosine phosphorylation, and also bind to the zona pellucida (ZP) when using Sr(2+)-instead of Ca(2+)-containing media. However, the spontaneous AR typical of hamster sperm does not take place in Sr(2+)-medium, even if sperm are previously capacitated with Ca(2+). Nevertheless, Sr(2+) was able to sustain AR when cells were treated with thapsigargin or depolarized with K(+) in Na(+)-depleted medium. Considering that the absence of Na(+) increased spontaneous AR in Sr(2+)-medium, we tested whether Na(+)-transport systems could be involved in the inability of Sr(2+)-incubated sperm to undergo AR. We found that when sperm incubated in Sr(2+)-medium are treated with amiloride to inhibit epithelial Na(+) channel (ENaC), they are able to undergo spontaneous AR. The same result was obtained when analyzing AR on the ZP. On the contrary, addition of ouabain (a Na(+)/K(+)-ATPase inhibitor) or DIDS (a Na(+)/HCO3(-) co-transporter inhibitor) showed no effect. These results suggest that, differing from what happens in Ca(2+)-incubated sperm, cells incubated in Sr(2+)-modified medium would have an active ENaC.

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One of the most important processes in fertilization is the fusion of egg and sperm; however, the molecular mechanisms involved in this process are not well understood. So far, using genetic approaches, only two proteins have been demonstrated to be necessary for this process: Izumo in sperm and CD9 in the egg. Here we demonstrate that sperm produced by Tssk6 (Sstk)-null mice present defects that prevent the successful fertilization of eggs in vitro and the fusion to zona-pellucida-free eggs. Tssk6 is a member of the testis-specific serine kinase family of proteins and is expressed postmeiotically in male germ cells. In order for fusion to occur, during the process known as acrosome reaction Izumo needs to relocate from the anterior head to other regions, including the postacrosomal compartment. Tssk6-null sperm fails to relocate Izumo during the acrosome reaction. Agents that interfere with actin dynamics blocked the acrosome-reaction-associated translocation of Izumo that is required for fusion in wild-type sperm. Additionally, actin polymerization was compromised in Tssk6-null sperm. Taken together, our results indicate that Tssk6 is involved in sperm-egg fusion through the regulation of actin polymerization and changes in Izumo localization.

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Mammalian sperm become fertile after completing capacitation, a process associated with cholesterol loss and changes in the biophysical properties of the sperm membranes that prepares the sperm to undergo the acrosome reaction. Different laboratories have hypothesized that cholesterol efflux can influence the extent and/or movement of lipid raft microdomains. In a previous study, our laboratory investigated the identity of sperm proteins putatively associated with rafts. After extraction with Triton X-100 and ultracentrifugation in sucrose gradients, proteins distributing to the light buoyant-density fractions were cored from polyacrylamide gels and microsequenced. In this study, a subset of these proteins (TEX101, basigin, hexokinase 1, facilitated glucose transporter 3, IZUMO, and SPAM1) and other molecules known to be enriched in membrane rafts (caveolin 2, flotillin 1, flotillin 2, and the ganglioside GM3) were selected to investigate their localization in the sperm and their behavior during capacitation and the acrosome reaction. These molecules localize to multiple sperm domains, including the acrosomal cap (IZUMO, caveolin 2, and flotillin 2), equatorial segment (GM3), cytoplasmic droplet (TEX101), midpiece (basigin, facilitated glucose transporter 3, and flotillin 2), and principal piece (facilitated glucose transporter 3). Some of these markers modified their immunofluorescence pattern after sperm incubation under capacitating conditions, and these changes correlated with the occurrence of the acrosome reaction. While GM3 and caveolin 2 were not detected after the acrosome reaction, flotillin 2 was found in the equatorial segment of acrosome-reacted sperm, and IZUMO distributed along the sperm head, reaching the post- and para-acrosomal areas. Taking into consideration the requirement of the acrosome reaction for sperm to become fusogenic, these results suggest that membrane raft dynamics may have a role in sperm-egg membrane interaction.

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The glycosidase-recognizing N-acetylglucosamine terminal residue, N-acetylglucosaminidase (NAG), has been repetitively implicated in fertilization. Nevertheless, its role in the multiple steps comprising this process is a matter of debate because it has been involved in zona pellucida (ZP) binding and penetration and polyspermy block. In this study, the involvement of NAG during sperm interaction with the ZP was analysed. Soluble ZP was able to inhibit sperm NAG activity, suggesting that it can be recognized as a ligand by this enzyme. Sperm-ZP binding assays were carried out under conditions where acrosome reaction (AR) could not take place (salt-stored oocytes and a modified medium where Ca(2+) was replaced by Sr(2+)). Different NAG-specific reagents-an inhibitor (2-acetamido-2-deoxy-D-glucono-1,5-lactone), a substrate (p-nitrophenyl-N-acetylglucosaminide) and an anti-NAG antibody-were able to impair sperm binding to the ZP when present during these assays. The lactone was also able to inhibit oocyte penetration during IVF assays, although not when present after primary binding had taken place. This result was not related to the interference of lactone with AR or zona penetrability. Exogenous NAG also inhibited sperm-oocyte interaction when present during binding and IVF assays or used for oocyte pre-incubation. These results suggest the participation of NAG in sperm primary binding to the ZP.

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CD59 is a GPI-linked membrane protein that inhibits formation of the membrane attack complex of complement. We reported recently that mice have two CD59 genes (termed mCd59a and mCd59b), and that the targeted deletion of mCd59b (mCd59b-/-) results in spontaneous hemolytic anemia and progressive loss of male fertility. Further studies of the reproductive abnormalities in mCd59b-/- mice reported in this study revealed the presence of abnormal multinucleated cells and increased apoptotic cells within the walls of the seminiferous tubules, and a decrease in the number, motility, and viability of sperm associated with a significant increase in abnormal sperm morphologies. Both the capacitation-associated tyrosine phosphorylation and the ionophore-induced acrosome reaction as well as luteinizing hormone, follicle-stimulating hormone, and testosterone serum levels were similar in mCd59b-/- and mCd59b+/+. Surprisingly, the functional deficiency of the complement protein C3 did not rescue the abnormal reproductive phenotype of mCd59b-/-, although it was efficient in rescuing their hemolytic anemia. These results indicate that the male reproductive abnormalities in mCd59b-/- are complement-independent, and that mCd59 may have a novel function in spermatogenesis that is most likely unrelated to its function as an inhibitor of membrane attack complex formation.

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Mammalian sperm acquire fertilization capacity after residing in the female tract during a process known as capacitation. The present study examined whether cholesterol efflux during capacitation alters the biophysical properties of the sperm plasma membrane by potentially reducing the extent of lipid raft domains as analyzed by the isolation of detergent-resistant membrane fractions using sucrose gradients. In addition, this work investigated whether dissociation of the detergent-resistant membrane fraction during capacitation alters resident sperm raft proteins. Mouse sperm proteins associated with such fractions were studied by silver staining, tandem mass spectrometry, and Western blot analysis. Caveolin 1 was identified in sperm lipid rafts in multimeric states, including a high-molecular-weight oligomer that is sensitive to degradation under reducing conditions at high pH. Capacitation resulted in reduction of the light buoyant-density, detergent-resistant membrane fraction and decreased the array of proteins isolated within this fraction, including loss of the high-molecular-weight caveolin 1 oligomers. Proteomic analysis of sperm proteins isolated in the light buoyant-density fraction identified several proteins, including hexokinase 1, testis serine proteases 1 and 2, TEX101, hyaluronidase (PH20, SPAM1), facilitated glucose transporter 3, lactate dehydrogenase A, carbonic anhydrase IV, IZUMO, pantophysin, basigin, and cysteine-rich inhibitory secretory protein 1. Capacitation also resulted in a significant reduction of sperm labeling by the fluorescent lipid-analog DiIC16, indicating that capacitation alters the liquid-ordered domains in the sperm plasma membrane. The observations that capacitation alters the protein composition of the detergent-resistant membrane fractions is consistent with the hypothesis that cholesterol efflux during capacitation dissociates lipid raft constituents, initiating signaling events that lead to sperm capacitation.

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Glycoproteins and lectin-like proteins mediate sperm-zona pellucida interaction. The present study analysed the participation of carbohydrates in the different stages of sperm interaction with the zona pellucida in hamster, by determining the effects of different monosaccharides. N-acetylglucosamine (GlcNAc, 1 mM) reduced sperm ability to bind to the zona pellucida. Surprisingly, spontaneous acrosome reaction (AR) was also inhibited by this sugar. In order to analyse the effect of GlcNAc on sperm-zona pellucida binding, independent of its effect on the AR, strontium (Sr) was used as a calcium (Ca) replacement in the sperm capacitation and co-incubation medium. Sr seemed to be able to replace Ca for sperm capacitation, at least when measured as the ability to bind to the zona pellucida, and undergo AR when Ca is provided. Moreover, sperm-zona pellucida binding could also take place in a Sr-modified medium. When binding assays were carried out in the Sr medium, GlcNAc also produced an inhibitory effect. This could be reproduced when sperm, but not oocytes, were pre-incubated with the monosaccharide. IVF assays were also carried out to analyse the participation of GlcNAc in the different steps of sperm-oocyte interaction. Taken together, the results support the involvement of the GlcNAc residues of the zona pellucida in the early steps of the interaction with sperm.

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PROBLEM: To determine the ability of IgGs isolated from follicular fluids (hFFIgGs) to induce the acrosome reaction (AR) in human spermatozoa and to inhibit sperm-zona pellucida (ZP) interaction. METHOD OF STUDY: Incubation of capacitated spermatozoa with hFFIgGs (n = 40) and assessment of their effect on the AR or hemizona (HZ) assay in a condition that allows sperm-ZP interaction, avoiding acrosomal exocytosis. RESULTS: hFFIgGs from different women varied in their ability of inducing the AR. Those hFFIgGs with the highest AR-inducing capacity evoked the exocytotic response in most of the different sperm donors tested [high Induction Frequency (IF)]. Some of these antibodies were also able of inhibiting sperm binding to ZP [low HZ Index (HZI)]. A significant correlation was found between the IF and the HZI for each hFFIgG. CONCLUSIONS: Human follicular fluid contains antibodies capable of inducing the AR and inhibiting sperm-ZP binding, suggesting that they could be directed towards ZP receptors. hFFIgGs would constitute a tool for the identification of sperm entities involved in fertilization.

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Mammalian spermatozoa fertilize only after capacitation. The removal of decapacitation factors that inhibit the acrosome reaction (AR) is one of the events taking place during capacitation. In this report, human sperm were capacitated by 18-h incubation in Biggers, Whitten & Whittingham medium (BWW) medium and the proteins, on release, were analysed. After gel filtration by high-performance liquid chromatography a main peak with an approximate native molecular weight of 130 kDa was recognized by an antinormal seminal plasma antibody. This fraction was able to inhibit the follicular fluid as well as the progesterone-induced AR, when added to capacitated spermatozoa. Additionally, it reacted with an antibody directed against seminal plasma from vasectomized donors but not with an antibody against epididymal proteins. The AR inhibitory activity was heat-denatured, could be partially destroyed when treated with proteases, and bound to Concanavalin-A and wheat germ lectins. These results suggest that during in vitro capacitation, human spermatozoa release a glycoproteic decapacitation factor produced by accessory sex glands.

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OBJECTIVE: To identify human sperm proteins involved in homologous zona pellucida (ZP) interaction. DESIGN: Prospective study. SETTINGS: Basic research laboratory. PATIENT(S): Semen samples from normozoospermic donors, tissue sections from surgical pieces, and ZP from nonfertilized oocytes. INTERVENTION(S): Antibodies for sperm proteins (HSE; high salt extract) were developed (anti-HSE) and partially characterized. Participation of sperm proteins on ZP-interaction was tested with the hemizona assay (HZA). Antigens were immunolocalized in sperm and tissues. MAIN OUTCOME MEASURE(S): Sperm and tissue immunostaining; Western blotting; and number of sperm bound to the ZP. RESULT(S): Anti-HSE antibodies recognized several polypeptides in HSE (9 to 200 kd). Specific antibodies for 49 and 66 kd proteins (p49, p66) were obtained. Both (anti-p49 and anti-p66) stained the head of ejaculated and capacitated sperm. In the HZA, sperm preincubation with a mixture of anti-p49 and anti-p66 (100 micro g/mL) resulted in a decrease in the number of spermatozoa bound to the ZP. Presence of p66 (10 micro g/mL) inhibited sperm-ZP interaction. In contrast, p49 did not alter sperm binding to the ZP. Immunohistochemical analysis showed that p66 is present in the epididymis. No staining was observed in testicular sections. CONCLUSION(S): We found that p66 is an epididymal protein that participates in human sperm interaction with homologous ZP.

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OBJECTIVE: To determine the effect of human sperm incubation at room temperature (20 degrees C) upon capacitation-related events. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Semen samples were obtained from normozoospermic volunteers. Human follicular fluid (hFF) was collected from women undergoing assisted reproductive treatment. INTERVENTION(S): Spermatozoa were incubated for up to 18 hours at 20 degrees C and/or 37 degrees C. MAIN OUTCOME MEASURE(S): Protein tyrosine phosphorylation patterns, development of hyperactivated motility, and induction of acrosome reaction (AR) in response to hFF. RESULT(S): Spermatozoa incubated for 18 hours at 20 degrees C showed an array of tyrosine phosphorylated proteins similar to noncapacitated cells. After incubation at 20 degrees C, the percentage of spermatozoa displaying hyperactivated motility and undergoing acrosomal loss in response to hFF was significantly lower when compared with cells kept the same time at 37 degrees C. Conversely, spermatozoa incubated overnight at 37 degrees C could respond to hFF, either at 37 degrees C or 20 degrees C. When preincubation at 20 degrees C was followed by sperm exposure to 37 degrees C, capacitation-related events could be activated. In capacitated cells (16 hours at 37 degrees C), 2-hour incubation at 20 degrees C led to a significant decrease in acrosome reaction inducibility, suggesting sperm decapacitation. CONCLUSION(S): Human sperm incubation at room temperature does not allow capacitation, although it does not affect hFF-induced acrosome reaction in capacitated cells. The blocking effect is overcome when spermatozoa are exposed to 37 degrees C.

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La interacción inicial entre las gametas es mediada por proteínas de superficie de la cabeza del espermatozoide y de la matriz extracelular del ovocito, la zona pellucida (ZP). El presente trabajo tuvo como objetivo la identificación de antígenos de superficie de espermatozoides humanos potencialmente involucrados en el reconocimiento de la ZP. Para ello se obtuvo un extracto de proteínas periféricas de espermatozoides humanos por tratamiento de las células con solución de alta fuerza iónica (HSE - High Salt Extract) (Buffer Pipes 100mM, pH 7,4; NaCl1M, sacarosa 0,25 M). Se desarrollaron anticuerpos policlonales (anti-HSE) que reconocieron numerosas proteínas en HSE (9-200 KDa). Las proteínas fueron separadas por electroforesis en geles de poliacrilamida, transferidas a membranas de nitrocelulosa y utilizadas para adsorber inmunoglobulinas de anti-HSE. Con éste método se obtuvieron anticuerpos contra dos polipéptidos mayoritarios de 49 (p49) y 66 (p66) kDa. Ambos anticuerpos (anti-p49 y anti-p66) reconocieron epitopes localizados en la cabeza y el flagelo de espermatozoides eyaculados y capacitados...

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La interacción inicial entre las gametas es mediada por proteínas de superficie de la cabeza del espermatozoide y de la matriz extracelular del ovocito, la zona pellucida (ZP). El presente trabajo tuvo como objetivo la identificación de antígenos de superficie de espermatozoides humanos potencialmente involucrados en el reconocimiento de la ZP. Para ello se obtuvo un extracto de proteínas periféricas de espermatozoides humanos por tratamiento de las células con solución de alta fuerza iónica (HSE - High Salt Extract) (Buffer Pipes 100mM, pH 7,4; NaCl1M, sacarosa 0,25 M). Se desarrollaron anticuerpos policlonales (anti-HSE) que reconocieron numerosas proteínas en HSE (9-200 KDa). Las proteínas fueron separadas por electroforesis en geles de poliacrilamida, transferidas a membranas de nitrocelulosa y utilizadas para adsorber inmunoglobulinas de anti-HSE. Con éste método se obtuvieron anticuerpos contra dos polipéptidos mayoritarios de 49 (p49) y 66 (p66) kDa. Ambos anticuerpos (anti-p49 y anti-p66) reconocieron epitopes localizados en la cabeza y el flagelo de espermatozoides eyaculados y capacitados...(AU)

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