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Sample records for major sperm protein

  1. Major Sperm Protein Genes from Globodera rostochiensis

    PubMed Central

    Novitski, Charles E.; Brown, Shiela; Chen, Ru; Corner, Adam S.; Atkinson, Howard J.; McPherson, Michael J.

    1993-01-01

    Three genes in the major sperm protein (MSP) gene family from the potato cyst nematode Globodera rostochiensis were cloned and sequenced. In contrast to the absence of introns in Caenorhabditis elegans MSP genes, these genes in G. rostochiensis contained a 57 nucleotide intron, with normal exon-intron boundaries, in the same relative location as the intron in Onchocerca volvulus. The MSP genes of G. rostochiensis had putative CAAT, TATA, and polyadenylation signals. The predicted G. rostochiensis MSP gene product is 126 amino acids long, one residue shorter than the products in the other species. The comparison of MSP amino acid sequences from four diverse nematode species suggests that O. volvulus, Ascaris suum, and C. elegans may be more closely related to each other than they are to G. rostochiensis. PMID:19279808

  2. Deficiency in Sperm-Egg Protein Interaction as a Major Cause of Fertilization Failure.

    PubMed

    Sabetian, Soudabeh; Shamsir, Mohd Shahir

    2017-04-01

    Complete elucidation of fertilization process at molecular level is one of the unresolved challenges in sexual reproduction studies, and understanding the molecular mechanism is crucial in overcoming difficulties in infertility and unsuccessful in vitro fertilization. Sperm-oocyte interaction is one of the most remarkable events in fertilization process, and deficiency in protein-protein interactions which mediate this interaction is a major cause of unexplained infertility. Due to detection of how the various defects of sperm-oocyte interaction can affect fertilization failure, different experimental methods have been applied. This review summarizes the current understanding of sperm-egg interaction mechanism during fertilization and also accumulates the different types of sperm-egg interaction abnormalities and their association with infertility. Several detection approaches regarding sperm-egg protein interactions and the associated defects are reviewed in this paper.

  3. Dephosphorylation of Major Sperm Protein (MSP) Fiber Protein 3 by Protein Phosphatase 2A during Cell Body Retraction in the MSP-based Amoeboid Motility of Ascaris Sperm

    PubMed Central

    Yi, Kexi; Wang, Xu; Emmett, Mark R.; Marshall, Alan G.; Stewart, Murray

    2009-01-01

    The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Pharmacological and depletion-add back assays showed that retraction was triggered by a putative protein phosphatase 2A (PP2A, a Ser/Thr phosphatase activated by tyrosine dephosphorylation). Immunofluorescence showed that PP2A was present in the cell body and was concentrated at the base of the lamellipod where the force for retraction is generated. PP2A targeted MSP fiber protein 3 (MFP3), a protein unique to nematode sperm that binds to the MSP filaments in the motility apparatus. Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated filament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward. PMID:19458186

  4. Expansion and functional diversification of a leucyl aminopeptidase family that encodes the major protein constituents of Drosophila sperm

    PubMed Central

    2011-01-01

    Background The evolutionary diversification of gene families through gene creation (and loss) is a dynamic process believed to be critical to the evolution of functional novelty. Previous identification of a closely related family of eight annotated metalloprotease genes of the M17 Merops family in the Drosophila sperm proteome (termed, Sperm-LeucylAminoPeptidases, S-LAPs 1-8) led us to hypothesize that this gene family may have experienced such a diversification during insect evolution. Results To assess putative functional activities of S-LAPs, we (i) demonstrated that all S-LAPs are specifically expressed in the testis, (ii) confirmed their presence in sperm by two-dimensional gel electrophoresis and mass spectrometry, (iii) determined that they represent a major portion of the total protein in sperm and (iv) identified aminopeptidase enzymatic activity in sperm extracts using LAP-specific substrates. Functionally significant divergence at the canonical M17 active site indicates that the largest phylogenetic group of S-LAPs lost catalytic activity and likely acquired novel, as yet undetermined, functions in sperm prior to the expansion of the gene family. Conclusions Comparative genomic and phylogenetic analyses revealed the dramatic expansion of the S-LAP gene family during Drosophila evolution and copy number heterogeneity in the genomes of related insects. This finding, in conjunction with the loss of catalytic activity and potential neofunctionalization amongst some family members, extends empirical support for pervasive "revolving door" turnover in the evolution of reproductive gene family composition and function. PMID:21466698

  5. Localized Depolymerization of the Major Sperm Protein Cytoskeleton Correlates with the Forward Movement of the Cell Body in the Amoeboid Movement of Nematode Sperm

    PubMed Central

    Italiano, Joseph E.; Stewart, Murray; Roberts, Thomas M.

    1999-01-01

    The major sperm protein (MSP)-based amoeboid motility of Ascaris suum sperm requires coordinated lamellipodial protrusion and cell body retraction. In these cells, protrusion and retraction are tightly coupled to the assembly and disassembly of the cytoskeleton at opposite ends of the lamellipodium. Although polymerization along the leading edge appears to drive protrusion, the behavior of sperm tethered to the substrate showed that an additional force is required to pull the cell body forward. To examine the mechanism of cell body movement, we used pH to uncouple cytoskeletal polymerization and depolymerization. In sperm treated with pH 6.75 buffer, protrusion of the leading edge slowed dramatically while both cytoskeletal disassembly at the base of the lamellipodium and cell body retraction continued. At pH 6.35, the cytoskeleton pulled away from the leading edge and receded through the lamellipodium as its disassembly at the cell body continued. The cytoskeleton disassembled rapidly and completely in cells treated at pH 5.5, but reformed when the cells were washed with physiological buffer. Cytoskeletal reassembly occurred at the lamellipodial margin and caused membrane protrusion, but the cell body did not move until the cytoskeleton was rebuilt and depolymerization resumed. These results indicate that cell body retraction is mediated by tension in the cytoskeleton, correlated with MSP depolymerization at the base of the lamellipodium. PMID:10477761

  6. Major regulatory mechanisms involved in sperm motility.

    PubMed

    Pereira, Rute; Sá, Rosália; Barros, Alberto; Sousa, Mário

    2017-01-01

    The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies.

  7. Major regulatory mechanisms involved in sperm motility

    PubMed Central

    Pereira, Rute; Sá, Rosália; Barros, Alberto; Sousa, Mário

    2017-01-01

    The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies. PMID:26680031

  8. Protein and carbohydrate intake influence sperm number and fertility in male cockroaches, but not sperm viability

    PubMed Central

    Bunning, Harriet; Rapkin, James; Belcher, Laurence; Archer, C. Ruth; Jensen, Kim; Hunt, John

    2015-01-01

    It is commonly assumed that because males produce many, tiny sperm, they are cheap to produce. Recent work, however, suggests that sperm production is not cost-free. If sperm are costly to produce, sperm number and/or viability should be influenced by diet, and this has been documented in numerous species. Yet few studies have examined the exact nutrients responsible for mediating these effects. Here, we quantify the effects of protein (P) and carbohydrate (C) intake on sperm number and viability in the cockroach Nauphoeta cinerea, as well as the consequences for male fertility. We found the intake of P and C influenced sperm number, being maximized at a high intake of diets with a P : C ratio of 1 : 2, but not sperm viability. The nutritional landscapes for male fertility and sperm number were closely aligned, suggesting that sperm number is the major determinant of male fertility in N. cinerea. Under dietary choice, males regulate nutrient intake at a P : C ratio of 1 : 4.95, which is midway between the ratios needed to maximize sperm production and pre-copulatory attractiveness in this species. This raises the possibility that males regulate nutrient intake to balance the trade-off between pre- and post-copulatory traits in this species. PMID:25608881

  9. Protein and carbohydrate intake influence sperm number and fertility in male cockroaches, but not sperm viability.

    PubMed

    Bunning, Harriet; Rapkin, James; Belcher, Laurence; Archer, C Ruth; Jensen, Kim; Hunt, John

    2015-03-07

    It is commonly assumed that because males produce many, tiny sperm, they are cheap to produce. Recent work, however, suggests that sperm production is not cost-free. If sperm are costly to produce, sperm number and/or viability should be influenced by diet, and this has been documented in numerous species. Yet few studies have examined the exact nutrients responsible for mediating these effects. Here, we quantify the effects of protein (P) and carbohydrate (C) intake on sperm number and viability in the cockroach Nauphoeta cinerea, as well as the consequences for male fertility. We found the intake of P and C influenced sperm number, being maximized at a high intake of diets with a P : C ratio of 1 : 2, but not sperm viability. The nutritional landscapes for male fertility and sperm number were closely aligned, suggesting that sperm number is the major determinant of male fertility in N. cinerea. Under dietary choice, males regulate nutrient intake at a P : C ratio of 1 : 4.95, which is midway between the ratios needed to maximize sperm production and pre-copulatory attractiveness in this species. This raises the possibility that males regulate nutrient intake to balance the trade-off between pre- and post-copulatory traits in this species. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  10. Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

    PubMed

    de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

    2016-11-01

    Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p < 0.05). When epididymal sperm were treated with milk followed by seminal plasma, coating of sperm with BSP proteins was not significantly reduced (57.6 %; p > 0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

  11. Evolution and function of mammalian binder of sperm proteins.

    PubMed

    Plante, Geneviève; Prud'homme, Bruno; Fan, Jinjiang; Lafleur, Michel; Manjunath, Puttaswamy

    2016-01-01

    Binder of sperm (BSP) proteins are ubiquitous among mammals and have been extensively investigated over the last three decades. They were first characterized in bull seminal plasma and have now been identified in more than 15 different mammalian species where they represent a superfamily. In addition to sharing a common structure, BSP proteins share many characteristics. They are expressed by seminal vesicles and epididymides, interact with similar ligands and bind to the outer leaflet of sperm membranes via an interaction with choline phospholipids. In addition to playing a major role in sperm capacitation, they are implicated as molecular chaperones in sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume and possibly in the interaction between sperm and oocytes, making them crucial multifunctional proteins. Furthermore, BSP proteins can bind to egg yolk low-density lipoproteins and milk components, an interaction important for the protection of sperm during semen preservation in liquid or frozen state. Our current knowledge of BSP proteins strongly emphasizes their fundamental importance in male fertility and in the optimization of semen preservation techniques. Much work is still ahead in order to fully understand all the mysteries of BSP proteins.

  12. Changes in exposed membrane proteins during in vitro capacitation of boar sperm

    SciTech Connect

    Berger, T. )

    1990-11-01

    Exposed plasma membrane proteins were labeled with {sup 125}I before and after incubation of boar sperm under capacitating conditions. Labeled protein profiles were compared to the ability of the sperm to penetrate zona-free hamster ova. Quantitatively, the labeled sperm membrane proteins were primarily low Mr prior to capacitation. The majority of the labeled seminal plasma protein was also low Mr. After capacitation, two new proteins (64,000 Mr and 78,000 Mr) were labeled. Sperm did not exhibit these exposed membrane proteins when incubated under noncapacitating conditions. Appearance of these proteins was not correlated to the percentage of acrosome-reacted sperm. Although the 64,000 Mr protein was not consistently observed, the relative labeling of the 78,000 Mr protein was highly correlated with the ability of sperm to fuse with zona-free hamster ova. The 78,000 Mr protein may be a sperm protein involved in fusion with the egg plasma membrane.

  13. Moonlighting proteins in sperm-egg interactions.

    PubMed

    Petit, François M; Serres, Catherine; Auer, Jana

    2014-12-01

    Sperm-egg interaction is a highly species-specific step during the fertilization process. The first steps consist of recognition between proteins on the sperm head and zona pellucida (ZP) glycoproteins, the acellular coat that protects the oocyte. We aimed to determine which sperm head proteins interact with ZP2, ZP3 and ZP4 in humans. Two approaches were combined to identify these proteins: immunoblotting human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far-Western blotting of human sperm proteins overlaid by each of the human recombinant ZP (hrZP) proteins. We used a proteomic approach with 2D electrophoretic separation of sperm protein revealed using either ASAs eluted from infertile patients or recombinant human ZP glycoproteins expressed in Chinese-hamster ovary (CHO) cells. Only spots highlighted by both methods were analysed by MALDI-MS/MS for identification. We identified proteins already described in human spermatozoa, but implicated in different metabolic pathways such as glycolytic enzymes [phosphokinase type 3 (PK3), enolase 1 (ENO1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase A (ALDOA) and triose phosphate isomerase (TPI)], detoxification enzymes [GST Mu (GSTM) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) 4], ion channels [voltage-dependent anion channel 2 (VDAC2)] or structural proteins (outer dense fibre 2). Several proteins were localized on the sperm head by indirect immunofluorescence, and their interaction with ZP proteins was confirmed by co-precipitation experiments. These results confirm the complexity of the sperm-ZP recognition process in humans with the implication of different proteins interacting with the main three ZP glycoproteins. The multiple roles of these proteins suggest that they are multifaceted or moonlighting proteins.

  14. Functional features and protein network of human sperm-egg interaction.

    PubMed

    Sabetian, Soudabeh; Shamsir, Mohd Shahir; Abu Naser, Mohammed

    2014-12-01

    Elucidation of the sperm-egg interaction at the molecular level is one of the unresolved problems in sexual reproduction, and understanding the molecular mechanism is crucial in solving problems in infertility and failed in vitro fertilization (IVF). Many molecular interactions in the form of protein-protein interactions (PPIs) mediate the sperm-egg membrane interaction. Due to the complexity of the problem such as difficulties in analyzing in vivo membrane PPIs, many efforts have failed to comprehensively elucidate the fusion mechanism and the molecular interactions that mediate sperm-egg membrane fusion. The main purpose of this study was to reveal possible protein interactions and associated molecular function during sperm-egg interaction using a protein interaction network approach. Different databases have been used to construct the human sperm-egg interaction network. The constructed network revealed new interactions. These included CD151 and CD9 in human oocyte that interact with CD49 in sperm, and CD49 and ITGA4 in sperm that interact with CD63 and CD81, respectively, in the oocyte. These results showed that the different integrins in sperm may be involved in human sperm-egg interaction. It was also suggested that sperm ADAM2 plays a role as a protein candidate involved in sperm-egg membrane interaction by interacting with CD9 in the oocyte. Interleukin-4 receptor activity, receptor signaling protein tyrosine kinase activity, and manganese ion transmembrane transport activity are the major molecular functions in sperm-egg interaction protein network. The disease association analysis indicated that sperm-egg interaction defects are also reflected in other disease networks such as cardiovascular, hematological, and breast cancer diseases. By analyzing the network, we identified the major molecular functions and disease association genes in sperm-egg interaction protein. Further experimental studies will be required to confirm the significance of these new

  15. Proteomic identification of rainbow trout sperm proteins.

    PubMed

    Nynca, Joanna; Arnold, Georg J; Fröhlich, Thomas; Otte, Kathrin; Ciereszko, Andrzej

    2014-06-01

    Proteomics represents a powerful tool for the analysis of fish spermatozoa, since these cells are transcriptionally inactive. The aim of the present study was to generate an inventory of the most prominent rainbow trout sperm proteins by SDS-PAGE prefractionation combined with nano-LC-MS/MS based identification. This study provides the first in-depth analysis of the rainbow trout sperm proteome, with a total of 206 identified proteins. We found that rainbow trout spermatozoa are equipped with functionally diverse proteins related to energetic metabolism, signal transduction, protein turnover, transport, cytoskeleton, oxidative injuries, and stress and reproduction. The availability of a catalog of rainbow trout sperm proteins provides a crucial tool for the understanding of fundamental molecular processes in fish spermatozoa, for the ongoing development of novel markers of sperm quality and for the optimization of short- and long-term sperm preservation procedures. The MS data are available at ProteomeXchange with the dataset identifier PXD000355 and DOI 10.6019/PXD000355. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  17. Alterations to the Bull Sperm Surface Proteins That Bind Sperm to Oviductal Epithelium1

    PubMed Central

    Hung, Pei-hsuan; Suarez, Susan S.

    2012-01-01

    ABSTRACT Three Binder of SPerm proteins (BSP1, BSP3, BSP5) are secreted by bovine seminal vesicles into seminal plasma and adsorbed onto sperm. When sperm inseminated into the female reach the oviduct, the BSP proteins bind them to its epithelial lining, forming a sperm storage reservoir. Previously, we reported that binding of capacitated sperm to oviductal epithelium in vitro is lower than that of uncapacitated sperm and we proposed that reduced binding was due to loss of BSP proteins during capacitation. Because of differences in amino acid sequences, we predicted that each BSP would respond differently to capacitating conditions. To test whether all three BSP proteins were lost from sperm during capacitation and whether the kinetics of loss differed among the three BSP proteins, ejaculated bull sperm were incubated under various capacitating conditions, and then the amounts of BSP proteins remaining on the sperm were assayed by Western blotting. Capacitation was assayed by analysis of protein tyrosine phosphorylation. While loss of BSP1 was not detected, most of the BSP5 was lost from sperm during incubation in TALP medium, even without addition of the capacitation enhancers heparin and dbcAMP-IBMX. Surprisingly, a smaller molecular mass was detected by anti-BSP3 antibodies in extracts of incubated sperm. Its identity was confirmed as BSP3 by mass spectrometry, indicating that BSP3 undergoes modification on the sperm surface. These changes in the composition of BSP proteins on sperm could play a role in releasing sperm from the storage reservoir by modifying sperm interactions with the oviductal epithelium. PMID:22837481

  18. Detecting coevolution in mammalian sperm-egg fusion proteins.

    PubMed

    Claw, Katrina G; George, Renee D; Swanson, Willie J

    2014-06-01

    Interactions between sperm and egg proteins can occur physically between gamete surface-binding proteins, and genetically between gamete proteins that work in complementary pathways in which they may not physically interact. Physically interacting sperm-egg proteins have been functionally identified in only a few species, and none have been verified within mammals. Candidate genes on both the sperm and egg surfaces exist, but gene deletion studies do not support functional interactions between these sperm-egg proteins; interacting sperm-egg proteins thus remain elusive. Cooperative gamete proteins undergo rapid evolution, and it is predicted that these sperm-egg proteins will also have correlated evolutionary rates due to compensatory changes on both the sperm and egg. To explore potential physical and genetic interactions in sperm-egg proteins, we sequenced four candidate genes from diverse primate species, and used regression and likelihood methods to test for signatures of coevolution between sperm-egg gene pairs. With both methods, we found that the egg protein CD9 coevolves with the sperm protein IZUMO1, suggesting a physical or genetic interaction occurs between them. With regression analysis, we found that CD9 and CRISP2 have correlated rates of evolution, and with likelihood analysis, that CD9 and CRISP1 have correlated rates. This suggests that the different tests may reflect different levels of interaction, be it physical or genetic. Coevolution tests thus provide an exploratory method for detecting potentially interacting sperm-egg protein pairs.

  19. Proteins associated with critical sperm functions and sperm head shape are differentially expressed in morphologically abnormal bovine sperm induced by scrotal insulation.

    PubMed

    Shojaei Saadi, Habib A; van Riemsdijk, Evine; Dance, Alysha L; Rajamanickam, Gayathri D; Kastelic, John P; Thundathil, Jacob C

    2013-04-26

    The objective was to investigate expression patterns of proteins in pyriform sperm, a common morphological abnormality in bull sperm. Ejaculates were collected from sexually mature Holstein bulls (n=3) twice weekly for 10 weeks (pre-thermal insult samples). Testicular temperature was elevated in all bulls by scrotal insulation for 72 consecutive hours during week 2. Total sperm proteins were extracted from pre- and post-thermal insult sperm samples and subjected to two-dimensional gel electrophoresis. Among the protein spots detected, 131 spots were significantly expressed (False Detection Rate <0.01) with ≥ 2 fold changes between normal and pyriform sperm. Among them, 25 spots with ≥ 4 fold difference in expression patterns were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Expression of several proteins involved in sperm capacitation, sperm-egg interaction and sperm cytoskeletal structure was decreased in pyriform sperm, whereas proteins regulating antioxidant activity, apoptosis and metabolic activity were increased. Contents of reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm. In addition to understanding the molecular basis of functional deficiencies in sperm with specific morphological abnormalities, comparing normal versus morphologically abnormal sperm appeared to be a suitable experimental model for identifying important sperm functional proteins. To our knowledge, this study is the first report on differential expression of proteins in pyriform bovine sperm versus morphologically normal sperm. We report that expression of several proteins involved in sperm capacitation, sperm-egg interaction and sperm cytoskeletal structure was decreased in pyriform sperm, whereas proteins which regulate antioxidant activity, apoptosis and metabolic activity were increased. Contents of reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm. In addition to understanding

  20. Protein restriction affects sperm number but not sperm viability in male ants.

    PubMed

    Dávila, Francisco; Aron, Serge

    2017-07-01

    Sperm cells are costly to produce; diet should therefore affect sperm number and/or viability. In non-social insects and vertebrates, there is compelling evidence that diet influences sperm production. Less is known about this relationship in eusocial hymenopterans (all ants and some bees and wasps), whose mating systems impose unique selective pressures on sperm production. Males face physiological constraints: they acquire all of the resources they will use in future reproductive efforts as larvae and emerge from the pupal stage with a fixed, non-renewable amount of sperm. Furthermore, males die shortly after copulation, but their genetic material persists for years since their spermatozoa are stored in their mates' spermathecae. We examined the effects of protein restriction during larval development on sperm number and viability in the Argentine ant Linepithema humile. We also looked at its impact on male development, adult mass, and adult fluctuating asymmetry. We found that protein restriction during larval development significantly reduced sperm production, but not sperm viability. It did not affect the number of males reared, male mass, or male asymmetry. However, males from protein-restricted nests developed much more slowly than males from protein-supplemented nests. These results suggest investing in sperm quality and in somatic growth, which enhances a male's ability to disperse and find a mate, are critical to successful male reproduction. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

    SciTech Connect

    Lee, C.Y.G.; Huang, Y.S.; Hu, P.C.; Gomel, V.; Menge, A.C.

    1982-06-01

    A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 +/- 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility.

  2. Egg jelly proteins stimulate directed motility in Xenopus laevis sperm.

    PubMed

    Burnett, Lindsey A; Sugiyama, Hitoshi; Bieber, Allan L; Chandler, Douglas E

    2011-06-01

    Previously we have shown that extracts from Xenopus egg jelly (egg water) increase the passage of sperm through a porous membrane in a dose-dependent manner. Although this assay has shown that sperm accumulation occurs only in the presence of an egg water gradient, it has not revealed the dynamic features of how Xenopus sperm swim in such gradients. Here, we use video microscopic observations to trace sperm trajectories in a Zigmond chamber. Our results show that Xenopus sperm swim in linear and gently curving paths and only infrequently perform turns. In the presence of an egg water gradient, however, the percent of sperm swimming up the gradient axis and the net distance traveled by each sperm along this axis was increased significantly. There was no change in curvilinear velocity. Rather, the orientation of sperm travel was shifted to more closely match that of the gradient axis. In addition, using a porous filter assay, we demonstrate that the egg water protein allurin, in both purified and recombinant forms, stimulates directed motility of sperm. Finally, we use Oregon Green 488-conjugated allurin to show that this protein binds primarily to the sperm midpiece; binding of allurin to the entire head was observed in a minor subpopulation of sperm. Dose dependence of allurin binding occurred over the 0-1 µg/ml range and correlated well with previously published dose-dependent sperm attraction data. Binding was rapid with a half-time of about 10 sec. These data suggest that egg water proteins bind to sperm and modify sperm-orienting behavior.

  3. Boar seminal plasma exosomes: effect on sperm function and protein identification by sequencing.

    PubMed

    Piehl, Lidia L; Fischman, M Laura; Hellman, Ulf; Cisale, Humberto; Miranda, Patricia V

    2013-04-15

    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

  4. Proteomics of ionomycin-induced ascidian sperm reaction: Released and exposed sperm proteins in the ascidian Ciona intestinalis.

    PubMed

    Nakazawa, Shiori; Shirae-Kurabayashi, Maki; Otsuka, Kei; Sawada, Hitoshi

    2015-12-01

    Sperm proteins mediating sperm-egg interaction should be exhibited on the sperm surface, or exposed or released when sperm approach an egg. In ascidians (protochordates), sperm undergo a sperm reaction, characterized by enhanced sperm motility and mitochondrial swelling and shedding on contact with the vitelline coat (VC) or by treatment with Ca(2+) ionophore. Here, proteomic analysis was conducted on sperm exudates and sperm surface proteins using ionomycin-induced sperm reaction and cell-impermeable labeling in Ciona intestinalis type A (C. robusta). In the exudate from sperm treated with ionomycin, membrane proteins including a possible VC receptor CiUrabin were abundant, indicating the release of membranous compartments during sperm reaction. Among the surface proteins XP_009859314.1 (uncharacterized protein exhibiting homology to HrTTSP-1) was most abundant before the sperm reaction, but XP_004227079.1 (unknown Ig superfamily protein) appears to be most abundantly exposed by the sperm reaction. Moreover, proteins containing a notable set of domains, astacin-like metalloprotease domain and thrombospondin type 1 repeat(s), were found in this fraction. Possible roles in fertilization as well as localizations and behaviors of these proteins are discussed.

  5. The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

    PubMed Central

    Pantano, Lorena; Jodar, Meritxell; Bak, Mads; Ballescà, Josep Lluís; Tommerup, Niels; Oliva, Rafael; Vavouri, Tanya

    2015-01-01

    At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline. PMID:25904136

  6. Association of the protein D and protein E forms of rat CRISP1 with epididymal sperm.

    PubMed

    Roberts, Kenneth P; Ensrud-Bowlin, Kathy M; Piehl, Laura B; Parent, Karlye R; Bernhardt, Miranda L; Hamilton, David W

    2008-12-01

    Cysteine-rich secretory protein 1 (CRISP1) is a secretory glycoprotein produced by the rat epididymal epithelium in two forms, referred to as proteins D and E. CRISP1 has been implicated in sperm-egg fusion and has been shown to suppress capacitation in rat sperm. Several studies have suggested that CRISP1 associates transiently with the sperm surface, whereas others have shown that at least a portion of CRISP1 persists on the surface. In the present study, we demonstrate that protein D associates transiently with the sperm surface in a concentration-dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation-inhibiting activity. In contrast, protein E persists on the sperm surface after all exogenous protein D has been dissociated. Comparison of caput and cauda sperm reveal that protein E becomes bound to the sperm in the cauda epididymidis. We show that protein E associates with caput sperm, which do not normally have it on their surfaces, in vitro in a time- and temperature-dependent manner. These studies demonstrate that most CRISP1 interacts with sperm transiently, possibly with a specific receptor on the sperm surface, consistent with its action in suppressing capacitation during epididymal storage of sperm. These studies also confirm a tightly bound population of protein E that could act in the female tract.

  7. Egg Activation at Fertilization by a Soluble Sperm Protein.

    PubMed

    Swann, Karl; Lai, F Anthony

    2016-01-01

    The most fundamental unresolved issue of fertilization is to define how the sperm activates the egg to begin embryo development. Egg activation at fertilization in all species thus far examined is caused by some form of transient increase in the cytoplasmic free Ca(2+) concentration. What has not been clear, however, is precisely how the sperm triggers the large changes in Ca(2+) observed within the egg cytoplasm. Here, we review the studies indicating that the fertilizing sperm stimulates a cytosolic Ca(2+) increase in the egg specifically by delivering a soluble factor that diffuses into the cytosolic space of the egg upon gamete membrane fusion. Evidence is primarily considered in species of eggs where the sperm has been shown to elicit a cytosolic Ca(2+) increase by initiating Ca(2+) release from intracellular Ca(2+) stores. We suggest that our best understanding of these signaling events is in mammals, where the sperm triggers a prolonged series of intracellular Ca(2+) oscillations. The strongest empirical studies to date suggest that mammalian sperm-triggered Ca(2+) oscillations are caused by the introduction of a sperm-specific protein, called phospholipase C-zeta (PLCζ) that generates inositol trisphosphate within the egg. We will discuss the role and mechanism of action of PLCζ in detail at a molecular and cellular level. We will also consider some of the evidence that a soluble sperm protein might be involved in egg activation in nonmammalian species.

  8. Ram seminal plasma proteins contribute to sperm capacitation and modulate sperm-zona pellucida interaction.

    PubMed

    Luna, C; Colás, C; Casao, A; Serrano, E; Domingo, J; Pérez-Pé, R; Cebrián-Pérez, J A; Muiño-Blanco, T

    2015-03-01

    Incubation of ram spermatozoa in capacitating conditions with cAMP-elevating agents promotes a progressive time-dependent increase in the capacitated sperm subpopulation. In this study, the fertilizing capacity of ram spermatozoa (ability to bind to the zona pellucida, ZBA rate) capacitated in these conditions was determined. The results showed an increase (P < 0.001) in ZBA rate related to control samples in basal medium that contained BSA, calcium, and bicarbonate (1.97 ± 0.19 vs. 1.31 ± 0.09 sperm bound/oocyte, respectively). A significant correlation between protein tyrosine phosphorylation and ZBA rate (P < 0.05, r = 0.501) corroborated that incubation in a "high-cAMP" environment improves the fertilizing ability of ram spermatozoa. Likewise, the presence of two seminal plasma (SP) proteins able to protect sperm against cold shock (RSVP14 and RSVP20) was evidenced in both SP and the ram sperm surface, and their influence in the fertilizing ability of spermatozoa capacitated in basal medium or with cAMP-elevating agents was determined. The results verified that RSVP14 and RSVP20 act as decapacitating factors given that their addition to SP-free sperm samples previously to capacitation maintained high proportions of the noncapacitated sperm pattern with no increase in protein tyrosine phosphorylation. However, the obtained ZBA rate in the high-cAMP-containing samples was increased in the presence of RSVP20 (P < 0.05). These findings would indicate that the stimulating effect exerted by this protein on the sperm-oocyte binding occurs downstream from the cAMP generation and that the mechanisms by which RSVP20 promotes the zona pellucida binding might be independent of protein tyrosine phosphorylation.

  9. Seminal plasma proteins interacting with sperm surface revert capacitation indicators in frozen-thawed ram sperm.

    PubMed

    Ledesma, Alba; Fernández-Alegre, Estela; Cano, Adriana; Hozbor, Federico; Martínez-Pastor, Felipe; Cesari, Andreína

    2016-10-01

    This study was conducted to evaluate the effects of interacting seminal plasma proteins (iSPP) obtained by AV or EE on frozen-thawed ram sperm in order to test the hypothesis whether this fraction could be sufficient to emulate the effect of complete seminal plasma (SP). Additionally, we evaluated whether these proteins have a differential effect between spermatozoa from high and low fertility rams and between breeding and non-breeding seasons. We assessed sperm motility, quality parameters (intracellular reactive oxygen species, membrane fluidity, plasma membrane permeability and mitochondrial activity) and capacitation status. The main findings from this work were: i) iSPP had no effect on sperm motility, whereas SP (AV or EE) addition produced the highest values of total motility (74.13±2.99 and 72.27±2.99 for AV and EE, respectively) and progressive motility (64.97±2.64 and 63.73±2.64 for AV and EE, respectively); ii) iSPP had no effect on sperm quality parameters (p>0.05), but whole SP improved all parameters evaluated. Moreover, SP collected by AV yielded significantly higher viability (44.60±2.87) and sperm with stable plasma membrane (44.56±2.49) comparing with the addition of SP collected by EE (35.80±2.47 and 36.67±1.71, respectively); iii) iSPP and SP collected by EE, but not by AV, reverted molecular signals of capacitation as protein tyrosine phosphorylation caused by freezing temperatures; iv) there were no effects of fertility or season in sperm quality parameters evaluated. This study demonstrated that, although the iSPP have a clear decapacitating effect, including the ability to revert cryo-capacitation indicators, they are not sufficient to emulate the effects of complete SP regarding sperm functional parameters.

  10. Differential extraction and enrichment of human sperm surface proteins in a proteome: identification of immunocontraceptive candidates.

    PubMed

    Shetty, J; Diekman, A B; Jayes, F C; Sherman, N E; Naaby-Hansen, S; Flickinger, C J; Herr, J C

    2001-08-01

    The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two-dimensional (2-D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim-up sperm were either solubilized directly or solubilized after surface labeling with sulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin). Comparisons were made of proteins extracted with four lysis buffers: (i) Celis buffer containing 9.8 M urea and 2% Igepal CA-630; (ii) 1% Triton X (TX)-100; (iii) 1.7% TX-114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high-resolution 2-D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains: the sperm surface (SAGA-1), the acrosome (SP-10), and the cytoskeleton (alpha-tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20-s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX-100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX-100, TX-114, and 1 M NaCl. Extraction with TX-114 followed by phase-partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.

  11. Two-dimensional polyacrylamide gel electrophoresis of membrane proteins from flow cytometrically sorted ram sperm.

    PubMed

    Leahy, T; Marti, J I; Crossett, B; Evan, G; Maxwell, W M C

    2011-03-15

    Membrane proteins orchestrate key events required for participation of sperm in fertilisation. These proteins may be removed or altered due to the mechanical and dilution stressors associated with sex-sorting of sperm. Ram sperm were incubated with Hoechst 33342 and flow-sorted. Sex-selected (viable, orientated) and waste (separated into non-viable or non-orientated) sperm populations were collected, or sperm were not sorted. Sperm membrane proteins were extracted and characterised by one- and two-dimensional PAGE. Densiometric analysis of protein bands separated by one-dimensional PAGE showed proteins of 30 and 28 kDa as doublet bands on non-sorted sperm, and single bands on sex-sorted sperm, and the proportion of a 14 kDa protein was 3-fold higher in non-sorted compared to sorted sperm. Proteins in the 14 kDa band were identified by mass spectroscopy as a bovine Fibronectin type-2 protein (Fn-2), cytochrome oxidase 5a (Cox5a) and a sperm membrane associated protein (SLLP1). The abundance of these proteins in the two-dimensional gels was lowest in the sorted sperm population identified as viable during sorting (orientated and non-orientated sperm) and highest in the non-viable sperm population (P < 0.001). We concluded that the membrane protein profile was different for sex-sorted compared with non-sorted sperm, due to the selection of plasma membrane-intact cells in the flow-sorted population. This provided further evidence that sex-sorting selected a homogenous population of sperm with superior function to non-sorted sperm. Furthermore, this was apparently the first time sperm membrane acrosome associated protein was reported in ram sperm, and it was demonstrated that seminal plasma proteins remained on the sperm membrane after sex-sorting. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Activation of proacrosin accompanies upregulation of sp32 protein tyrosine phosphorylation in pig sperm.

    PubMed

    Sun, P L; Yang, L X; Cui, J-J; Tian, Y; Liu, Y; Jin, Y

    2013-12-11

    This study investigated the relationship between acrosin activation and pig sperm proacrosin binding protein (sp32) phosphorylation levels. Differently processed pig spermatozoa (fresh semen sperm, capacitation sperm, acrosome reaction sperm, capacitation-like sperm, and thawed sperm) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. The fresh semen and capacitation sperm groups both produced proacrosin protein bands of 55 kDa; however, the result of the fresh semen sperm group was clearer than that of the capacitation sperm group. The thawed sperm group showed a shallow strip at 55 kDa. The capacitation and acrosome reaction sperm groups produced obvious proacrosin protein bands at 35 kDa, and the strips of the capacitation sperm group were again clearer. A faint band was visible at 32 kDa in the acrosome reaction sperm group. The capacitation, thawed, and acrosome reaction sperm groups showed significant strips in sp32, and the bands of the acrosome reaction sperm group were shallower than those of the 2 other groups. The capacitation and thawed sperm groups produced significant strips at 40 kDa, and the capacitation sperm group produced an additional strip at 55 kDa. In conclusion, sp32 phosphorylation levels can promote proacrosin activation into the active acrosin.

  13. Sustained High Protein-tyrosine Phosphatase 1B Activity in the Sperm of Obese Males Impairs the Sperm Acrosome Reaction*

    PubMed Central

    Shi, Lei; Zhang, Qipeng; Xu, Binqiang; Jiang, Xiaohong; Dai, Yutian; Zhang, Chen-Yu; Zen, Ke

    2014-01-01

    Evidence of a causal link between male obesity and subfertility or infertility has been demonstrated previously. However, the mechanism underlying this link is incompletely understood. Here, we report that sustained high protein-tyrosine phosphatase 1B (PTP1B) activity in sperm of obese donors plays an essential role in coupling male obesity and subfertility or infertility. First, PTP1B level and activity were significantly higher in sperm from ob/ob mice than in wild-type littermates. High PTP1B level and activity in sperm was also observed in obese patients compared with non-obese donors. The enhanced sperm PTP1B level and activity in ob/ob mice and obese patients correlated with a defect of the sperm acrosome reaction (AR). Second, treating sperm from male ob/ob mice or obese men with a specific PTP1B inhibitor largely restored the sperm AR. Finally, blockade of sperm AR by enhanced PTP1B activity in male ob/ob mice or obese men was due to prolonged dephosphorylation of N-ethylmaleimide-sensitive factor by PTP1B, leading to the inability to reassemble the trans-SNARE complexes, which is a critical step in sperm acrosomal exocytosis. In summary, our study demonstrates for the first time that a sustained high PTP1B level or activity in the sperm of obese donors causes a defect of sperm AR and that PTP1B is a novel potential therapeutic target for male infertility treatment. PMID:24519936

  14. The localization of protein carboxyl-methylase in sperm tails

    PubMed Central

    1980-01-01

    Protein carboxyl-methylase (PCM), an enzyme known to be involved in exocytotic secretion and chemotaxis, has been studied in rat and rabbit spermatozoa. PCM activity and its substrate methyl acceptor protein(s) (MAP) were demonstrated in the supernate after solubilization of the sperm cell membrane by detergent (Triton X-100). A protein methylesterase that hydrolyzes methyl ester bonds created by PCM was demonstrated in rabbit but not in rat spermatozoa. This enzyme was not solubilized by nonionic detergent. The specific activities of PCM in rat spermatozoa from caput and cauda epididymis were similar and lower than that found in testis. By contrast, MAP substrates were low in testis and increased in parallel with sperm maturation in the epididymis. Multiple MAP were demonstrated in spermatozoa by polyacrylamide gel electrophoresis. The pattern of these proteins was similar in spermatozoa from different portions of the reproductive tract. Fractionation of heads and tails of rat spermatozoa on sucrose gradients indicated that PCM was found exclusively in the tail fraction, whereas MAP was detected both in head and tail fractions. The presence of all the components of the protein carboxyl-methylation system in spermatozoa and the localization of PCM and some of its substrates in the sperm tail are consistent with their involvement in sperm cell motility. PMID:7400214

  15. Heterogenous turnover of sperm and seminal vesicle proteins in the mouse revealed by dynamic metabolic labeling.

    PubMed

    Claydon, Amy J; Ramm, Steven A; Pennington, Andrea; Hurst, Jane L; Stockley, Paula; Beynon, Robert

    2012-06-01

    Plasticity in ejaculate composition is predicted as an adaptive response to the evolutionary selective pressure of sperm competition. However, to respond rapidly to local competitive conditions requires dynamic modulation in the production of functionally relevant ejaculate proteins. Here we combine metabolic labeling of proteins with proteomics to explore the opportunity for such modulation within mammalian ejaculates. We assessed the rate at which proteins are synthesized and incorporated in the seminal vesicles of male house mice (Mus musculus domesticus), where major seminal fluid proteins with potential roles in sperm competition are produced. We compared rates of protein turnover in the seminal vesicle with those during spermatogenesis, the timing of which is well known in mice. The subjects were fed a diet containing deuterated valine ([(2)H(8)]valine) for up to 35 days, and the incorporation of dietary-labeled amino acid into seminal vesicle- or sperm-specific proteins was assessed by liquid chromatography-mass spectrometry of samples recovered from the seminal vesicle lumen and cauda epididymis, respectively. Analyses of epididymal contents were consistent with the known duration of spermatogenesis and sperm maturation in this species and in addition revealed evidence for a subset of epididymal proteins subject to rapid turnover. For seminal vesicle proteins, incorporation of the stable isotope was evident from day 2 of labeling, reaching a plateau of labeling by day 24. Hence, even in the absence of copulation, the seminal vesicle proteins and certain epididymal proteins demonstrate considerable turnover, a response that is consonant with the capacity to rapidly modulate protein production. These techniques can now be used to assess the extent of phenotypic plasticity in mammalian ejaculate production and allocation according to social and environmental cues of sperm competition.

  16. Valosin-containing protein/p97 interacts with sperm-activating and sperm-attracting factor (SAAF) in the ascidian egg and modulates sperm-attracting activity.

    PubMed

    Kondoh, Eri; Konno, Aru; Inaba, Kazuo; Oishi, Tohru; Murata, Michio; Yoshida, Manabu

    2008-10-01

    Sperm chemotaxis toward an egg is observed in many animals, and the control of sperm-attracting activity is thought to play an important role in ensuring fertilization. However, the mechanism underlying the release of a sperm attractant from an egg is still obscure. In this study, we examined the systems involved in the release of sperm-activating and sperm-attracting factor (SAAF), which is the sperm attractant of the ascidian Ciona intestinalis. Here, we show that the egg acquires sperm-attracting activity after germinal vesicle breakdown. Further, since the cytoplasmic extracts of immature oocytes exhibit no sperm-attracting activity, the SAAF in oocytes may be activated after germinal vesicle breakdown. We found 13 SAAF-binding proteins in an egg plasma membrane extract and identified five proteins by proteomic analysis: valosin-containing protein (VCP)/p97, proteasome alpha 2 subunit, MGC97756 protein, proteasome subunit Y, and beta-tubulin. In particular, the interaction between VCP/p97 and SAAF was confirmed by a pull-down assay. VCP/p97 is initially localized in the germinal vesicle, and during oocyte maturation, it shifts to the endoplasmic reticulum in the cortical regions. Thus, VCP/p97 is a potential modulator of SAAF release from the egg.

  17. Comparison of semen variables, sperm DNA damage and sperm membrane proteins in two male layer breeder lines.

    PubMed

    M, Shanmugam; T R, Kannaki; A, Vinoth

    2016-09-01

    Semen variables are affected by the breed and strain of chicken. The present study was undertaken to compare the semen quality in two lines of adult chickens with particular reference to sperm chromatin condensation, sperm DNA damage and sperm membrane proteins. Semen from a PD3 and White Leghorn control line was collected at 46 and 47 weeks and 55 weeks of age. The semen was evaluated for gross variables and sperm chromatin condensation by aniline blue staining. Sperm DNA damage was assessed by using the comet assay at 47 weeks of age and sperm membrane proteins were assessed at 55 weeks of age. The duration of fertility was studied by inseminating 100 million sperm once into the hens of the same line as well as another line. The eggs were collected after insemination for 15days and incubated. The eggs were candled on 18th day of incubation for observing embryonic development. The White Leghorn control line had a greater sperm concentration and lesser percentage of morphologically abnormal sperm at the different ages where assessments occurred. There was no difference in sperm chromatin condensation, DNA damage and membrane proteins between the lines. Only low molecular weight protein bands of less than 95kDa were observed in samples of both lines. The line from which semen was used had no effect on the duration over which fertility was sustained after insemination either when used in the same line or another line. Thus, from the results of the present study it may be concluded that there was a difference in gross semen variables between the lines that were studied, however, the sperm chromatin condensation, DNA damage, membrane proteins and duration over which fertility was sustained after insemination did not differ between the lines. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro

    PubMed Central

    LIU, Jinghui; MAREY, Mohamed A.; KOWSAR, Rasoul; HAMBRUCH, Nina; SHIMIZU, Takashi; HANEDA, Shingo; MATSUI, Motozumi; SASAKI, Motoki; HAYAKAWA, Hiroyuki; PFARRER, Christiane; MIYAMOTO, Akio

    2014-01-01

    We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E2 (PGE2) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE2 at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct. PMID:24931131

  19. Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage.

    PubMed

    Bernardini, A; Hozbor, F; Sanchez, E; Fornés, M W; Alberio, R H; Cesari, A

    2011-08-01

    Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. AM67, a secretory component of the guinea pig sperm acrosomal matrix, is related to mouse sperm protein sp56 and the complement component 4-binding proteins.

    PubMed

    Foster, J A; Friday, B B; Maulit, M T; Blobel, C; Winfrey, V P; Olson, G E; Kim, K S; Gerton, G L

    1997-05-09

    The guinea pig sperm acrosomal matrix is the dense core of the acrosome and is likely to be important in acrosome biogenesis and fertilization. Isolated acrosomal matrices are composed of a limited number of major bands when analyzed by SDS-polyacrylamide gel electrophoresis, among which is a Mr 67,000 protein that we have termed AM67. Indirect immunofluorescence demonstrated that AM67 is localized to the apical segment of the cauda epididymal sperm acrosome. Immunoelectron microscopy further refined the localization of AM67 to the M1 (dorsal bulge) domain within the acrosome. Using a polymerase chain reaction product based upon tryptic peptide sequences from AM67, a lambdagt11 guinea pig testis cDNA library was screened to yield two cDNA clones that encode the AM67 peptides. Northern analysis revealed that AM67 is transcribed as a 1. 9-kilobase testis-specific mRNA. The complete AM67 sequence encodes a prepropolypeptide of 533 amino acids with a calculated Mr of 59, 768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have a Mr of 56,851 and seven consensus sites for asparagine-linked glycosylation. The deduced amino acid sequence of AM67 is most similar to those of the mouse sperm protein sp56 and the alpha-subunits of complement component 4-binding proteins from various mammalian species. Although mouse sp56 has been reported to be a cell-surface receptor for the murine zona pellucida glycoprotein ZP3, standard immunoelectron microscopy using the anti-sp56 monoclonal antibody 7C5 detected sp56 within the mouse sperm acrosome, but failed to detect sp56 on the surface of acrosome-intact mouse sperm. Furthermore, acrosomal labeling was detected in mouse sperm prepared for immunofluorescence using paraformaldehyde fixation, but was not observed with live unfixed sperm. Thus, the finding that sp56 is present within the acrosome provides further support that sp56 and AM67 are orthologues and suggests that sp56 may function in

  1. Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking

    SciTech Connect

    Bleil, J.D.; Wassarman, P.M. )

    1990-07-01

    During the process of fertilization in mammals, sperm bind in a relatively species-specific manner to the zona pellucida (ZP) of ovulated eggs. ZP3, a glycoprotein found in the mouse egg zona pellucida, serves as receptor for sperm during gamete adhesion. We report here that a Mr 56,000 protein found on mouse sperm has properties expected for a sperm component that recognizes and binds to ZP3. This sperm protein is radiolabeled preferentially by a photoactivatable heterobifunctional crosslinker (Denny-Jaffee reagent) covalently linked to purified ZP3, binds very tightly to ZP3-affinity columns, and is localized to heads of acrosome-intact but not acrosome-reacted sperm. These and other findings suggest that this protein may be a ZP3-binding protein that, together with the sperm receptor, supports species-specific binding of mouse sperm to unfertilized eggs.

  2. Inhibition of tyrosine phosphorylation of sperm flagellar proteins, outer dense fiber protein-2 and tektin-2, is associated with impaired motility during capacitation of hamster spermatozoa.

    PubMed

    Mariappa, Daniel; Aladakatti, Ravindranath H; Dasari, Santosh K; Sreekumar, Arun; Wolkowicz, Michael; van der Hoorn, Frans; Seshagiri, Polani B

    2010-02-01

    In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

  3. An ATP-binding cassette transporter is a major glycoprotein of sea urchin sperm membranes.

    PubMed

    Mengerink, Kathryn J; Vacquier, Victor D

    2002-10-25

    Sperm are terminally differentiated cells that undergo several membrane-altering events before fusion with eggs. One event, the sea urchin sperm acrosome reaction (AR), is blocked by the lectin wheat germ agglutinin (WGA). In an effort to identify proteins involved in the AR induction, the peptide sequence was obtained from a 220-kDa WGA-binding protein. Degenerate PCR and library screening resulted in the full-length deduced amino acid sequence of an ATP-binding cassette transporter, suABCA. The protein of 1,764 residues has two transmembrane regions, two nucleotide-binding domains, and is most closely related to the human ABC subfamily A member 3 transporter (ABCA3). Sequence analysis suggests a large extracellular loop between transmembrane spanning segments 7 and 8, with five N-linked glycosylation sites. An antibody made to the loop region binds to non-permeabilized cells, supporting that this region is extracellular. suABCA is found in sperm membrane vesicles, it can be solubilized with nonionic detergents, and it shifts from 220 to 200 kDa upon protein:N-glycanase F digestion. suABCA localizes to the entire surface of sperm in a punctate pattern, but is not detected in lipid rafts. Based on its relationship to subfamily A, suABCA is most likely involved in phospholipid or cholesterol transport. This is the first investigation of an ABC transporter in animal sperm.

  4. Heat Shock Protein 90 Has Roles in Intracellular Calcium Homeostasis, Protein Tyrosine Phosphorylation Regulation, and Progesterone-Responsive Sperm Function in Human Sperm

    PubMed Central

    Chen, Aijun; Jiang, Youfang; Xie, Haifeng; Shi, Qixian; Zhang, Songying; Ni, Ya

    2014-01-01

    Heat shock protein 90 plays critical roles in client protein maturation, signal transduction, protein folding and degradation, and morphological evolution; however, its function in human sperm is not fully understood. Therefore, our objective in this study was to elucidate the mechanism by which heat shock protein 90 exerts its effects on human sperm function. By performing indirect immunofluorescence staining, we found that heat shock protein 90 was localized primarily in the neck, midpiece, and tail regions of human sperm, and that its expression increased with increasing incubation time under capacitation conditions. Geldanamycin, a specific inhibitor of heat shock protein 90, was shown to inhibit this increase in heat shock protein 90 expression in western blotting analyses. Using a multifunctional microplate reader to examine Fluo-3 AM-loaded sperm, we observed for the first time that inhibition of heat shock protein 90 by using geldanamycin significantly decreased intracellular calcium concentrations during capacitation. Moreover, western blot analysis showed that geldanamycin enhanced tyrosine phosphorylation of several proteins, including heat shock protein 90, in a dose-dependent manner. The effects of geldanamycin on human sperm function in the absence or presence of progesterone was evaluated by performing chlortetracycline staining and by using a computer-assisted sperm analyzer. We found that geldanamycin alone did not affect sperm capacitation, hyperactivation, and motility, but did so in the presence of progesterone. Taken together, these data suggest that heat shock protein 90, which increases in expression in human sperm during capacitation, has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-stimulated sperm function. In this study, we provide new insights into the roles of heat shock protein 90 in sperm function. PMID:25541943

  5. [Progress in researches on A-kinase anchor proteins in sperm].

    PubMed

    Li, Yang; Liu, Jihong

    2004-09-01

    This article reviews the advances in the studies of A-kinase anchor proteins (AKAPs) in sperm, including their classification, structure and mechanism. The influence of AKAPs that are involved on sperm mobility and acrosome reaction is emphasized. We hope it could play a directive role in the studies of AKAPs that are involved in regulating sperm mobility and acrosome reaction.

  6. Sperm and spermatids contain different proteins and bind distinct egg factors.

    PubMed

    Teperek, Marta; Miyamoto, Kei; Simeone, Angela; Feret, Renata; Deery, Michael J; Gurdon, John B; Jullien, Jerome

    2014-09-19

    Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.

  7. Proteins involved in motility and sperm-egg interaction evolve more rapidly in mouse spermatozoa.

    PubMed

    Vicens, Alberto; Lüke, Lena; Roldan, Eduardo R S

    2014-01-01

    Proteomic studies of spermatozoa have identified a large catalog of integral sperm proteins. Rapid evolution of these proteins may underlie adaptive changes of sperm traits involved in different events leading to fertilization, although the selective forces underlying such rapid evolution are not well understood. A variety of selective forces may differentially affect several steps ending in fertilization, thus resulting in a compartmentalized adaptation of sperm proteins. Here we analyzed the evolution of genes associated to various events in the sperm's life, from sperm formation to sperm-egg interaction. Evolutionary analyses were performed on gene sequences from 17 mouse strains whose genomes have been sequenced. Four of these are derived from wild Mus musculus, M. domesticus, M. castaneus and M. spretus. We found a higher proportion of genes exhibiting a signature of positive selection among those related to sperm motility and sperm-egg interaction. Furthermore, sperm proteins involved in sperm-egg interaction exhibited accelerated evolution in comparison to those involved in other events. Thus, we identified a large set of candidate proteins for future comparative analyses of genotype-phenotype associations in spermatozoa of species subjected to different sexual selection pressures. Adaptive evolution of proteins involved in motility could be driven by sperm competition, since this selective force is known to increase the proportion of motile sperm and their swimming velocity. On the other hand, sperm proteins involved in gamete interaction could be coevolving with their egg partners through episodes of sexual selection or sexual conflict resulting in species-specific sperm-egg interactions and barriers preventing interspecies fertilization.

  8. Atlantic salmon eggs favour sperm in competition that have similar major histocompatibility alleles

    PubMed Central

    Yeates, Sarah E.; Einum, Sigurd; Fleming, Ian A.; Megens, Hendrik-Jan; Stet, René J.M.; Hindar, Kjetil; Holt, William V.; Van Look, Katrien J.W.; Gage, Matthew J.G.

    2008-01-01

    Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran in vitro fertilization competitions that mimicked the gametic microenvironment, and replicated a paired-male experimental design that allowed us to compare differences in sperm competition success among males when their sperm compete for eggs from females that were genetically either similar or dissimilar at the MH class I locus. Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm–egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation. PMID:18854296

  9. Identification of Bovine Sperm Acrosomal Proteins that Interact with a 32kDa Acrosomal Matrix Protein

    PubMed Central

    Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

    2016-01-01

    Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction is comprised of three major (54, 50, and 45kDa) and several minor (38–19kDa) polypeptides. The set of minor polypeptides (38–19kDa) termed “OMCrpf polypeptides” is selectively solubilized by high-pH extraction (pH 10.5) while the three major polypeptides (55, 50 and 45kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38–19kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment; whereas, IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidyl choline (LPC

  10. Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

    PubMed

    Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

    2016-03-01

    Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome

  11. Polymeric ADAM protein mimics interrogate mammalian sperm-egg binding.

    PubMed

    Lee, Younjoo; Sampson, Nicole S

    2009-03-23

    The sperm proteins ADAM2 and ADAM3, members of the ADAM family of proteins, have been implicated in mammalian sperm-egg binding. However, elucidating their roles is complex because of the interdependence of ADAM protein expression in the testis. Hence, multivalent probes containing the three-amino acid binding sequence of ADAM2, glutamate-cysteine-aspartate (ECD), and ADAM3, glutamine-cysteine-aspartate (QCD), were designed, synthesized, and tested to investigate gamete interactions. In this work, ECD polymer mimics were synthesized by ring-opening metathesis polymerization with a faster initiating ruthenium catalyst than previously used. Polymers containing 100 copies of the ECD peptide mimic were found to be the best inhibitors of fertilization. The multivalent QCD polymers were also tested as inhibitors of fertilization. The structure-activity profile was the same as ECD polymers, but the overall potency was lower. Both ECD and QCD polymers require the presence of beta(1) integrin to inhibit fertilization. Next, triblock ABA and ABC copolymers containing both ECD and QCD ligands were synthesized with 96 monomer spacers as their B blocks. Although these polymers had lower densities of ECD and QCD peptides, their potencies correlated with the potencies of their corresponding homopolymers. In addition, no synergy between ECD and QCD mimics was observed. All the data suggest that QCD and ECD bind to the same complex of proteins that includes beta(1) integrin.

  12. Protein deubiquitination during oocyte maturation influences sperm function during fertilisation, antipolyspermy defense and embryo development.

    PubMed

    Yi, Young-Joo; Sutovsky, Miriam; Song, Won-Hee; Sutovsky, Peter

    2015-11-01

    Ubiquitination is a covalent post-translational modification of proteins by the chaperone protein ubiquitin. Upon docking to the 26S proteasome, ubiquitin is released from the substrate protein by deubiquitinating enzymes (DUBs). We hypothesised that specific inhibitors of two closely related oocyte DUBs, namely inhibitors of the ubiquitin C-terminal hydrolases (UCH) UCHL1 (L1 inhibitor) and UCHL3 (L3 inhibitor), would alter porcine oocyte maturation and influence sperm function and embryo development. Aberrant cortical granule (CG) migration and meiotic spindle defects were observed in oocytes matured with the L1 or L3 inhibitor. Embryo development was delayed or blocked in oocytes matured with the general DUB inhibitor PR-619. Aggresomes, the cellular stress-inducible aggregates of ubiquitinated proteins, formed in oocytes matured with L1 inhibitor or PR-619, a likely consequence of impaired protein turnover. Proteomic analysis identified the major vault protein (MVP) as the most prominent protein accumulated in oocytes matured with PR-619, suggesting that the inhibition of deubiquitination altered the turnover of MVP. The mitophagy/autophagy of sperm-contributed mitochondria inside the fertilised oocytes was hindered by DUB inhibitors. It is concluded that DUB inhibitors alter porcine oocyte maturation, fertilisation and preimplantation embryo development. By regulating the turnover of oocyte proteins and mono-ubiquitin regeneration, the DUBs may promote the acquisition of developmental competence during oocyte maturation.

  13. Cryptic female choice favours sperm from major histocompatibility complex-dissimilar males.

    PubMed

    Løvlie, Hanne; Gillingham, Mark A F; Worley, Kirsty; Pizzari, Tommaso; Richardson, David S

    2013-10-22

    Cryptic female choice may enable polyandrous females to avoid inbreeding or bias offspring variability at key loci after mating. However, the role of these genetic benefits in cryptic female choice remains poorly understood. Female red junglefowl, Gallus gallus, bias sperm use in favour of unrelated males. Here, we experimentally investigate whether this bias is driven by relatedness per se, or by similarity at the major histocompatibility complex (MHC), genes central to vertebrate acquired immunity, where polymorphism is critical to an individual's ability to combat pathogens. Through experimentally controlled natural matings, we confirm that selection against related males' sperm occurs within the female reproductive tract but demonstrate that this is more accurately predicted by MHC similarity: controlling for relatedness per se, more sperm reached the eggs when partners were MHC--dissimilar. Importantly, this effect appeared largely owing to similarity at a single MHC locus (class I minor). Further, the effect of MHC similarity was lost following artificial insemination, suggesting that male phenotypic cues might be required for females to select sperm differentially. These results indicate that postmating mechanisms that reduce inbreeding may do so as a consequence of more specific strategies of cryptic female choice promoting MHC diversity in offspring.

  14. Cryptic female choice favours sperm from major histocompatibility complex-dissimilar males

    PubMed Central

    Løvlie, Hanne; Gillingham, Mark A. F.; Worley, Kirsty; Pizzari, Tommaso; Richardson, David S.

    2013-01-01

    Cryptic female choice may enable polyandrous females to avoid inbreeding or bias offspring variability at key loci after mating. However, the role of these genetic benefits in cryptic female choice remains poorly understood. Female red junglefowl, Gallus gallus, bias sperm use in favour of unrelated males. Here, we experimentally investigate whether this bias is driven by relatedness per se, or by similarity at the major histocompatibility complex (MHC), genes central to vertebrate acquired immunity, where polymorphism is critical to an individual's ability to combat pathogens. Through experimentally controlled natural matings, we confirm that selection against related males' sperm occurs within the female reproductive tract but demonstrate that this is more accurately predicted by MHC similarity: controlling for relatedness per se, more sperm reached the eggs when partners were MHC-dissimilar. Importantly, this effect appeared largely owing to similarity at a single MHC locus (class I minor). Further, the effect of MHC similarity was lost following artificial insemination, suggesting that male phenotypic cues might be required for females to select sperm differentially. These results indicate that postmating mechanisms that reduce inbreeding may do so as a consequence of more specific strategies of cryptic female choice promoting MHC diversity in offspring. PMID:24004935

  15. Sperm protein 17 and AKAP-associated sperm protein cancer/testis antigens are expressed in ciliated hepatic foregut cysts.

    PubMed

    Grizzi, Fabio; Franceschini, Barbara; Di Biccari, Sonia; Musardo, Stefano; Pedretti, Elisa; Chiriva-Internati, Maurizio; Osipov, Vladimir; Fernández-Aceñero, Maria J

    2015-09-01

    Ciliated hepatic foregut cysts (CHFCs) are retained benign lesions of the liver. However, a case of squamous cell metaplasia and five cases of squamous cell carcinoma arising from a CHFC have been described. The potential of malignant transformation makes the identification of new biomarkers necessary. As the cancer/testis antigen sperm protein 17 (Sp17) has been detected in oral and oesophageal squamous cell carcinomas, the aim of this study was to investigate the expression of Sp17 and AKAP-associated sperm protein (ASP), which has a shared N-terminal sequence with Sp17, in four surgically resected CHFCs. CHFC specimens were taken from two patients who attended the Medical College of Wisconsin, Milwaukee, USA and two patients who attended the Fundación Jiménez Díaz, Madrid, Spain. CHFCs were found to be immunopositive for Sp17 and ASP. Both proteins were localized to the cytoplasm of ciliated cells lining the cysts, and their cilia. Confocal microscopy demonstrated that Sp17 and ASP overlapped in the same region of the cell. Sp17 and ASP cancer/testis antigens were found in ciliated cells of four CHFCs. Further characterization of Sp17 and ASP in patients with CHFCs may provide significant clues for understanding the molecular mechanisms underlying their predisposition to develop squamous cell carcinomas. © 2015 John Wiley & Sons Ltd.

  16. Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization.

    PubMed

    Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C

    2005-08-01

    This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

  17. Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen's egg yolk.

    PubMed

    Manjunath, Puttaswamy; Nauc, Veronica; Bergeron, Annick; Ménard, Martin

    2002-10-01

    Over the past 60 years, egg yolk (EY) has been routinely used in both liquid semen extenders and those used to cryopreserve sperm. However, the mechanism by which EY protects sperm during liquid storage or from freezing damage is unknown. Bovine seminal plasma contains a family of proteins designated BSP-A1/-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins). These proteins are secretory products of seminal vesicles that are acquired by sperm at ejaculation, modifying the sperm membrane by inducing cholesterol efflux. Because cholesterol efflux is time and concentration dependent, continuous exposure to seminal plasma (SP) that contains BSP proteins may be detrimental to the sperm membrane, which may adversely affect the ability of sperm to be preserved. In this article, we show that the BSP proteins bind to the low-density fraction (LDF), a lipoprotein component of the EY extender. The binding is rapid, specific, saturable, and stable even after freeze-thawing of semen. Furthermore, LDF has a very high capacity for BSP protein binding. The binding of BSP proteins to LDF may prevent their detrimental effect on sperm membrane, and this may be crucial for sperm storage. Thus, we propose that the sequestration of BSP proteins of SP by LDF may represent the major mechanism of sperm protection by EY.

  18. Evolutionary conservation of mammalian sperm proteins associates with overall, not tyrosine, phosphorylation in human spermatozoa.

    PubMed

    Schumacher, Julia; Ramljak, Sanja; Asif, Abdul R; Schaffrath, Michael; Zischler, Hans; Herlyn, Holger

    2013-12-06

    We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein-protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility.

  19. Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster

    SciTech Connect

    Clark, A.G.; Prout, T.; Harshman, L.G.

    1995-01-01

    Genes that influence mating and/or fertilization success may be targets for strong natural selection. If females remate frequently relative to the duration of sperm storage and rate of sperm use, sperm displacement may be an important component of male reproductive success. Although it has long been known that mutant laboratory stocks of Drosophila differ in sperm displacement, the magnitude of the naturally occurring genetic variation in this character has not been systematically quantified. Here we report the results of a screen for variation in sperm displacement among 152 lines of Drosophila melanogaster that were made homozygous for second and/or third chromosomes recovered from natural populations. Sperm displacement was assayed by scoring the progeny of cn;bw females that had been mated sequentially to cn;bw and tested males in either order. Highly significant differences were seen in both the ability to displace sperm that is resident in the female`s reproductive tract and in the ability to resist displacement by subsequent sperm. Most lines exhibited nearly complete displacement, having nearly all progeny sired by the second male, but several lines had as few as half the progeny fathered by the second male. Lines that were identified in the screen for naturally occurring variation in sperm displacement were also characterized for single-strand conformation polymorphisms (SSCP) at seven accessory gland protein (Acp) genes. Significant associations were found between particular Acp alleles at four different loci (Acp26Aa/Ab, Acp29B, Acp36DE and Acp53E) and the ability of males to resist displacement by subsequent sperm. There was no correlation between the ability to displace resident sperm and the ability to resist being displaced by subsequent sperm. This lack of correlation, and the association of Acp alleles with resisting subsequent sperm only, suggests that different mechanisms mediate the two components of sperm displacement. 36 refs., 4 figs., 7 tabs.

  20. Sperm surface hyaluronan binding protein (HABP1) interacts with zona pellucida of water buffalo (Bubalus bubalis) through its clustered mannose residues.

    PubMed

    Ghosh, Ilora; Datta, Kasturi

    2003-02-01

    Sperm-oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between zona pellucida (ZP) glycoproteins and sperm surface receptor. d-mannosylated glycoproteins, the major constituents of ZP are considered to serve as ligands for sperm binding. The presence of hyaluronan binding protein 1 (HABP1) on sperm surface of different mammals including cattle and its possible involvement in sperm function is already reported. Recently, we have demonstrated the specificity of clustered mannose as another ligand for HABP1 (Kumar et al., 2001: J Biosci 26:325-332). Here, we report that only N-linked mannosylated zona-glycoproteins bind to sperm surface HABP1. Labeled HABP1 interacts with ZP of intact oocyte of Bubalus bubalis, which can be competed with unlabeled HABP1 or excess d-mannosylated albumin (DMA). This data suggests the specific interaction of HABP1 with ZP, through clustered mannose residues. In order to examine the physiological significance of such an interaction, the capacity of sperm binding to oocytes under in vitro fertilization plates was examined either in presence of DMA alone or in combination with HABP1. The number of sperms, bound to oocytes was observed to reduce significantly in presence of DMA, which could be reversed by the addition of purified recombinant HABP1 (rHABP1) in the same plate. This suggests that sperm surface HABP1 may act as mannose binding sites for zona recognition. Copyright 2003 Wiley-Liss, Inc.

  1. Identification of Bovine Sperm Surface Proteins Involved in Carbohydrate-mediated Fertilization Interactions*

    PubMed Central

    2016-01-01

    Glycan-protein interactions play a key role in mammalian fertilization, but data on the composition and identities of protein complexes involved in fertilization events are scarce, with the added complication that the glycans in such interactions tend to differ among species. In this study we have used a bovine model to detect, characterize and identify sperm lectins relevant in fertilization. Given the complexity of the sperm-toward-egg journey, two important aspects of the process, both primarily mediated by protein-sugar interactions, have been addressed: (1) formation of the sperm reservoir in the oviductal epithelium, and (2) gamete recognition (oocyte-sperm interaction). Using whole sperm cells and a novel affinity capture method, several groups of proteins with different glycan specificities, including 58 hitherto unreported as lectins, have been identified in sperm surface, underscoring both the efficacy of our selective approach and the complex composition and function of sperm. Based on these results and previous data, we suggest that sperm surface proteins play significant roles in fertilization events such as membrane remodeling, transport, protection and function, thus supporting the hypothesis that rather than a simple lock-and-key model, mammalian fertilization relies on a complex interactome involving multiple ligands/receptors and recognition/binding events. PMID:27094474

  2. Sperm nuclear basic proteins of tunicates and the origin of protamines.

    PubMed

    Saperas, Núria; Ausió, Juan

    2013-08-01

    Sperm nuclear basic proteins (SNBPs) are the chromosomal proteins that are found associated with DNA in sperm nuclei at the end of spermiogenesis. These highly specialized proteins can be classified into three major types: histone type (H-type), protamine-like type (PL-type), and protamine type (P-type). A hypothesis from early studies on the characterization of SNBPs proposed a mechanism for the vertical evolution of these proteins that involved an H1 → PL → P transition. However, the processes and mechanisms involved in such a transition were not understood. In particular, it was not clear how a molecular transition from a lysine-rich protein precursor (H1 histone) to the arginine-rich protamines might have taken place. In deuterostomes, the presence of SNBPs of the H-type in echinoderms and of protamines in the higher phylogenetic groups of vertebrates had long been known. The initial work on the characterization of tunicate SNBPs attempted to define the types and range of SNBPs that characterize this phylogenetically intermediate group. It was found that tunicate SNBPs belong to the PL-type. In this work we discuss how the study of SNBPs in the tunicates has been key to providing support to the H1 → PL → P transition. Most significantly, it was in tunicates that a potential molecular mechanism to explain the lysine-to-arginine transition was first reported.

  3. Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins.

    PubMed

    Tanphaichitr, Nongnuj; Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark

    2015-01-01

    The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm-ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm-ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm-ZP binding step.

  4. Sperm cryopreservation of the Indian major carp, Labeo calbasu: effects of cryoprotectants, cooling rates and thawing rates on egg fertilization.

    PubMed

    Nahiduzzaman, Md; Hassan, Md Mahbubul; Roy, Pankoz Kumar; Hossain, Md Akhtar; Hossain, Mostafa Ali Reza; Tiersch, Terrence R

    2012-12-01

    A sperm cryopreservation protocol for the Indian major carp, Labeo calbasu, was developed for long-term preservation and artificial fertilization. Milt collected from mature male fish were placed in Alsever's solution (296mOsmolkg(-1)) to immobilize the sperm. Cryoprotectant toxicity was evaluated by motility assessment with dimethyl sulfoxide (DMSO) and methanol at 5, 10 and 15% concentrations. DMSO was more toxic at higher concentrations than methanol, and consequently 15% DMSO was excluded from further study. A one-step cooling protocol (from 5 to 80°C) with two cooling rates (5 and 10°C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL(®) CL-3300; Australia). Based on post-thaw motility, the 10°C/min cooling rate with either 10% DMSO or 10% methanol yielded significantly higher (P=0.011) post-thaw motility than the other rate and cryoprotectant concentrations. Sperm thawed at 40°C for 15s and fresh sperm were used to fertilize freshly collected L. calbasu eggs and significant differences were observed (P=0.001) in percent fertilization between cryopreserved and fresh sperm as well as among different sperm-to-egg ratios (P=0.001). The highest fertilization and hatching rates were observed for thawed sperm at a sperm-to-egg ratio of 4.1×10(5):1. The cryopreservation protocol developed can facilitate hatchery operations and long-term conservation of genetic resources of L. calbasu.

  5. LOCALIZATION OF THE SPERM PROTEIN SP22 AND INHIBITION OF FERTILITY IN VIVO AND IN VITRO

    EPA Science Inventory

    We previously established that the levels sperm membrane protein SP22 are highly correlated with the fertility of sperm from the cauda epididymidis of rats exposed to both epididymal and testicular toxicants, and that a testis-specific SP22 transcript is expressed in post-meiotic...

  6. Detection of boar sperm plasma membrane protein using Rhodamine 640; implications for cryobiology and physiology

    USDA-ARS?s Scientific Manuscript database

    Rhodamine 640 (R640) was used to detect changes in boar sperm plasma membrane protein (PMP) during cryopreservation; a poorly understood phenomenon. The protocol was adapted for boar sperm so that semen samples (n = 17) could be analyzed for PMP (R640 positive) and plasma membrane integrity (PMI; Y...

  7. LOCALIZATION OF THE SPERM PROTEIN SP22 AND INHIBITION OF FERTILITY IN VIVO AND IN VITRO

    EPA Science Inventory

    We previously established that the levels sperm membrane protein SP22 are highly correlated with the fertility of sperm from the cauda epididymidis of rats exposed to both epididymal and testicular toxicants, and that a testis-specific SP22 transcript is expressed in post-meiotic...

  8. Toxicant-induced acceleration of epididymal sperm transit: androgen-dependent proteins may be involved.

    PubMed

    Klinefelter, G R; Suarez, J D

    1997-01-01

    protein profile in homogenates of the caput/corpus epididymidis revealed treatment-related diminutions in two proteins CC9 (M(r) = 42 kDa, pI = 4.2) and CC34 (M(r) = 35 kDa, pI = 5.5), and the level of each of these proteins in the caput/corpus was significantly correlated with the decrease in caput/corpus sperm number. Thus, both CEMS and HFLUT accelerate sperm transit through the proximal segment of the epididymis; and, while this effect is not dependent on the testis, it may involve a lesion in androgen-dependent epididymal function.

  9. Seminal plasma proteins of adult boars and correlations with sperm parameters.

    PubMed

    González-Cadavid, Verónica; Martins, Jorge A M; Moreno, Frederico B; Andrade, Tiago S; Santos, Antonio C L; Monteiro-Moreira, Ana Cristina O; Moreira, Renato A; Moura, Arlindo A

    2014-09-15

    The present study was conducted to identify the major seminal plasma protein profile of boars and its associations with semen criteria. Semen samples were collected from 12 adult boars and subjected to evaluation of sperm parameters (motility, morphology, vitality, and percent of cells with intact acrosome). Seminal plasma was obtained by centrifugation, analyzed by two-dimensional SDS-PAGE, and proteins identified by mass spectrometry (electrospray ionization quadrupole time-of-flight). We tested regression models using spot intensities related to the same proteins as independent variables and semen parameters as dependent variables (P ≤ 0.05). One hundred twelve spots were identified in the boar seminal plasma gels, equivalent to 39 different proteins. Spermadhesin porcine seminal protein (PSP)-I and PSP-II, as well as spermadhesins AQN-1, AQN-3 and AWN-1 represented 45.2 ± 8% of the total intensity of all spots. Other proteins expressed in the boar seminal plasma included albumin, complement proteins (complement factor H precursor, complement C3 precursor and adipsin/complement factor D), immunoglobulins (IgG heavy chain precursor, IgG delta heavy chain membrane bound form, IgG gamma-chain, Ig lambda chain V-C region PLC3, and CH4 and secreted domains of swine IgM), IgG-binding proteins, epididymal-specific lipocalin 5, epididymal secretory protein E1 precursor, epididymal secretory glutathione peroxidase precursor, transferrin, lactotransferrin and fibronectin type 1 (FN1). On the basis of the regression analysis, the percentage of sperm with midpiece defects was related to the amount of CH4 and secreted domains of swine IgM and FN1 (r² = 0.58, P = 0.006), IgG-binding protein (r² = 0.41, P = 0.024), complement factor H precursor (r² = 0.61, P = 0.014) and lactadherin (r² = 0.45, P = 0.033). The percentage of sperm with tail defects was also related to CH4 and secreted domains of swine IgM and FN1 (r² = 0.40, P = 0.034), IgG-binding protein (r² = 0

  10. Sperm proteome of Mytilus galloprovincialis: Insights into the evolution of fertilization proteins in marine mussels.

    PubMed

    Zhang, Yanjie; Mu, Huawei; Lau, Stanley C K; Zhang, Zhifeng; Qiu, Jian-Wen

    2015-12-01

    Cataloging the sperm proteome of an animal can improve our understanding of its sperm-egg interaction and speciation, but such data are available for only a few free-spawning invertebrates. This study aimed to identify the sperm proteome of Mytilus galloprovincialis, a free-spawning marine mussel. We integrated public transcriptome datasets by de novo assembly, and applied SDS-PAGE coupled LC-MS/MS analysis to profile the sperm proteome, resulting in the identification of 550 proteins. Comparing the homologous sperm protein coding genes between M. galloprovincialis and its closely related species M. edulis revealed that fertilization proteins have the highest mean nonsynonymous substitution rate (Ka/Ks = 0.62) among 11 functional groups, consistent with previous reports of positive selection of several fertilization proteins in Mytilus. Moreover, 78 sperm proteins in different functional groups have Ka/Ks values > 0.5, indicating the presence of many candidate sperm proteins for further analysis of rapid interspecific divergence. The MS data are available in ProteomeXchange with the identifier PXD001665.

  11. D-penicillamine prevents ram sperm agglutination by reducing the disulphide bonds of a copper-binding sperm protein.

    PubMed

    Leahy, T; Rickard, J P; Aitken, R J; de Graaf, S P

    2016-05-01

    Head-to-head agglutination of ram spermatozoa is induced by dilution in the Tyrode's capacitation medium with albumin, lactate and pyruvate (TALP) and ameliorated by the addition of the thiol d-penicillamine (PEN). To better understand the association and disassociation of ram spermatozoa, we investigated the mechanism of action of PEN in perturbing sperm agglutination. PEN acts as a chelator of heavy metals, an antioxidant and a reducing agent. Chelation is not the main mechanism of action, as the broad-spectrum chelator ethylenediaminetetraacetic acid and the copper-specific chelator bathocuproinedisulfonic acid were inferior anti-agglutination agents compared with PEN. Oxidative stress is also an unlikely mechanism of sperm association, as PEN was significantly more effective in ameliorating agglutination than the antioxidants superoxide dismutase, ascorbic acid, α-tocopherol and catalase. Only the reducing agents cysteine and DL-dithiothreitol displayed similar levels of non-agglutinated spermatozoa at 0 h compared with PEN but were less effective after 3 h of incubation (37 °C). The addition of 10 µM Cu(2+) to 250 µM PEN + TALP caused a rapid reversion of the motile sperm population from a non-agglutinated state to an agglutinated state. Other heavy metals (cobalt, iron, manganese and zinc) did not provoke such a strong response. Together, these results indicate that PEN prevents sperm association by the reduction of disulphide bonds on a sperm membrane protein that binds copper. ADAM proteins are possible candidates, as targeted inhibition of the metalloproteinase domain significantly increased the percentage of motile, non-agglutinated spermatozoa (52.0% ± 7.8) compared with TALP alone (10.6% ± 6.1).

  12. Regulation of sperm-specific proteins by IFE-1, a germline-specific homolog of eIF4E, in C. elegans.

    PubMed

    Kawasaki, Ichiro; Jeong, Myung-Hwan; Shim, Yhong-Hee

    2011-02-01

    ABSTEACT: IFE-1 is one of the five C. elegans homologs of eIF4E, which is the mRNA 5' cap-binding component of the translation initiation complex eIF4F. Depletion of IFE-1 causes defects in sperm, suggesting that IFE-1 regulates a subset of genes required for sperm functions. To further understand the molecular function of IFE-1, proteomic analysis was performed to search for sperm proteins that are downregulated in ife-1(ok1978); fem-3(q20) mutants relative to the fem-3(q20) control. The fem-3(q20) mutant background was used because it only produces sperm at restrictive temperature. Total worm proteins were subjected to 2D-DIGE, and differentially expressed protein spots were further identified by MALDI-TOF mass spectrometry. Among the identified proteins, GSP-3 and Major Sperm Proteins (MSPs) were found to be significantly down-regulated in the ife-1(ok1978) mutant. Moreover, RNAi of gsp-3 caused an ife-1-like phenotype. These results suggest that IFE-1 is required for efficient expression of some sperm-specific proteins, and the fertilization defect of ife-1 mutant is caused mainly by a reduced level of GSP-3.

  13. Effect of Pseudomonas aeruginosa on sperm capacitation and protein phosphorylation of boar spermatozoa.

    PubMed

    Sepúlveda, Lilian; Bussalleu, Eva; Yeste, Marc; Bonet, Sergi

    2016-05-01

    Several studies have reported the detrimental effects that bacteriospermia causes on boar sperm quality, but little is known about its effects on IVC. Considering that, the present study sought to evaluate the effects of different concentrations of Pseudomonas aeruginosa on different indicators of capacitation status (sperm viability, membrane lipid disorder, sperm motility kinematics, and protein phosphorylation of boar spermatozoa) after IVC. Flow cytometry and computer assisted sperm analysis (CASA) revealed that the presence of P aeruginosa in boar sperm samples, mostly at concentrations greater than 10(6) CFU/mL, is associated with a significant (P < 0.05) decrease in the percentages of both sperm membrane integrity and sperm with low membrane lipid disorder, and also with a reduction in sperm motility kinetic parameters when compared with results obtained from the control sample, which presented the typical motility pattern of capacitated-like boar spermatozoa. Moreover, Western blot results also showed significant (P < 0.05) changes in the levels of tyrosine, serine, and threonine protein phosphorylation because of bacterial contamination, the decrease in phosphotyrosine levels of p32, a well-known marker of IVC achievement in boar sperm, being the most relevant. Indeed, after 3 hours of IVC, phosphotyrosine levels of p32 in the control sample were 3.13 ± 0.81, whereas in the tubes with 10(6) and 10(8) CFU/mL were 1.05 ± 0.20 and 0.36 ± 0.07, respectively. Therefore, the present study provides novel data regarding the effects of bacterial contamination on boar sperm, suggesting that the presence of P aeruginosa affects the fertilizing ability of boar sperm by altering its ability to accomplish IVC.

  14. Equatorial segment protein (ESP) is a human alloantigen involved in sperm-egg binding and fusion.

    PubMed

    Wolkowicz, M J; Digilio, L; Klotz, K; Shetty, J; Flickinger, C J; Herr, J C

    2008-01-01

    The equatorial segment of the sperm head is known to play a role in fertilization; however, the specific sperm molecules contributing to the integrity of the equatorial segment and in binding and fusion at the oolemma remain incomplete. Moreover, identification of molecular mediators of fertilization that are also immunogenic in humans is predicted to advance both the diagnosis and treatment of immune infertility. We previously reported the cloning of Equatorial Segment Protein (ESP), a protein localized to the equatorial segment of ejaculated human sperm. ESP is a biomarker for a subcompartment of the acrosomal matrix that can be traced through all stages of acrosome biogenesis (Wolkowicz et al, 2003). In the present study, ESP immunoreacted on Western blots with 4 (27%) of 15 antisperm antibody (ASA)-positive serum samples from infertile male patients and 2 (40%) of 5 ASA-positive female sera. Immunofluorescent studies revealed ESP in the equatorial segment of 89% of acrosome-reacted sperm. ESP persisted as a defined equatorial segment band on 100% of sperm tightly bound to the oolemma of hamster eggs. Antisera to recombinant human ESP inhibited both oolemmal binding and fusion of human sperm in the hamster egg penetration assay. The results indicate that ESP is a human alloantigen involved in sperm-egg binding and fusion. Defined recombinant sperm immunogens, such as ESP, may offer opportunities for differential diagnosis of immune infertility.

  15. Mouse sperm basic nuclear protein. Electrophoretic characterization and fate after fertilization

    PubMed Central

    1975-01-01

    Mouse sperm were labeled in vivo with [3H]arginine. The sperm were then followed autoradiographically from the time of label incorporation until after fertilization. The label was completely lost from the sperm head after fertilization, during the oocyte's second meiotic division. That the [3H]arginine was incorporated into a sperm-specific basic protein was demonstrated by fractionating acid extracts of epididymal and ejaculated sperm with polyacrylamide gel electrophoresis. All the histone fractions were resolved in the epididymal extracts, but in addition a band was present that migrated faster than histone F2al and slower than the salmon protamine used as a marker. This new fraction (proposed name: musculine) was also present in ejaculated sperm; it was shown to be the only fraction that was labeled. Musculine therefore represents the end product of a histone transition in mice. It is, however, according to our electrophoretic characterization, not identical to the classical fish protamines. Rather, musculine resembles bovine sperm nuclear protein. Since the loss of this fraction from the sperm head was coincident with the rearrangement of the male genome, before its resumption of transcription, it is suggested that musculine is involved in the control of chromatin that accompanies spermiogenesis and fertilization. PMID:1141382

  16. Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster.

    PubMed

    Clark, A G; Aguadé, M; Prout, T; Harshman, L G; Langley, C H

    1995-01-01

    Genes that influence mating and/or fertilization success may be targets for strong natural selection. If females remate frequently relative to the duration of sperm storage and rate of sperm use, sperm displacement may be an important component of male reproductive success. Although it has long been known that mutant laboratory stocks of Drosophila differ in sperm displacement, the magnitude of the naturally occurring genetic variation in this character has not been systematically quantified. Here we report the results of a screen for variation in sperm displacement among 152 lines of Drosophilia melanogaster that were made homozygous for second and/or third chromosomes recovered from natural populations. Sperm displacement was assayed by scoring the progeny of cn;bw females that had been mated sequentially to cn;bw and tested males in either order. Highly significant differences were seen in both the ability to displace sperm that is resident in the female's reproductive tract and in the ability to resist displacement by subsequent sperm. Most lines exhibited nearly complete displacement, having nearly all progeny sired by the second male, but several lines had as few as half the progeny fathered by the second male. Lines that were identified in the screen for naturally occurring variation in sperm displacement were also characterized for single-strand conformation polymorphisms (SSCP) at seven accessory gland protein (Acp) genes, Glucose dehydrogenase (Gld), and Esterase-6 (Est-6). Acp genes encode proteins that are in some cases known to be transmitted to the female in the seminal fluid and are likely candidates for genes that might mediate the phenomenon of sperm displacement. Significant associations were found between particular Acp alleles at four different loci (Acp26Aa/Ab, Acp29B, Acp36DE and Acp53E) and the ability of males to resist displacement by subsequent sperm. There was no correlation between the ability to displace resident sperm and the ability

  17. Membrane proteins associated with sperm-oocyte interaction: A proteomic comparison between Kedah Kelantan (Bos indicus) and Mafriwal (Bos taurus × Bos indicus) sperm

    NASA Astrophysics Data System (ADS)

    Ashrafzadeh, Ali; Nathan, Sheila; Othman, Iekhsan; Yee, Tee Ting; Karsani, Saiful Anuar

    2013-11-01

    Production performance of European cattle breeds has significantly improved through various breeding programs. However, European breeds are more susceptible to heat stress compared to zebu cattle (Bos indicus) as their conception rate can range between 20 to 30% in hot seasons compared to winter. To identify cattle sperm proteins associated with zebu cattle higher fertility and heat tolerance in tropical environments, we utilised a proteomics-based approach to compare sperm from the highly fertile Malaysian indigenous breed, Kedah Kelantan (Bos indicus), with sperm from the sub-fertile crossbreed, Mafriwal (Bos taurus × Bos indicus). Frozen semen of three high performance bulls from each breed was processed to obtain live and pure sperm. Proteins were separated and gel bands were processed by in-gel tryptic digestion. For each breed, mass spectrometry data was acquired over 11 replicates. The analyzed data identified peptides with different expression levels (99% confidence level) and protein identification was determined by targeted MS/MS. Among the identified proteins associated with sperm-oocyte interaction, two proteins were up-regulated in Kedah Kelantan sperm and 7 proteins were up-regulated in or specific to Mafriwal. Our results suggest that the higher fertility of zebu cattle in tropical areas may not be related to more efficient sperm-oocyte interaction. Further analysis of the other regulated proteins in these two breeds may contribute further knowledge on the physiological reason/s for higher fertility and heat tolerance of Zebu cattle in tropical areas.

  18. Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.

    PubMed

    Marey, Mohamed A; Liu, Jinghui; Kowsar, Rasoul; Haneda, Shingo; Matsui, Motozumi; Sasaki, Motoki; Takashi, Shimizu; Hayakawa, Hiroyuki; Wijayagunawardane, Missaka P B; Hussein, Fekry M; Miyamoto, Akio

    2014-02-01

    This study aimed to investigate the presence of polymorphonuclear neutrophils (PMNs) in bovine oviduct fluid under physiological conditions and to determine the possible role of bovine oviduct epithelial cells (BOECs) in the regulation of the phagocytic activity of PMNs for sperm. During the pre-ovulatory stage, PMNs were identified in the bovine oviduct fluid in relatively constant numbers. In our experiments, PMNs were incubated for 4 h with the supernatant of cultured BOECs stimulated for 24 h by LH (10 ng/ml). Phagocytosis was then assayed by co-incubation of these PMNs with sperm treated to induce capacitation. The BOEC supernatant significantly suppressed sperm phagocytosis by PMNs, and the LH-stimulated BOEC supernatant further suppressed phagocytosis. Importantly, in the BOEC culture, LH stimulated the secretion of prostaglandin E2 (PGE2), which dose-dependently (10(-6), 10(-7), and 10(-8) M) suppressed sperm phagocytosis by PMNs. Furthermore, a PGEP2 receptor antagonist significantly abrogated the inhibition of phagocytosis by the LH-stimulated BOEC supernatant. Additionally, using scanning electron microscopy, incubation of PMNs with either PGE2 or LH-stimulated BOEC supernatant before phagocytosis was found to prevent the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results indicate that sperm are exposed to PMNs in the oviduct and PGE2 released into the oviduct fluid after LH stimulation may play a major role in the suppression of the phagocytic activity of PMNs for sperm via interaction with EP2 receptors. Thus, the bovine oviduct provides a PGE2-rich microenvironment to protect sperm from phagocytosis by PMNs, thereby supporting sperm survival in the oviduct. Free Japanese abstract A Japanese translation of this abstract is freely available at http://www.reproduction-online.org/content/147/2/211/suppl/DC1.

  19. Binding of Sperm to the Zona Pellucida Mediated by Sperm Carbohydrate-Binding Proteins is not Species-Specific in vitro between Pigs and Cattle

    PubMed Central

    Takahashi, Kazuya; Kikuchi, Kazuhiro; Uchida, Yasuomi; Kanai-Kitayama, Saeko; Suzuki, Reiichiro; Sato, Reiko; Toma, Kazunori; Geshi, Masaya; Akagi, Satoshi; Nakano, Minoru; Yonezawa, Naoto

    2013-01-01

    Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm–oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for β-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from β-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm–oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle. PMID:24970158

  20. Protein differences between normal and oligospermic human sperm demonstrated by two-dimensional gel electrophoresis.

    PubMed

    Morgentaler, A; Schopperle, W M; Crocker, R H; DeWolf, W C

    1990-11-01

    Protein expression by sperm obtained from men with normal semen analysis and men with oligospermia were evaluated by two-dimensional gel electrophoresis. Proteins were solubilized in a 9.5 M urea/2% Nonidet-P40 (LKB, Bromma, Sweden) lysis buffer and underwent second dimension separation on 10 to 16% polyacrylamide gradient gels. A set of 36 invariant proteins was identified in all normospermic samples, whereas 8 of 10 evaluable oligospermic samples lacked 1 or more of the invariant proteins. Proteins absent in oligospermic samples may be critical to normal sperm function and may serve as markers for infertility.

  1. Modeling how reproductive ecology can drive protein diversification and result in linkage disequilibrium between sperm and egg proteins.

    PubMed

    Tomaiuolo, Maurizio; Levitan, Don R

    2010-07-01

    Gamete-recognition proteins determine whether sperm and eggs are compatible at fertilization, and they often evolve rapidly. The source of selection driving the evolution of these proteins is still debated. It has been suggested that sexual conflict can result in proliferation of genetic variation and possibly linkage disequilibrium between sperm and egg proteins. Empirical evidence suggests that both male and female reproductive success can be predicted by their sperm ligand genotype, but why female success can be predicted by a protein expressed only in males is unknown. Here we use mathematical modeling to investigate the interaction between reproductive behavior and sperm availability on the evolution of sperm ligands and egg receptors. We consider haploid and diploid expression in gametes in two possible ecological scenarios, monogamous spawning and competitive spawning. Reproductive behavior plays an important role in determining possible outcomes resulting from sexual conflict. Sperm limitation selects for common genotypes regardless of mating behavior. Under conditions of sperm abundance, competitive spawning provides conditions for the persistence of allelic variation and gametic disequilibrium. With monogamous spawning, such conditions are more restrictive.

  2. Cryopreservation Of Nili-Ravi Buffalo Bull Sperm in Cryodiluent Supplemented with Lolium perenne Protein Preparations.

    PubMed

    Qadeer, S; Khan, M A; Ansari, M S; Rakha, B A; Ejaz, R; Husna, A U; Azam, A; Ullah, N; Walker, V K; Akhter, S

    Semen from the Nili-Ravi buffalo bull, Bubalus bubalis, shows poor survival after freeze storage compared to bovine (Bos taurus and Bos indicus) semen. Freeze-susceptibility distinctions in these two genera have been attributed to differences in sperm membranes. We measured the impact of protein preparations derived from a frost-resistant perennial grass, Lolium perenne, with ice recrystallization inhibition activity on the low temperature storage of B. bubalis semen. When the L. perenne preparations (0.1, 1, 10 µg/mL) were added to buffalo semen [2 ejaculates per bull (N=3) per replicate (r=3)] in Tris-citrate extender (50×10(6)sperm mL(-1)), there was no impact on semen quality, as measured by sperm motility and plasma membrane integrity, after storage at 4 degree C (P>0.05). However, when semen supplemented with the grass proteins (0.1 and 1 µg mL(-1)) was evaluated after freezing and storage in liquid nitrogen for 24 h, post-thaw sperm progressive motility and plasma membrane integrity was higher (P<0.05) than in control samples. Post-thaw sperm viability and sperm acrosome integrity was similar (P > 0.05) to controls. The improvement in cryopreserved buffalo sperm progressive motility and plasma membrane integrity suggests that the use of these easily-made preparations may improve fertility after cryopreservation and offers the prospect of improved conception rates after artificial insemination with cryopreservation.

  3. How nematode sperm crawl.

    PubMed

    Bottino, Dean; Mogilner, Alexander; Roberts, Tom; Stewart, Murray; Oster, George

    2002-01-15

    Sperm of the nematode, Ascaris suum, crawl using lamellipodial protrusion, adhesion and retraction, a process analogous to the amoeboid motility of other eukaryotic cells. However, rather than employing an actin cytoskeleton to generate locomotion, nematode sperm use the major sperm protein (MSP). Moreover, nematode sperm lack detectable molecular motors or the battery of actin-binding proteins that characterize actin-based motility. The Ascaris system provides a simple 'stripped down' version of a crawling cell in which to examine the basic mechanism of cell locomotion independently of other cellular functions that involve the cytoskeleton. Here we present a mechanochemical analysis of crawling in Ascaris sperm. We construct a finite element model wherein (a) localized filament polymerization and bundling generate the force for lamellipodial extension and (b) energy stored in the gel formed from the filament bundles at the leading edge is subsequently used to produce the contraction that pulls the rear of the cell forward. The model reproduces the major features of crawling sperm and provides a framework in which amoeboid cell motility can be analyzed. Although the model refers primarily to the locomotion of nematode sperm, it has important implications for the mechanics of actin-based cell motility.

  4. The role of ezrin-associated protein network in human sperm capacitation

    PubMed Central

    Wang, Lei; Chen, Wen; Zhao, Chun; Huo, Ran; Guo, Xue-Jiang; Lin, Min; Huang, Xiao-Yan; Mao, Yun-Dong; Zhou, Zuo-Min; Sha, Jia-Hao

    2010-01-01

    Membrane modifications in sperm cells represent a key step in sperm capacitation; however, the molecular basis of these modifications is not fully understood. Ezrin is the best-studied member of the ezrin/radixin/merlin family. As a cross-linker between the cortical cytoskeleton and plasma membrane proteins, ezrin contributes to remodeling of the membrane surface structure. Furthermore, activated ezrin and the Rho dissociation inhibitor, RhoGDI, promote the formation of cortical cytoskeleton-polymerized actin through Rho activation. Thus, ezrin, actin, RhoGDI, Rho and plasma membrane proteins form a complicated network in vivo, which contributes to the assembly of the structure of the membrane surface. Previously, we showed that ezrin and RhoGDI1 are expressed in human testes. Thus, we sought to determine whether the ezrin–RhoGDI1–actin–membrane protein network has a role in human sperm capacitation. Our results by Western blot indicate that ezrin is activated by phosphorylation of the threonine567 residue during capacitation. Co-immunoprecipitation studies revealed that, during sperm capacitation, the interaction between ezrin and RhoGDI1 increases, and phosphostaining of two dimensional electrophoresis gels showed that RhoGDI1 is phosphorylated, suggesting that RhoGDI1 dissociates from RhoA and leads to actin polymerization on the sperm head. We speculate that activated ezrin interacts with polymerized actin and the glycosylated membrane protein cd44 after capacitation. Blocking sperm capacitation using ezrin- or actin-specific monoclonal antibodies decreases their acrosome reaction (AR) rate, but has no effect on the AR alone. Taken together, our results show that a network consisting of ezrin, RhoGDI1, RhoA, F-actin and membrane proteins functions to influence the modifications that occur on the membrane of the sperm head during human sperm capacitation. PMID:20711218

  5. Targeted Disruption of the Transition Protein 2 Gene Affects Sperm Chromatin Structure and Reduces Fertility in Mice†

    PubMed Central

    Zhao, Ming; Shirley, Cynthia R.; Yu, Y. Eugene; Mohapatra, Bhagyalaxmi; Zhang, Yun; Unni, Emmanual; Deng, Jian M.; Arango, Nelson A.; Terry, Nicholas H. A.; Weil, Michael M.; Russell, Lonnie D.; Behringer, Richard R.; Meistrich, Marvin L.

    2001-01-01

    During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation. PMID:11585907

  6. Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes

    SciTech Connect

    Lafleur, Michel; Courtemanche, Lesley; Karlsson, Goeran; Edwards, Katarina; Schwartz, Jean-Louis; Manjunath, Puttaswamy

    2010-08-27

    Research highlights: {yields} Binder-of-sperm protein 1 (BSP1) modifies the morphology of lipidic vesicles inducing bead necklace-like and thread-like structures. {yields} In the presence of multilamellar liposomes, BSP1 leads to the formation of long nanotubes. {yields} The insertion of BSP1 in the external lipid leaflet of membranes induces local changes in bilayer curvature. -- Abstract: Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed with phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process.

  7. The single and synergistic effects of the major tea components caffeine, epigallocatechin-3-gallate and L-theanine on rat sperm viability.

    PubMed

    Dias, Tânia R; Alves, Marco G; Casal, Susana; Silva, Branca M; Oliveira, Pedro F

    2016-03-01

    Caffeine, epigallocatechin-3-gallate (EGCG) and L-theanine are the major components of tea (Camellia sinensis L.) and the main representatives of the classes of methylxanthines, catechins and free amino acids present in this beverage. There are many studies reporting tea's health benefits, however it is not clear if those effects are mediated by a single component or a synergistic action. This study aimed to evaluate the individual and synergistic effects of tea's major components on rat epididymal spermatozoa survival and oxidative profile during 3-day storage at room temperature (RT). For that, spermatozoa were incubated with caffeine (71 μg mL(-1)), EGCG (82 μg mL(-1)), or L-theanine (19 μg mL(-1)), alone or in combination. Spermatozoa viability was assessed by the eosin-nigrosin staining technique. The oxidative profile was established by evaluating the levels of carbonyl groups, protein nitration and lipid peroxidation. Supplementation of sperm storage medium with the three compounds together improved sperm viability, after 24, 48 and 72 h of incubation, relative to the control and the groups incubated with each component individually. However, at the end of the 72 h of incubation, there was an increase in protein oxidation in the group exposed to the three compounds, illustrating that the combined treatment triggers different alterations in sperm proteins during their maturational process in the epididymis. This study highlights the importance of the synergism between tea components for the beneficial effects usually attributed to this beverage, particularly in sperm storage at RT.

  8. Seminal plasma proteins modify the distribution of sperm subpopulations in cryopreserved semen of rams with lesser fertility.

    PubMed

    Ledesma, Alba; Zalazar, Lucía; Fernández-Alegre, Estela; Hozbor, Federico; Cesari, Andreina; Martínez-Pastor, Felipe

    2017-09-01

    Any physiological mechanism involved in sperm selection and semen improvement has effects on heterogeneous sperm populations. This is mainly due to the fact that sperm populations within a single ejaculate have considerable heterogeneity for many variables, such as motility which is meaningful in terms of understanding how some sperm cells possess fertility advantages as compared with other cells. In the present research, initially there was a multivariate and clustering analysis used to assess sperm motility data from cryopreserved ram semen to identify subpopulations and compare the distribution of these clusters between rams with lesser and greater fertility. There were four classifications made of sperm subpopulations (clusters): CL1 fast/linear/progressive sperm; CL2 fast/non-linear sperm; CL3 very fast/linear sperm with vigorous beating and CL4 slow/non-linear sperm. Rams with greater fertility had a lesser proportion of sperm considered as "hyperactivated" (CL2) and a greater proportion of slow and non-linear sperm (CL4) than sperm of rams with lesser fertility. In addition, the effects were assessed for the capacity of seminal plasma (SP) and interacting SP proteins (iSPP) that were present during different seasons of the year to improve the distribution of sperm within subpopulations of semen from rams with lesser fertility. The iSPP and SP were obtained by artificial vagina (AV) and electroejaculation (EE) during breeding and non-breeding seasons and added to thawed semen. All the aggregates had a significant effect on the distribution of sperm subpopulations and effects differed among seasons of the year and depending on collection method used. Even though, future studies are needed to assess the contribution of each subpopulation on ram sperm fertility, it is important that a multivariate analysis be used to evaluate the effect of a treatment on sperm quality variables. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation

    PubMed Central

    Lobo, Vivian; Rao, Parimala; Gajbhiye, Rahul; Kulkarni, Vijay; Parte, Priyanka

    2015-01-01

    GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant

  10. Yolk protein is expressed in the insect testis and interacts with sperm

    PubMed Central

    Bebas, Piotr; Kotwica, Joanna; Joachimiak, Ewa; Giebultowicz, Jadwiga M

    2008-01-01

    Background Male and female gametes follow diverse developmental pathways dictated by their distinct roles in fertilization. While oocytes of oviparous animals accumulate yolk in the cytoplasm, spermatozoa slough off most of their cytoplasm in the process of individualization. Mammalian spermatozoa released from the testis undergo extensive modifications in the seminal ducts involving a variety of glycoproteins. Ultrastructural studies suggest that glycoproteins are involved in sperm maturation in insects; however, their characterization at the molecular level is lacking. We reported previously that the circadian clock controls sperm release and maturation in several insect species. In the moth, Spodoptera littoralis, the secretion of glycoproteins into the seminal fluid occurs in a daily rhythmic pattern. The purpose of this study was to characterize seminal fluid glycoproteins in this species and elucidate their role in the process of sperm maturation. Results We collected seminal fluid proteins from males before and after daily sperm release. These samples were separated by 2-D gel electrophoresis, and gels were treated with a glycoprotein-detecting probe. We observed a group of abundant glycoproteins in the sample collected after sperm release, which was absent in the sample collected before sperm release. Sequencing of these glycoproteins by mass spectroscopy revealed peptides bearing homology with components of yolk, which is known to accumulate in developing oocytes. This unexpected result was confirmed by Western blotting demonstrating that seminal fluid contains protein immunoreactive to antibody against yolk protein YP2 produced in the follicle cells surrounding developing oocytes. We cloned the fragment of yp2 cDNA from S. littoralis and determined that it is expressed in both ovaries and testes. yp2 mRNA and YP2 protein were detected in the somatic cyst cells enveloping sperm inside the testis. During the period of sperm release, YP2 protein appears in

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

    PubMed

    Gibbs, Gerard M; Orta, Gerardo; Reddy, Thulasimala; Koppers, Adam J; Martínez-López, Pablo; de la Vega-Beltràn, José Luis; Lo, Jennifer C Y; Veldhuis, Nicholas; Jamsai, Duangporn; McIntyre, Peter; Darszon, Alberto; O'Bryan, Moira K

    2011-04-26

    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.

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

    PubMed Central

    Gibbs, Gerard M.; Orta, Gerardo; Reddy, Thulasimala; Koppers, Adam J.; Martínez-López, Pablo; Luis de la Vega-Beltràn, José; Lo, Jennifer C. Y.; Veldhuis, Nicholas; Jamsai, Duangporn; McIntyre, Peter; Darszon, Alberto; O'Bryan, Moira K.

    2011-01-01

    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. PMID:21482758

  13. Localization and identification of sumoylated proteins in human sperm: excessive sumoylation is a marker of defective spermatozoa.

    PubMed

    Vigodner, Margarita; Shrivastava, Vibha; Gutstein, Leah Elisheva; Schneider, Jordana; Nieves, Edward; Goldstein, Marc; Feliciano, Miriam; Callaway, Myrasol

    2013-01-01

    Sumoylation is a type of post-translational modification that is implicated in the regulation of numerous cellular events. However, its role in the function of human sperm has not yet been characterized. In this study, both immunofluorescence and electron microscopy revealed that small ubiquitin-like modifiers (SUMO) SUMO1 and SUMO2/3 were highly enriched in the neck area of human sperm that is associated with the redundant nuclear envelope and were also detectable in the flagella and some head regions. Similar localization patterns of SUMO were also observed in mouse and fly sperm. Nonmotile, two-tailed, curled tailed, misshapen, microcephalic (small head) and aciphalic (no head) sperm exhibited abnormally high levels of sumoylation in their neck and tail regions relative to normal sperm. Numerous sumoylated proteins, ranging from 20 to 260 kDa, were detected via western blotting and identified by mass spectrometry, and 55 SUMO targets that were present specifically in human sperm, and not in the control fraction, corresponded to flagella proteins, proteins involved in the maturation and differentiation of sperm, heat shock proteins and important glycolytic and mitochondrial enzymes. The targets that were identified included proteins with specific functions in germ cells and sperm, such as heat shock-related 70-kDa protein 2, outer dense fiber protein 3, A-kinase anchor proteins 3 and 4, L-lactate dehydrogenase C, sperm protein associated with the nucleus on the X chromosome B/F, valosin-containing protein, seminogelins, histone H4 and ubiquitin. Coimmunoprecipitation experiments confirmed the sumoylation of semenogelin and indicated that some sperm proteins are modified by sumoylation and ubiquitination simultaneously. Numerous proteins are modified by sumoylation in human sperm; excessive sumoylation is a marker of defective spermatozoa.

  14. Comparative analysis of Mafriwal (Bos taurus × Bos indicus) and Kedah Kelantan (Bos indicus) sperm proteome identifies sperm proteins potentially responsible for higher fertility in a tropical climate.

    PubMed

    Ashrafzadeh, Ali; Nathan, Sheila; Karsani, Saiful Anuar

    2013-07-30

    The fertility of zebu cattle (Bos indicus) is higher than that of the European purebred (Bos taurus) and crossbred (Bos taurus × Bos indicus) cattle in tropical areas. To identify proteins related to the higher thermo-tolerance and fertility of Zebu cattle, this study was undertaken to identify differences in sperm proteome between the high fertile Malaysian indigenous zebu cattle (Kedah Kelantan) and the sub-fertile crossbred cattle (Mafriwal). Frozen semen from three high performance bulls from each breed were processed to obtain live and pure sperm. Sperm proteins were then extracted, and two-dimensional gel electrophoresis performed to compare proteome profiles. Gel image analysis identified protein spots of interest which were then identified by liquid chromatography mass spectrometry quadrupole time-of-flight (LC MS/MS Q-TOF). STRING network analysis predicted interactions between at least 20 of the identified proteins. Among the identified proteins, a number of motility and energy related proteins were present in greater abundance in Kedah Kelantan. Sperm motility evaluation by Computer Assisted Semen Analysis (CASA) confirmed significantly higher motility in Kedah Kelantan. While results from this study do identify proteins that may be responsible for the higher fertility of Kedah Kelantan, functional characterization of these proteins is warranted to reinforce our understanding of their roles in sperm fertility.

  15. Comparative Analysis of Mafriwal (Bos taurus × Bos indicus) and Kedah Kelantan (Bos indicus) Sperm Proteome Identifies Sperm Proteins Potentially Responsible for Higher Fertility in a Tropical Climate

    PubMed Central

    Ashrafzadeh, Ali; Nathan, Sheila; Karsani, Saiful Anuar

    2013-01-01

    The fertility of zebu cattle (Bos indicus) is higher than that of the European purebred (Bos taurus) and crossbred (Bos taurus × Bos indicus) cattle in tropical areas. To identify proteins related to the higher thermo-tolerance and fertility of Zebu cattle, this study was undertaken to identify differences in sperm proteome between the high fertile Malaysian indigenous zebu cattle (Kedah Kelantan) and the sub-fertile crossbred cattle (Mafriwal). Frozen semen from three high performance bulls from each breed were processed to obtain live and pure sperm. Sperm proteins were then extracted, and two-dimensional gel electrophoresis performed to compare proteome profiles. Gel image analysis identified protein spots of interest which were then identified by liquid chromatography mass spectrometry quadrupole time-of-flight (LC MS/MS Q-TOF). STRING network analysis predicted interactions between at least 20 of the identified proteins. Among the identified proteins, a number of motility and energy related proteins were present in greater abundance in Kedah Kelantan. Sperm motility evaluation by Computer Assisted Semen Analysis (CASA) confirmed significantly higher motility in Kedah Kelantan. While results from this study do identify proteins that may be responsible for the higher fertility of Kedah Kelantan, functional characterization of these proteins is warranted to reinforce our understanding of their roles in sperm fertility. PMID:23903046

  16. Female major histocompatibility complex type affects male testosterone levels and sperm number in the horse (Equus caballus)

    PubMed Central

    Burger, D.; Dolivo, G.; Marti, E.; Sieme, H.; Wedekind, C.

    2015-01-01

    Odours of vertebrates often contain information about the major histocompatibility complex (MHC), and are used in kin recognition, mate choice or female investment in pregnancy. It is, however, still unclear whether MHC-linked signals can also affect male reproductive strategies. We used horses (Equus caballus) to study this question under experimental conditions. Twelve stallions were individually exposed either to an unfamiliar MHC-similar mare and then to an unfamiliar MHC-dissimilar mare, or vice versa. Each exposure lasted over a period of four weeks. Peripheral blood testosterone levels were determined weekly. Three ejaculates each were collected in the week after exposure to both mares (i.e. in the ninth week) to determine mean sperm number and sperm velocity. We found high testosterone levels when stallions were kept close to MHC-dissimilar mares and significantly lower ones when kept close to MHC-similar mares. Mean sperm number per ejaculate (but not sperm velocity) was positively correlated to mean testosterone levels and also affected by the order of presentation of mares: sperm numbers were higher if MHC-dissimilar mares were presented last than if MHC-similar mares were presented last. We conclude that MHC-linked signals influence testosterone secretion and semen characteristics, two indicators of male reproductive strategies. PMID:25904670

  17. Female major histocompatibility complex type affects male testosterone levels and sperm number in the horse (Equus caballus).

    PubMed

    Burger, D; Dolivo, G; Marti, E; Sieme, H; Wedekind, C

    2015-05-22

    Odours of vertebrates often contain information about the major histocompatibility complex (MHC), and are used in kin recognition, mate choice or female investment in pregnancy. It is, however, still unclear whether MHC-linked signals can also affect male reproductive strategies. We used horses (Equus caballus) to study this question under experimental conditions. Twelve stallions were individually exposed either to an unfamiliar MHC-similar mare and then to an unfamiliar MHC-dissimilar mare, or vice versa. Each exposure lasted over a period of four weeks. Peripheral blood testosterone levels were determined weekly. Three ejaculates each were collected in the week after exposure to both mares (i.e. in the ninth week) to determine mean sperm number and sperm velocity. We found high testosterone levels when stallions were kept close to MHC-dissimilar mares and significantly lower ones when kept close to MHC-similar mares. Mean sperm number per ejaculate (but not sperm velocity) was positively correlated to mean testosterone levels and also affected by the order of presentation of mares: sperm numbers were higher if MHC-dissimilar mares were presented last than if MHC-similar mares were presented last. We conclude that MHC-linked signals influence testosterone secretion and semen characteristics, two indicators of male reproductive strategies.

  18. Microgravity alters protein phosphorylation changes during initiation of sea urchin sperm motility

    NASA Technical Reports Server (NTRS)

    Tash, J. S.; Bracho, G. E.

    1999-01-01

    European Space Agency (ESA) studies demonstrated that bull sperm swim with higher velocity in microgravity (microG) than at 1 G. Coupling between protein phosphorylation and sperm motility during activation in microG and at 1 G was examined in the ESA Biorack on two space shuttle missions. Immotile sperm were activated to swim (86-90% motility) at launch +20 h by dilution into artificial seawater (ASW). Parallel ground controls were performed 2 h after the flight experiment. Activation after 0, 30, and 60 s was terminated with electrophoresis sample buffer and samples analyzed for phosphoamino acids by Western blotting. Phosphorylation of a 130-kDa phosphothreonine-containing protein (FP130) occurred three to four times faster in microG than at 1 G. A 32-kDa phosphoserine-containing protein was significantly stimulated at 30 s but returned to 1 G control levels at 60 s. The rate of FP130 phosphorylation in microG was attenuated by D2O, suggesting that changes in water properties participate in altering signal transduction. Changes in FP130 phosphorylation triggered by the egg peptide speract were delayed in microG. These results demonstrate that previously observed effects of microG on sperm motility are coupled to changes in phosphorylation of specific flagellar proteins and that early events of sperm activation and fertilization are altered in microG.

  19. Microgravity alters protein phosphorylation changes during initiation of sea urchin sperm motility

    NASA Technical Reports Server (NTRS)

    Tash, J. S.; Bracho, G. E.

    1999-01-01

    European Space Agency (ESA) studies demonstrated that bull sperm swim with higher velocity in microgravity (microG) than at 1 G. Coupling between protein phosphorylation and sperm motility during activation in microG and at 1 G was examined in the ESA Biorack on two space shuttle missions. Immotile sperm were activated to swim (86-90% motility) at launch +20 h by dilution into artificial seawater (ASW). Parallel ground controls were performed 2 h after the flight experiment. Activation after 0, 30, and 60 s was terminated with electrophoresis sample buffer and samples analyzed for phosphoamino acids by Western blotting. Phosphorylation of a 130-kDa phosphothreonine-containing protein (FP130) occurred three to four times faster in microG than at 1 G. A 32-kDa phosphoserine-containing protein was significantly stimulated at 30 s but returned to 1 G control levels at 60 s. The rate of FP130 phosphorylation in microG was attenuated by D2O, suggesting that changes in water properties participate in altering signal transduction. Changes in FP130 phosphorylation triggered by the egg peptide speract were delayed in microG. These results demonstrate that previously observed effects of microG on sperm motility are coupled to changes in phosphorylation of specific flagellar proteins and that early events of sperm activation and fertilization are altered in microG.

  20. Characterisation of Caenorhabditis elegans sperm transcriptome and proteome

    PubMed Central

    2014-01-01

    Background Although sperm is transcriptionally and translationally quiescent, complex populations of RNAs, including mRNAs and non-coding RNAs, exist in sperm. Previous microarray analysis of germ cell mutants identified hundreds of sperm genes in Caenorhabditis elegans. To take a more comprehensive view on C. elegans sperm genes, here, we isolate highly pure sperm cells and employ high-throughput technologies to obtain sperm transcriptome and proteome. Results First, sperm transcriptome consists of considerable amounts of non-coding RNAs, many of which have not been annotated and may play functional roles during spermatogenesis. Second, apart from kinases/phosphatases as previously reported, ion binding proteins are also enriched in sperm, underlying the crucial roles of intracellular ions in post-translational regulation in sperm. Third, while the majority of sperm genes/proteins have low abundance, a small number of sperm genes/proteins are hugely enriched in sperm, implying that sperm only rely on a small set of proteins for post-translational regulation. Lastly, by extensive RNAi screening of sperm enriched genes, we identified a few genes that control fertility. Our further analysis reveals a tight correlation between sperm transcriptome and sperm small RNAome, suggesting that the endogenous siRNAs strongly repress sperm genes. This leads to an idea that the inefficient RNAi screening of sperm genes, a phenomenon currently with unknown causes, might result from the competition between the endogenous RNAi pathway and the exogenous RNAi pathway. Conclusions Together, the obtained sperm transcriptome and proteome serve as valuable resources to systematically study spermatogenesis in C. elegans. PMID:24581041

  1. Hexavalent chromium affects sperm motility by influencing protein tyrosine phosphorylation in the midpiece of boar spermatozoa.

    PubMed

    Zhen, Linqing; Wang, Lirui; Fu, Jieli; Li, Yuhua; Zhao, Na; Li, Xinhong

    2016-01-01

    Hexavalent chromium reportedly induces reproductive toxicity and further inhibits male fertility in mammals. In this study, we investigated the molecular mechanism by which hexavalent chromium affects motility signaling in boar spermatozoa in vitro. The results indicated that Cr(VI) decreased sperm motility, protein phosphorylation, mitochondrial membrane potential (ΔΨm) and metabolic enzyme activity starting at 4μmol/mL following incubation for 1.5h. Notably, all parameters were potently inhibited by 10μmol/mL Cr, while supplementation with the dibutyryl-cAMP (dbcAMP) and the 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibition of protein phosphorylation. Interestingly, high concentrations of Cr (>10μmol/mL) increased the tyrosine phosphorylation of some high-molecular-weight proteins in the principle piece but decreased that in the middle piece associated with an extreme reduction of sperm motility. These results suggest that chromium affects boar sperm motility by impairing tyrosine phosphorylation in the midpiece of sperm by blocking the cAMP/PKA pathway in boar sperm in vitro.

  2. Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

    PubMed

    Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

    2015-07-01

    Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

  3. Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

    PubMed Central

    Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

    2016-01-01

    Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

  4. INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22

    EPA Science Inventory

    INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22. SC Jeffay*, SD Perreault, KL Bobseine*, JE Welch*, GR Klinefelter, US EPA, Research Triangle Park, NC.
    SP22, a rat sperm membrane protein that is highly-correlated w...

  5. INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22

    EPA Science Inventory

    INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22. SC Jeffay*, SD Perreault, KL Bobseine*, JE Welch*, GR Klinefelter, US EPA, Research Triangle Park, NC.
    SP22, a rat sperm membrane protein that is highly-correlated w...

  6. Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L.

    PubMed

    Li, P; Hulak, M; Koubek, P; Sulc, M; Dzyuba, B; Boryshpolets, S; Rodina, M; Gela, D; Manaskova-Postlerova, P; Peknicova, J; Linhart, O

    2010-08-01

    Damage to spermatozoa during cryopreservation is regarded as a major obstacle to the expansion of sperm storage technology. The authors used two-dimensional polyacrylamide gel electrophoresis and matrix-associated laser desorption/ionization time-of-flight mass spectrometry to explore whether the protein profile of common carp (Cyprinus carpio) spermatozoa is affected by cryopreservation. Fourteen protein spots were significantly altered following cryopreservation. Eleven of these were identified: three as specific membrane proteins (N-ethylmaleimide-sensitive fusion protein attachment protein alpha, cofilin 2, and annexin A4) involved in membrane trafficking, organization, and cell movement; six as cytoplasmic enzymes (S-Adenosylhomocysteine hydrolase, Si:dkey-180p18.9 protein, lactate dehydrogenase B, phosphoglycerate kinase 1, transaldolase 1, and esterase D/formylglutathione hydrolase) involved in cell metabolism, oxidoreductase activity, and signal transduction; and two as transferrin variant C and F. Based on these findings, the authors hypothesize that transferrin in cryopreserved sperm may protect spermatozoa against oxidative damage during the freeze-thaw process. Cryopreservation caused changes in spermatozoa protein profiles that may lead to decreased spermatozoa velocity, motility, and fertilization success, and to subsequent ova hatching rate.

  7. Aquaporin inhibition changes protein phosphorylation pattern following sperm motility activation in fish.

    PubMed

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Cabrita, Elsa; Storelli, Carlo; Vilella, Sebastiano

    2011-09-01

    Our previous studies demonstrated that osmolality is the key signal in sperm motility activation in Sparus aurata spermatozoa. In particular, we have proposed that the hyper-osmotic shock triggers water efflux from spermatozoa via aquaporins. This water efflux determines the cell volume reduction and, in turn, the rise in the intracellular concentration of ions. This increase could lead to the activation of adenylyl cyclase and of the cAMP-signaling pathway, causing the phosphorylation of sperm proteins and then the initiation of sperm motility. This study confirms the important role of sea bream AQPs (Aqp1a and Aqp10b) in the beginning of sperm motility. In fact, when these proteins are inhibited by HgCl(2), the phosphorylation of some proteins (174 kDa protein of head; 147, 97 and 33 kDa proteins of flagella), following the hyper-osmotic shock, was inhibited (totally or partially). However, our results also suggest that more than one transduction pathways could be activated when sea bream spermatozoa were ejaculated in seawater, since numerous proteins showed an HgCl(2)(AQPs)-independent phosphorylation state after motility activation. The role played by each different signal transduction pathways need to be clarified.

  8. Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding.

    PubMed

    Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark A; Tanphaichitr, Nongnuj

    2015-02-01

    The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

  9. Loss of heat shock protein 70 from apical region of buffalo (Bubalus bubalis) sperm head after freezing and thawing.

    PubMed

    Varghese, Tincy; Divyashree, Bannur C; Roy, Sudhir C; Roy, Kajal S

    2016-03-15

    The post-thaw fertility of frozen-thawed mammalian spermatozoa is substantially low as compared with that of fresh sperm. Furthermore, the post-thaw fertility of the cryopreserved buffalo sperm has been reported to be poor as compared with that of cattle sperm. Recently, heat shock protein 70 (HSP70) has been found to play a critical role in mammalian fertilization and early embryonic development in boar and cattle. However, the presence of such fertility-related HSP70 in buffalo sperm and its status after cryopreservation has not been reported so far. Thus, a study was conducted to determine the effect of cryopreservation on the level and distribution pattern of HSP70 molecule in buffalo sperm after cryopreservation. Buffalo semen samples, after dilution in semen extender, were aliquoted in straws and divided into two groups. One group was not cryopreserved, and the other group was cryopreserved for 60 days. Sperm proteins were extracted from both non-cryopreserved (NC) and cryopreserved (C) sperm and subjected to Western blot analysis for detection of HSP70 using a monoclonal anti-HSP70 antibody. The distribution pattern of these proteins in buffalo sperm was also monitored before and after cryopreservation using indirect immunofluorescence technique. A prominent 70-kDa protein band of HSP70 protein was detected in protein extracts of both NC and C buffalo sperm. Densitometry analysis revealed that the intensity of 70-kDa HSP70 protein band of cryopreserved sperm decreased significantly (P < 0.05) compared with that of NC sperm. However, the level of HSP70 in cryopreserved extended seminal plasma (ESP) did not change as compared with that of NC samples indicating a possible degradation of HSP70 in the spermatozoa itself rather than leakage of the protein into the ESP. Furthermore, Western blot also confirmed that several HSP70 immunoreactive protein bands detected in the ESP were contributed by the egg yolk that was added to the extender. Immunocytochemistry

  10. Sperm nuclear DNA fragmentation rate is associated with differential protein expression and enriched functions in human seminal plasma.

    PubMed

    Intasqui, Paula; Camargo, Mariana; Del Giudice, Paula T; Spaine, Deborah M; Carvalho, Valdemir M; Cardozo, Karina H M; Zylbersztejn, Daniel S; Bertolla, Ricardo P

    2013-10-01

    To analyse the proteomic profile of seminal plasma with the aim of identifying the proteins and post-genomic pathways associated with sperm DNA fragmentation. A cross-sectional study including 89 subjects from a human reproduction service was carried out. All semen samples were assessed for sperm DNA fragmentation using a comet assay. Results from 60 sperm were analysed using Komet 6.0.1 software and the 'Olive tail moment' variable was used to stratify these into low and high sperm DNA fragmentation groups. Seminal plasma proteins from the two groups were pooled and used for proteomic analysis. Quantitative data were used for functional enrichment studies. Seventy-two proteins were identified or quantified in seminal plasma. Of these, nine were differentially expressed in the low group and 21 in the high group. Forty-two proteins were conserved between these groups. Functional enrichment analysis indicated that sperm DNA fragmentation was related to functions such as lipoprotein particle remodelling and regulation, fatty acid binding and immune response. Proteins found exclusively in the low group may be involved in correcting spermatogenesis and/or improving sperm function. Proteins in the high group were associated with increased innate immune response, sperm motility and/or maturation and inhibition of mitochondrial apoptosis. Protein expression and post-genomic pathways of seminal plasma differ according to the rate of sperm DNA integrity. © 2013 The Authors. BJU International © 2013 BJU International.

  11. Upregulation of uncoupling protein Ucp2 through acute cold exposure increases post-thaw sperm quality in zebrafish.

    PubMed

    Wang, Gongfa; Kang, Ning; Gong, Hongmei; Luo, Yan; Bai, Chenglian; Chen, Yuanhong; Ji, Xiaoping; Huang, Changjiang; Dong, Qiaoxiang

    2015-12-01

    Oxidative stress plays an important role in sperm damage during cryopreservation. Mild mitochondrial uncoupling has been shown to reduce excessive reactive oxygen species (ROS) and thus mitigate oxidative stress. Uncoupling protein (Ucp2) regulates mitochondrial uncoupling and can be induced by temperature fluctuation. In the present study, we explored a novel approach of acute cold exposure on Ucp2 activation and its association with oxidative damage and post-thaw sperm quality in zebrafish. Our study revealed that acute cold exposure of zebrafish at 18 °C for 24 h led to significant increase of ucp2 mRNA and Ucp2 protein in zebrafish fresh sperm as well as thawed sperm after cryopreservation. Although cold exposure had no effect on fresh sperm quality except for decreasing lipid peroxidation, sperm collected from cold-exposed zebrafish exhibited higher resistance to cryodamage, which was demonstrated by increased post-thaw motility, decreased lipid peroxidation, increased ATP production, and ultimately increased fertilization success. However, except for reduced lipid peroxidation, we did not observe any significant ROS reduction associated with increased Ucp2 activation in cold-exposed group, suggesting mechanisms other than mitochondrial uncoupling could have contributed to cold exposure associated benefits in post-thaw sperm survival. Nevertheless, our findings indicate that acute cold exposure prior to sperm cryopreservation is beneficial for post-thaw sperm survival in zebrafish, and this novel approach may be used to improve post-thaw sperm quality for other aquatic species.

  12. In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins.

    PubMed

    del Valle, Luis J

    2005-06-01

    Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg-jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø(o) (sperm-specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca(2+), pH, and this activity could be a serine-proteinase. Thermal denaturalization of the oocyte extracts (80 degrees C, 10-15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø(o)) will be a first condition in the process of sperm chromatin remodeling.

  13. Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

    PubMed

    McGinnis, Lynda K; Luo, Jinping; Kinsey, William H

    2013-04-01

    Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

  14. Tyrosine Phosphorylation Pattern in Sperm Proteins Isolated from Normospermic and Teratospermic Men

    PubMed Central

    Jabbari, Sepideh; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mahdi; Ebrahim Habibi, Azadeh; Amirjanati, Naser; Lakpour, Niknam; Asgharpour, Lima; Ardekani, Ali M.

    2009-01-01

    Introduction In mammalian system, spermatozoa are not able to fertilize the oocyte immediately upon ejaculation, thus they undergo a series of biochemical and molecular changes which is termed capacitation. During sperm capacitation, signal transduction pathways are activated which lead to protein tyrosine phosphorylation. Tyrosine phosphorylated proteins have an important role in sperm capacitation such as hyperactive motility, interaction with zona pellucida and acrosome reaction. Evaluation of tyrosine phosphorylation pattern is important for further understanding of molecular mechanisms of fertilization and the etiology of sperm dysfunctions and abnormalities such as teratospermia. The goal of this study is to characterize tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic infertile men attending Avicenna Infertility Clinic in Tehran. Materials and Methods Semen samples were collected and the spermatozoa were isolated using Percoll gradient centrifugation. Then the spermatozoa were incubated up to 6h at 37°C with 5% CO2 in 3% Bovine Serum Albumin-supplemented Ham's F-10 for capacitation to take place. The total proteins from spermatozoa were extracted and were subjected to SDS-PAGE before and after capacitation. To evaluate protein tyrosine phosphorylation pattern, western blotting with specific antibody against phosphorylated tyrosines was performed. Results The results upon western blotting showed: 1) at least six protein bands were detected before capacitation in the spermatozoa from normospermic samples. However, comparable levels of tyrosine phosphorylation was not observed in the spermatozoa from teratospermic samples. 2) The intensity of protein tyrosine phosphorylation appears to have been increased during capacitation in the normospermic relative to the teratospermic group. Conclusion For the first time, these findings demonstrate and suggest that the differences in the types of proteins and diminished

  15. Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein.

    PubMed

    Miwa, Naofumi

    2015-05-22

    Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP), which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

  16. Oviductal expression of avidin, avidin-related protein-2 and progesterone receptor in turkey hens in relation to sperm storage: effects of oviduct tissue type, sperm presence, and turkey line

    USDA-ARS?s Scientific Manuscript database

    The turkey hen’s sperm storage tubules (SST), located in the uterovaginal junction (UVJ) of the oviduct, maintain viable sperm for up to10 weeks after a single insemination. The mechanisms of this in vivo sperm storage are poorly understood. Our objective was to evaluate mRNA and protein expression...

  17. Major chimpanzee-specific structural changes in sperm development-associated genes.

    PubMed

    Kim, Ryong Nam; Kim, Dae-Won; Choi, Sang-Haeng; Chae, Sung-Hwa; Nam, Seong-Hyeuk; Kim, Dong-Wook; Kim, Aeri; Kang, Aram; Park, Kun-Hyang; Lee, Yong Seok; Hirai, Momoki; Suzuki, Yutaka; Sugano, Sumio; Hashimoto, Katsuyuki; Kim, Dae-Soo; Park, Hong-Seog

    2011-09-01

    A comprehensive analysis of transcriptional structures of chimpanzee sperm development-associated genes is of significant interest for deeply understanding sperm development and male reproductive process. In this study, we sequenced 7,680 clones from a chimpanzee testis full-length cDNA library and obtained 1,933 nonredundant high-quality full-length cDNA sequences. Comparative analysis between human and chimpanzee showed that 78 sperm development-associated genes, most of which were yet uncharacterized, had undergone severe structural changes (mutations at the start/stop codons, INDELs, alternative splicing variations and fusion forms) on genomic and transcript levels throughout chimpanzee evolution. Specifically, among the 78 sperm development-associated genes, 39 including ODF2, UBC, and CD59 showed markedly chimpanzee-specific structural changes. Through dN/dS analysis, we found that 56 transcripts (including seven sperm development-associated genes) had values of greater than one when comparing human and chimpanzee DNA sequences, whereas the values were less than one when comparing humans and orangutans. Gene ontology annotation and expression profiling showed that the chimpanzee testis transcriptome was enriched with genes that are associated with chimpanzee male germ cell development. Taken together, our study provides the first comprehensive molecular evidence that many chimpanzee sperm development-associated genes had experienced severe structural changes over the course of evolution on genomic and transcript levels.

  18. Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics.

    PubMed

    Abiko, Mafumi; Furuta, Kensyo; Yamauchi, Yoshio; Fujita, Chiharu; Taoka, Masato; Isobe, Toshiaki; Okamoto, Takashi

    2013-01-01

    In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms.

  19. Identification of Proteins Enriched in Rice Egg or Sperm Cells by Single-Cell Proteomics

    PubMed Central

    Abiko, Mafumi; Furuta, Kensyo; Yamauchi, Yoshio; Fujita, Chiharu; Taoka, Masato; Isobe, Toshiaki; Okamoto, Takashi

    2013-01-01

    In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms. PMID:23936051

  20. Histones and nucleosomes in Cancer sperm (Decapod: Crustacea) previously described as lacking basic DNA-associated proteins: a new model of sperm chromatin.

    PubMed

    Kurtz, Kathryn; Martínez-Soler, Fina; Ausió, Juan; Chiva, Manel

    2008-10-01

    To date several studies have been carried out which indicate that DNA of crustacean sperm is neither bound nor organized by basic proteins and, contrary to the rest of spermatozoa, do not contain highly packaged chromatin. Since this is the only known case of this type among metazoan cells, we have re-examined the composition, and partially the structure, of the mature sperm chromatin of Cancer pagurus, which has previously been described as lacking basic DNA-associated proteins. The results we present here show that: (a) sperm DNA of C. pagurus is bound by histones forming nucleosomes of 170 base pairs, (b) the ratio [histones/DNA] in sperm of two Cancer species is 0.5 and 0.6 (w/w). This ratio is quite lower than the proportion [proteins/DNA] that we found in other sperm nuclei with histones or protamines, whose value is from 1.0 to 1.2 (w/w), (c) histone H4 is highly acetylated in mature sperm chromatin of C. pagurus. Other histones (H3 and H2B) are also acetylated, though the level is much lower than that of histone H4. The low ratio of histones to DNA, along with the high level of acetylation of these proteins, explains the non-compact, decondensed state of the peculiar chromatin in the sperm studied here. In the final section we offer an explanation for the necessity of such decondensed chromatin during gamete fertilization of this species. (c) 2008 Wiley-Liss, Inc.

  1. Heavy ion radiation can promote greater motility and enolase protein expression in ram sperm in in vitro liquid storage.

    PubMed

    He, Yuxuan; Li, Hongyan; He, Jianhua; Zhao, Xingxu

    2014-08-01

    The aim of the study was to determine the effects of heavy ion radiation (HIR) on ram sperm quality during 24h of in vitro liquid storage at 15°C, and identify the most appropriate dose which did not injure, but actually improved sperm quality and confirmed the relationship between highly expressed enolase and ram sperm quality during storage in vitro. Six Dorset ram (Ovis aries) semen pools from five mature and healthy rams were each divided into seven experimental groups with different doses of HIR (0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5Gy) under the same experimental conditions. Sperm motility, viability, ATP content, and the gene and protein expression of enolase were measured at 24h of storage. Irradiated semen which had been stored for 24h, retained not only greater sperm motility, viability, and ATP content, but had greater enolase protein expression. This was evidenced by increased amounts of mRNA for this enzyme and amount of enolase protein as compared with semen from control rams, especially for the 0.1Gy group (P<0.001). These results indicate that HIR can promote enhanced motility and viability during in vitro liquid storage, and the 0.1Gy may be a suitable dose for improving sperm quality. Greater amounts of enolase and ATP content may results from enhanced sperm glycolysis by HIR. HIR enhances sperm glycolysis to generate sufficient ATP for maintaining sperm motility during storage.

  2. Electroejaculation increases low molecular weight proteins in seminal plasma modifying sperm quality in Corriedale rams.

    PubMed

    Ledesma, A; Manes, J; Cesari, A; Alberio, R; Hozbor, F

    2014-04-01

    This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation. © 2014 Blackwell Verlag GmbH.

  3. LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22

    EPA Science Inventory

    LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22. GR Klinefelter1, JE Welch*1, HDM Moore*2, K Bobseine*1, J Suarez*1 ,N Roberts*1 ,R Zucker *1 1U.S. EPA, NHEERL, Reproductive Toxicology Division, RTP, NC and 2University of Sheffield...

  4. LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22

    EPA Science Inventory

    LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22. GR Klinefelter1, JE Welch*1, HDM Moore*2, K Bobseine*1, J Suarez*1 ,N Roberts*1 ,R Zucker *1 1U.S. EPA, NHEERL, Reproductive Toxicology Division, RTP, NC and 2University of Sheffield...

  5. Chronic restraint stress induces sperm acrosome reaction and changes in testicular tyrosine phosphorylated proteins in rats

    PubMed Central

    Arun, Supatcharee; Burawat, Jaturon; Sukhorum, Wannisa; Sampannang, Apichakan; Maneenin, Chanwit; Iamsaard, Sitthichai

    2016-01-01

    Background: Stress is a cause of male infertility. Although sex hormones and sperm quality have been shown to be low in stress, sperm physiology and testicular functional proteins, such as phosphotyrosine proteins, have not been documented. Objective: To investigate the acrosome status and alterations of testicular proteins involved in spermatogenesis and testosterone synthesis in chronic stress in rats. Materials and Methods: In this experimental study, male rats were divided into 2 groups (control and chronic stress (CS), n=7). CS rats were immobilized (4 hr/day) for 42 consecutive days. The blood glucose level (BGL), corticosterone, testosterone, acrosome status, and histopathology were examined. The expressions of testicular steroidogenic acute regulatory (StAR), cytochrome P450 side chain cleavage (CYP11A1), and phosphorylated proteins were analyzed. Results: Results showed that BGL (71.25±2.22 vs. 95.60±3.36 mg/dl), corticosterone level (24.33±4.23 vs. 36.9±2.01 ng/ml), acrosome reacted sperm (3.25±1.55 vs. 17.71±5.03%), and sperm head abnormality (3.29±0.71 vs. 6.21±1.18%) were significantly higher in CS group in comparison with control. In contrast, seminal vesicle (0.41±0.05 vs. 0.24±0.07 g/100g), testosterone level (3.37±0.79 vs. 0.61±0.29 ng/ml), and sperm concentration (115.33±7.70 vs. 79.13±3.65×106 cells/ml) of CS were significantly lower (p<0.05) than controls. Some atrophic seminiferous tubules and low sperm mass were apparent in CS rats. The expression of CYP11A1 except StAR protein was markedly decreased in CS rats. In contrast, a 55 kDa phosphorylated protein was higher in CS testes. Conclusion: CS decreased the expression of CYP11A, resulting in decreased testosterone, and increased acrosome-reacted sperm, assumed to be the result of an increase of 55 kDa phosphorylated protein. PMID:27525328

  6. The spe-42 gene is required for sperm-egg interactions during C. elegans fertilization and encodes a sperm-specific transmembrane protein.

    PubMed

    Kroft, Tim L; Gleason, Elizabeth J; L'Hernault, Steven W

    2005-10-01

    Fertilization, the union of sperm and egg to form a new organism, is a critical process that bridges generations. Although the cytological and physiological aspects of fertilization are relatively well understood, little is known about the molecular interactions that occur between gametes. C. elegans has emerged as a powerful system for the identification of genes that are necessary for fertilization. C. elegans spe-42 mutants are sterile, producing cytologically normal spermatozoa that fail to fertilize oocytes. Indeed, male mating behavior, sperm transfer to hermaphrodites, sperm migration to the spermatheca, which is the site of fertilization and sperm competition are normal in spe-42 mutants. spe-42 mutant sperm make direct contact with oocytes in the spermatheca, suggesting that SPE-42 plays a role during sperm-egg interactions just prior to fertilization. No other obvious defects were observed in spe-42 mutant worms. Cloning and sequence analysis revealed that SPE-42 is a novel predicted 7-pass integral membrane protein with homologs in many metazoan species, suggesting that its mechanism of action could be conserved.

  7. Association between the presence of protein bands in ram seminal plasma and sperm tolerance to freezing.

    PubMed

    Goularte, K L; Gastal, G D A; Schiavon, R S; Gonçalves, A O; Schneider, J R; Corcini, C D; Lucia, T

    2014-05-01

    This study evaluated associations between the presence of protein bands in ram seminal plasma and the quality of sperm frozen with distinct extenders. Ejaculates were frozen in a Tris-egg yolk based extender, including either 5% glycerol or 100mM trehalose. Seminal plasma samples were submitted to unidimensional electrophoresis. Pre-freezing and post-thawing sperm quality was similar between extenders (P>0.05). A total of 26 bands were identified in ram seminal plasma. Pre-freezing sperm motility was increased when the 15, 19 and 80kDa bands were present in seminal plasma (P<0.05). The presence of an 11kDa band in seminal plasma was associated with reduced pre-freezing membrane integrity (P<0.05). After thawing, both sperm motility and membrane integrity were reduced when a 24kDa band was present in seminal plasma (P<0.05). Post-thawing acrosome integrity was greater in the presence of a 31kDa band in seminal plasma (P<0.05). Regardless of the cryoprotectant included in the freezing extender, these six bands may be potential markers for ram sperm tolerance to freezing.

  8. Putative sperm fusion protein IZUMO and the role of N-glycosylation

    SciTech Connect

    Inoue, Naokazu; Ikawa, Masahito; Okabe, Masaru

    2008-12-19

    IZUMO is the mouse sperm protein proven to be essential for fusion with eggs. It contains one immunoglobulin-like domain with a conserved glycosylation site within. In the present paper, we produced transgenic mouse lines expressing unglycosylated IZUMO (N204Q-IZUMO) in Izumo1 -/- background. The expression of N204Q-IZUMO rescued the infertile phenotype of IZUMO disrupted mice, indicating glycosylation is not essential for fusion-facilitating activity of IZUMO. The N204Q-IZUMO was produced in testis in comparable amounts to wild-type IZUMO, but the amount of N204Q-IZUMO on sperm was significantly decreased by the time sperm reached the cauda epididymis. These data suggest that glycosylation is not essential for the function of IZUMO, but has a role in protecting it from fragmentation in cauda epididymis.

  9. Reduction in the level of hyaluronan binding protein 1 (HABP1) is associated with loss of sperm motility.

    PubMed

    Ghosh, Ilora; Bharadwaj, Archana; Datta, Kasturi

    2002-01-01

    Hyaluronan binding protein 1 (HABP1) was reported to be present on human sperm surface and its involvement in fertilization has already been elucidated (Mol. Repro. Dev. 38 (1994) 69). In the present communication, we report a significant reduction in the level of this protein in sperms from asthenozoospermic and oligozoospermic patients as compared to normozoospermic one. Further evidence of the absence of HABP1 in sperms, having motility <20% is documented, which again is a determining factor for fertilization. HABP1 was quantitatively determined using anti-HABP1 antibody from sperm extracts isolated from semen samples of both the fertile and infertile groups demonstrating low sperm motility. Sperm samples with low motility revealed a significant reduction in the level of HABP1 in immunoblot detection as well as immunolocalization experiment. It suggests that decreased HABP1 level may be associated with low motility of sperms, which in turn might cause infertility in the patient. Thus, the sperm surface HABP1 level can be correlated with the degree of sperm motility, an important criteria for fertilization.

  10. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    EPA Science Inventory

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM
    IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2

    * Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue1
    1The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  11. The heterotrimeric motor protein kinesin-II localizes to the midpiece and flagellum of sea urchin and sand dollar sperm.

    PubMed

    Henson, J H; Cole, D G; Roesener, C D; Capuano, S; Mendola, R J; Scholey, J M

    1997-01-01

    We have utilized immunoblotting and light microscopic immunofluorescent staining methods to examine the expression and localization of sea urchin kinesin-II, a heterotrimeric plus end-directed microtubule motor protein (previously referred to as KRP(85/95)), in sea urchin and sand dollar sperm. We demonstrate the presence of the 85 K and 115 K subunits of kinesin-II in sperm and localize these proteins to the sperm flagella and midpiece. The kinesin-II localization pattern is punctate and discontinuous, and in the flagella it is quite distinct from the continuous labeling present in sperm labeled with anti-flagellar dynein. The kinesin-II staining is largely insensitive to prefixation detergent extraction, suggesting that it is not associated with membranous elements in the sperm. In the midpiece the kinesin-II staining is similar to the pattern present in sperm labeled with an anti-centrosomal antibody. To our knowledge, this is the first localization of kinesin-like proteins in mature sperm and corroborates the recent identification and localization of kinesin-like proteins in the flagella and basal body of the unicellular green alga Chlamydomonas. We hypothesize that kinesin-II in the sperm may play functional roles in intraflagellar transport and/or the formation of flagella during spermatogenesis.

  12. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    EPA Science Inventory

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM
    IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2

    * Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue1
    1The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  13. Effect of oviductal proteins on structural and functional characteristics of cryopreserved sperm in Murrah buffaloes.

    PubMed

    Imam, S; Ansari, M R; Kumar, A; Singh, C; Bharti, V K; Kumaresan, A

    2010-04-01

    The present study was undertaken to elucidate the effect of non-luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non-luteal based on ovarian morphology. Non-luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10,000 g at 4 degrees C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at -20 degrees C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris-citrate-egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non-luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5 degrees C, filled in 0.5 ml French straws, exposed to LN(2) vapour, plunged into LN(2) and then stored at -196 degrees C. The equilibrated and frozen-thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo-osmotic sperm swelling test (HOST). In frozen-thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non-luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze-thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen-thawed semen. It was inferred that incorporation of non-luteal whole oviductal fluid

  14. Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele.

    PubMed

    Fariello, Roberta Maria; Pariz, Juliana Risso; Spaine, Deborah Montagnini; Gozzo, Fábio César; Pilau, Eduardo Jorge; Fraietta, Renato; Bertolla, Ricardo Pimenta; Andreoni, Cassio; Cedenho, Agnaldo Pereira

    2012-11-01

    What are the effects of smoking on the functional aspects of the sperm, the levels of lipid peroxidation and the protein profile of seminal plasma in patients with varicocele? In men with varicocele, smoking is associated with altered semen quality, decreased sperm functional integrity and seminal oxidative stress. Alterations in seminal plasma protein profiles are also present and may explain the altered semen phenotype. Varicocele is a major cause of male infertility. It reduces testicular blood renewal with a consequent accumulation of toxic substances. Thus, it can potentiate the toxic effects of environmental exposure to genotoxic substances such as those found in cigarette smoke. A cross-sectional study was performed in 110 patients presenting with variococele to the Human Reproduction Section of the Sao Paulo Federal University (2006-2010). The patients were divided into a control group of non-smokers, a moderate smokers group and a heavy smokers group. Semen parameters were analysed by standard methods. Sperm DNA integrity and mitochondrial activity were assessed by Comet assays and by 3,3'-diaminobenzidine deposition, respectively. The level of lipid peroxidation in semen was determined by malondialdehyde quantification. Proteomic studies were performed by 2D-electrophoresis and mass spectrometry. Both groups of smokers showed reduced semen quality in comparison with the control group. In the groups of smokers, sperm DNA integrity and mitochondrial activity were also decreased and lipid peroxidation levels were increased. Proteomic analyses revealed 20 proteins differentially expressed between the study groups. A study including smokers without varicocele is still warranted as these results apply only to smokers who present varicocele. Patients with varicocele who are exposed to tobacco smoking present more important alterations to semen quality and sperm functional integrity and show changes in the seminal plasma proteome. This suggests testicular, and

  15. Luteinizing hormone modulates intracellular calcium, protein tyrosine phosphorylation and motility during human sperm capacitation.

    PubMed

    López-Torres, Aideé S; González-González, María E; Mata-Martínez, Esperanza; Larrea, Fernando; Treviño, Claudia L; Chirinos, Mayel

    2017-02-05

    In order to fertilize, spermatozoa must undergo physiological and biochemical changes during their transit along the female reproductive tract before reaching and fusing with the oocyte, process known as capacitation. Sperm modifications associated with capacitation are modulated by their interaction with molecules present in the female reproductive tract. During the woman fertile window, some reproductive hormones reach their maximum concentrations in serum, such as the luteinizing hormone (LH). Since spermatozoa preparing to fertilize may be exposed to LH, the purpose of this work was to study the effects of this hormone on intracellular Ca(2+) concentrations ([Ca(2+)]i), protein tyrosine phosphorylation, sperm motility and acrosome reaction under capacitating conditions. The results showed that LH increases the duration and amplitude of Ca(2+) oscillations. Furthermore, motility analysis indicated that LH decreases rapid progressive motility and that sperm hyperactivation as well as several kinetic parameters augment in the presence of 0.5 and 1 μg/ml of the hormone. In addition, these two hormone concentrations also consistently promoted protein tyrosine phosphorylation. However, no effects on acrosome reaction were observed. In conclusion, the evidence indicates that LH modulates several sperm function variables involved in capacitation, suggesting that may have an important and unexplored role during human fertilization. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Elucidation of the Involvement of p14, a Sperm Protein during Maturation, Capacitation and Acrosome Reaction of Caprine Spermatozoa

    PubMed Central

    Nandi, Pinki; Ghosh, Swatilekha; Jana, Kuladip; Sen, Parimal C.

    2012-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event. PMID:22291985

  17. Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa.

    PubMed

    Nandi, Pinki; Ghosh, Swatilekha; Jana, Kuladip; Sen, Parimal C

    2012-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

  18. Impact of antifreeze proteins and antifreeze glycoproteins on bovine sperm during freeze-thaw.

    PubMed

    Prathalingam, N S; Holt, W V; Revell, S G; Mirczuk, S; Fleck, R A; Watson, P F

    2006-11-01

    There are no reports on the use of antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) for the use of bull sperm cryopreservation despite studies in the ram, mouse and chimpanzee. The effect of freezing and thawing on bull sperm viability, osmotic resistance and acrosome integrity were observed following the addition of AFP1, AFPIII and AFGP at four concentrations (0.1, 1, 10 and 100 microg/ml). In a second part of the experiment, fluorescein was conjugated to the AFPs and AFGP and observations were made using fluorescence microscopy to determine whether binding occurred between the sperm cell membranes and the proteins. In the final part of the study the cryopreservation media were cooled in the presence of the AFPs and AFGPs at the four concentrations on a cryomicroscope to mimic similar cooling curves as those used in the presence of sperm. Following freeze-thaw, AFPI resulted in increased osmotic resistant cells at 0.1-10 microg/ml compared to the control (P<0.01). AFPI and AFPIII did bind to the sperm cells. There was no visual difference in ice structure between the control, AFPIII and AFGP but AFPI resulted in parallel crystals at 0.1, 1 and 10 microg/ml. We suggest that the increased osmotic resistance in the spermatozoa cryopreserved in AFPI is due to the cells orientating between the ice crystals, reducing mechanical stress to the cell membrane. Previous research has shown that osmotic resistance correlates with bull fertility, suggesting that bull spermatozoa cryopreserved in the presence of AFPI may have increased fertility in vivo.

  19. LYZL6, an acidic, bacteriolytic, human sperm-related protein, plays a role in fertilization

    PubMed Central

    Huang, Peng; Li, Wenshu; Yang, Zhifang; Zhang, Ning; Xu, Yixin; Bao, Jianying; Jiang, Deke; Dong, Xianping

    2017-01-01

    Lysozyme-like proteins (LYZLs) belong to the c-type lysozyme/α-lactalbumin family and are selectively expressed in the mammalian male reproductive tract. Two members, human sperm lysozyme-like protein (SLLP) -1 and mouse LYZL4, have been reported to contribute to fertilization but show no bacteriolytic activity. Here, we focused on the possible contribution of LYZL6 to immunity and fertilization. In humans, LYZL6 was selectively expressed by the testis and epididymis and became concentrated on spermatozoa. Native LYZL6 isolated from sperm extracts exhibited bacteriolytic activity against Micrococcus lysodeikticus. Recombinant LYZL6 (rLYZL6) reached its peak activity at pH 5.6 and 15 mM of Na+, and could inhibit the growth of Gram-positive, but not Gram-negative bacteria. Nevertheless, the bacteriolytic activity of rLYZL6 proved to be much lower than that of human lysozyme under physiological conditions. Immunodetection with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa. Immunoneutralization of LYZL6 significantly decreased the numbers of human spermatozoa fused with zona-free hamster eggs in a dose-dependent manner in vitro. Thus, we report here for the first time that LYZL6, an acidic, bacteriolytic and human sperm-related protein, is likely important for fertilization but not for the innate immunity of the male reproductive tract. PMID:28182716

  20. Evaluation of antifreeze protein III for cryopreservation of Nili-Ravi (Bubalus bubalis) buffalo bull sperm.

    PubMed

    Qadeer, S; Khan, M A; Ansari, M S; Rakha, B A; Ejaz, R; Husna, A U; Ashiq, M; Iqbal, R; Ullah, N; Akhter, S

    2014-07-01

    Lower fertility in buffaloes with frozen-thawed semen is attributed to sperm damage that is believed to be due to formation of ice crystals during freeze/thaw process. It was hypothesized that antifreeze proteins in the extender may improve the post thaw quality of buffalo bull sperm. For this purpose, two separate experiments were conducted to evaluate antifreeze proteins III (AFP III) at 0 (control), 0.1, 1 and 10 μg/mL (Experiment I) and 0 (control), 0.01, 0.1 and 1 μg/mL (Experiment II) for its effect on post thaw quality of buffalo bull semen. Semen was collected from three Nili-Ravi buffalo (Bubalus bubalis) bulls with artificial vagina (42 °C) for three weeks (replicate) per experiment. For each experiment, qualifying ejaculates (6 ejaculates/bull) were divided into four aliquots and diluted (at 37 °C having 50 × 10(6) sperm/mL) in tris-citric acid extender containing above mentioned concentrations of AFP III. Diluted semen was cooled to 4 °C in 2 h, equilibrated for 4 h, filled in 0.5 mL straws, kept over liquid nitrogen vapors for 10 min and plunged in the liquid nitrogen. After 24 h of storage, semen straws were thawed at 37 °C for 30 s to assess sperm progressive motility (SM), plasma membrane integrity (PMI), viability (live sperm with intact acrosome) and normal epical ridge (NAR). In experiment I, improvement (P<0.05) in percentage SM and sperm PMI was recorded in extender containing 0.1 μg/mL AFP III compared to control, the higher concentrations (1 μg/mL and 10 μg/mL) being inefficient. While evaluating the lower concentration (experiment II), 0.01 μg/mL of AFP III in the extender it was found to be ineffective to improve semen quality parameters, while 0.1 μg/mL AFP III in extender was found better in terms of progressive motility and plasma membrane integrity of buffalo bull semen compared to control. Sperm viability and NAR remained similar (P>0.05) in extenders containing different concentrations of AFP III and control in both of

  1. Heterologous recombinant protein with decapacitating activity prevents and reverts cryodamage in ram sperm: An emerging biotechnological tool for cryobiology.

    PubMed

    Zalazar, L; Ledesma, A; Hozbor, F; Cesari, A

    2016-01-01

    During the last decades fundamental and applied aspects of mammalian ram sperm cryopreservation have been increasingly explored by scientists and biotechnologists. Many works report modifications in the composition of the freezing extenders and explore the beneficial and detrimental effects of seminal plasma or seminal plasma components in cryopreservation. Seminal plasma is known to contain stabilizing proteins, thereby this is a good start point to study the maintenance of membrane stability based on the basic knowledge of sperm physiology. However, seminal plasma composition is variable among rams and also the introduction of exogenous seminal plasma or its fractions to commercial semen can be associated with the transmission of viral diseases. Our work shows that a mouse protein, called SPINK3 (Serine Protease Inhibitor Kazal type 3) with decapacitating activity interacts with heterologous ram sperm when it is produced as a recombinant molecule. By immunocytochemistry assays we demonstrate that this protein (naturally expressed by mouse seminal vesicle under androgenic control) binds to the apical portion of both fresh and frozen ram sperm, the same localization described in mouse homologous sperm. Furthermore, it significantly improves sperm progressive motility compared to non-treated samples when it is added to freezing extenders and to dilution media after thawing. On the contrary, addition of SPINK3 does not modify sperm viability. The percentage of sperm with intact acrosome after ionophore induction was also significantly higher in sperm frozen in the presence of SPINK3 compared to control samples and the addition of SPINK3 after thawing significantly reduced both induced and non induced acrosomal loss, indicating that heterologous SPINK3 might act as a calcium inhibitor transport as described in mouse. Based on our results SPINK3 may find a place as a desirable biotechnological tool to achieve a higher proportion of competent sperm to fertilize

  2. Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis).

    PubMed

    Tulake, Kuerban; Wang, Xuguang; Chen, Yong; Yu, Chucai; Jing, Binyu; Li, Heping

    2015-03-01

    High efficiency of in vitro capacitation of deer sperm has not yet been achieved as low sperm penetration rates were reported in in vitro fertilization studies. Our main goal in this study was to identify the changes of frozen-thawed sperm of the rare red deer Tarim wapiti (Cervus elaphus yarkandensis) and detect the effect of bovine serum albumin (BSA), serum, and heparin on the protein tyrosine phosphorylation of frozen-thawed sperm. The frozen-thawed sperm of Tarim wapiti was suspended in improved modified tyrode-albumin-lactate-pyruvate medium and cultured in 5% CO2 at 38.5°C, and the status of protein tyrosine phosphorylation of sperm was detected by Western blotting. Although the results showed that the type number and expression of protein tyrosine phosphorylation of frozen-thawed wapiti sperm were decreased, the tyrosine-phosphorylated proteins such as 10, 14, 40, 47, and 55kDa were increased significantly during the process of capacitation culture (1-2h). In addition, tyrosine-phosphorylated proteins were promoted by BSA rather than serum, and estrus sheep serum (ESS) rather than estrus deer serum. When ESS and heparin were used together at 4h after capacitation, four main tyrosine phosphorylation proteins (10±2, 14±2, 25±3, and 47±3kDa) had a significantly higher expression than that at 2h after capacitation. We demonstrated that these proteins were involved in wapiti sperm in vitro capacitation, heparin in the incubation media was necessary for the capacitation and tyrosine phosphorylation protein was promoted by ESS.

  3. High-protein paternal diet confers an advantage to sons in sperm competition

    PubMed Central

    2017-01-01

    Parental environment can widely influence offspring phenotype, but paternal effects in the absence of parental care remain poorly understood. We asked if protein content in the larval diet of fathers affected paternity success and gene expression in their sons. We found that males reared on high-protein diet had sons that fared better during sperm competition, suggesting that postcopulatory sexual selection is subject to transgenerational paternal effects. Moreover, immune response genes were downregulated in sons of low-protein fathers, while genes involved in metabolic and reproductive processes were upregulated. PMID:28202685

  4. Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice.

    PubMed

    Aarabi, Mahmoud; Balakier, Hanna; Bashar, Siamak; Moskovtsev, Sergey I; Sutovsky, Peter; Librach, Clifford L; Oko, Richard

    2014-10-01

    Mammalian zygotic development is initiated by sperm-mediated intracellular calcium oscillations, followed by activation of metaphase II-arrested oocytes. Sperm postacrosomal WW binding protein (PAWP) fulfils the criteria set for an oocyte-activating factor by inducing oocyte activation and being stored in the perinuclear theca, the sperm compartment whose content is first released into oocyte cytoplasm during fertilization. However, proof that PAWP initiates mammalian zygotic development relies on demonstration that it acts upstream of oocyte calcium oscillations. Here, we show that PAWP triggers calcium oscillations and pronuclear formation in human and mouse oocytes similar to what is observed during intracytoplasmic sperm injection (ICSI). Most important, sperm-induced calcium oscillations are blocked by coinjection of a competitive inhibitor, derived from the WWI domain-binding motif of PAWP, implying the requirement of sperm PAWP and an oocyte-derived WWI domain protein substrate of PAWP for successful fertilization. Sperm-delivered PAWP is, therefore, a unique protein with a nonredundant role during human and mouse fertilization, required to trigger zygotic development. Presented data confirm our previous findings in nonmammalian models and suggest potential applications of PAWP in the diagnosis and treatment of infertility.- © FASEB.

  5. Correlation between Sperm Parameters and Protein Expression of Antioxidative Defense Enzymes in Seminal Plasma: A Pilot Study

    PubMed Central

    Macanovic, Biljana; Vucetic, Milica; Jankovic, Aleksandra; Buzadzic, Biljana; Garalejic, Eliana; Otasevic, Vesna

    2015-01-01

    Background. Semen analysis is the cornerstone in the evaluation of male (in)fertility. However, there are men with normal semen tests but with impaired fertilizing ability, as well as fertile men with poor sperm characteristics. Thus, there is rising interest to find novel parameters that will help to predict and define the functional capacity of spermatozoa. Methods. We examined whether there is a correlation between semen parameters (count, progressive motility, and morphology) and protein expression/activity of antioxidative defense enzymes in seminal plasma from 10 normospermic subjects. Results. Sperm progressive motility was in positive correlation with seminal plasma protein expression of both superoxide dismutase (SOD) isoforms (MnSOD and CuZnSOD) and catalase. Also, positive correlation was observed between sperm count and MnSOD protein expression, as well as between sperm morphology and protein expression of catalase in seminal plasma. In contrast, protein expression of glutathione peroxidase was not in correlation with any sperm parameter, while its activity negatively correlated with sperm morphology and motility. Conclusions. These data suggest that evaluation of protein expression of antioxidative defense enzymes in seminal plasma might be of importance in the evaluation of male fertility status and that could be used as an additional biomarker along with classic semen analysis in assessment of semen quality. PMID:25691806

  6. Identification of the SP22 sperm protein in Santa Inês and Dorper rams.

    PubMed

    Favareto, A P A; Rodello, L; Taconeli, C A; Bicudo, S D; Klinefelter, G R; Kempinas, W G

    2010-04-01

    The sperm membrane protein referred to as SP22 has been identified in different species and, at least in rats, is highly correlated with fertility. The goals of this study were to identify and to quantify the SP22 protein on spermatozoa from adult rams (Dorper and Santa Inês breeds), and to correlate its levels to morphological and kinematics parameters. SP22 on ram sperm was effectively quantified by both enzyme-linked immunosorbent assay (ELISA) and fluorescein isothiocyanate immunostaining analysis and the two methods were significantly correlated (R(2) = 0.70). Clustering analysis of motility parameters obtained by computer-assisted semen analysis system was used to establish that three distinct kinematic subpopulations with different vigour and progressiveness coexistent within ejaculate. While there were significant differences in the distribution of the three subpopulations in the rams, there was no significant correlation between the proportion of each subpopulation in the rams and the SP22 levels. Quantification of SP22 immunostaining intensity was not correlated with any of the sperm parameters. However, SP22 levels obtained by ELISA were negatively correlated with morphological abnormalities and positively correlated with membrane integrity (three variable R(2) = 0.47). Future breeding studies are now needed to validate that this protein is a biomarker of fertility in this species.

  7. Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

    PubMed Central

    Kost, Nils; Kaiser, Sophie; Ostwal, Yogesh; Riedel, Dietmar; Stützer, Alexandra; Nikolov, Miroslav; Rathke, Christina; Renkawitz-Pohl, Renate; Fischle, Wolfgang

    2015-01-01

    Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure. PMID:25735749

  8. Assortative mating drives linkage disequilibrium between sperm and egg recognition protein loci in the sea urchin Strongylocentrotus purpuratus.

    PubMed

    Stapper, Andres Plata; Beerli, Peter; Levitan, Don R

    2015-04-01

    Sperm and eggs have interacting proteins on their surfaces that influence their compatibility during fertilization. These proteins are often polymorphic within species, producing variation in gamete affinities. We first demonstrate the fitness consequences of various sperm bindin protein (Bindin) variants in the sea urchin Strongylocentrotus purpuratus, and assortative mating between males and females based on their sperm Bindin genotype. This empirical finding of assortative mating based on sperm Bindin genotype could arise by linkage disequilibrium (LD) between interacting sperm and egg recognition loci. We then examine sequence variation in eight exons of the sea urchin egg receptor for sperm Bindin (EBR1). We find little evidence of LD among the eight exons of EBR1, yet strong evidence for LD between sperm Bindin and EBR1 overall, and varying degrees of LD between sperm Bindin among the eight exons. We reject the alternate hypotheses of LD driven by shared evolutionary histories, population structure, or close physical linkage between these interacting loci on the genome. The most parsimonious explanation for this pattern of LD is that it represents selection driven by assortative mating based on interactions among these sperm and egg loci. These findings indicate the importance of ongoing sexual selection in the maintenance of protein polymorphisms and LD, and more generally highlight how LD can be used as an indication of current mate choice, as opposed to historic selection. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility.

    PubMed

    Liu, Y; Qu, F; Cao, X; Chen, G; Guo, Q; Ying, X; Guo, W; Lu, L; Ding, Z

    2012-04-01

    Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.

  10. Osmotic stress stimulates phosphorylation and cellular expression of heat shock proteins in rhesus macaque sperm.

    PubMed

    Cole, Julie A; Meyers, Stuart A

    2011-01-01

    The cryosurvival of sperm requires cell signaling mechanisms to adapt to anisotonic conditions during the freezing and thawing process. Chaperone proteins heat shock protein 70 (HSP 70) and heat shock protein 90 (HSP 90; recently renamed HSPA and HSPC, respectively) facilitate some of these cell signaling events in somatic cells. Sperm were evaluated for their cellular expression and levels of phosphorylation of both HSP 70 and HSP 90 under anisotonic conditions as a potential model for cell signaling during the cryopreservation of macaque spermatozoa. In order to monitor the level of stress, the motility and viability parameters were evaluated at various time points. Cells were then either prepared for phosphoprotein enrichment or indirect immunocytochemistry. As controls, the phosphoserine, phosphothreonine, and phosphotyrosine levels were measured under capacitation and cryopreservation conditions and were compared with the phosphoprotein levels expressed under osmotic conditions. As expected, there was an increase in the level of tyrosine phosphorylation under capacitation and cryopreservation conditions. There was also a significant increase in the level of all phosphoproteins under hyperosmotic conditions. There was no change in the level of expression of HSP 70 or 90 under osmotic stress conditions as measured by Western blot. The enrichment of phosphoproteins followed by Western immunoblotting revealed an increase in the phosphorylation of HSP 70 but not HSP 90 under osmotic stress conditions. Indirect immunofluorescence localized HSP 70 to the postacrosomal region of sperm, and the level of membrane expression of HSP 70 was significantly affected by anisotonic conditions, as measured by flow cytometry. Taken together, these results suggest a differential role for HSP 70 and HSP 90 during osmotic stress conditions in rhesus macaque sperm.

  11. The Drosophila chromosomal protein Mst77F is processed to generate an essential component of mature sperm chromatin

    PubMed Central

    2016-01-01

    In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function in vivo. Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro, but its role during spermiogenesis remains unclear. Here, we show that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35Ba/b. We demonstrate that Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals. PMID:27810970

  12. Mitogenic properties of major extracellular proteins.

    PubMed

    Lévesque, J P; Hatzfeld, A; Hatzfeld, J

    1991-08-01

    The major plasma and extracellular matrix proteins are multifunctional molecules. Some, such as fibrinogen or C3, have one domain that binds adhesion receptors and another that specifically binds and activates a separate, mitogenic receptor. In this review, Jean-Pierre Lévesque, Antoinette Hatzfeld and Jacques Hatzfeld describe adhesion and mitogenic receptors that bind to distinct domains of the same extracellular matrix protein and discuss the possibility of common ancestral genes for cell adhesion molecules, extracellular matrix proteins, integrins, immunoglobulins, growth factors and their receptors.

  13. Expression of α-gustducin and α-transducin, G proteins coupled with taste receptors, in boar sperm.

    PubMed

    Spinaci, M; Bucci, D; Mazzoni, M; Giaretta, E; Bernardini, C; Vallorani, C; Tamanini, C; Clavenzani, P; Galeati, G

    2014-07-01

    During the transit in the female genital tract, spermatozoa are exposed to an environment that varies in composition from the vagina to the oviduct. Because G proteins, α-gustducin and α-transducin, are accepted as markers of chemosensitive cells, this study was aimed at assessing whether these proteins are expressed in boar germ cells. Ejaculated sperm extracts were analyzed by Western blot, and indirect immunofluorescence was performed on testis sections, smears of epididymal and ejaculated spermatozoa, sperm cells after in vitro induction of capacitation and acrosome reaction (IVAR), and in sperm cells bound to zona pellucida during IVF. Based on immunoblot results, both G proteins are present in boar sperm. In the testicular tissue sections, α-gustducin and α-transducin positivity was recorded in the germinal cells near the tubular lumen, whereas no positive signal was evident in spermatogonia located in the outer region of the seminiferous tubules. α-Gustducin expression in epididymal and ejaculated spermatozoa was mainly detectable in both the acrosome and the principal piece of the tail, whereas α-transducin was confined to the acrosome and the midpiece. No changes after in vitro induction of capacitation and IVAR were observed, except for the disappearance of acrosomal positivity in reacted spermatozoa. In sperm bound to zona pellucida, the G protein signal was congruent with that observed in IVAR cells. To the best of our knowledge, this is the first description of α-transducin in mammalian sperm and the first description of α-gustducin in boar sperm. Further studies are needed to clarify the possible role of these G proteins in sperm physiology.

  14. Partial characterization of protein extracted from Terminalia catappa seed behaving as lectin that is capable of mouse sperm agglutination

    NASA Astrophysics Data System (ADS)

    Haryanto, Hery; Kamilah, Santi Nurul

    2017-05-01

    The objective of this research is to partially characterize proteins extracted from Terminalia catappa seeds that behave like lectin, being capable of mouse sperm agglutination. Cotyledons of the seed were extracted in homogenizing PBS buffer. The supernatant was precipitated with ammonium sulfate to a final concentration of 50% (w/v), then dialyzed with 2 kinds of dialysis tubes, i.e. 6000 and 12000 MWCO. The precipitated proteins were then diluted in 0.9% (w/v) NaCl. Next, dialysis fractions of the precipitated protein were used for the mouse sperm agglutination test. Protein from the dialysis fraction of 6000 MWCO tube could agglutinate mouse sperm. The quantitation of protein was carried out by using the Bradford Coomassie kit from crude extract to dialyzed fractions. Both dialyzed fractions were also run on SDS-PAGE to see the protein profiles.

  15. Hypercholesterolemia Impaired Sperm Functionality in Rabbits

    PubMed Central

    Monclus, Maria A.; Cabrillana, Maria E.; Clementi, Marisa A.; Espínola, Leandro S.; Cid Barría, Jose L.; Vincenti, Amanda E.; Santi, Analia G.; Fornés, Miguel W.

    2010-01-01

    Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a “folded head”-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events. PMID:20976152

  16. Characterization of secretory proteins from cultured cauda epididymal cells that significantly sustain bovine sperm motility in vitro.

    PubMed

    Reyes-Moreno, Carlos; Boilard, Mathieu; Sullivan, Robert; Sirard, Marc-André

    2002-12-01

    Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture

  17. A Novel Cysteine Knot Protein for Enhancing Sperm Motility That Might Facilitate the Evolution of Internal Fertilization in Amphibians.

    PubMed

    Yokoe, Misato; Takayama-Watanabe, Eriko; Saito, Yoko; Kutsuzawa, Megumi; Fujita, Kosuke; Ochi, Haruki; Nakauchi, Yuni; Watanabe, Akihiko

    2016-01-01

    Internal fertilization ensures successful reproduction of tetrapod vertebrates on land, although how this mode of reproduction evolved is unknown. Here, we identified a novel gene encoding sperm motility-initiating substance (SMIS), a key protein for the internal fertilization of the urodele Cynops pyrrhogaster by Edman degradation of an isolated protein and subsequent reverse transcription polymerase chain reaction. The SMIS gene encoded a 150 amino-acid sequence including the cysteine knot (CK) motif. No gene with substantial similarity to the SMIS was in the data bank of any model organisms. An active site of the SMIS was in the C-terminal region of the 2nd loop of CK motif. A synthetic peptide including the active site sequence bound to the midpiece and initiated/enhanced the circular motion of C. pyrrhogaster sperm, which allows penetration of the egg jelly specialized for the internal fertilization of this species. The synthetic peptide bound to whole sperm of Rhacophorus arboreus and enhanced the rotary motion, which is adapted to propel the sperm through egg coat matrix specialized for arboreal reproduction, while it bound to the tip of head and tail of Bufo japonicus sperm, and enhanced the vibratory motion, which is suited to sperm penetration through the egg jelly specialized for the reproduction of that species in freshwater. The polyclonal antibody against the active site of the SMIS specifically bound to egg coat matrix of R. arboreus. These findings suggest that diversification of amphibian reproductive modes accompanies the specialization of egg coat and the adaptation of sperm motility to penetrate the specialized egg coat, and SMIS acts as the sperm motility enhancer of anurans and urodeles that might facilitate to adaptively optimize sperm motility for allowing the establishment of internal fertilization.

  18. A Novel Cysteine Knot Protein for Enhancing Sperm Motility That Might Facilitate the Evolution of Internal Fertilization in Amphibians

    PubMed Central

    Yokoe, Misato; Takayama-Watanabe, Eriko; Saito, Yoko; Kutsuzawa, Megumi; Fujita, Kosuke; Ochi, Haruki; Nakauchi, Yuni; Watanabe, Akihiko

    2016-01-01

    Internal fertilization ensures successful reproduction of tetrapod vertebrates on land, although how this mode of reproduction evolved is unknown. Here, we identified a novel gene encoding sperm motility-initiating substance (SMIS), a key protein for the internal fertilization of the urodele Cynops pyrrhogaster by Edman degradation of an isolated protein and subsequent reverse transcription polymerase chain reaction. The SMIS gene encoded a 150 amino-acid sequence including the cysteine knot (CK) motif. No gene with substantial similarity to the SMIS was in the data bank of any model organisms. An active site of the SMIS was in the C-terminal region of the 2nd loop of CK motif. A synthetic peptide including the active site sequence bound to the midpiece and initiated/enhanced the circular motion of C. pyrrhogaster sperm, which allows penetration of the egg jelly specialized for the internal fertilization of this species. The synthetic peptide bound to whole sperm of Rhacophorus arboreus and enhanced the rotary motion, which is adapted to propel the sperm through egg coat matrix specialized for arboreal reproduction, while it bound to the tip of head and tail of Bufo japonicus sperm, and enhanced the vibratory motion, which is suited to sperm penetration through the egg jelly specialized for the reproduction of that species in freshwater. The polyclonal antibody against the active site of the SMIS specifically bound to egg coat matrix of R. arboreus. These findings suggest that diversification of amphibian reproductive modes accompanies the specialization of egg coat and the adaptation of sperm motility to penetrate the specialized egg coat, and SMIS acts as the sperm motility enhancer of anurans and urodeles that might facilitate to adaptively optimize sperm motility for allowing the establishment of internal fertilization. PMID:27579691

  19. Calcium regulates motility and protein phosphorylation by changing cAMP and ATP concentrations in boar sperm in vitro.

    PubMed

    Li, Xinhong; Wang, Lirui; Li, Yuhua; Zhao, Na; Zhen, Linqing; Fu, Jieli; Yang, Qiangzhen

    2016-09-01

    Considering the importance of calcium (Ca(2+)) in regulating sperm capacitation, hyperactivation and acrosome reaction, little is known about the molecular mechanism of action of this ion in this process. In the present study, assessment of the molecular mechanism from the perspective of energy metabolism occurred. Sperm motility variables were determined using computer-assisted sperm analysis (CASA) and the phosphorylation of PKA substrates, tyrosine residues and AMP-activated protein kinase (AMPK) were analyzed by Western blot. Moreover, intracellular sperm-specific glyceraldehyde 3-phosphatedehydrogenase (GAPDH) activity, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenosine 5'-triphosphate (ATP) concentrations were assessed in boar sperm treated with Ca(2+). Results of the present study indicated that, under greater extracellular Ca(2+)concentrations (≥3.0mM), sperm motility and protein phosphorylation were inhibited. Interestingly, these changes were correlated with that of GAPDH activity, AMPK phosphorylation, cAMP and ATP concentrations. The negative effects of Ca(2+) on these intracellular processes were attenuated by addition of the calmodulin (CaM) inhibitor W7 and the inhibitor of calmodulin-dependent protein kinase (CaMK), KN-93. In the presence of greater extracellular Ca(2+), however, the phosphorylation pathway was suppressed by H-89. Taken together, these results suggested that Ca(2+) had a dual role in regulating boar sperm motility and protein phosphorylation due to the changes of cAMP and ATP concentrations, in response to cAMP-mediated signal transduction and the Ca(2+) signaling cascade. The present study provided some novel insights into the molecular mechanism underlying the effects of Ca(2+) on boar sperm as well as the involvement of energy metabolism in this mechanism.

  20. Procurement of exogenous ammonia by the swallowtail butterfly, Papilio polytes, for protein biosynthesis and sperm production.

    PubMed

    Honda, Keiichi; Takase, Hiroyuki; Ômura, Hisashi; Honda, Hiroshi

    2012-09-01

    How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with (15)N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly would foster better understanding of the foraging and reproductive strategies in insects.

  1. Procurement of exogenous ammonia by the swallowtail butterfly, Papilio polytes, for protein biosynthesis and sperm production

    NASA Astrophysics Data System (ADS)

    Honda, Keiichi; Takase, Hiroyuki; Ômura, Hisashi; Honda, Hiroshi

    2012-09-01

    How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with 15N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly would foster better understanding of the foraging and reproductive strategies in insects.

  2. Mice expressing aberrant sperm-specific protein PMIS2 produce normal-looking but fertilization-incompetent spermatozoa.

    PubMed

    Yamaguchi, Ryo; Fujihara, Yoshitaka; Ikawa, Masahito; Okabe, Masaru

    2012-07-01

    Eight kinds of gene-disrupted mice (Clgn, Calr3, Pdilt, Tpst2, Ace, Adam1a, Adam2, and Adam3) show impaired sperm transition into the oviducts and defective sperm binding to the zona pellucida. All of these knockout strains are reported to lack or show aberrant expression of a disintegrin and metallopeptidase domain 3 (ADAM3) on the sperm membrane. We performed proteomic analyses of the proteins of these infertile spermatozoa to clarify whether the abnormal function is caused exclusively by a deficiency in ADAM3 expression. Two proteins, named PMIS1 and PMIS2, were missing in spermatozoa from Clgn-disrupted mice. To study their roles, we generated two gene-disrupted mouse lines. Pmis1-knockout mice were fertile, but Pmis2-knockout males were sterile because of a failure of sperm transport into the oviducts. Pmis2-deficient spermatozoa also failed to bind to the zona pellucida. However, they showed normal fertilizing ability when eggs surrounded with cumulus cells were used for in vitro fertilization. Further analysis revealed that these spermatozoa lacked the ADAM3 protein, but the amount of PMIS2 was also severely reduced in Adam3-deficient spermatozoa. These results suggest that PMIS2 might function both as the ultimate factor regulating sperm transport into the oviducts and in modulating sperm-zona binding.

  3. Expression of uncharacterized male germ cell-specific genes and discovery of novel sperm-tail proteins in mice.

    PubMed

    Kwon, Jun Tae; Ham, Sera; Jeon, Suyeon; Kim, Youil; Oh, Seungmin; Cho, Chunghee

    2017-01-01

    The identification and characterization of germ cell-specific genes are essential if we hope to comprehensively understand the mechanisms of spermatogenesis and fertilization. Here, we searched the mouse UniGene databases and identified 13 novel genes as being putatively testis-specific or -predominant. Our in silico and in vitro analyses revealed that the expressions of these genes are testis- and germ cell-specific, and that they are regulated in a stage-specific manner during spermatogenesis. We generated antibodies against the proteins encoded by seven of the genes to facilitate their characterization in male germ cells. Immunoblotting and immunofluorescence analyses revealed that one of these proteins was expressed only in testicular germ cells, three were expressed in both testicular germ cells and testicular sperm, and the remaining three were expressed in sperm of the testicular stages and in mature sperm from the epididymis. Further analysis of the latter three proteins showed that they were all associated with cytoskeletal structures in the sperm flagellum. Among them, MORN5, which is predicted to contain three MORN motifs, is conserved between mouse and human sperm. In conclusion, we herein identify 13 authentic genes with male germ cell-specific expression, and provide comprehensive information about these genes and their encoded products. Our finding will facilitate future investigations into the functional roles of these novel genes in spermatogenesis and sperm functions.

  4. The effect of removing surface-associated proteins from viable chicken spermatozoa on sperm function in vivo and in vitro.

    PubMed

    Steele, M G; Wishart, G J

    1996-12-02

    When chicken spermatozoa were diluted in isotonic or hypertonic solutions, sperm surface-associated proteins were lost to the medium, quantitatively more protein being lost to medium of higher osmotic strength. The proteins which were removed from the spermatozoa were also found within chicken seminal plasma, but at different relative concentrations, thus demonstrating selective association of only certain seminal proteins with the spermatozoa. The removal of these proteins occurred with only minimal damage to the spermatozoa, as judged by sperm motility, ATP content and ability to exclude eosin. Spermatozoa treated with hypertonic solutions were unable to reach the freshly ovulated egg in the infundibulum and could not be found within the uterovaginal sperm storage tubules after uterovaginal insemination. However, if inseminated directly into the uterovaginal junction, spermatozoa were able to enter the sperm storage tubules, suggesting that the treatment limited their ability to migrate through the vagina. Loss of sperm fertilizing ability following simple centrifugation and washing treatments appears to result from removal of surface-associated proteins.

  5. Discriminant analysis indicates a single sperm protein (SP22) is predictive of fertility following exposure to epididymal toxicants.

    PubMed

    Klinefelter, G R; Laskey, J W; Ferrell, J; Suarez, J D; Roberts, N L

    1997-01-01

    In a previous study, we found that ethane dimethanesulphonate (EDS) compromised the fertilizing ability of proximal cauda epididymal sperm from the rat within 4 days of exposure, an effect that persisted in castrated, testosterone (T)-implanted animals, establishing direct action on the epididymis. This EDS-induced reduction in fertilizing ability was highly correlated with a quantitative decrease in specific sperm protein. Here we sought to determine whether the fertility of proximal cauda epididymal sperm recovered from animals exposed to a variety of male reproductive toxicants could be predicted by assessing quantitative changes in specific sperm protein(s), or whether more common endpoints (e.g., sperm motility, sperm morphology, serum and epididymal tissue T, cauda epididymal sperm reserves) also are required to predict fertility. Intact adult male rats were dosed with EDS (25 or 50 mg/kg), chloroethylmethanesulphonate (CEMS; 12.5 or 18.75 mg/kg), or epichlorohydrin (EPI; 3 or 6 mg/kg) daily for 4 days. Castrated, T-implanted rats were dosed with hydroxyflutamide (HFLUT; 12.5 or 25 mg/kg) daily for 5 days. On day 5, proximal cauda epididymal sperm were inseminated in utero into receptive, cervically stimulated adult females, and on day 9, fertility (implants/corpora lutea) was assessed. Fertility-was decreased by the higher dose of each toxicant (P < 0.05) and also by the lower dose of EPI and HFLUT. Likewise, an acidic 22 kDa sperm protein (SP22) was decreased quantitatively (P < 0.05) in silver-stained two-dimensional gels by the higher dose of each toxicant as well as by the lower dose of EPI and HFLUT. Although sperm motility and serum T were altered by specific exposures, these endpoints were not useful in predicting fertility. In contrast, SP22 was highly correlated (P < 0.0001; r2 = 0.83) with fertility. Indeed, the amount of SP22 correctly predicted 90% and 94% of the fertile (> 50% fertility) and subfertile (< 50 fertility) animals, respectively

  6. Low seminal zinc bound to high molecular weight proteins in asthenozoospermic patients: evidence of increased sperm zinc content in oligoasthenozoospermic patients.

    PubMed

    Carpino, A; Siciliano, L; Petroni, M F; De Stefano, C; Aquila, S; Andó, S; Petrone, M F

    1998-01-01

    Total seminal zinc concentration, seminal zinc fraction bound to high molecular weight proteins (HMW-Zn%) and zinc content in spermatozoa were assayed in the ejaculates of 90 asthenozoospermic patients subdivided into two study groups: normoasthenozoospermics (group I: n = 50) and oligoasthenozoospermics (group II: n = 40). The zinc concentrations of patients were compared with those of a control group of donors showing normal semen parameters. All samples were also investigated for their sperm membrane functional integrity by the hypo-osmotic swelling test (HOS). The results showed normal total zinc concentrations but very low HMW-Zn% values (P < 0.001) in seminal plasma of the two groups of asthenozoospermic patients compared to the controls. Furthermore higher zinc amounts (P < 0.001) were measured in spermatozoa of oligoasthenozoospermic patients compared to group I and to the control group. Oligoasthenozoospermics also displayed a lower HOS score (P < 0.001) compared to the other two groups. These data suggest that the increased unbound seminal zinc could contribute to the decrease of sperm motility in normoasthenozoospermic and oligoasthenozoospermic patients. A further impairment in sperm motility could occur in the oligoasthenozoospermic patients where the increase of seminal free zinc was followed by a major zinc uptake by spermatozoa. The higher intrasperm zinc content in these patients could be a reflection of their low sperm membrane functionality.

  7. Opisthorchis viverrini: analysis of the sperm-specific rhophilin associated tail protein 1-like.

    PubMed

    Rattanachan, Sitthichon; Grams, Rudi; Tesana, Smarn; Smooker, Peter M; Grams, Suksiri Vichasri

    2014-12-01

    Concurrent deficiency of rhophilin associated tail protein (ROPN1) and ROPN1-like (ROPN1L) in mice causes structural abnormalities and immotility of sperm and thereby infertility. In the present research, ROPN1L of the human liver fluke Opisthorchis viverrini was molecularly characterized and showed unexpected potential as a diagnostic tool. ROPN1L transcripts were detected in 2-week-old juveniles by RT-PCR. Immunohistochemical analysis of the adult worm localized the protein in testis lobes, seminal vesicle and receptacle and immunoelectron microscopic analysis revealed its location on the tail of spermatozoa. Interestingly, sera of experimentally infected hamsters and sera of individuals suffering from opisthorchiasis showed reactivity to recombinant OvROPN1L (rOvROPN1L). The protein shows modest conservation to the human homolog at 47.2% sequence identity and a mouse anti-rOvROPN1L antiserum was not reactive with sperm protein extracts from hamsters, mice and rats. Unsurprisingly, conservation is higher in trematodes, e.g. 78.4% and 71.2% identity to Fasciola gigantica and Schistosoma haematobium, respectively and evaluation of diagnostic specificity is required using sera of individuals suffering from different trematodiases in Thailand.

  8. Improvement of the post-thaw qualities of Okinawan native Agu pig sperm frozen in an extender supplemented with antiapoptotic PTD-FNK protein.

    PubMed

    Shimokawa, Kazuki; Oshiro, Ryuko; Yamanaka, Kenichi; Ashizawa, Koji; Ohta, Shigeo; Tatemoto, Hideki

    2012-10-15

    The technical establishment of boar sperm cryopreservation is indispensable for effective breeding of the scarce Okinawan native Agu pig. The objective was to determine whether an artificial anticell death protein (PTD-FNK protein) was capable of improving the quality of cryopreserved Agu sperm. Ejaculated Agu sperm frozen in an extender supplemented with 0, 100, 200, 300, or 400 nm PTD-FNK protein was thawed, and mitochondrial integrity and other sperm characteristics were evaluated. Treatment with 300 nm PTD-FNK protein had the most beneficial effect (P < 0.05) on mitochondrial integrity (45-59%) and sperm motility (56-67%) after freezing-thawing. In particular, the proportion of post-thaw sperm with activated caspase-9 and -3 but not caspase-8 was markedly reduced among sperm frozen in the presence of PTD-FNK protein (P < 0.05), implying protection against apoptotic-cell death in response to mitochondrial damage. There were high levels of intracellular ATP (9.4-10.5 nmol/10(8) sperm) in post-thaw sperm treated with PTD-FNK protein, and the inhibitory effect of PTD-FNK protein on activation of caspases influenced the increase in the number of sperm with intact DNA (36-53%; P < 0.05). Furthermore, the addition of PTD-FNK protein to the freezing extender strongly preserved the ability of the sperm to penetrate to mature oocytes in all individuals (60-80%; P < 0.05). In conclusion, treatment with PTD-FNK protein in the freezing extender effectively improved post-thaw qualities of fragile Agu sperm through prevention of mitochondrial dysfunction leading to apoptotic-cell death during cryopreservation.

  9. Major proteins of yam bean tubers.

    PubMed

    Gomes, A V; Sirju-Charran, G; Barnes, J A

    1997-09-01

    The tuberous roots of the Mexican yam bean, jicama, (Pachyrhizus erosus L. Urban) contained large quantities of two acidic glycoproteins which accounted for more than 70% of the total soluble proteins (about 3 g per 100 g of tuber on a dry weight basis). The two major proteins, tentatively named YBG1 and YBG2, had apparent M(r)s of 28,000 and 26,000, respectively, by SDS-PAGE. A third protein named YBP22 which accounted for 2-5% of the total soluble proteins had an M(r) of 22,000. YBG1 and YBG2 exhibited great similarity on the basis of their amino acid composition and had identical N-terminal amino acid sequences. The first 23 amino acids in the N-terminal region of YBG2 were DDLPDYVDWRDYGAVTRIKNQGQ which showed strong homology with the papain class of cysteine proteases. YBG1 and YBG2 were found to bind to a Concanavalin A-Sepharose column and were also stained positively by a sensitive glycoprotein stain. Both glycoproteins exhibited cysteine proteolytic activity. In contrast, YBP22 showed sequence homology with several known protease inhibitors, and a polyclonal antibody raised against this protein cross reacted with soybean trypsin inhibitor.

  10. Evaluation of an animal protein-free semen extender for cryopreservation of epididymal sperm from North American bison (Bison bison).

    PubMed

    Krishnakumar, S; Whiteside, D P; Elkin, B; Thundathil, J C

    2011-07-15

    The objective was to evaluate the suitability of an animal protein-free semen extender for cryopreservation of epididymal sperm from the two subspecies of North American bison: plains (Bison bison bison) and wood (Bison bison athabascae) bison. Both cauda epididymides (from six plains and five wood bison) were minced and incubated in Sp-TALPH buffer for approximately 2 h at 37 °C to release actively motile sperm. Sperm suspensions were filtered, centrifuged and the sperm pellet from each bull was divided into two fractions and diluted either in egg yolk containing extender, Triladyl, or in an animal protein-free extender, Andromed, and equilibrated for 20 min at 37 °C. Thereafter, samples were chilled and cryopreserved. Frozen-thawed sperm were evaluated for motility (computer assisted sperm analysis), viability (SYBR 14 and propidium iodide), acrosome integrity (FITC conjugated PSA), cryocapacitation (tyrosine phosphorylation of sperm proteins as a biomarker), and fertilizing ability (in a heterologous IVF system). There was no significant difference for progressive motility, viability, and acrosome integrity between the two extenders for plains bison (36.8 ± 9.0, 60.5 ± 17.4, and 77.3 ± 4.6%; overall mean ± SD) as well as for wood bison (11.7 ± 8.1, 13.7 ± 5.6, and 73.4 ± 4.2%). Levels of tyrosine phosphorylation did not differ for sperm preserved in the two extenders for both subspecies, although an inter-bull variability in the response to tyrosine phosphorylation between extenders was suggested for plains bison. Fertilization percent did not differ significantly between extenders for plains bison (84.16 ± 9.92%, overall mean ± SD) and for wood bison (59.53 ± 19.99%). In conclusion, in the absence of significant difference between extenders in post-thaw sperm characteristics, we inferred that Andromed (animal protein-free) was suitable for cryopreservation of epididymal sperm from North American bison.

  11. New insights into the mechanisms of ram sperm protection by seminal plasma proteins.

    PubMed

    Mendoza, Noelia; Casao, Adriana; Pérez-Pé, Rosaura; Cebrián-Pérez, José A; Muiño-Blanco, Teresa

    2013-06-01

    To provide new insights into the mechanisms through which seminal plasma proteins (SPP) are able to protect spermatozoa, we tested the hypothesis that apoptosis can contribute to the negative effect of refrigeration on ram spermatozoa, and that SPP prevent this damage. Having proved the presence of key constituents of apoptosis-related pathways in ram sperm protein extracts, we carried out a comparative analysis of the effects of the addition of SPP before refrigeration (15 °C, 30 min) and induced-apoptosis with betulinic acid or fibroblast-associated receptor ligand, assessing sperm quality parameters and apoptotic markers. The protective effect of SPP on plasma membrane integrity and potential, motility and mitochondrial inner membrane potential, and surface (cardiolipin content) was evidenced in refrigerated and induced-apoptosis samples. The addition of SPP resulted in lower values of phosphatidylserine externalization, DNA damage, and caspase activity. Therefore, apoptosis in fresh or refrigerated ram spermatozoa can occur due to activation of both the extrinsic and the intrinsic mediated pathway, and SPP might interfere with both pathways. The addition of SPP also resulted in higher proportions of viable, noncapacitated sperm and fertilizing ability (ZBA rate). This report demonstrates that SPP support survival of ram spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, and proposes the possibility that SPP might interfere with the extrinsic and the intrinsic apoptotic pathways. This opens new, interesting perspectives for the study of cellular regulatory mechanisms in spermatozoa that could be crucial for the improvement of ram semen preservation protocols.

  12. Profiling signaling proteins in human spermatozoa: biomarker identification for sperm quality evaluation.

    PubMed

    Silva, Joana Vieira; Freitas, Maria João; Correia, Bárbara Regadas; Korrodi-Gregório, Luís; Patrício, António; Pelech, Steven; Fardilha, Margarida

    2015-10-01

    To determine the correlation between semen basic parameters and the expression and activity of signaling proteins. In vitro studies with human spermatozoa. Academic research institute. Thirty-seven men provided semen samples for routine analysis. None. Basic semen parameters tracked included sperm DNA fragmentation (SDF), the expression levels of 75 protein kinases, and the phosphorylation/cleavage patterns of 18 signaling proteins in human spermatozoa. The results indicated that the phosphorylated levels of several proteins (Bad, GSK-3β, HSP27, JNK/SAPK, mTOR, p38 MAPK, and p53), as well as cleavage of PARP (at D214) and Caspase-3 (at D175), were significantly correlated with motility parameters. Additionally, the percentage of morphologically normal spermatozoa demonstrated a significant positive correlation with the phosphorylated levels of p70 S6 kinase and, in turn, head defects and the teratozoospermia index (TZI) showed a significant negative correlation with the phosphorylated levels of Stat3. There was a significant positive correlation between SDF and the teratozoospermia index, as well as the presence of head defects. In contrast, SDF negatively correlated with the percentage of morphologically normal spermatozoa and the phosphorylation of Akt and p70 S6 kinase. Subjects with varicocele demonstrated a significant negative correlation between head morphological defects and the phosphorylated levels of Akt, GSK3β, p38 MAPK, and Stat1. Additionally, 34 protein kinases were identified as expressed in their total protein levels in normozoospermic samples. This study contributed toward establishing a biomarker "fingerprint" to assess sperm quality on the basis of molecular parameters. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  13. Acrosin-binding protein (ACRBP) and triosephosphate isomerase (TPI) are good markers to predict boar sperm freezing capacity.

    PubMed

    Vilagran, Ingrid; Castillo, Judit; Bonet, Sergi; Sancho, Sílvia; Yeste, Marc; Estanyol, Josep M; Oliva, Rafael

    2013-09-15

    Sperm cryopreservation is the most efficient method for storing boar sperm samples for a long time. However, one of the inconveniences of this method is the large variation between and within boars in the cryopreservation success of their sperm. The aim of the present work was thus to find reliable and useful predictive biomarkers of the good and poor capacity to withstand the freeze-thawing process in boar ejaculates. To find these biomarkers, the amount of proteins present in the total proteome in sperm cells were compared between good freezability ejaculates (GFE) and poor freezability ejaculates (PFE) using the two-dimensional difference gel electrophoresis technique. Samples were classified as GFE and PFE using progressive motility and viability of the sperm at 30 and 240 minutes after thawing, and the proteomes from each group, before starting cryopreservation protocols, were compared. Because two proteins, acrosin binding protein (ACRBP) and triosephosphate isomerase (TPI), presented the highest significant differences between GFE and PFE groups in two-dimensional difference gel electrophoresis assessment, Western blot analyses for ACRBP and TPI were also performed for validation. ACRBP normalized content was significantly lower in PFE than in GFE (P < 0.05), whereas the TPI amounts were significantly lower in GFE (P < 0.05) than in PFE. The association of ACRBP and TPI with postthaw sperm viability and motility was confirmed using Pearson's linear correlation. In conclusion, ACRBP and TPI can be used as markers of boar sperm freezability before starting the cryopreservation procedure, thereby avoiding unnecessary costs involved in this practice.

  14. Certain Strongylocentrotus purpuratus sperm mitochondrial proteins co-purify with low density detergent-insoluble membranes and are PKA or PKC-substrates possibly involved in sperm motility regulation.

    PubMed

    Loza-Huerta, Arlet; Vera-Estrella, Rosario; Darszon, Alberto; Beltrán, Carmen

    2013-11-01

    Sea urchin sperm motility is regulated by Speract, a sperm-activating peptide (SAP) secreted from the outer egg coat. Upon binding to its receptor in the sperm flagellum, Speract induces a series of ionic and metabolic changes in Strongylocentrotus purpuratus spermatozoa that regulate their motility. Among these events, protein phosphorylation is one of the most relevant and evidence indicates that some proteins of the Speract signaling cascade localize in low density detergent-insoluble membranes (LD-DIM). LD-DIM-derived proteins from immotile, motile or Speract-stimulated S. purpuratus sperm were resolved in 2-D gels and the PKA and PKC substrates detected with specific antibodies were identified by LC-MS/MS. Differential PKA and PKC substrate phosphorylation levels among the LD-DIM isolated from sperm in different motility conditions were found and identified by mass spectrometry as: ATP synthase, creatine kinase, NADH dehydrogenase (ubiquinone) flavoprotein 2, succinyl-CoA ligase and the voltage-dependent anion channel 2 (VDAC2), which are mitochondrial proteins, as well as, the cAMP-dependent protein kinase type II regulatory (PKA RII) subunit, Tubulin β chain and Actin Cy I changed their phosphorylation state. Some mitochondrial proteins regulated by PKA or PKC may influence sea urchin sperm motility. The fact that a high percentage (66%) of the PKA or PKC substrates identified in LD-DIM are mitochondrial proteins suggests that the phosphorylation of these proteins modulates sea urchin sperm motility via Speract stimulation by providing sufficient energy to sperm physiology. Those mitochondrial proteins are indeed PKA- or PKC-substrates in the sea urchin spermatozoa. © 2013.

  15. Analysis of protein thiol changes occurring during rat sperm epididymal maturation.

    PubMed

    Baker, Mark A; Weinberg, Anita; Hetherington, Louise; Villaverde, Ana Izabel S B; Velkov, Tony

    2015-01-01

    The maturation of spermatozoa throughout the epididymal environment occurs in the complete absence of nuclear protein biosynthesis. As such, these cells rely heavily on posttranslational modifications of existing proteins in order to obtain the potential for fertilization. We have used an OxiCat approach to label both free and oxidized cysteine residues in rat sperm proteins and compared the ratio of reduced:oxidized peptides as these cells undergo epididymal transit. In all, 20 peptides, corresponding to 15 proteins, underwent a change in their redox status. Included in this list were A-kinase anchoring protein 4 and fatty acid-binding protein 9. Both of these proteins undergo intradisulfide bonding, leading to reduced solubility and, in the case of the latter, is likely to cause a loss of protein function. Interestingly, two glycolytic enzymes, hexokinase-1 and lactate dehydrogenase, also display increased cysteine oxidation during epididymal transit, which may be involved in the regulation of the enzyme activities. © 2015 by the Society for the Study of Reproduction, Inc.

  16. Monoclonal antibodies against boar sperm zona pellucida-binding protein AWN-1. Characterization of a continuous antigenic determinant and immunolocalization of AWN epitopes in inseminated sows.

    PubMed

    Calvete, J J; Ensslin, M; Mburu, J; Iborra, A; Martínez, P; Adermann, K; Waberski, D; Sanz, L; Töpfer-Petersen, E; Weitze, K F; Einarsson, S; Rodríguez-Martínez, H

    1997-10-01

    Boar spermadhesin AWN-1 is a sperm surface-associated 14.7-kDa lectin and a major protein of porcine seminal plasma. AWN-1 binds to beta-galactosides and to porcine zona pellucida glycoproteins, suggesting that this protein might play a role in the primary binding of spermatozoa to the egg's external glycoprotein matrix. We have produced a collection of murine monoclonal antibodies against purified AWN-1. Five monoclonal antibodies recognized sequential antigenic determinants. All these epitopes were located at the C-terminal region of AWN-1 (residues 109-123) by competitive ELISA using overlapping synthetic peptides that cover the complete 133 amino acid sequence of the lectin. In a structural model of spermadhesin AWN-1, the polypeptide stretch 109-123 is fully solvent-exposed, providing a reasonable explanation for its high immunogenicity. In addition to epitope mapping, we have employed anti-AWN monoclonal antibodies for immunolocalization of the protein in the genital tract of inseminated sows. Clusters of AWN epitopes were occasionally found attached to the epithelium of the uterotubal junction and the adjacent lower isthmus. However, neither AWN-1 nor other seminal plasma proteins were found in the isthmic fluid collected 10-26 h after insemination. These results suggest that the whole amount of seminal plasma proteins are absorbed by the epithelium of the female genital tract, supporting the claim that removal of seminal plasma components from spermatozoa might be a major event in both in vitro and in vivo sperm capacitation.

  17. Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans.

    PubMed

    Liu, Zhiyu; Wang, Bin; He, Ruijun; Zhao, Yanmei; Miao, Long

    2014-02-01

    In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

  18. Major intrinsic proteins in biomimetic membranes.

    PubMed

    Nielsen, Claus Hélix

    2010-01-01

    Biological membranes define the structural and functional boundaries in living cells and their organelles. The integrity of the cell depends on its ability to separate inside from outside and yet at the same time allow massive transport of matter in and out the cell. Nature has elegantly met this challenge by developing membranes in the form of lipid bilayers in which specialized transport proteins are incorporated. This raises the question: is it possible to mimic biological membranes and create a membrane based sensor and/or separation device? In the development of a biomimetic sensor/separation technology, a unique class of membrane transport proteins is especially interesting-the major intrinsic proteins (MIPs). Generally, MIPs conduct water molecules and selected solutes in and out of the cell while preventing the passage of other solutes, a property critical for the conservation of the cells internal pH and salt concentration. Also known as water channels or aquaporins they are highly efficient membrane pore proteins some of which are capable of transporting water at very high rates up to 10(9) molecules per second. Some MIPs transport other small, uncharged solutes, such as glycerol and other permeants such as carbon dioxide, nitric oxide, ammonia, hydrogen peroxide and the metalloids antimonite, arsenite, silicic and boric acid depending on the effective restriction mechanism of the protein. The flux properties of MIPs thus lead to the question ifMIPs can be used in separation devices or as sensor devices based on, e.g., the selective permeation of metalloids. In principle a MIP based membrane sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but water or the solute in question. In practice, however, a biomimetic support matrix will generally have finite permeabilities to both electrolytes and non-electrolytes. The feasibility of a biomimetic MIP device thus depends on the relative transport

  19. Redox control of surface protein sulphhydryls in bovine spermatozoa reversibly modulates sperm adhesion to the oviductal epithelium and capacitation.

    PubMed

    Gualtieri, Roberto; Mollo, Valentina; Duma, Gennaro; Talevi, Riccardo

    2009-07-01

    Oviductal fluid molecules, such as sulphated glycosaminoglycans and disulphide-reductants, may represent periovulatory signals for the release of spermatozoa from the oviductal reservoir in the bovine species. Disulphide-reductants release spermatozoa through the reduction of sperm-surface disulphides to sulphhydryls (SH). Herein, we studied sperm-surface protein SH through labelling with maleimidylpropionyl biocytin in the initial sperm suspension, in the subpopulations able and unable to adhere to the in vitro cultured oviductal epithelium, and in spermatozoa released either through the disulphide-reductant penicillamine (PEN) or the sulphated glycosaminoglycan heparin (HEP). Adhesion assays were performed to study the ability of released spermatozoa to readhere to the oviductal epithelium. Results showed that the level of SH in sperm-surface proteins was: 1) low in adhering spermatozoa; 2) high in spermatozoa unable to adhere; and 3) markedly increased in released spermatozoa. Adhesion assays showed that: 1) PEN-released spermatozoa promptly recovered adhesion after removal of the disulphide-reductant and could be released again in response to PEN; 2) conversely, a limited number of HEP-released spermatozoa was able to readhere to the oviductal epithelium and this ability was not affected by HEP removal. Recovery of adhesion was associated to reoxidation of sperm-surface protein SH and to the reversal of capacitation. In conclusion, redox modulation of sperm-surface protein SH is involved in the release of spermatozoa adhering to the oviduct in vitro; the reversible action of disulphide-reductants might be responsible for intermittent phases of adhesions and releases; and the irreversible action of HEP indicates that it may represent a terminal releasing signal.

  20. A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphates and calcium.

    PubMed

    Vijayaraghavan, S; Trautman, K D; Goueli, S A; Carr, D W

    1997-06-01

    Sperm motility is regulated by protein phosphorylation. We have recently shown that a serine/threonine phosphatase system is involved in motility regulation. Two of the components of the phosphatase system, GSK-3 and PP1gamma2, are regulated by tyrosine phosphorylation. During our investigation of sperm tyrosine-phosphorylated proteins we discovered a 55-kDa protein whose tyrosine phosphorylation correlates closely to the motility state of sperm. This protein is tyrosine phosphorylated to a much higher degree in motile caudal than in immotile caput epididymal sperm. Motility inhibition of caudal epididymal sperm by protein kinase A (PKA) anchoring inhibition or by ionomycin-induced calcium overload led to the virtual disappearance of tyrosine phosphorylation of the 55-kDa protein. Conversely, treatment of sperm with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. The protein was present in the soluble 100 000 x g supernatants of sperm extracts and was heat labile. Chromatography through diethylaminoethyl-cellulose and Western blot analysis showed that this 55-kDa protein is not a regulatory subunit of PKA or alpha-tubulin. Our results represent the identification of a soluble protein whose tyrosine phosphorylation varies directly with motility and suggest that motility regulation may involve cross talk between PKA, calcium, and tyrosine kinase pathways.

  1. PLCzeta(zeta): a sperm protein that triggers Ca2+ oscillations and egg activation in mammals.

    PubMed

    Swann, K; Saunders, C M; Rogers, N T; Lai, F A

    2006-04-01

    At fertilization in mammals, the sperm activates development by causing a prolonged series of intracellular Ca(2+) oscillations that are generated by increased production of inositol trisphosphate (InsP(3)). It appears that the sperm initiates InsP(3) generation via the introduction of a sperm factor into the egg after gamete membrane fusion. We recently identified a sperm-specific form of phospholipase C (PLC), referred to as PLCzeta(zeta). We review the evidence that PLCzeta represents the sperm factor that activates development of the egg and discuss the characteristics of PLCzeta that distinguish it from the somatic forms of PLC.

  2. A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis.

    PubMed

    McPartlin, L A; Littell, J; Mark, E; Nelson, J L; Travis, A J; Bedford-Guaus, S J

    2008-03-15

    Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating medium) yielded high rates of protein tyrosine phosphorylation. Either HCO3(-) or BSA was required to support these changes, with the combination of both providing the most intense results. When a membrane-permeable form of cAMP and a phosphodiesterase inhibitor (IBMX) were added to MW in the absence of HCO3(-) and BSA, the tyrosine phosphorylation results obtained in our capacitating conditions could not be replicated, suggesting either effects apart from cAMP were responsible for tyrosine phosphorylation, or that stallion sperm might respond differently to these reagents as compared to sperm from other mammals. Sperm incubation in capacitating conditions was also associated with high percentages (Psperm incubated in medium devoid of BSA and NaHCO3. Our results were novel in that we report protein tyrosine phosphorylation in stallion sperm incubated in defined conditions, coupled with significant percentages of acrosome reacted sperm. The continuation of these studies might help to elucidate the conditions and pathways supporting sperm capacitation in the horse.

  3. Primary, secondary, and tertiary structure of the core of a histone H1-like protein from the sperm of Mytilus.

    PubMed

    Jutglar, L; Borrell, J I; Ausió, J

    1991-05-05

    We have analyzed the structure of the trypsin-resistant core of the protein PL-II* of the sperm from Mytilus californianus. The peptide has a molecular mass of 8436 Da and its primary sequence is ATGGAKKP STLSMIVAAIQAMKNRKGSSVQAIRKYILANNKG INTSRLGSAMKLAFAKGLKSGVLVRPKTSAGA SGATGSFRVG. This sequence bears an enormous homology and fulfills the constraints of the consensus sequence of the trypsin-resistant peptides of the proteins of the histone H1 family. Secondary structure analysis using Fourier-transform infared spectroscopy as well as predictive methods indicate the presence of 20-30% beta-structure and approximately 25% alpha-helix for this peptide. As in the case of histone H1 proteins, the protein PL-II* core exhibits a compact globular structure as deduced from hydrodynamic measurements. The presence of a histone H1 protein with protamine-like features, seems to be thus, a common general feature of the chromatin composition in the sperm of the bivalve molluscs.

  4. Gamete Therapeutics: Recombinant Protein Adsorption by Sperm for Increasing Fertility via Artificial Insemination

    PubMed Central

    Alvarez-Gallardo, Horacio; Kjelland, Michael E.; Moreno, Juan F.; Welsh, Thomas H.; Randel, Ronald D.; Lammoglia, Miguel A.; Pérez-Martínez, Mario; Lara-Sagahón, Alma V.; Esperón-Sumano, A. Enrique; Romo, Salvador

    2013-01-01

    A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs), specifically the fertility associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system’s response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA) and recombinant TIMP-2 (rTIMP-2) to bovine semen before cryopreservation for use in an artificial insemination (AI) program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus) heifers were selected based on their estrus cycle, body condition score (BCS), of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal) measurements. Heifers were synchronized using estradiol benzoate (EB), Celosil® (PGF2α) (Shering-Plough) and a controlled internal drug release (CIDR) device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL) and rTIMP-2 (25 µg/mL). In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037). Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented. PMID:23762288

  5. Spectroscopic and electrochemical studies of the interaction between oleuropein, the major bio-phenol in olives, and salmon sperm DNA

    NASA Astrophysics Data System (ADS)

    Mohamadi, Maryam; Afzali, Daryoush; Esmaeili-Mahani, Saeed; Mostafavi, Ali; Torkzadeh-Mahani, Masoud

    2015-09-01

    Interaction of oleuropein, the major bio-phenol in olive leaf and fruit, with salmon sperm double-stranded DNA was investigated by employing electronic absorption titrations, fluorescence quenching spectroscopy, competitive fluorescence spectroscopy, thermal denaturation and voltammetric studies. Titration of oleuropein with the DNA caused a hypochromism accompanied with a red shift indicating an intercalative mode of interaction. Binding constant of 1.4 × 104 M-1 was obtained for this interaction. From the curves of fluorescence titration of oleuropein with the DNA, binding constant and binding sites were calculated to be 8.61 × 103 M-1 and 1.05, respectively. Competitive studies with ethidium bromide (a well-known DNA intercalator) showed that the bio-phenol could take the place of ethidium bromide in the DNA intercalation sites. The interaction of oleuropein with DNA was also studied electrochemically. In the presence of the DNA, the anodic and cathodic peak currents of oleuropein decreased accompanied with increases in peak-to-peak potential separation and formal potential, indicating the intercalation of oleuropein into the DNA double helix. Moreover, melting temperature of the DNA was found to increase in the presence of oleuropein, indicating the stabilization of the DNA double helix due to an intercalative interaction.

  6. New Insights into the Phylogeny and Gene Context Analysis of Binder of Sperm Proteins (BSPs)

    PubMed Central

    Arruga, Diana; Pérez-Pé, Rosaura; Sánchez-Ferrer, Álvaro; Muiño-Blanco, Teresa; Cebrián-Pérez, José A.

    2015-01-01

    Seminal plasma (SP) proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm) proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases. The update view shows for the first time an equally distributed number of sequences between the three families: BSP, and their homologs 1 (BSPH1) and 2 (BSPH2). The BSP family is divided in four subfamilies, BSP1 subfamily being the predominant, followed by subfamilies BSP3, BSP5 and BSP2. BSPH proteins were found among placental mammals (Eutheria) belonging to the orders Proboscidea, Primates, Lagomorpha, Rodentia, Chiroptera, Perissodactyla and Cetartiodactyla. However, BSPH2 proteins were also found in the Scandentia order and Metatheria clade. This phylogenetic analysis, when combined with a gene context analysis, showed a completely new evolutionary scenario for the BSP superfamily of proteins with three defined different gene patterns, one for BSPs, one for BSPH1/BSPH2/ELSPBP1 and another one for BSPH1/BSPH2 without ELSPBP1. In addition, the study has permitted to define concise conserved blocks for each family (BSP, BSPH1 and BSPH2), which could be used for a more reliable assignment for the incoming sequences, for data curation of current databases, and for cloning new BSPs, as the one described in this paper, ram seminal vesicle 20 kDa protein (RSVP20, Ovis aries BSP5b). PMID:26333091

  7. Identification of Motile Sperm Domain–Containing Protein 2 as Regulator of Human Monocyte Migration

    PubMed Central

    Yacov, Niva; Salem, Yaniv; Propheta-Meiran, Oshrat; Ishai, Eti; Breitbart, Eyal

    2017-01-01

    Binding of chemokines to their cognate receptors on monocytes instigates a cascade of events that directs these cells to migrate to sites of inflammation and cancerous tissues. Although targeting of selected chemokine receptors on monocytes exhibited preclinical efficacy, attempts to translate these studies to the clinic have failed thus far, possibly due to redundancy of the target receptor. We reveal that motile sperm domain–containing protein 2 (MOSPD2), a protein with a previously unknown function, regulates monocyte migration in vitro. This protein was found to be expressed on the cytoplasmic membrane of human monocytes. Silencing or neutralizing MOSPD2 in monocytes restricted their migration when induced by different chemokines. Mechanistically, silencing MOSPD2 inhibited signaling events following chemokine receptor ligation. When tested for expression in other immune cell subsets, MOSPD2 was apparent also, though less abundantly, in neutrophils, but not in lymphocytes. Thus, in the presence of neutralizing Abs, neutrophil migration was inhibited to some extent whereas lymphocyte migration remained intact. In view of these results, we suggest MOSPD2 as a potential target protein for treating diseases in which monocyte and neutrophil accumulation is correlated with pathogenesis. PMID:28137892

  8. Identification of Motile Sperm Domain-Containing Protein 2 as Regulator of Human Monocyte Migration.

    PubMed

    Mendel, Itzhak; Yacov, Niva; Salem, Yaniv; Propheta-Meiran, Oshrat; Ishai, Eti; Breitbart, Eyal

    2017-03-01

    Binding of chemokines to their cognate receptors on monocytes instigates a cascade of events that directs these cells to migrate to sites of inflammation and cancerous tissues. Although targeting of selected chemokine receptors on monocytes exhibited preclinical efficacy, attempts to translate these studies to the clinic have failed thus far, possibly due to redundancy of the target receptor. We reveal that motile sperm domain-containing protein 2 (MOSPD2), a protein with a previously unknown function, regulates monocyte migration in vitro. This protein was found to be expressed on the cytoplasmic membrane of human monocytes. Silencing or neutralizing MOSPD2 in monocytes restricted their migration when induced by different chemokines. Mechanistically, silencing MOSPD2 inhibited signaling events following chemokine receptor ligation. When tested for expression in other immune cell subsets, MOSPD2 was apparent also, though less abundantly, in neutrophils, but not in lymphocytes. Thus, in the presence of neutralizing Abs, neutrophil migration was inhibited to some extent whereas lymphocyte migration remained intact. In view of these results, we suggest MOSPD2 as a potential target protein for treating diseases in which monocyte and neutrophil accumulation is correlated with pathogenesis. Copyright © 2017 by The American Association of Immunologists, Inc.

  9. Seminal vesicle protein SVS2 is required for sperm survival in the uterus.

    PubMed

    Kawano, Natsuko; Araki, Naoya; Yoshida, Kaoru; Hibino, Taku; Ohnami, Naoko; Makino, Maako; Kanai, Seiya; Hasuwa, Hidetoshi; Yoshida, Manabu; Miyado, Kenji; Umezawa, Akihiro

    2014-03-18

    In mammals, sperm migrate through the female reproductive tract to reach the egg; however, our understanding of this journey is highly limited. To shed light on this process, we focused on defining the functions of seminal vesicle secretion 2 (SVS2). SVS2(-/-) male mice produced sperm but were severely subfertile, and formation of a copulatory plug to cover the female genital opening did not occur. Surprisingly, even when artificial insemination was performed with silicon as a substitute for the plug, sperm fertility in the absence of SVS2 remained severely reduced because the sperm were already dead in the uterus. Thus, our results provide evidence that the uterus induces sperm cell death and that SVS2 protects sperm from uterine attack.

  10. Seminal vesicle protein SVS2 is required for sperm survival in the uterus

    PubMed Central

    Kawano, Natsuko; Araki, Naoya; Yoshida, Kaoru; Hibino, Taku; Ohnami, Naoko; Makino, Maako; Kanai, Seiya; Hasuwa, Hidetoshi; Yoshida, Manabu; Miyado, Kenji; Umezawa, Akihiro

    2014-01-01

    In mammals, sperm migrate through the female reproductive tract to reach the egg; however, our understanding of this journey is highly limited. To shed light on this process, we focused on defining the functions of seminal vesicle secretion 2 (SVS2). SVS2−/− male mice produced sperm but were severely subfertile, and formation of a copulatory plug to cover the female genital opening did not occur. Surprisingly, even when artificial insemination was performed with silicon as a substitute for the plug, sperm fertility in the absence of SVS2 remained severely reduced because the sperm were already dead in the uterus. Thus, our results provide evidence that the uterus induces sperm cell death and that SVS2 protects sperm from uterine attack. PMID:24591616

  11. Sperm proteome and reproductive technologies in mammals.

    PubMed

    Li, Chun-Jin; Wang, Dong; Zhou, Xu

    2016-10-01

    Sperm is highly differentiated cell that can be easily obtained and purified. Mature sperm is considered to be transcriptionally and translationally silent and incapable of protein synthesis. Recently, a large number of proteins have been identified in sperm from different species by using the proteomic approaches. Clinically, sperm proteins can be used as markers for male infertility due to different protein profiles identified in sperm from fertile and infertile male animals. Recent evidences have shown that the conditions of sperm preservation in vitro can also change the sperm protein profiles. This paper reviews the recent scientific publications available to address sperm proteome and their relationship with sperm cryopreservation, capacitation, fertilization, and separation of X and Y sperm. Future directions in the application of sperm proteomics to develop or optimize reproductive technologies in mammals are also discussed.

  12. Bactericidal/permeability-increasing protein in the reproductive system of male mice may be involved in the sperm-oocyte fusion.

    PubMed

    Li, Kun; Liu, Yue; Xia, Xiaoyu; Wang, Li; Lu, Meige; Hu, Yanqin; Xu, Chen

    2013-08-01

    Bactericidal/permeability-increasing protein (BPI) is a 455-residue (∼55 kDa) protein found mainly in the primary (azurophilic) granules of human neutrophils. BPI is an endogenous antibiotic protein that belongs to the family of mammalian lipopolysaccharide (LPS)-binding and lipid transport proteins. Its major function is to kill Gram-negative bacteria, thereby protecting the host from infection. In addition, BPI can inhibit angiogenesis, suppress LPS-mediated platelet activation, increase DNA synthesis, and activate ERK/Akt signaling. In this study, we found that Bpi was expressed in the testis and epididymis but not in the seminal vesicles, prostate, and solidification glands. BPI expression in the epididymis increased upon upregulation of testosterone, caused by injection of GNRH. In orchidectomized mice, BPI expression was significantly reduced, but its expression was restored to 30% of control levels in orchidectomized mice that received supplementary testosterone. The number of sperm fused per egg significantly decreased after incubation with anti-BPI antiserum. These results suggest that BPI may take part in the process of sperm-oocyte fusion and play a unique and significant role in reproduction.

  13. Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins

    PubMed Central

    Kalra, Shalini; Pradeep, Mangottil Ayyappan; Mohanty, Ashok K.; Kaushik, Jai K.

    2016-01-01

    Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38–50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3–2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as

  14. Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins.

    PubMed

    Kalra, Shalini; Pradeep, Mangottil Ayyappan; Mohanty, Ashok K; Kaushik, Jai K

    2016-01-01

    Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38-50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3-2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as

  15. Rat sperm immobilisation effects of a protein from Ricinus communis (Linn.): an in vitro comparative study with nonoxynol-9.

    PubMed

    Nithya, R S; Anuja, M M; Rajamanickam, C; Indira, M

    2012-12-01

    Previous study conducted in our department showed that 50% ethanolic extract of the root of Ricinus communis possess reversible antifertility effect and a 62-kDa protein (Rp) from this extract is responsible for the antifertility effects. In this study, we compared the spermicidal effect of this Rp with nonoxynol-9 (N-9) in vitro. The sperm immobilisation studies showed that 100 μg ml(-1) of Rp was able to immobilise the sperms completely within 30 s. Sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in Rp and N-9 treated groups in comparison with the control. In Rp and N-9 treated groups, the number of acrosome-reacted cells was found to be high and also caused agglutination of the spermatozoa, indicating the loss of intactness of the plasma membrane, which was further supported by the significant reduction in the activity of membrane bound 5'-nucleotidase, acrosomal acrosin. In short, the protein Rp possesses spermicidal activity in vitro and its effects are similar to that of nonoxynol 9. © 2012 Blackwell Verlag GmbH.

  16. Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination.

    PubMed

    Kennedy, Chelsey E; Krieger, Kari Beth; Sutovsky, Miriam; Xu, Wei; Vargovič, Peter; Didion, Bradley A; Ellersieck, Mark R; Hennessy, Madison E; Verstegen, John; Oko, Richard; Sutovsky, Peter

    2014-05-01

    Post-acrosomal WW-domain binding protein (PAWP) is a signaling molecule located in the post-acrosomal sheath (PAS) of mammalian spermatozoa. We hypothesized that the proper integration of PAWP in the sperm PAS is reflective of bull-sperm quality and fertility. Cryopreserved semen samples from 298 sires of acceptable, but varied, fertility used in artificial insemination services were analyzed using immunofluorescence microscopy and flow cytometry for PAWP protein. In normal spermatozoa, PAWP fluorescence formed a regular band around the proximal PAS. Anomalies of PAWP labeling in defective spermatozoa were reflected in flow cytometry by varied intensities of PAWP-induced fluorescence. Distinct sperm phenotypes were also identified, including morphologically normal and some defective spermatozoa with moderate levels of PAWP; grossly defective spermatozoa with low/no PAWP; and defective spermatozoa with high PAWP. Analysis by ImageStream flow cytometry confirmed the prevalence of abnormal sperm phenotypes in the spermatozoa with abnormal PAWP content. Live/dead staining and video recording showed that some abnormal spermatozoa are viable and capable of progressive motility. Conventional flow-cytometric measurements of PAWP correlated significantly with semen quality and fertility parameters that reflect the sires' artificial insemination fertility, including secondary sperm morphology, conception rate, non-return rate, and residual value. A multiplex, flow-cytometric test detecting PAWP, aggresomes (ubiquitinated protein aggregates), and acrosomal integrity (peanut-agglutinin-lectin labeling) had a predictive value for conception rate, as demonstrated by step-wise regression analysis. We conclude that PAWP correlates with semen/fertility parameters used in the cattle artificial insemination industry, making PAWP a potential biomarker of bull fertility. © 2014 Wiley Periodicals, Inc.

  17. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

    PubMed Central

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K.; Kline, Douglas W.; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  18. Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm.

    PubMed

    Cataldo, L; Baig, K; Oko, R; Mastrangelo, M A; Kleene, K C

    1996-11-01

    The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.

  19. Identification of protein tyrosine phosphatases and dual-specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation.

    PubMed

    González-Fernández, L; Ortega-Ferrusola, C; Macias-Garcia, B; Salido, G M; Peña, F J; Tapia, J A

    2009-06-01

    Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in boar and stallion sperm lysates, we unequivocally identified two PTPs (PTPRB and PTPN11) and two DSPs (DUSP3 and DUSP4). In dog sperm lysates, only PTPN11, DUSP3, and DUSP4 were detected. In all these species, we did not detect the specific signal with anti-PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, or PTPRZ antibodies. Positive matches were further investigated by indirect immunofluorescence and confocal microscopy. Results showed that PTPRB was associated with the plasma membrane in the head and tail of boar and stallion spermatozoa. In agreement with Western blotting results, PTPRB antibodies did not show immunoreactivity in dog sperm analyzed by immunofluorescence. In the three species, DUSP4 was mainly found in the tail of spermatozoa, with little or no immunoreactivity in the head. PTPN11 was mainly located in the postacrosomal region in the head, whereas DUSP3 immunoreactivity was extended within the acrosome. PTPN11 and DUSP3 showed immunoreactivity in the tail that was restricted to the midpiece. Finally, we incubated boar, stallion, and dog spermatozoa with pervanadate and sodium orthovanadate, two PTP inhibitors, and analyzed overall protein tyrosine phosphorylation and assessed sperm motility. Sodium orthovanadate and pervanadate showed concentration-dependent inhibition of sperm motility that was

  20. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  1. Sperm postacrosomal WW domain-binding protein is not required for mouse egg activation.

    PubMed

    Satouh, Yuhkoh; Nozawa, Kaori; Ikawa, Masahito

    2015-10-01

    To begin embryonic development, the zygote must resume the cell cycle correctly after stimulation by sperm-borne oocyte-activating factors (SOAFs). The postacrosomal WW domain-binding protein (PAWP) is one of the strongest SOAF candidates and is widely conserved among eutherian mammals. It has been reported that the microinjection of recombinant PAWP protein can trigger not only Ca(2+) oscillations in mammalian eggs but also intracellular Ca(2+) release in amphibian eggs. It was also suggested that PAWP is involved in the formation of high-quality spermatozoa. On the other hand, negligible SOAF activity for PAWP cRNA has also been reported. In this study, we generated PAWP null mice and examined the fertilizing ability of male mice. Electron microscopy showed no aberrant morphology in spermatogenesis. Intracytoplasmic injection of a single spermatozoon from the null mouse line showed that depletion of PAWP elicited no quantitative differences in Ca(2+) oscillations or in subsequent development of the embryos. We conclude that PAWP does not play an essential role in mouse fertilization.

  2. Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling pathway.

    PubMed

    Miraglia, Erica; De Angelis, Federico; Gazzano, Elena; Hassanpour, Hossain; Bertagna, Angela; Aldieri, Elisabetta; Revelli, Alberto; Ghigo, Dario

    2011-01-01

    Nitric oxide (NO), a modulator of several physiological processes, is involved in different human sperm functions. We have investigated whether NO may stimulate the motility of human spermatozoa via activation of the soluble guanylate cyclase (sGC)/cGMP pathway. Sperm samples obtained by masturbation from 70 normozoospermic patients were processed by the swim-up technique. The kinetic parameters of the motile sperm-rich fractions were assessed by computer-assisted sperm analysis. After a 30-90  min incubation, the NO donor S-nitrosoglutathione (GSNO) exerted a significant enhancing effect on progressive motility (77, 78, and 78% vs 66, 65, and 62% of the control at the corresponding time), straight linear velocity (44, 49, and 48 μm/s vs 34, 35, and 35.5 μm/s), curvilinear velocity (81, 83, and 84 μm/s vs 68 μm/s), and average path velocity (52, 57, and 54 μm/s vs 40, 42, and 42 μm/s) at 5 μM but not at lower concentrations, and in parallel increased the synthesis of cGMP. A similar effect was obtained with the NO donor spermine NONOate after 30 and 60  min. The GSNO-induced effects on sperm motility were abolished by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (a specific sGC inhibitor) and mimicked by 8-bromo-cGMP (8-Br-cGMP; a cell-permeating cGMP analog); the treatment with Rp-8-Br-cGMPS (an inhibitor of cGMP-dependent protein kinases) prevented both the GSNO- and the 8-Br-cGMP-induced responses. On the contrary, we did not observe any effect of the cGMP/PRKG1 (PKG) pathway modulators on the onset of hyperactivated sperm motility. Our results suggest that NO stimulates human sperm motility via the activation of sGC, the subsequent synthesis of cGMP, and the activation of cGMP-dependent protein kinases.

  3. An Epididymis-Specific Secretory Protein HongrES1 Critically Regulates Sperm Capacitation and Male Fertility

    PubMed Central

    Zhou, Yuchuan; Zheng, Min; Shi, Qixian; Zhang, Li; Zhen, Wei; Chen, Wenying; Zhang, Yonglian

    2008-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilizion. Although progress had been made in understanding the physiology and biochemistry of capacitation, little is known about the potential roles of epididymal proteins during this process. Here we report that HongrES1, a new member of the SERPIN (serine proteinase inhibitor) family exclusively expressed in the rat cauda epididymis and up-regulated by androgen, is secreted into the lumen and covers the sperm head. Co-culture of caudal sperms with HongrES1 antibody in vitro resulted in a significant increase in the percentage of capacitated spermatozoa. Furthermore, the percentage of capacitated spermatozoa clearly increased in rats when HongrES1 was down-regulated by RNAi in vivo. Remarkably, knockdown of HongrES1 in vivo led to reduced fertility accompanied with deformed appearance of fetuses and pups. These results identify HongrES1 as a novel and critical molecule in the regulation of sperm capacitation and male fertility. PMID:19116669

  4. Sperm content of postacrosomal WW binding protein is related to fertilization outcomes in patients undergoing assisted reproductive technology.

    PubMed

    Aarabi, Mahmoud; Balakier, Hanna; Bashar, Siamak; Moskovtsev, Sergey I; Sutovsky, Peter; Librach, Clifford L; Oko, Richard

    2014-08-01

    To determine the levels of postacrosomal WW binding protein (PAWP) in the spermatozoa of men that were used clinically for intracytoplasmic sperm injection (ICSI) and to correlate them with infertility treatment outcomes. Prospective clinical and laboratory study. University-based laboratory and infertility clinic. Men undergoing ICSI for the treatment of couples' infertility (n=110). Quantitative analysis of sperm PAWP levels by flow cytometry and developmental analysis of PAWP expression by immunoblotting, immunofluorescence, and immunohistochemistry. PAWP flow-cytometric levels and immunolocalization in spermatozoa. A strong positive correlation was found between PAWP expression levels and fertilization rates after ICSI, with high levels of PAWP being associated with higher fertilization rates; the positive correlation was independent of age, DNA fragmentation index, and other sperm parameters. PAWP expression levels were correlated with embryonic development, with high levels of PAWP being associated with a lower number of arrested embryos within 3-5 days post-ICSI. PAWP expression was detected during the late stages of human spermiogenesis in elongating spermatids, confirming previous findings in various animal models. Our clinical data from infertile couples demonstrate significant correlations between sperm PAWP levels and both fertilization rates and normal embryonic development after ICSI. Considering its proposed role in the initiation of oocyte activation, we suggest that PAWP could have potential applications in the diagnosis and treatment of infertility. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  5. Proteolytic processing of a protein involved in sperm-egg fusion correlates with acquisition of fertilization competence

    PubMed Central

    1990-01-01

    A protein located on the surface of guinea pig sperm (PH-30) has been implicated in the process of sperm-egg fusion (Primakoff, P., H. Hyatt, and J. Tredick-Kline. 1987. J. Cell Biol. 104:141-149). In this paper we have assessed basic biochemical properties of PH-30 and have analyzed the molecular forms of PH-30 present at different stages of sperm maturation. We show the following: (a) PH-30 is an integral membrane glycoprotein; (b) it is composed of two tightly associated and immunologically distinct subunits; (c) both subunits are made as larger precursors; (d) processing of the two subunits occurs at different developmental stages; (e) the final processing step occurs in the region of the epididymis where sperm become fertilization competent; (f) processing can be mimicked in vitro; (g) processing exposes at least two new epitopes on PH-30-one of the newly exposed epitopes is recognized by a fusion-inhibitory monoclonal antibody. These results are discussed in terms of the possible role of PH-30 in mediating fusion with the egg plasma membrane. PMID:2114412

  6. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions

    PubMed Central

    Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

    2016-01-01

    Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca2+, or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca2+. Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca2+ entry in sperm through the Ca2+/CaM/CaMKKs/CaMKI pathway. The Ca2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca2+ entry in the cells

  7. Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases

    PubMed Central

    Ickowicz, Debby; Finkelstein, Maya; Breitbart, Haim

    2012-01-01

    Mammalian sperm must undergo a series of biochemical and physiological modifications, collectively called capacitation, in the female reproductive tract prior to the acrosome reaction (AR). The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca2+ during both capacitation and the AR. In the present review, we summarize some of the signaling events that are involved in capacitation. During the capacitation process, phosphatidyl-inositol-3-kinase (PI3K) is phosphorylated/activated via a protein kinase A (PKA)-dependent cascade, and downregulated by protein kinase C α (PKCα). PKCα is active at the beginning of capacitation, resulting in PI3K inactivation. During capacitation, PKCα as well as PP1γ2 is degraded by a PKA-dependent mechanism, allowing the activation of PI3K. The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate (cAMP) produced by the bicarbonate-dependent soluble adenylyl cyclase. This activation of PKA leads to an increase in actin polymerization, an essential process for the development of hyperactivated motility, which is necessary for successful fertilization. Actin polymerization is mediated by PIP2 in two ways: first, PIP2 acts as a cofactor for phospholipase D (PLD) activation, and second, as a molecule that binds and inhibits actin-severing proteins such as gelsolin. Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase (SFK) is also important for its inactivation. Prior to the AR, gelsolin is released from PIP2 and undergoes dephosphorylation/activation, resulting in fast F-actin depolymerization, leading to the AR. PMID:23001443

  8. ZP Domain Proteins in the Abalone Egg Coat Include a Paralog of VERL under Positive Selection That Binds Lysin and 18-kDa Sperm Proteins

    PubMed Central

    Aagaard, Jan E.; Vacquier, Victor D.; MacCoss, Michael J.; Swanson, Willie J.

    2010-01-01

    Identifying fertilization molecules is key to our understanding of reproductive biology, yet only a few examples of interacting sperm and egg proteins are known. One of the best characterized comes from the invertebrate archeogastropod abalone (Haliotis spp.), where sperm lysin mediates passage through the protective egg vitelline envelope (VE) by binding to the VE protein vitelline envelope receptor for lysin (VERL). Rapid adaptive divergence of abalone lysin and VERL are an example of positive selection on interacting fertilization proteins contributing to reproductive isolation. Previously, we characterized a subset of the abalone VE proteins that share a structural feature, the zona pellucida (ZP) domain, which is common to VERL and the egg envelopes of vertebrates. Here, we use additional expressed sequence tag sequencing and shotgun proteomics to characterize this family of proteins in the abalone egg VE. We expand 3-fold the number of known ZP domain proteins present within the VE (now 30 in total) and identify a paralog of VERL (vitelline envelope zona pellucida domain protein [VEZP] 14) that contains a putative lysin-binding motif. We find that, like VERL, the divergence of VEZP14 among abalone species is driven by positive selection on the lysin-binding motif alone and that these paralogous egg VE proteins bind a similar set of sperm proteins including a rapidly evolving 18-kDa paralog of lysin, which may mediate sperm–egg fusion. This work identifies an egg coat paralog of VERL under positive selection and the candidate sperm proteins with which it may interact during abalone fertilization. PMID:19767347

  9. Duplicate Abalone Egg Coat Proteins Bind Sperm Lysin Similarly, but Evolve Oppositely, Consistent with Molecular Mimicry at Fertilization

    PubMed Central

    Aagaard, Jan E.; Springer, Stevan A.; Soelberg, Scott D.; Swanson, Willie J.

    2013-01-01

    Sperm and egg proteins constitute a remarkable paradigm in evolutionary biology: despite their fundamental role in mediating fertilization (suggesting stasis), some of these molecules are among the most rapidly evolving ones known, and their divergence can lead to reproductive isolation. Because of strong selection to maintain function among interbreeding individuals, interacting fertilization proteins should also exhibit a strong signal of correlated divergence among closely related species. We use evidence of such molecular co-evolution to target biochemical studies of fertilization in North Pacific abalone (Haliotis spp.), a model system of reproductive protein evolution. We test the evolutionary rates (d N/d S) of abalone sperm lysin and two duplicated egg coat proteins (VERL and VEZP14), and find a signal of co-evolution specific to ZP-N, a putative sperm binding motif previously identified by homology modeling. Positively selected residues in VERL and VEZP14 occur on the same face of the structural model, suggesting a common mode of interaction with sperm lysin. We test this computational prediction biochemically, confirming that the ZP-N motif is sufficient to bind lysin and that the affinities of VERL and VEZP14 are comparable. However, we also find that on phylogenetic lineages where lysin and VERL evolve rapidly, VEZP14 evolves slowly, and vice versa. We describe a model of sexual conflict that can recreate this pattern of anti-correlated evolution by assuming that VEZP14 acts as a VERL mimic, reducing the intensity of sexual conflict and slowing the co-evolution of lysin and VERL. PMID:23408913

  10. The Papillomavirus Major Capsid Protein L1

    PubMed Central

    Buck, Christopher B.; Day, Patricia M.; Trus, Benes L.

    2013-01-01

    The elegant icosahedral surface of the papillomavirus virion is formed by a single protein called L1. Recombinant L1 proteins can spontaneously self-assemble into a highly immunogenic structure that closely mimics the natural surface of native papillomavirus virions. This has served as the basis for two highly successful vaccines against cancer-causing human papillomaviruses (HPVs). During the viral life cycle, the capsid must undergo a variety of conformational changes, allowing key functions including the encapsidation of the ~8 kb viral genomic DNA, maturation into a more stable state to survive transit between hosts, mediating attachment to new host cells, and finally releasing the viral DNA into the newly infected host cell. This brief review focuses on conserved sequence and structural features that underlie the functions of this remarkable protein. PMID:23800545

  11. Growth, testis size, spermatogenesis, semen parameters and seminal plasma and sperm membrane protein profile during the reproductive development of male goats supplemented with de-oiled castor cake.

    PubMed

    Oliveira, C H A; Silva, A M; Silva, L M; van Tilburg, M F; Fernandes, C C L; Velho, A L M C; Moura, A A; Moreno, F B M B; Monteiro-Moreira, A C O; Moreira, R A; Lima, I M T; Rondina, D

    2015-06-01

    The present study was conducted to evaluate the effect of de-oiled castor cake on reproductive traits of crossbreed goats. Fourteen males were grouped into two lots (n = 7/group), as described: group without de-oiled castor cake (WCC) and group fed with de-oiled castor cake (CC). Goats received two diets containing a mixture of Bermudagrass hay and concentrates with the same energy (73% total digestive nutrients) and protein content (15% crude protein) during 150 days, corresponding to ages from 40 (puberty) to 60 weeks. Blood plasma concentrations of urea, albumin, lactate dehydrogenase, creatinine, alanine aminotransferase and testosterone were determined. We also evaluated scrotal circumference, sperm parameters, quantitative aspects of spermatogenesis and daily sperm production (DSP), as well as the proteome of seminal plasma and sperm membrane. Seminal fluid and sperm proteins were analyzed by 2D SDS-PAGE and mass spectrometry. After 150 days of castor cake feeding, animals had no changes in the biochemical composition of blood plasma, suggesting the absence of intoxication by ingestion of ricin. There were no alterations in dry mater intake, weight gain, testis size, peripheral concentrations of testosterone, sperm concentration, motility and morphology. Sertoli and germ cell populations in the testis and DSP were not affected either. However, there were significant variations in the expression of five seminal plasma proteins and four sperm membrane proteins. In conclusion, the replacement of soybean meal by castor cake (with ricin concentrations of 50mg/kg) did not interfere with the growth and core reproductive development of male goats. However, the diet with ricin altered the expression of certain seminal plasma and sperm membrane proteins, which play roles in sperm function and fertilization. Lower expression of these proteins may impair the ricin-fed animals to perform as high-fertility sires.

  12. Phosphorylation state of a Tob/BTG protein, FOG-3, regulates initiation and maintenance of the Caenorhabditis elegans sperm fate program.

    PubMed

    Lee, Myon-Hee; Kim, Kyung Won; Morgan, Clinton T; Morgan, Dyan E; Kimble, Judith

    2011-05-31

    FOG-3, the single Caenorhabditis elegans Tob/BTG protein, directs germ cells to adopt the sperm fate at the expense of oogenesis. Importantly, FOG-3 activity must be maintained for the continued production of sperm that is typical of the male sex. Vertebrate Tob proteins have antiproliferative activity and ERK phosphorylation of Tob proteins has been proposed to abrogate "antiproliferative" activity. Here we investigate FOG-3 phosphorylation and its effect on sperm fate specification. We found both phosphorylated and unphosphorylated forms of FOG-3 in nematodes. We then interrogated the role of FOG-3 phosphorylation in sperm fate specification. Specifically, we assayed FOG-3 transgenes for rescue of a fog-3 null mutant. Wild-type FOG-3 rescued both initiation and maintenance of sperm fate specification. A FOG-3 mutant with its four consensus ERK phosphorylation sites substituted to alanines, called FOG-3(4A), rescued partially: sperm were made transiently but not continuously in both sexes. A different FOG-3 mutant with its sites substituted to glutamates, called FOG-3(4E), had no rescuing activity on its own, but together with FOG-3(4A) rescue was complete. Thus, when FOG-3(4A) and FOG-3(4E) were both introduced into the same animals, sperm fate specification was not only initiated but also maintained, resulting in continuous spermatogenesis in males. Our findings suggest that unphosphorylated FOG-3 initiates the sperm fate program and that phosphorylated FOG-3 maintains that program for continued sperm production typical of males. We discuss implications of our results for Tob/BTG proteins in vertebrates.

  13. Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42

    PubMed Central

    2011-01-01

    Background The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure. Results We made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants. Conclusions The results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain. PMID:21345212

  14. Characterization of proteins in the bovine epididymal and seminal fluid and proteins attached to epididymal and ejaculated sperm

    USDA-ARS?s Scientific Manuscript database

    During final maturation sperm lose their ability to biosynthesize, repair, grow, and divide. They have minimal metabolic function, and thus become completely dependent on their environment. In the epididymis, sperm are stored for a long period of time, but upon ejaculation motility is increased and...

  15. Male Age Affects Female Mate Preference, Quantity of Accessory Gland Proteins, and Sperm Traits and Female Fitness in D. melanogaster.

    PubMed

    Rezaei, Abolhasan; Krishna, Mysore Siddaiah; Santhosh, Hassan T

    2015-01-01

    For species in which mating is resource-independent and offspring do not receive parental care, theoretical models of age-based female mate preference predict that females should prefer to mate with older males as they have demonstrated ability to survive. Thus, females should obtain a fitness benefit from mating with older males. However, male aging is often associated with reductions in quantity of sperm. The adaptive significance of age-based mate choice is therefore unclear. Various hypotheses have made conflicting predictions concerning this issue, because published studies have not investigated the effect of age on accessory gland proteins and sperm traits. D. melanogaster exhibits resource-independent mating, and offspring do not receive parental care, making this an appropriate model for studying age-based mate choice. In the present study, we found that D. melanogaster females of all ages preferred to mate with the younger of two competing males. Young males performed significantly greater courtship attempts and females showed least rejection for the same than middle-aged and old males. Young males had small accessory glands that contained very few main cells that were larger than average. Nevertheless, compared with middle-aged or old males, the young males transferred greater quantities of accessory gland proteins and sperm to mated females. As a result, females that mated with young male produced more eggs and progeny than those that mated with older males. Furthermore, mating with young male reduced female's lifespan. These studies indicate that quantity of accessory gland proteins and sperm traits decreased with male age and females obtain direct fitness benefit from mating with preferred young males.

  16. Functional interaction of phospholipid hydroperoxide glutathione peroxidase with sperm mitochondrion-associated cysteine-rich protein discloses the adjacent cysteine motif as a new substrate of the selenoperoxidase.

    PubMed

    Maiorino, Matilde; Roveri, Antonella; Benazzi, Louise; Bosello, Valentina; Mauri, Pierluigi; Toppo, Stefano; Tosatto, Silvio C E; Ursini, Fulvio

    2005-11-18

    The mitochondrial capsule is a selenium- and disulfide-rich structure enchasing the outer mitochondrial membrane of mammalian spermatozoa. Among the proteins solubilized from the sperm mitochondrial capsule, we confirmed, by using a proteomic approach, the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPx) as a major component, and we also identified the sperm mitochondrion-associated cysteine-rich protein (SMCP) and fragments/aggregates of specific keratins that previously escaped detection (Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., and Flohé, L. (1999) Science 285, 1393-1396). The evidence for a functional association between PHGPx, SMCP, and keratins is further supported by the identification of a sequence motif of regularly spaced Cys-Cys doublets common to SMCP and high sulfur keratin-associated proteins, involved in bundling hair shaft keratin by disulfide cross-linking. Following the oxidative polymerization of mitochondrial capsule proteins, catalyzed by PHGPx, two-dimensional redox electrophoresis analysis showed homo- and heteropolymers of SMCP and PHGPx, together with other minor components. Adjacent cysteine residues in SMCP peptides are oxidized to cystine by PHGPx. This unusual disulfide is known to drive, by reshuffling oxidative protein folding. On this basis we propose that oxidative polymerization of the mitochondrial capsule is primed by the formation of cystine on SMCP, followed by reshuffling. Occurrence of reshuffling is further supported by the calculated thermodynamic gain of the process. This study suggests a new mechanism where selenium catalysis drives the cross-linking of structural elements of the cytoskeleton via the oxidation of a keratin-associated protein.

  17. Etiologies of sperm oxidative stress

    PubMed Central

    Sabeti, Parvin; Pourmasumi, Soheila; Rahiminia, Tahereh; Akyash, Fatemeh; Talebi, Ali Reza

    2016-01-01

    Sperm is particularly susceptible to reactive oxygen species (ROS) during critical phases of spermiogenesis. However, the level of seminal ROS is restricted by seminal antioxidants which have beneficial effects on sperm parameters and developmental potentials. Mitochondria and sperm plasma membrane are two major sites of ROS generation in sperm cells. Besides, leukocytes including polymer phonuclear (PMN) leukocytes and macrophages produce broad category of molecules including oxygen free radicals, non-radical species and reactive nitrogen species. Physiological role of ROS increase the intracellular cAMP which then activate protein kinase in male reproductive system. This indicates that spermatozoa need small amounts of ROS to acquire the ability of nuclear maturation regulation and condensation to fertilize the oocyte. There is a long list of intrinsic and extrinsic factors which can induce oxidative stress to interact with lipids, proteins and DNA molecules. As a result, we have lipid peroxidation, DNA fragmentation, axonemal damage, denaturation of the enzymes, over generation of superoxide in the mitochondria, lower antioxidant activity and finally abnormal spermatogenesis. If oxidative stress is considered as one of the main cause of DNA damage in the germ cells, then there should be good reason for antioxidant therapy in these conditions. PMID:27351024

  18. Bovine sperm raft membrane associated Glioma Pathogenesis-Related 1-like protein 1 (GliPr1L1) is modified during the epididymal transit and is potentially involved in sperm binding to the zona pellucida.

    PubMed

    Caballero, Julieta; Frenette, Gilles; D'Amours, Olivier; Belleannée, Clémence; Lacroix-Pepin, Nicolas; Robert, Claude; Sullivan, Robert

    2012-12-01

    Glioma pathogenesis-related 1-like protein1 (GliPr1L1) was identified by liquid chromatography-tandem mass spectrometry analyses of proteins associated to bovine sperm lipid raft membrane domains. This protein belongs to the CAP superfamily including cysteine-rich secretory proteins, Antigen 5 and pathogenesis-related 1 protein. PCR analysis revealed that GliPr1L1 is expressed in testis and, at a much lower level, all along the epididymis. Western blotting showed a similar distribution of GliPr1L1 in testicular and epididymal tissue extracts. In the epididymal lumen, GliPr1L1 was associated with the maturing spermatozoa and epididymosomes all along the excurrent duct but was undetectable in the soluble fraction of epididymal fluid. The protein was detectable as multiple isoforms with a higher MW form in the testis and proximal caput. Treatments with PNGase F revealed that N-glycosylation was responsible of multiple bands detected on Western blots. These results suggest that the N-glycosylation moiety of GliPr1L1 is processed during the transit in the caput. Western blots demonstrated that GliPr1L1 was associated with the sperm plasma membrane preparation. GliPr1L1 is glycosyl phosphatidyl inositol (GPI) anchored to caput and cauda spermatozoa as demonstrated by the ability of phosphatidylinositol specific phospholipase C to release GliPr1L1 from intact sperm cells. Lipid raft membrane domains were separated from caput and cauda epididymal spermatozoa. GliPr1L1 was immunodetectable in the low buoyant density fractions where lipid rafts are distributed. GliPr1L1 was localized on sperm equatorial segment and neck. In vitro fertilization performed in presence of anti-GliPr1L1 showed that this protein is involved in sperm-zona pellucida interaction.

  19. KIFC1-Like Motor Protein Associates with the Cephalopod Manchette and Participates in Sperm Nuclear Morphogenesis in Octopus tankahkeei

    PubMed Central

    Tan, Fu-Qing; Yang, Wan-Xi

    2010-01-01

    Background Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery. Methodology/Principal Findings We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level. Conclusions/Significance The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod. PMID:21187923

  20. The gap junctional protein INX-14 functions in oocyte precursors to promote C. elegans sperm guidance

    PubMed Central

    Edmonds, Johnathan W.; McKinney, Shauna L.; Prasain, Jeevan K.; Miller, Michael A.

    2011-01-01

    Innexins are the subunits of invertebrate gap junctions. Here we show that the innexin INX-14 promotes sperm guidance to the fertilization site in the C. elegans hermaphrodite reproductive tract. inx-14 loss causes cell nonautonomous defects in sperm migration velocity and directional velocity. Results from genetic and immunocytochemical analyses provide strong evidence that INX-14 acts in transcriptionally active oocyte precursors in the distal gonad, not in transcriptionally inactive oocytes that synthesize prostaglandin sperm-attracting cues. Somatic gonadal sheath cell interaction is necessary for INX-14 function, likely via INX-8 and INX-9 expressed in sheath cells. However, electron microscopy has not identified gap junctions in oocyte precursors, suggesting that INX-14 acts in a channel-independent manner or INX-14 channels are difficult to document. INX-14 promotes prostaglandin signaling to sperm at a step after F-series prostaglandin synthesis in oocytes. Taken together, our results support the model that INX-14 functions in a somatic gonad/germ cell signaling mechanism essential for sperm function. We propose that this mechanism regulates the transcription of a factor(s) that modulates prostaglandin metabolism, transport, or activity in the reproductive tract. PMID:21889935

  1. Proteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface Proteins on Human Embryonic Stem Cells and Sperms

    PubMed Central

    Gu, Bin; Zhang, Jiarong; Wu, Ying; Zhang, Xinzong; Tan, Zhou; Lin, Yuanji; Huang, Xiao; Chen, Liangbiao; Yao, Kangshou; Zhang, Ming

    2011-01-01

    Background It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells. Methods and Principal Findings Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed. Conclusions/Significance Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells. PMID:21559292

  2. cDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm-egg adhesion and the wide distribution of its gene among mammals

    PubMed Central

    1990-01-01

    Sperm binding to the egg zona pellucida in mammals is a cell-cell adhesion process that is generally species specific. The guinea pig sperm protein PH-20 has a required function in sperm adhesion to the zona pellucida of guinea pig eggs. PH-20 is located on both the sperm plasma membrane and acrosomal membrane. We report here the isolation and sequence of a full-length cDNA for PH-20 (available from EMBL/GenBank/DDBJ under accession number X56332). The derived amino acid sequence shows a mature protein of 468 amino acids containing six N-linked glycosylation sites and twelve cysteines, eight of which are tightly clustered near the COOH terminus. The sequence indicates PH-20 is a novel protein with no relationship to the mouse sperm adhesion protein galactosyl transferase and no significant homology with other known proteins. The two PH-20 populations, plasma membrane and acrosomal membrane, could arise because one form of PH-20 is encoded and differentially targeted at different spermatogenic stages. Alternatively, two different forms of PH-20 could be encoded. Our evidence thus far reveals only one sequence coding for PH-20: Southern blots of guinea pig genomic DNA indicated there is a single PH-20 gene, Northern blots showed a single size PH-20 message (approximately 2.2 kb), and no sequence variants were found among the sequenced cDNA clones. Cross-species Southern blots reveal the presence of a homologue of the PH-20 gene in mouse, rat, hamster, rabbit, bovine, monkey, and human genomic DNA, showing the PH-20 gene is conserved among mammals. Since genes for zona glycoproteins are also conserved among mammals, the general features of sperm and zona proteins involved in mammalian sperm-egg adhesion may have been evolutionarily maintained. Species specificity may result from limited changes in these molecules, either in their binding domains or in other regions that affect the ability of the binding domains to interact. PMID:2269661

  3. Synthesis and application of peptide immunogens related to the sperm tail protein tpx-1, a member of the CRISP superfamily of proteins.

    PubMed

    Keah, H H; O'Bryan, M K; de Kretser, D M; Hearn, M T

    2001-01-01

    The synthesis of peptides containing 0, 1 and 2 cysteine residues related to the human sperm tail protein, tpx-1, is described. These synthetic peptides, following conjugation to keyhole limpet hemocyanin modified with maleimidobenzoic acid N-hydroxysuccinimide ester, were used as immunogens to generate polyclonal antibodies in female New Zealand white rabbits. The binding characteristics of the derived antipeptide sera were evaluated using indirect and competitive ELISA procedures. Western immunoblot experiments also confirmed that these synthetic peptide immunogens are able to generate high-titer polyclonal antibodies capable of cross-reacting with the mature tpx-1 protein present in crude rat sperm tail/testis preparations as well as in outer dense fiber preparations. Consequently, these synthetic peptides represent promising candidates for investigations into the role of tpx-1 in the immunoregulation of sperm function in the rat and other mammalian models, with the derived antisera also providing an avenue to explore possible sites of expression of tpx-1 proteins in other tissues.

  4. Semen variables and sperm membrane protein profile of Saanen bucks ( Capra hircus) in dry and rainy seasons of the northeastern Brazil (3°S)

    NASA Astrophysics Data System (ADS)

    van Tilburg, M. F.; Salles, M. G. F.; Silva, M. M.; Moreira, R. A.; Moreno, F. B.; Monteiro-Moreira, A. C. O.; Martins, J. A. M.; Cândido, M. J. D.; Araújo, A. A.; Moura, A. A. A.

    2015-05-01

    The Saanen is a highly productive breed, and for this reason, it has been raised in Brazil, but mostly under climate conditions completely different from where the breed originated. The objective of this study was to investigate variations in semen parameters and sperm membrane proteins from Saanen bucks ( n = 7) raised in Northeastern Brazil, during dry season (September, October, and November) and rainy season (March, April, and May). We showed that during the dry season, sperm motility, concentration, and the percentage of normal sperm decreased as compared to the rainy season. Rectal temperatures of bucks had no significant ( p > 0.05) variations during the dry and rainy seasons. However, temperatures of left and right skin testis were higher ( p < 0.05) during the dry as compared to the rainy season. Expression of three proteins (lysine-specific demethylase 5D, adenosine triphosphate (ATP) synthase subunit d, and radial spoke head protein 9 homolog) in sperm membrane were more intense in rainy season and only one protein (cytosol aminopeptidase) had greater expression in the dry season of the year. Our results show that mechanisms of testicular thermoregulation of Saanen bucks did not prevent a decrease in seminal parameters during the dry season. This deterioration may be related to reduced expression of proteins associated with important functions in sperm membrane.

  5. Semen variables and sperm membrane protein profile of Saanen bucks (Capra hircus) in dry and rainy seasons of the northeastern Brazil (3°S).

    PubMed

    van Tilburg, M F; Salles, M G F; Silva, M M; Moreira, R A; Moreno, F B; Monteiro-Moreira, A C O; Martins, J A M; Cândido, M J D; Araújo, A A; Moura, A A A

    2015-05-01

    The Saanen is a highly productive breed, and for this reason, it has been raised in Brazil, but mostly under climate conditions completely different from where the breed originated. The objective of this study was to investigate variations in semen parameters and sperm membrane proteins from Saanen bucks (n = 7) raised in Northeastern Brazil, during dry season (September, October, and November) and rainy season (March, April, and May). We showed that during the dry season, sperm motility, concentration, and the percentage of normal sperm decreased as compared to the rainy season. Rectal temperatures of bucks had no significant (p > 0.05) variations during the dry and rainy seasons. However, temperatures of left and right skin testis were higher (p < 0.05) during the dry as compared to the rainy season. Expression of three proteins (lysine-specific demethylase 5D, adenosine triphosphate (ATP) synthase subunit d, and radial spoke head protein 9 homolog) in sperm membrane were more intense in rainy season and only one protein (cytosol aminopeptidase) had greater expression in the dry season of the year. Our results show that mechanisms of testicular thermoregulation of Saanen bucks did not prevent a decrease in seminal parameters during the dry season. This deterioration may be related to reduced expression of proteins associated with important functions in sperm membrane.

  6. Src Kinase Is the Connecting Player between Protein Kinase A (PKA) Activation and Hyperpolarization through SLO3 Potassium Channel Regulation in Mouse Sperm.

    PubMed

    Stival, Cintia; La Spina, Florenza A; Baró Graf, Carolina; Arcelay, Enid; Arranz, Silvia E; Ferreira, Juan J; Le Grand, Sibylle; Dzikunu, Victor A; Santi, Celia M; Visconti, Pablo E; Buffone, Mariano G; Krapf, Dario

    2015-07-24

    Plasma membrane hyperpolarization is crucial for mammalian sperm to acquire acrosomal responsiveness during capacitation. Among the signaling events leading to mammalian sperm capacitation, the immediate activation of protein kinase A plays a pivotal role, promoting the subsequent stimulation of protein tyrosine phosphorylation that associates with fertilizing capacity. We have shown previously that mice deficient in the tyrosine kinase cSrc are infertile and exhibit improper cauda epididymis development. It is therefore not clear whether lack of sperm functionality is due to problems in epididymal maturation or to the absence of cSrc in sperm. To further address this problem, we investigated the kinetics of cSrc activation using anti-Tyr(P)-416-cSrc antibodies that only recognize active cSrc. Our results provide evidence that cSrc is activated downstream of PKA and that inhibition of its activity blocks the capacitation-induced hyperpolarization of the sperm plasma membrane without blocking the increase in tyrosine phosphorylation that accompanies capacitation. In addition, we show that cSrc inhibition also blocks the agonist-induced acrosome reaction and that this inhibition is overcome by pharmacological hyperpolarization. Considering that capacitation-induced hyperpolarization is mediated by SLO3, we evaluated the action of cSrc inhibitors on the heterologously expressed SLO3 channel. Our results indicate that, similar to SLO1 K(+) channels, cSrc blockers significantly decreased SLO3-mediated currents. Together, these results are consistent with findings showing that hyperpolarization of the sperm plasma membrane is necessary and sufficient to prepare the sperm for the acrosome reaction and suggest that changes in sperm membrane potential are mediated by cSrc activation.

  7. Recombinant mouse sperm ZP3-binding protein (ZP3R/sp56) forms a high order oligomer that binds eggs and inhibits mouse fertilization in vitro.

    PubMed

    Buffone, Mariano G; Zhuang, Tiangang; Ord, Teri S; Hui, Ling; Moss, Stuart B; Gerton, George L

    2008-05-02

    Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

  8. Involvement of epidermal growth factor receptors and mitogen-activated protein kinase in progestin-induction of sperm hypermotility in Atlantic croaker through membrane progestin receptor-alpha.

    PubMed

    Tan, Wenxian; Thomas, Peter

    2015-10-15

    The intracellular pathways mediating rapid, nongenomic progestin stimulation of sperm motility remain unclear. The role of epidermal growth factor receptors (Egfr and ErbB2) and mitogen-activated protein kinase (Mapk) in membrane progestin receptor-alpha (mPRα)-mediated progestin stimulation of sperm hypermotility was examined in a teleost, Atlantic croaker. Inhibition of upstream regulators of Egfr, intracellular tyrosine kinase (Src) with PP2, and matrix metalloproteinase (MMP) with Ilomastat, abolished progestin-initiated sperm hypermotility by 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S; 20 nM) and a specific mPRα agonist, Org OD 02-0 (20 nM). Pretreatment of croaker sperm with EGFR inhibitors, AG1478 (5 μM) and RG13022 (50 μM), the ErbB2 inhibitor, AG879 (5 nM), or the MEK1/2 inhibitor, U0126 (500 nM) blocked progestin stimulation of sperm motility. Levels of phosphorylated extracellular-related kinase 1 and 2 (P-Erk1/2) were increased after 20β-S treatment. These results demonstrate that progestin-mediated hypermotility via mPRα in croaker sperm involves activation of the Egfr, ErbB2 and Mapk pathways. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. MicroRNA-29a inhibited epididymal epithelial cell proliferation by targeting nuclear autoantigenic sperm protein (NASP).

    PubMed

    Ma, Wubin; Xie, Shengsong; Ni, Minjie; Huang, Xingxu; Hu, Shuanggang; Liu, Qiang; Liu, Aihua; Zhang, Jinsong; Zhang, Yonglian

    2012-03-23

    Cell proliferation often decreases gradually during postnatal development of some organs. However, the underlying molecular mechanisms remain unclear. Epididymis, playing important roles in sperm maturation, is a typical organ of this type, which displays a decreased proliferation during postnatal development and even ceased at the adult stage. Here, epididymis was employed as a model to explore the underlying mechanisms. We profiled the microRNA and mRNA expression of newborn (1 day) and adult (90 day) rat epididymis by microarray analysis, and found that the level of miR-29a was dramatically up-regulated during postnatal development of rat epididymis. Subsequent investigations demonstrated that overexpression of miR-29a inhibited the proliferation of epididymal epithelial cells in vitro. The nuclear autoantigenic sperm protein (NASP), a novel target of miR-29a, was significantly down-regulated during postnatal development of rat epididymis. Further analysis showed that silence of NASP mimicked the anti-proliferation effect of miR-29a, whereas overexpression of this protein attenuated the effect of miR-29a. As in rat epididymis, miR-29a was up-regulated and Nasp was down-regulated during postnatal development of mouse epididymis, heart, liver, and lung. Moreover, miR-29a can also inhibit the proliferation of cancer cells by targeting Nasp. Thus, an increase of miR-29a, and hence decrease of Nasp, may contribute to inhibit cell proliferation during postnatal organ development.

  10. Proteins of human milk. I. Identification of major components

    SciTech Connect

    Anderson, N.G.; Powers, M.T.; Tollaksen, S.L.

    1982-04-01

    Traditionally, human milk proteins are identified largely by reference to bovine milk. Hence, to identify the major proteins in human milk, we subjected human and bovine milk, in parallel, to high-resolution two-dimensional electrophoresis. Isoelectric precipitation at pH 4.6 was our criterion for distinguishing whey proteins from those of the casein complex. The ..cap alpha..- and..beta..-caseins were identified on the basis of relative abundance, relative molecular mass, and relative isoelectric points. No protein disappeared from ISO-DALT patterns of human milk after rennin treatment, and no new protein comparable to bovine para K-casein appeared in the BASO-DALT patterns; this suggests that K-casein is absent from human milk. The proteins identified in human milk patterns include the ..cap alpha.. and ..beta.. casein families, lactalbumin, albumin, transferrin, IgA, and lactoferrin. Numerous additional proteins seen in patterns for human milk remain to be identified.

  11. Sperm development and motility are regulated by PP1 phosphatases in Caenorhabditis elegans.

    PubMed

    Wu, Jui-ching; Go, Aiza C; Samson, Mark; Cintra, Thais; Mirsoian, Susan; Wu, Tammy F; Jow, Margaret M; Routman, Eric J; Chu, Diana S

    2012-01-01

    Sperm from different species have evolved distinctive motility structures, including tubulin-based flagella in mammals and major sperm protein (MSP)-based pseudopods in nematodes. Despite such divergence, we show that sperm-specific PP1 phosphatases, which are required for male fertility in mouse, function in multiple processes in the development and motility of Caenorhabditis elegans amoeboid sperm. We used live-imaging analysis to show the PP1 phosphatases GSP-3 and GSP-4 (GSP-3/4) are required to partition chromosomes during sperm meiosis. Postmeiosis, tracking fluorescently labeled sperm revealed that both male and hermaphrodite sperm lacking GSP-3/4 are immotile. Genetic and in vitro activation assays show lack of GSP-3/4 causes defects in pseudopod development and the rate of pseudopodial treadmilling. Further, GSP-3/4 are required for the localization dynamics of MSP. GSP-3/4 shift localization in concert with MSP from fibrous bodies that sequester MSP at the base of the pseudopod, where directed MSP disassembly facilitates pseudopod contraction. Consistent with a role for GSP-3/4 as a spatial regulator of MSP disassembly, MSP is mislocalized in sperm lacking GSP-3/4. Although a requirement for PP1 phosphatases in nematode and mammalian sperm suggests evolutionary conservation, we show PP1s have independently evolved sperm-specific paralogs in separate lineages. Thus PP1 phosphatases are highly adaptable and employed across a broad range of sexually reproducing species to regulate male fertility.

  12. Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm.

    PubMed

    Rodríguez-Villamil, P; Hoyos-Marulanda, V; Martins, J A M; Oliveira, A N; Aguiar, L H; Moreno, F B; Velho, A L M C S; Monteiro-Moreira, A C; Moreira, R A; Vasconcelos, I M; Bertolini, M; Moura, A A

    2016-02-01

    The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 μg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 μg/mL BSP1 (77.8 ± 3.1%), or 20 μg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 μg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 μg/mL BSP1 (22.4 ± 2.9%) and 40 μg/mL BSP1 (19.3 ± 4.1%; P < 0.05). In the second experiment, cumulus-oocyte complexes (n = 1213) were incubated with frozen-thawed cauda epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 μg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P < 0.05). Also, cleavage was better after Fert-TALP medium incubation with 40 μg/mL BSP1 (79.0 ± 1.1%) than with heparin (68.5 ± 1.3%; P < 0.05). Embryo development was higher (P < 0.05) after treatment with 20 μg/mL BSP1 (35.6 ± 2.5%) and 40 μg/mL (41.1 ± 2%) than after incubations with heparin (24.7 ± 3.2%) or without heparin (27.3 ± 1.6%). Interestingly, BSP1 did not cause reductions in blastocyst rates after fertilization with epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and

  13. Characterization and comparison of proteins in the sperm storage tubules of female chickens to bovine epididymal fluid

    USDA-ARS?s Scientific Manuscript database

    Female birds are able to store sperm in crypts called sperm storage tubules (SSTs) in their reproductive tracts for between two and six weeks. Comparatively, sperm in a cow’s reproductive tract remain viable for between 18 and 24 hours. The objective of this experiment was to try to identify and co...

  14. Human sperm protein encyclopedia and alloantigen index: mining novel allo-antigens using sera from ASA-positive infertile patients and vasectomized men.

    PubMed

    Shetty, Jagathpala; Bronson, Richard A; Herr, John C

    2008-01-01

    Anti-sperm antibodies (ASA) are an important cause of immunological infertility. The objective of this study was to identify immunodominant sperm antigens recognized by anti-sperm antibodies (ASA) in serum samples of infertile men, women and vasectomized men. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing (IEF) or nonequilibrium pH gradient electrophoresis (NEPHGE), followed by PAGE and Western blotting. Serum samples from five infertile male and five infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT), were analyzed by Western blotting using NEPHGE gels followed by enhanced chemiluminescence (ECL) to identify the basic sperm antigens reactive to the sera. Serum samples from five fertile male and five fertile female subjects that were ASA-negative by IBT were used as controls. Serum samples from six vasectomized men collected before vasectomy and at different time intervals until 6 months after vasectomy were analyzed by Western blotting using IEF gels. The ECL blots were analyzed to compare immunoreactivity between serum samples from fertile and infertile subjects and identify antigens unique to sera of the infertile subjects. Similarly, immunoreactivity between serum samples from pre- and post-vasectomy was compared to identify antigens unique to sera collected following vasectomy. Five allo-antigenic basic protein spots were recognized by sera from infertile males but not from fertile subjects. Five sperm iso-antigenic basic spots were recognized by infertile female subjects. Two among six of the vasectomized men's sera showed a difference in the Western blot profile 6 months after vasectomy, recognizing at least one new protein spot in each case when compared to pre-vasectomy sera. The acrosomal protein SP-10 was identified as an alloantigen recognized by a post-vasectomy serum. Molecular identities of the known allo- and iso

  15. Oviductal fluid proteins associated with the bovine zona pellucida and the effect on in vitro sperm-egg binding, fertilization and embryo development.

    PubMed

    Gonçalves, R F; Staros, A L; Killian, G J

    2008-12-01

    Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

  16. Immunoglobulin G Expression in Human Sperm and Possible Functional Significance

    PubMed Central

    Yan, Meiling; Zhang, Xiaoyu; Pu, Qinxue; Huang, Tao; Xie, Qingdong; Wang, Yan; Li, Jing; Wang, Yun; Gu, Huan; Huang, Tianhua; Li, Zhiling; Gu, Jiang

    2016-01-01

    Immunoglobulin G (IgG), the major molecule of the immune system, which was traditionally thought to be produced by differentiated B-lymphocytes, had recently been found in non-immune cells including spermatozoa of rabbit testis. To study if human sperms could produce IgG that might play a role in fertilization, we employed immunofluorescent staining, Western blot, in situ hybridization, RT-PCR (reverse transcription polymerase chain reaction) and immunoelectron microscope and found that human sperms were capable of synthesizing IgG. IgG protein and mRNA were detected in the cytoplasm, mainly the neck region of the sperm and IgG immunoreactivity was found to cover the entire sperm cell. The essential enzymes necessary for IgG synthesis and class switching, RAG1 (recombination activating gene 1), RAG2 (recombination activating gene 2) and AID (activation-induced cytidine deaminase), were also detected in the sperm cells. Furthermore, we found that anti-IgG antibody could inhibit sperm from penetrating Zona-free hamster egg with statistical significance. These discoveries suggested that immunoglobulin G could be produced by human sperms and it might play a role during fertilization. PMID:26833114

  17. The 5’-AMP-Activated Protein Kinase (AMPK) Is Involved in the Augmentation of Antioxidant Defenses in Cryopreserved Chicken Sperm

    PubMed Central

    Nguyen, Thi Mong Diep; Seigneurin, François; Froment, Pascal; Combarnous, Yves; Blesbois, Elisabeth

    2015-01-01

    Semen cryopreservation is a unique tool for the management of animal genetic diversity. However, the freeze-thaw process causes biochemical and physical alterations which make difficult the restoration of sperm energy-dependent functions needed for fertilization. 5’-AMP activated protein kinase (AMPK) is a key sensor and regulator of intracellular energy metabolism. Mitochondria functions are known to be severely affected during sperm cryopreservation with deleterious oxidative and peroxidative effects leading to cell integrity and functions damages. The aim of this study was thus to examine the role of AMPK on the peroxidation/antioxidant enzymes defense system in frozen-thawed sperm and its consequences on sperm functions. Chicken semen was diluted in media supplemented with or without AMPK activators (AICAR or Metformin [MET]) or inhibitor (Compound C [CC]) and then cryopreserved. AMPKα phosphorylation, antioxidant enzymes activities, mitochondrial potential, ATP, citrate, viability, acrosome reaction ability (AR) and various motility parameters were negatively affected by the freeze-thaw process while reactive oxygen species (ROS) production, lipid peroxidation (LPO) and lactate concentration were dramatically increased. AICAR partially restored superoxide dismutase (SOD), Glutathione Peroxidase (GPx) and Glutathione Reductase (GR), increased ATP, citrate, and lactate concentration and subsequently decreased the ROS and LPO (malondialdehyde) in frozen-thawed semen. Motility parameters were increased (i.e., + 23% for motility, + 34% for rapid sperm) as well as AR (+ 100%). MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased. CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters. Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation, thus

  18. Onion (Allium cepa L.) peel extract (OPE) regulates human sperm motility via protein kinase C-mediated activation of the human voltage-gated proton channel.

    PubMed

    Chae, M R; Kang, S J; Lee, K P; Choi, B R; Kim, H K; Park, J K; Kim, C Y; Lee, S W

    2017-09-01

    Onion (Allium cepa L.) and quercetin protect against oxidative damage and have positive effects on multiple functional parameters of spermatozoa, including viability and motility. However, the associated underlying mechanisms of action have not yet been identified. The aim of this study was to investigate the effect of onion peel extract (OPE) on voltage-gated proton (Hv1) channels, which play a critical role in rapid proton extrusion. This process underlies a wide range of physiological processes, particularly male fertility. The whole-cell patch-clamp technique was used to record the changes in Hv1 currents in HEK293 cells transiently transfected with human Hv1 (HVCN1). The effects of OPE on human sperm motility were also analyzed. OPE significantly activated the outward-rectifying proton currents in a concentration-dependent manner, with an EC50 value of 30 μg/mL. This effect was largely reversible upon washout. Moreover, OPE induced an increase in the proton current amplitude and decreased the time constant of activation at 0 mV from 4.9 ± 1.7 to 0.6 ± 0.1 sec (n = 6). In the presence of OPE, the half-activation voltage (V1/2 ) shifted in the negative direction, from 20.1 ± 5.8 to 5.2 ± 8.7 mV (n = 6), but the slope was not significantly altered. The OPE-induced current was profoundly inhibited by 10 μm Zn(2+) , the most potent Hv1 channel inhibitor, and was also inhibited by treatment with GF109203X, a specific protein kinase C (PKC) inhibitor. Furthermore, sperm motility was significantly increased in the OPE-treated groups. OPE exhibits protective effects on sperm motility, at least partially via regulation of the proton channel. Moreover, similar effects were exerted by quercetin, the major flavonoid in OPE. These results suggest OPE, which is rich in the potent Hv1 channel activator quercetin, as a possible new candidate treatment for human infertility. © 2017 American Society of Andrology and European Academy of Andrology.

  19. [Glycine-14C and methionine-35S incorporation into liver and gastric mucosa proteins during sperm oil treatment of experimental stomach ulcer].

    PubMed

    Sarkisian, E L; Budantseva, S I; Kalinichenko, N I

    1977-01-01

    The effect of a course-wise introduction of the sperm oil on the intensity of the proteinic synthesis in experimental gastric ulcer was investigated. Tests were conducted on albino male-rats in which an experimental gastric ulcer failing to heal for a long time had been induced through introduction of a 5% acetic acid solution into the subserous membrane of the stomach. The intensity of the proteinic synthesis in the liver and gastric mucosa was assessed by the rate of the glycine-14C and methionine-35S incorporation in the total, mitochondrial and nucleonic proteins on the 7th, 14th, 21st and 28th days of the experimental ulcer course. The sperm oil secured a more effective production and thereby created propicious conditions for an intensified proteinic synthesis. It also contributed to an earlier normalization of the proteinic synthesis rates and to a quicker cicatrization of the ulcerous defect.

  20. Signature proteins for the major clades of Cyanobacteria

    PubMed Central

    2010-01-01

    Background The phylogeny and taxonomy of cyanobacteria is currently poorly understood due to paucity of reliable markers for identification and circumscription of its major clades. Results A combination of phylogenomic and protein signature based approaches was used to characterize the major clades of cyanobacteria. Phylogenetic trees were constructed for 44 cyanobacteria based on 44 conserved proteins. In parallel, Blastp searches were carried out on each ORF in the genomes of Synechococcus WH8102, Synechocystis PCC6803, Nostoc PCC7120, Synechococcus JA-3-3Ab, Prochlorococcus MIT9215 and Prochlor. marinus subsp. marinus CCMP1375 to identify proteins that are specific for various main clades of cyanobacteria. These studies have identified 39 proteins that are specific for all (or most) cyanobacteria and large numbers of proteins for other cyanobacterial clades. The identified signature proteins include: (i) 14 proteins for a deep branching clade (Clade A) of Gloebacter violaceus and two diazotrophic Synechococcus strains (JA-3-3Ab and JA2-3-B'a); (ii) 5 proteins that are present in all other cyanobacteria except those from Clade A; (iii) 60 proteins that are specific for a clade (Clade C) consisting of various marine unicellular cyanobacteria (viz. Synechococcus and Prochlorococcus); (iv) 14 and 19 signature proteins that are specific for the Clade C Synechococcus and Prochlorococcus strains, respectively; (v) 67 proteins that are specific for the Low B/A ecotype Prochlorococcus strains, containing lower ratio of chl b/a2 and adapted to growth at high light intensities; (vi) 65 and 8 proteins that are specific for the Nostocales and Chroococcales orders, respectively; and (vii) 22 and 9 proteins that are uniquely shared by various Nostocales and Oscillatoriales orders, or by these two orders and the Chroococcales, respectively. We also describe 3 conserved indels in flavoprotein, heme oxygenase and protochlorophyllide oxidoreductase proteins that are specific for

  1. The molecular chaperone HSPA2 plays a key role in regulating the expression of sperm surface receptors that mediate sperm-egg recognition.

    PubMed

    Redgrove, Kate A; Nixon, Brett; Baker, Mark A; Hetherington, Louise; Baker, Gordon; Liu, De-Yi; Aitken, R John

    2012-01-01

    A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm-egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm-zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.

  2. Identification of Major Outer Surface Proteins of Streptococcus agalactiae

    PubMed Central

    Hughes, Martin J. G.; Moore, Joanne C.; Lane, Jonathan D.; Wilson, Rebecca; Pribul, Philippa K.; Younes, Zabin N.; Dobson, Richard J.; Everest, Paul; Reason, Andrew J.; Redfern, Joanne M.; Greer, Fiona M.; Paxton, Thanai; Panico, Maria; Morris, Howard R.; Feldman, Robert G.; Santangelo, Joseph D.

    2002-01-01

    To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates. PMID:11854208

  3. Epididymosomes transfer epididymal sperm binding protein 1 (ELSPBP1) to dead spermatozoa during epididymal transit in bovine.

    PubMed

    D'Amours, Olivier; Frenette, Gilles; Bordeleau, Louis-Jean; Allard, Nancy; Leclerc, Pierre; Blondin, Patrick; Sullivan, Robert

    2012-10-01

    Previously, we showed that epididymal sperm binding protein 1 (ELSPBP1) characterizes spermatozoa already dead before ejaculation in bovine. In this study, we investigated the presence of ELSPBP1 in bull genital tract as well as its acquisition by spermatozoa during epididymal transit. As assessed by real-time RT-PCR, ELSPBP1 was highly expressed in the caput and the corpus epididymis but was present in lower expression levels in the testis and the cauda epididymis. Immunohistochemistry revealed the same expression pattern. However, Western blot on tissue homogenates showed some discrepancies, as ELSPBP1 was found in a comparable concentration all along the epididymis. This difference was due to the presence of ELSPBP1 in the epididymal fluid. In both caput and cauda epididymal fluid, ELSPBP1 was associated with the epididymosomes, small membranous vesicles secreted by epithelial cells of the epididymis and implicated in the transfer of proteins to spermatozoa. As assessed by immunocytometry, ELSPBP1 was found on a subset of dead spermatozoa in caput epididymis but was found on all dead spermatozoa in cauda epididymis. To assess ELSPBP1 acquisition by spermatozoa, caput epididymal spermatozoa were incubated with cauda epididymosomes under various conditions. ELSPBP1 detection by immunocytometry assay revealed that only spermatozoa already dead before incubation were receptive to ELSPBP1 transfer by epididymosomes. This receptivity was enhanced by the presence of zinc in the incubation medium. This specificity for a sperm subpopulation suggests that an underlying mechanism is involved and that ELSPBP1 could be a tag for the recognition of dead spermatozoa during epididymal transit.

  4. Treatment with protein kinase C activator is effective for improvement of male pronucleus formation and further embryonic development of sperm-injected oocytes in pigs.

    PubMed

    Nakai, M; Ito, J; Kashiwazaki, N; Men, N T; Tanihara, F; Noguchi, J; Kaneko, H; Onishi, A; Kikuchi, K

    2016-03-01

    To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm-injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 μM) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-μM PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 μM, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a

  5. Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm

    PubMed Central

    1995-01-01

    The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin. PMID:7822422

  6. Microbial drug efflux proteins of the major facilitator superfamily.

    PubMed

    Saidijam, Massoud; Benedetti, Giulia; Ren, Qinghu; Xu, Zhiqiang; Hoyle, Christopher J; Palmer, Sarah L; Ward, Alison; Bettaney, Kim E; Szakonyi, Gerda; Meuller, Johan; Morrison, Scott; Pos, Martin K; Butaye, Patrick; Walravens, Karl; Langton, Kate; Herbert, Richard B; Skurray, Ronald A; Paulsen, Ian T; O'reilly, John; Rutherford, Nicholas G; Brown, Melissa H; Bill, Roslyn M; Henderson, Peter J F

    2006-07-01

    Drug efflux proteins are widespread amongst microorganisms, including pathogens. They can contribute to both natural insensitivity to antibiotics and to emerging antibiotic resistance and so are potential targets for the development of new antibacterial drugs. The design of such drugs would be greatly facilitated by knowledge of the structures of these transport proteins, which are poorly understood, because of the difficulties of obtaining crystals of quality. We describe a structural genomics approach for the amplified expression, purification and characterisation of prokaryotic drug efflux proteins of the 'Major Facilitator Superfamily' (MFS) of transport proteins from Helicobacter pylori, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Bacillus subtilis, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides and Streptomyces coelicolor. The H. pylori putative drug resistance protein, HP1092, and the S. aureus QacA proteins are used as detailed examples. This strategy is an important step towards reproducible production of transport proteins for the screening of drug binding and for optimisation of crystallisation conditions to enable subsequent structure determination.

  7. Heat shock proteins and their association with major pediatric malignancies.

    PubMed

    Skora, Dorota; Gorska, Magdalena

    2016-01-01

    Heat shock proteins belong to a group of molecular chaperones responsible for the regulation of many intracellular processes. HSPs play a pivotal role in the survival of cells under stressful conditions. Over-expression of these proteins have been found in both healthy and a great number of cancer cells. HSPs may be involved in numerous carcinogenic and chemoresistant processes. Due to that fact, they may be referred to as diagnostic biomarkers of oncogenesis and potential targets for anticancer drugs. Thus, we decided to review the involvement of major HSPs in the most malignant childhood cancers.

  8. The binding of drugs to major human milk whey proteins.

    PubMed Central

    Atkinson, H C; Begg, E J

    1988-01-01

    The binding of nine drugs of diverse physicochemical characteristics to major human milk whey proteins is reported. This group included acids, bases and neutral drugs. No drug bound to alpha-lactalbumin, which is the protein present in greatest concentrations in mature milk. Four drugs, diclofenac, phenytoin, prednisolone and warfarin, bound to albumin but to a much lesser extent than in plasma, consistent with quantitatively less albumin in milk. None of the basic drugs studied bound to albumin. Five drugs, atenolol, diclofenac, prednisolone, propranolol and warfarin, bound to lactoferrin though the extent was minimal except for diclofenac. This group included acids, bases and neutral drugs. PMID:3203054

  9. Increased receptor for advanced glycation end products in spermatozoa of diabetic men and its association with sperm nuclear DNA fragmentation.

    PubMed

    Karimi, J; Goodarzi, M T; Tavilani, H; Khodadadi, I; Amiri, I

    2012-05-01

    Although the majority of patients with diabetes have disorders in sexual function, associations between diabetes mellitus and sperm function at the molecular level are largely unknown. As receptor for advanced glycation end products plays a key role in many diabetic complications, we hypothesised that it may be involved in sperm nuclear DNA fragmentation. RAGE levels were determined using ELISA and western blot analysis in sperm samples from 32 diabetic and 35 nondiabetic men. Sperm DNA fragmentation was assessed using TUNEL assay. Diabetic men had significantly higher mean levels of RAGE protein (P < 0.001) and DNA fragmentation (P < 0.001) in spermatozoa. Sperm RAGE was directly correlated to sperm DNA fragmentation in diabetic men (r = 0.81, P < 0.001). The high positive correlation between RAGE levels and nuclear DNA fragmentation in spermatozoa of diabetic men suggests a central role of RAGE in disturbances in sexual function of diabetic men. © 2011 Blackwell Verlag GmbH.

  10. Identification of CRISP2 from human sperm as PSP94-binding protein and generation of CRISP2-specific anti-peptide antibodies.

    PubMed

    Anklesaria, Jenifer H; Kulkarni, Bhalchandra J; Pathak, Bhakti R; Mahale, Smita D

    2016-06-01

    Cysteine-rich secretory proteins (CRISPs) are mainly found in the mammalian male reproductive tract and reported to be involved at different stages of fertilization. CRISPs have been shown to interact with prostate secretory protein of 94 amino acids (PSP94) from diverse sources, and the binding of these evolutionarily conserved proteins across species is proposed to be of functional significance. Of the three mammalian CRISPs, PSP94-CRISP3 interaction is well characterized, and specific binding sites have been identified; whereas, CRISP2 has been shown to interact with PSP94 in vitro. Interestingly, human CRISP3 and CRISP2 proteins are closely related showing 71.4% identity. In this study, we identified CRISP2 as a potential binding protein of PSP94 from human sperm. Further, we generated antisera capable of specifically detecting CRISP2 and not CRISP3. In this direction, specific peptides corresponding to the least conserved ion channel regulatory region were synthesized, and polyclonal antibodies were generated against the peptide in rabbits. The binding characteristics of the anti-CRISP2 peptide antibody were evaluated using competitive ELISA. Immunoblotting experiments also confirmed that the peptide was able to generate antibodies capable of detecting the mature CRISP2 protein present in human sperm lysate. Furthermore, this anti-CRISP2 peptide antibody also detected the presence of native CRISP2 on sperm.Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  11. Do candidate genes mediating conspecific sperm precedence affect sperm competitive ability within species? A test case in Drosophila.

    PubMed

    Civetta, Alberto; Finn, Scott

    2014-07-16

    When females mate to multiple males, the last male to mate fathers the majority of progeny. When males of different species inseminate a female, the sperm of the male conspecific to the female is favored in fertilization in a process known as conspecific sperm precedence (CSP). A large number of studies in Drosophila have assayed the genetic basis of sperm competition, with a main focus on D. melanogaster and accessory gland protein genes. Only a few studies have attempted to disentangle the genetic basis of CSP between related species of Drosophila. Although there is no a priori reason to believe that genes influencing intraspecific sperm competitive ability might also mediate conspecific sperm precedence, no study has addressed the question. Here, we test a group of candidate CSP genes between D. simulans and D. mauritiana for their effect on sperm competition in D. melanogaster. The use of P-element insertion lines identified CG14891 gene disruption as the only one causing a significant decrease in second male paternity success relative to wild-type and ebony tester males. The gene disruption affected both sperm displacement and the sperm fertilizing ability. Out of five genes tested using RNA interference, only gene knockdown of CG6864(Mst89B) [corrected] significantly reduced the male's ability to father progeny when second to mate. Our results suggest that CG14891 and CG6468 might have been co-opted from an intraspecies gene function (i.e., sperm competition) into an interspecies avoidance phenotype (i.e., CSP). Alternatively, the dual role of these genes could be a consequence of their pleiotropic roles. Copyright © 2014 Civetta and Finn.

  12. Do Candidate Genes Mediating Conspecific Sperm Precedence Affect Sperm Competitive Ability Within Species? A Test Case in Drosophila

    PubMed Central

    Civetta, Alberto; Finn, Scott

    2014-01-01

    When females mate to multiple males, the last male to mate fathers the majority of progeny. When males of different species inseminate a female, the sperm of the male conspecific to the female is favored in fertilization in a process known as conspecific sperm precedence (CSP). A large number of studies in Drosophila have assayed the genetic basis of sperm competition, with a main focus on D. melanogaster and accessory gland protein genes. Only a few studies have attempted to disentangle the genetic basis of CSP between related species of Drosophila. Although there is no a priori reason to believe that genes influencing intraspecific sperm competitive ability might also mediate conspecific sperm precedence, no study has addressed the question. Here, we test a group of candidate CSP genes between D. simulans and D. mauritiana for their effect on sperm competition in D. melanogaster. The use of P-element insertion lines identified CG14891 gene disruption as the only one causing a significant decrease in second male paternity success relative to wild-type and ebony tester males. The gene disruption affected both sperm displacement and the sperm fertilizing ability. Out of five genes tested using RNA interference, only gene knockdown of CG6864 (Mst89B) significantly reduced the male’s ability to father progeny when second to mate. Our results suggest that CG14891 and CG6864 might have been co-opted from an intraspecies gene function (i.e., sperm competition) into an interspecies avoidance phenotype (i.e., CSP). Alternatively, the dual role of these genes could be a consequence of their pleiotropic roles. PMID:25031180

  13. Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.

    PubMed Central

    Putterman, D G; Casadevall, A; Boyle, P D; Yang, H L; Frangione, B; Day, L A

    1984-01-01

    The region of the Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, f1) genome. Nevertheless, the major coat protein is unique among the major coat proteins of fd and the other filamentous phages studied in that it lacks a signal sequence and appears to be a direct translation product and in that it has fewer basic amino acid residues than its equivalent of DNA phosphates in the virion. These features are relevant to considerations of both protein insertion into membranes and DNA structure in filamentous viruses. The single-stranded DNA-binding protein also has a sequence that is different from the sequences of single-stranded DNA-binding proteins from other filamentous viruses. Images PMID:6422463

  14. Identification and Characterization of a Bovine Sperm Acrosomal Matrix Protein and its Mechanism of Interaction with Acrosomal Hydrolases

    PubMed Central

    Nagdas, Subir K; Smith, Linda; Mcnamara, Allen; Hernandez–Encarnacion, Luisa; Medina-Ortiz, Ilza

    2015-01-01

    Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads is comprised of 54, 50, 45, and 38–19kDa polypeptides. The objective of this study is to identify and to characterize the 45kDa (OMC45) polypeptide and to define its role in binding acrosomal hydrolases and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI–TOF–TOF yielded 8 peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X–100–permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti–OMC45 and anti–TEKT3. The OMC45 polypeptide was solubilized by RIPA (radio-immunoprecipitation assay) buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti–OMC45 immunoprecipitation pellet. An identical blot stained with anti–TEKT3 exhibited the presence of TEKT3 polypeptide in the anti–OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide; that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N–linked and O–linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N–acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid

  15. Spermiogenesis and biflagellate spermatozoon of the teleost fish Lampanyctus crocodilus (Myctophiformes, Myctophidae): ultrastructure and characterisation of its sperm basic nuclear proteins.

    PubMed

    Ribes, E; Cheema, M; González-Romero, R; Lloris, D; Ausió, J; Saperas, N

    2015-08-01

    We undertook an ultrastructural study of the spermiogenesis of the lanternfish Lampanyctus crocodilus (Myctophiformes, Myctophidae) with special emphasis on the condensation of chromatin and the biochemical characterisation of its sperm nuclear basic proteins (SNBPs). The round head of the early spermatid of L. crocodilus develops into a curved conical-shaped head in the spermatozoon. Two flagella, present even in the spermatid, are inserted laterally at the convex side of the sperm head. Both flagella possess an axoneme with a 9 + 0 instead of the typical 9 + 2 axonemal structure. Mitochondria undergo a characteristic redistribution during spermiogenesis. A reduced number of them are present lying away from the centrioles at both ends of the concave side of the sperm head. During the chromatin condensation stages in spermiogenesis, fibrogranular structures with granules of 25 ± 5 and 50 ± 5 nm can be observed in the early spermatid and develop into larger granules of about 150 ± 50 nm in the middle spermatid. The latter granules coalesce during the transition to the advanced spermatid and spermatozoon giving rise to highly condensed chromatin in the sperm cell. Protamines are the main SNBPs associated with this chromatin; however, they are unusually large and correspond to the largest protamines described in fish to date. Small stoichiometric amounts of histones and other basic proteins coexist with these protamines in the spermatozoon.

  16. Identification and localization of the sperm CRISP family protein CiUrabin involved in gamete interaction in the ascidian Ciona intestinalis.

    PubMed

    Yamaguchi, Akira; Saito, Takako; Yamada, Lixy; Taniguchi, Hisaaki; Harada, Yoshito; Sawada, Hitoshi

    2011-07-01

    Ascidians are hermaphrodites, and most release sperm and eggs nearly simultaneously. Many species, including Halocynthia roretzi and Ciona intestinalis, are self-sterile. We previously reported that the interaction between a 12 EGF-like repeat-containing vitelline-coat (VC) protein, HrVC70, and a sperm GPI-anchored CRISP, HrUrabin, in lipid rafts plays a key role in self-/nonself-recognizable gamete interaction in H. roretzi. On the other hand, we recently identified two pairs of polymorphic genes responsible for self-incompatibility in C. intestinalis by positional cloning: The sperm polycystin 1-like receptors s-Themis-A/B and its fibrinogen-like ligand v-Themis-A/B on the VC. However, it is not known if the orthologs of HrVC70 and HrUrabin also participate in gamete interaction in C. intestinalis since they are from different orders. Here, we tested for a C. intestinalis ortholog (CiUrabin) of HrUrabin by searching the genome database and proteomes of sperm lipid rafts. The identified CiUrabin belongs to the CRISP family, with a PR domain and a GPI-anchor-attachment site. CiUrabin appears to be specifically expressed in the testis and localized at the surface of the sperm head, as revealed by Northern blotting and immunocytochemistry, respectively. The specific interaction between CiVC57, a C. intestinalis ortholog of HrVC70, and CiUrabin was confirmed by Far Western analysis, similarly to the interaction between HrVC70 and HrUrabin. The molecular interaction between CiVC57 and CiUrabin may be involved in the primary binding of sperm to the VC prior to the allorecognition process, mediated by v-Themis-A/B and s-Themis-A/B, during fertilization of C. intestinalis.

  17. Identification of major proteins associated with Dictyostelium discoideum endocytic vesicles.

    PubMed

    Adessi, C; Chapel, A; Vinçon, M; Rabilloud, T; Klein, G; Satre, M; Garin, J

    1995-10-01

    Magnetic isolation of endocytic vesicles from Dictyostelium discoideum was accomplished after feeding the amoebae with iron oxide particles. Proteins associated with the endocytic vesicles were resolved by SDS-PAGE and digested 'in-gel' with endoproteinase Lys-C or Asp-N to generate peptides for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles: namely, the A, B, D, E and 110 kDa subunits of a vacuolar type H(+)-ATPase, actin, a Rab 7-like GTPase, a p34 protein corresponding to a new cysteine proteinase and the 25 kDa product of a recently sequenced D. discoideum open reading frame.

  18. Sperm membrane proteome in wild Japanese macaque (Macaca fuscata) and Sika deer (Cervus nippon).

    PubMed

    Kawase, Osamu; Cao, Shinuo; Xuan, Xuenan

    2015-01-01

    Whereas recent advances in proteome-related techniques have accumulated a lot of information about sperm proteins in model animals, the information in non-model wildlife species is absolutely deficient, although this knowledge would be valuable to regulate wildlife overabundance. To characterize the repertoires of sperm membrane proteins in Japanese overpopulated wildlife, our study focuses on the following two species: Macaca fuscata and Cervus nippon. We enriched sperm membrane proteins by the phase partitioning with Triton X-114, and then separated them by two-dimensional electrophoresis, and, finally, they were comprehensively identified by peptide mass fingerprinting. Sperm membrane proteins were successfully enriched. They included some proteins with unknown function and fertility-related proteins that work in sperm development, motility, capacitation, transport, protection, acrosome reaction, and fertilization. Additionally, beta-defensin 126 and epithelial chloride channel were strongly detected in M. fuscata but not in C. nippon, whereas lactadherin and NADH-cytochrome b5 reductase 1 were strongly detected in C. nippon alone. This study is an initiative case showing that the sperm of wildlife conserve major fertility-related proteins, but express some proteins in a species-specific manner. In the development of a practical method for fertility control, this aspect may be taken into consideration. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Lactate and Pyruvate Are Major Sources of Energy for Stallion Sperm with Dose Effects on Mitochondrial Function, Motility, and ROS Production.

    PubMed

    Darr, Christa R; Varner, Dickson D; Teague, Sheila; Cortopassi, Gino A; Datta, Sandipan; Meyers, Stuart A

    2016-08-01

    Stallion sperm rely primarily on oxidative phosphorylation for production of ATP used in sperm motility and metabolism. The objective of the study was to identify which substrates included in Biggers, Whitten, and Whittingham (BWW) media are key to optimal mitochondrial function through measurements of sperm motility parameters, mitochondrial oxygen consumption, and cellular reactive oxygen species (ROS) production. It was expected that mitochondrial substrates, pyruvate and lactate, would support sperm motility and mitochondrial function better than the glycolytic substrate, glucose, due to direct utilization within the mitochondria. Measurements were performed after incubation in modified BWW media with varying concentrations of lactate, pyruvate, and glucose. The effects of media and duration of incubation on sperm motility, ROS production, and oxygen consumption were determined using a linear mixed-effects model. Duplicate ejaculates from four stallions were used in three separate experiments to determine the effects of substrate availability and concentration on sperm motility and mitochondrial function and the relationship of oxygen consumption with cellular ROS production. The present results indicate that lactate and pyruvate are the most important sources of energy for stallion sperm motility and velocity, and elicit a dose-dependent response. Additionally, lactate and pyruvate are ideal for maximal mitochondrial function, as sperm in these media operate at a very high level of their bioenergetic capability due to the high rate of energy metabolism. Moreover, we found that addition of glucose to the media is not necessary for short-term storage of equine sperm, and may even result in reduction of mitochondrial function. Finally, we have confirmed that ROS production can be the result of mitochondrial dysfunction as well as intense mitochondrial activity.

  20. Major intrinsic proteins (MIPs) in plants: a complex gene family with major impacts on plant phenotype.

    PubMed

    Forrest, Kerrie L; Bhave, Mrinal

    2007-10-01

    The ubiquitous cell membrane proteins called aquaporins are now firmly established as channel proteins that control the specific transport of water molecules across cell membranes in all living organisms. The aquaporins are thus likely to be of fundamental significance to all facets of plant growth and development affected by plant-water relations. A majority of plant aquaporins have been found to share essential structural features with the human aquaporin and exhibit water-transporting ability in various functional assays, and some have been shown experimentally to be of critical importance to plant survival. Furthermore, substantial evidence is now available from a number of plant species that shows differential gene expression of aquaporins in response to abiotic stresses such as salinity, drought, or cold and clearly establishes the aquaporins as major players in the response of plants to conditions that affect water availability. This review summarizes the function and regulation of these genes to develop a greater understanding of the response of plants to water insufficiency, and particularly, to identify tolerant genotypes of major crop species including wheat and rice and plants that are important in agroforestry.

  1. Targeted ablation of plasma membrane Ca2+-ATPase (PMCA) 1 and 4 indicates a major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4.

    PubMed

    Okunade, Gbolahan W; Miller, Marian L; Pyne, Gail J; Sutliff, Roy L; O'Connor, Kyle T; Neumann, Jonathan C; Andringa, Anastasia; Miller, Daniel A; Prasad, Vikram; Doetschman, Thomas; Paul, Richard J; Shull, Gary E

    2004-08-06

    The relative importance of plasma membrane Ca2+-ATPase (PMCA) 1 and PMCA4 was assessed in mice carrying null mutations in their genes (Atp2b1 and Atp2b4). Loss of both copies of the gene encoding PMCA1 caused embryolethality, whereas heterozygous mutants had no overt disease phenotype. Despite widespread and abundant expression of PMCA4, PMCA4 null (Pmca4-/-) mutants exhibited no embryolethality and appeared outwardly normal. Loss of PMCA4 impaired phasic contractions and caused apoptosis in portal vein smooth muscle in vitro; however, this phenotype was dependent on the mouse strain being employed. Pmca4-/- mice on a Black Swiss background did not exhibit the phenotype unless they also carried a null mutation in one copy of the Pmca1 gene. Pmca4-/- male mice were infertile but had normal spermatogenesis and mating behavior. Pmca4-/- sperm that had not undergone capacitation exhibited normal motility but could not achieve hyperactivated motility needed to traverse the female genital tract. Ultrastructure of the motility apparatus in Pmca4-/- sperm tails was normal, but an increased incidence of mitochondrial condensation indicated Ca2+ overload. Immunoblotting and immunohistochemistry showed that PMCA4 is the most abundant isoform in testis and sperm and that it is localized to the principle piece of the sperm tail, which is also the location of the major Ca2+ channel (CatSper) required for sperm motility. These results are consistent with an essential housekeeping or developmental function for PMCA1, but not PMCA4, and show that PMCA4 expression in the principle piece of the sperm tail is essential for hyperactivated motility and male fertility.

  2. Sperm dumping as a defense against meiotic drive

    PubMed Central

    Price, Tom; Lewis, Zenobia; Wedell, Nina

    2009-01-01

    Sperm from Drosophila simulans that carry a sex-ratio distorter is preferentially lost from females' sperm-storage organs. This suggests that sperm dumping is a major factor affecting sperm competition in this species, and may have evolved in response to sex-ratio distorters. PMID:19216726

  3. [Updated detection of the function of sperm plasma membrane].

    PubMed

    Zhou, Xin; Xia, Xin-Yi; Huang, Yu-Feng

    2010-08-01

    The sperm plasma membrane is rich in polyunsaturated fatty acids and a variety of proteins, and its function is associated with sperm capacitation, acrosome reaction and sperm-egg fusion. Sperm fertilizability can be predicted by detecting the function of the sperm plasma membrane, which is performed mainly with the following five techniques: sperm hypoosmotic swelling test, Eosin gamma water test, sperm membrane lipid peroxidation determination, seminal plasma superoxide dismutase determination, and flow cytometry. The evaluation of the function of sperm plasma membrane can be applied in detecting semen quality, selecting semen centrifugation, assessing the quality and fertilizability of sex-sorted sperm, improving cryopreservation, and guiding the optimization of intracytoplasmic sperm injection. This review presents an update on the principles, methods and steps of the detection of sperm plasma membrane function, as well as an overview of its status quo and application.

  4. ADM-1, a protein with metalloprotease- and disintegrin-like domains, is expressed in syncytial organs, sperm, and sheath cells of sensory organs in Caenorhabditis elegans.

    PubMed Central

    Podbilewicz, B

    1996-01-01

    A search was carried out for homologues of possible fusogenic proteins to study their function in a genetically tractable animal. The isolation, molecular, and cellular characterization of the Caenorhabditis elegans adm-1 gene (a disintegrin and metalloprotease domain) are described. A glycoprotein analogous to viral fusion proteins has been identified on the surface of guinea pig sperm (PH-30/fertilin) and is implicated in sperm-egg fusion. adm-1 is the first reported invertebrate gene related to PH-30 and a family of proteins containing snake venom disintegrin- and metalloprotease-like domains. ADM-1 shows a domain organization identical to PH-30. It contains prepro, metalloprotease, disintegrin, cysteine rich with putative fusion peptide, epidermal growth factor-like repeat, transmembrane, and cytoplasmic domains. Antibodies which recognize ADM-1 protein in immunoblots were generated. Using immunofluorescence and in situ hybridization, the products of adm-1 have been detected in specific cells during different stages of development. The localization of ADM-1 to the plasma membrane of embryonic cells and to the sheath cells of sensory organs suggests a function in cell adhesion. ADM-1 expression in the hypodermis, pharynx, vulva, and mature sperm is consistent with a putative role in somatic and gamete cell fusions. Images PMID:8970152

  5. Hydroxyproline in the major capsid protein VP1 of polyomavirus

    SciTech Connect

    Ludlow, J.W.; Consigli, R.A.

    1989-06-01

    Amino acid analysis of (/sup 3/H)proline-labeled polyomavirus major capsid protein VP1 by two-dimensional paper chromatography of the acid-hydrolyzed protein revealed the presence of /sup 3/H-labeled hydroxyproline. Addition of the proline analog L-azetidine-2-carboxylic acid to infected mouse kidney cell cultures prevented or greatly reduced hydroxylation of proline in VP1. Immunofluorescence analysis performed on infected cells over a time course of analog addition revealed that virus proteins were synthesized but that transport from the cytoplasm to the nucleus was impeded. A reduction in the assembly of progeny virions demonstrated by CsCl gradient purification of virus from (/sup 35/S)methionine-labeled infected cell cultures was found to correlate with the time of analog addition. These results suggest that incorporation of this proline analog into VP1, accompanied by reduction of the hydroxyproline content of the protein, influences the amount of virus progeny produced by affecting transport of VP1 to the cell nucleus for assembly into virus particles.

  6. Diversity of major urinary proteins (MUPs) in wild house mice

    PubMed Central

    Thoß, Michaela; Enk, Viktoria; Yu, Hans; Miller, Ingrid; Luzynski, Kenneth C.; Balint, Boglarka; Smith, Steve; Razzazi-Fazeli, Ebrahim; Penn, Dustin J.

    2016-01-01

    Major urinary proteins (MUPs) are often suggested to be highly polymorphic, and thereby provide unique chemical signatures used for individual and genetic kin recognition; however, studies on MUP variability have been lacking. We surveyed populations of wild house mice (Mus musculus musculus), and examined variation of MUP genes and proteins. We sequenced several Mup genes (9 to 11 loci) and unexpectedly found no inter-individual variation. We also found that microsatellite markers inside the MUP cluster show remarkably low levels of allelic diversity, and significantly lower than the diversity of markers flanking the cluster or other markers in the genome. We found low individual variation in the number and types of MUP proteins using a shotgun proteomic approach, even among mice with variable MUP electrophoretic profiles. We identified gel bands and spots using high-resolution mass spectrometry and discovered that gel-based methods do not separate MUP proteins, and therefore do not provide measures of MUP diversity, as generally assumed. The low diversity and high homology of Mup genes are likely maintained by purifying selection and gene conversion, and our results indicate that the type of selection on MUPs and their adaptive functions need to be re-evaluated. PMID:27922085

  7. Sperm-egg interaction.

    PubMed

    Evans, Janice P

    2012-01-01

    A crucial step of fertilization is the sperm-egg interaction that allows the two gametes to fuse and create the zygote. In the mouse, CD9 on the egg and IZUMO1 on the sperm stand out as critical players, as Cd9(-/-) and Izumo1(-/-) mice are healthy but infertile or severely subfertile due to defective sperm-egg interaction. Moreover, work on several nonmammalian organisms has identified some of the most intriguing candidates implicated in sperm-egg interaction. Understanding of gamete membrane interactions is advancing through characterization of in vivo and in vitro fertilization phenotypes, including insights from less robust phenotypes that highlight potential supporting (albeit not absolutely essential) players. An emerging theme is that there are varied roles for gamete molecules that participate in sperm-egg interactions. Such roles include not only functioning as fusogens, or as adhesion molecules for the opposite gamete, but also functioning through interactions in cis with other proteins to regulate membrane order and functionality.

  8. Use of heterospermic inseminations and paternity testing to evaluate the relative contributions of common sperm traits and seminal plasma proteins in boar fertility.

    PubMed

    Flowers, W L; Deller, F; Stewart, K R

    2016-11-01

    The objective of this study was to evaluate relationships between common semen quality estimates including sperm motility, sperm morphology, spontaneous capacitation status and seminal plasma proteins and boar fertility using heterospermic inseminations and subsequent paternity testing. All boars (n=12) used in the study had excellent semen quality (≥70% normal sperm) that resulted in average farrowing rates and litter sizes of 88.9±0.7% and 11.7±0.1 pigs, respectively. Their ejaculates were combined to make heterospermic insemination doses in such a way that each boar was tested against all of his contemporaries. The proportion of piglets sired by each individual was used to separate boars into three fertility groups: High (71.6±4.8%; n=3); Medium (51.6±3.8%; n=6); and Low (25.2%±5.3%; n=3). Ejaculates from High fertility boars had more motile sperm with normal acrosomes that moved faster in a straight-line and were more likely to undergo an acrosome reaction (p≤0.05) compared with their counterparts in the Low fertility group. Ejaculates from High fertility boars contained the greatest concentrations of three seminal plasma proteins (25.9kD/5.9pI; 55.1kD/4.8pI; and 70.1kD/5.2pI; p≤0.05), whereas concentrations of a 19.1kD/6.8pI were highest in semen from Low fertility boars (p≤0.05). Multiple regression analyses indicated that concentrations of the 25.9kD/5.9pI seminal plasma protein explained 66% of the variation observed in the proportion of pigs sired within a litter among boars (p≤0.00001). These results demonstrate that heterospermic inseminations and subsequent paternity testing is an effective technique for defining relationships between common semen quality tests and fertility, especially in situations where reproductive performance of all the boars is high. Motility, normal acrosome morphology, average linear velocity of motile sperm, and the proportion of sperm capable of an acrosome reaction were all positively associated with boar

  9. Compartmentalization of a Unique ADP/ATP Carrier Protein SFEC (Sperm Flagellar Energy Carrier, AAC4) with Glycolytic Enzymes in the Fibrous Sheath of the Human Sperm Flagellar Principal Piece

    PubMed Central

    Kim, Young-Hwan; Haidl, Gerhard; Schaefer, Martina; Egner, Ursula; Herr, John C.

    2007-01-01

    The longest part of the sperm flagellum, the principal piece, contains the fibrous sheath, a cytoskeletal element unique to spermiogenesis. We performed mass spectrometry proteomics on isolated human fibrous sheaths identifying a unique ADP/ATP carrier protein, SFEC [AAC4], seven glycolytic enzymes previously unreported in the human sperm fibrous sheath, and sorbitol dehydrogenase. SFEC, pyruvate kinase and aldolase were co-localized by immunofluorescence to the principal piece. A homology model constructed for SFEC predicted unique residues at the entrance to the nucleotide binding pocket of SFEC that are absent in other human ADP/ATP carriers, suggesting opportunities for selective drug targeting. This study provides the first evidence of a role for an ADP/ATP carrier family member in glycolysis. The co-localization of SFEC and glycolytic enzymes in the fibrous sheath supports a growing literature that the principal piece of the flagellum is capable of generating and regulating ATP independently from mitochondrial oxidation in the mid-piece. A model is proposed that the fibrous sheath represents a highly ordered complex, analogous to the electron transport chain, in which adjacent enzymes in the glycolytic pathway are assembled to permit efficient flux of energy substrates and products with SFEC serving to mediate energy generating and energy consuming processes in the distal flagellum, possibly as a nucleotide shuttle between flagellar glycolysis, protein phosphorylation and mechanisms of motility. PMID:17137571

  10. Insights into female sperm storage from the spermathecal fluid proteome of the honeybee Apis mellifera

    PubMed Central

    Baer, Boris; Eubel, Holger; Taylor, Nicolas L; O'Toole, Nicholas; Millar, A Harvey

    2009-01-01

    Background Female animals are often able to store sperm inside their body - in some species even for several decades. The molecular basis of how females keep non-own cells alive is largely unknown, but since sperm cells are reported to be transcriptionally silenced and, therefore, limited in their ability to maintain their own function, it is likely that females actively participate in sperm maintenance. Because female contributions are likely to be of central importance for sperm survival, molecular insights into the process offer opportunities to observe mechanisms through which females manipulate sperm. Results We used the honeybee, Apis mellifera, in which queens are highly polyandrous and able to maintain sperm viable for several years. We identified over a hundred proteins representing the major constituents of the spermathecal fluid, which females contribute to sperm in storage. We found that the gel profile of proteins from spermathecal fluid is very similar to the secretions of the spermathecal gland and concluded that the spermathecal glands are the main contributors to the spermathecal fluid proteome. A detailed analysis of the spermathecal fluid proteins indicate that they fall into a range of different functional groups, most notably enzymes of energy metabolism and antioxidant defense. A metabolic network analysis comparing the proteins detected in seminal fluid and spermathecal fluid showed a more integrated network is present in the spermathecal fluid that could facilitate long-term storage of sperm. Conclusions We present a large-scale identification of proteins in the spermathecal fluid of honeybee queens and provide insights into the molecular regulation of female sperm storage. PMID:19538722

  11. Interactions among the major and minor coat proteins of polyomavirus.

    PubMed Central

    Barouch, D H; Harrison, S C

    1994-01-01

    Murine polyomavirus contains two related minor coat proteins, VP2 and VP3, in addition to the major coat protein, VP1. The sequence of VP3 is identical to that of the carboxy-terminal two-thirds of VP2. VP2 may serve a role in uncoating of the virus, and both minor coat proteins may be important for viral assembly. In this study, we show that VP3 and a series of deletion mutants of VP3 can be expressed in Escherichia coli as fusion proteins to glutathione S-transferase and partially solubilized with a mild detergent. Using an in vitro binding assay, we demonstrate that a 42-amino-acid fragment near the carboxy terminus of VP3 (residues 140 to 181) is sufficient for binding to purified VP1 pentamers. This binding interaction is rapid, saturable, and specific for the common carboxy terminus of VP2 and VP3. The VP1-VP3 complex can be coimmunoprecipitated with an antibody specific to VP1, and a purified VP3 fragment can selectively extract VP1 from a crude cell lysate. The stoichiometry of the binding reaction suggests that each VP1 pentamer in the virus binds either one VP2 or one VP3, with the VP1-VP2/3 complex stabilized by hydrophobic interactions. These results, taken together with studies from other laboratories on the expression of polyomavirus capsid proteins in mouse and insect cells (S. E. Delos, L. Montross, R. B. Moreland, and R. L. Garcea, Virology, 194:393-398, 1993; J. Forstova, N. Krauzewicz, S. Wallace, A. J. Street, S. M. Dilworth, S. Beard, and B. E. Griffin, J. Virol. 67:1405-1413, 1993), support the idea that a VP1-VP2/3 complex forms in the cytoplasm and, after translocation into the nucleus, acts as the unit for viral assembly. Images PMID:8189532

  12. Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

    USDA-ARS?s Scientific Manuscript database

    Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein ...

  13. The Unique Catalytic Subunit of Sperm cAMP-dependent Protein Kinase Is the Product of an Alternative Cα mRNA Expressed Specifically in Spermatogenic Cells

    PubMed Central

    Agustin, Jovenal T. San; Wilkerson, Curtis G.; Witman, George B.

    2000-01-01

    cAMP-dependent protein kinase has a central role in the control of mammalian sperm capacitation and motility. Previous protein biochemical studies indicated that the only cAMP-dependent protein kinase catalytic subunit (C) in ovine sperm is an unusual isoform, termed Cs, whose amino terminus differs from those of published C isoforms of other species. Isolation and sequencing of cDNA clones encoding ovine Cs and Cα1 (the predominant somatic isoform) now reveal that Cs is the product of an alternative transcript of the Cα gene. Cs cDNA clones from murine and human testes also were isolated and sequenced, indicating that Cs is of ancient origin and widespread in mammals. In the mouse, Cs transcripts were detected only in testis and not in any other tissue examined, including ciliated tissues and ovaries. Finally, immunohistochemistry of the testis shows that Cs first appears in pachytene spermatocytes. This is the first demonstration of a cell type–specific expression for any C isoform. The conservation of Cs throughout mammalian evolution suggests that the unique structure of Cs is important in the subunit's localization or function within the sperm. PMID:10982398

  14. Sperm wars and the evolution of male fertility.

    PubMed

    Simmons, Leigh W; Fitzpatrick, John L

    2012-11-01

    Females frequently mate with several males, whose sperm then compete to fertilize available ova. Sperm competition represents a potent selective force that is expected to shape male expenditure on the ejaculate. Here, we review empirical data that illustrate the evolutionary consequences of sperm competition. Sperm competition favors the evolution of increased testes size and sperm production. In some species, males appear capable of adjusting the number of sperm ejaculated, depending on the perceived levels of sperm competition. Selection is also expected to act on sperm form and function, although the evidence for this remains equivocal. Comparative studies suggest that sperm length and swimming speed may increase in response to selection from sperm competition. However, the mechanisms driving this pattern remain unclear. Evidence that sperm length influences sperm swimming speed is mixed and fertilization trials performed across a broad range of species demonstrate inconsistent relationships between sperm form and function. This ambiguity may in part reflect the important role that seminal fluid proteins (sfps) play in affecting sperm function. There is good evidence that sfps are subject to selection from sperm competition, and recent work is pointing to an ability of males to adjust their seminal fluid chemistry in response to sperm competition from rival males. We argue that future research must consider sperm and seminal fluid components of the ejaculate as a functional unity. Research at the genomic level will identify the genes that ultimately control male fertility.

  15. A role for carbohydrate recognition in mammalian sperm-egg binding

    SciTech Connect

    Clark, Gary F.

    2014-08-01

    Highlights: • Mammalian sperm-egg binding as a carbohydrate dependent species recognition event. • The role of carbohydrate recognition in human, mouse and pig sperm-egg binding. • Historical perspective and future directions for research focused on gamete binding. - Abstract: Mammalian fertilization usually requires three sequential cell–cell interactions: (i) initial binding of sperm to the specialized extracellular matrix coating the egg known as the zona pellucida (ZP); (ii) binding of sperm to the ZP via the inner acrosomal membrane that is exposed following the induction of acrosomal exocytosis; and (iii) adhesion of acrosome-reacted sperm to the plasma membrane of the egg cell, enabling subsequent fusion of these gametes. The focus of this review is on the initial binding of intact sperm to the mammalian ZP. Evidence collected over the past fifty years has confirmed that this interaction relies primarily on the recognition of carbohydrate sequences presented on the ZP by lectin-like egg binding proteins located on the plasma membrane of sperm. There is also evidence that the same carbohydrate sequences that mediate binding also function as ligands for lectins on lymphocytes that can inactivate immune responses, likely protecting the egg and the developing embryo up to the stage of blastocyst hatching. The literature related to initial sperm-ZP binding in the three major mammalian models (human, mouse and pig) is discussed. Historical perspectives and future directions for research related to this aspect of gamete adhesion are also presented.

  16. Glycocalyx characterisation and glycoprotein expression of Sus domesticus epididymal sperm surface samples.

    PubMed

    Fàbrega, Anna; Puigmulé, Marta; Dacheux, Jean-Louis; Bonet, Sergi; Pinart, Elisabeth

    2012-01-01

    The sperm surface is covered with a dense coating of carbohydrate-rich molecules. Many of these molecules are involved in the acquisition of fertilising ability. In the present study, eight lectins (i.e. Arachis hypogae (peanut) agglutinin (PNA), Lens culimaris (lentil) agglutinin-A (LCA), Pisum sativum (pea) agglutin (PSA), Triticum vulgari (wheat) germ agglutinin (WGA), Helix pomatia agglutinin (HPA), Phaseolus vulgaris (red kidney bean) leucoagglutinin (PHA-L), Glycine max (soybean) agglutinin (SBA) and Ulex europaeus agglutinin I (UEA-I)) were investigated to identify changes in the nature and localisation of glycoproteins in boar spermatozoa migrating along the epididymal duct. Complementary procedures included measurement of global lectin binding over the surface of the viable sperm population by flow cytometry, analysis of lectin localisation on the membrane of individual spermatozoa using fluorescence microscopy and the electrophoretic characterisation of the major sperm surface glycoprotein receptors involved in lectin binding. A significant increase was found in sperm galactose, glucose/mannose and N-acetyl-d-glucosamine residues distally in the epididymis. Moreover, the sperm head, cytoplasmic droplet and midpiece were recognised by most of the lectins tested, whereas only HPA and WGA bound to the principal piece and end piece of the sperm tail. Fourteen sperm surface proteins were observed with different patterns of lectin expression between epididymal regions. The sperm glycocalyx modifications observed in the present study provide an insight into the molecular modifications associated with epididymal maturation, which may be correlated with the degree of maturation of ejaculated spermatozoa.

  17. Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation, catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme.

    PubMed

    Akama, Kuniko; Horikoshi, Tomoe; Sugiyama, Atsushi; Nakahata, Satoko; Akitsu, Aoi; Niwa, Nobuyoshi; Intoh, Atsushi; Kakui, Yasutaka; Sugaya, Michiko; Takei, Kazuo; Imaizumi, Noriaki; Sato, Takaya; Matsumoto, Rena; Iwahashi, Hitoshi; Kashiwabara, Shin-ichi; Baba, Tadashi; Nakamura, Megumi; Toda, Tosifusa

    2010-06-01

    In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.

  18. Localization of eosinophil granule major basic protein in human basophils

    PubMed Central

    1983-01-01

    In experiments using an immunofluorescent method to localize human eosinophil granule major basic protein (MBP) in cells and tissues, a small number of cells stained for MBP that subsequently could not be identified as eosinophils. Because the Charcot-Leyden crystal protein, another eosinophil protein, was recently identified in basophils, we tested whether MBP might also be a constituent of blood basophils. Highly purified, eosinophil-free basophil suspensions were prepared using the fluorescence-activated cell sorter (FACS) to sort basophil- containing mononuclear cell preparations stained with fluorescein- conjugated sheep IgG anti-human IgE antibody. Using these FACS-purified basophils, we found that: (a) enrichment for surface IgE-positive cells (greater than 95% basophils) by FACS also enriched for cells staining for MBP by immunofluorescence; (b) MBP appeared to be localized in the histamine-, heparin-containing granules; (c) significant quantities of MBP were measurable by radioimmunoassay (RIA) in freeze-thaw detergent extracts of purified basophils; and (d) RIA dose-response curves for extracts of purified eosinophils and basophils had identical slopes. The MBP content of basophils from normal individuals averaged 140 ng/10(6) cells, whereas purified eosinophils from normal donors and patients with the hyper-eosinophilic syndrome averaged 4,979 and 824 ng/10(6) cells, respectively. MBP was also detected by immunofluorescence and RIA in cells obtained from a patient with basophil leukemia, although the MBP content for basophil leukemia cells was lower than that for normal basophils. We conclude that basophils contain a protein that is immunochemically indistinguishable from eosinophil granule MBP. PMID:6350526

  19. Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours

    PubMed Central

    2011-01-01

    Background Sperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri. Results Large consort males transfer smaller (average total length = 73 μm) sperm to a female's internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 μm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males. Conclusions Here we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size. PMID:21831296

  20. Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization

    PubMed Central

    Theodoridou, Maria; Nomikos, Michail; Parthimos, Dimitris; Gonzalez-Garcia, J. Raul; Elgmati, Khalil; Calver, Brian L.; Sideratou, Zili; Nounesis, George; Swann, Karl; Lai, F. Anthony

    2013-01-01

    Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca2+ oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca2+ in eggs. To investigate how discrete protein domains contribute to Ca2+ release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca2+ oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca2+ sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca2+ oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca2+ releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca2+ sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca2+ oscillations in eggs, we have developed a mathematical model that incorporates Ca2+-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca2+ oscillatory activity associated with specific PLC variants. PMID:24152875

  1. Seminal plasma proteins and their relationship with percentage of morphologically normal sperm in 2-year-old Brahman (Bos indicus) bulls.

    PubMed

    Boe-Hansen, G B; Rego, J P A; Crisp, J M; Moura, A A; Nouwens, A S; Li, Y; Venus, B; Burns, B M; McGowan, M R

    2015-11-01

    The objective was to determine the relationship between seminal plasma proteins and sperm morphology in Bos indicus bulls of the Brahman breed. Fifty-six 24-month-old Australian Brahman bulls were electroejaculated and samples were examined to determine the percentage of morphologically normal sperm (PNS24) and the seminal plasma protein composition was identified and quantified by 2-D gel electrophoresis. The total integrated optical density of 152 seminal plasma protein spots (SPPs) across all gels was determined using the PDQuest software version 8.0 (Bio Rad, USA). Using a single regression mixed model with the density of individual spots as a covariate for PNS24, 17 SPPs were significantly associated with PNS24 (p<0.05). A multiple regression analyses of these SPPs, using three models; non-parametric Tree Model, Generalized Additive Model, and a step-wise selection method were conducted, and 6 SPPs could be used to predict PNS24; four SPPs had positive and two had negative association with PNS24. Together these spots explained 35% of the phenotypic variation in PNS24. Using mass spectrometry (MALDI-ToF and TripleToF-MS) the SPPs with positive relationship contained mainly apolipoprotein A-I (1310), protein DJ-1 and glutathione peroxidase 3 (2308), phosphoglycerate kinase 1 (6402) and apolipoprotein A-I and secretoglobin family 1D member (8008). The SPPs inversely associated with PNS24 were clusterin/seminal plasma protein A3 (1411) and epididymal secretory protein E1 (8108). This is the first comprehensive report on the association between seminal plasma protein composition in Bos indicus Brahman bulls and sperm morphology. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm.

    PubMed

    Flores, E; Ramió-Lluch, L; Bucci, D; Fernández-Novell, J M; Peña, A; Rodríguez-Gil, J E

    2011-11-01

    The main aim of this work is to gain insight into the mechanisms by which freezing-thawing alters the nucleoprotein structure of boar sperm. For this purpose, the freezing-thawing-related changes of structure and location of histones-DNA domains in the boar sperm head were analyzed through Western blot and immunocytochemistry. Afterwards, it was analyzed whether freezing-thawing induced changes in tyrosine phosphorylation levels of both protamine 1 and histone H1, through Western blot analyses in samples previously subjected to immunoprecipitation. This analysis was completed with the determination of the changes induced by freezing-thawing on the overall levels of sperm-head disulfide bonds through analysis of free-cysteine radicals levels. Freezing-thawing induced significant changes in the histones-DNA structures, which were manifested in the appearance of a freezing-thawing-linked histone H1-DNA aggregate of about a 35-kDa band and in the spreading of histone H1-positive markings from the caudal area of the sperm head to more cranial zones. Freezing-thawing did not have any significant effect on the tyrosine phosphorylation levels of either protamine 1 or histone H1. However, thawed samples showed a significant (P < 0.05) increase in the free cysteine radical content (from 3.1 ± 0.5 nmol/μg protein in fresh samples to 6.7 ± 0.8 nmol/μg protein). In summary, our results suggest that freezing-thawing causes significant alterations in the nucleoprotein structure of boar sperm head by mechanism/s linked with the rupture of disulfide bonds among the DNA. These mechanisms seem to be unspecific, affecting both the protamines-DNA unions and the histones-DNA bonds in a similar way. Furthermore, results suggest that the boar-sperm nuclear structure is heterogeneous suggesting the existence of a zonated pattern, differing in their total DNA density and the compactness of the precise nucleoprotein structures present in each zone. Copyright © 2011 Elsevier Inc. All

  3. Lactate Dehydrogenase C and Energy Metabolism in Mouse Sperm1

    PubMed Central

    Odet, Fanny; Gabel, Scott A.; Williams, Jason; London, Robert E.; Goldberg, Erwin; Eddy, Edward M.

    2011-01-01

    We demonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD+ cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC. PMID:21565994

  4. Identification of major rye secalins as coeliac immunoreactive proteins.

    PubMed

    Rocher, A; Calero, M; Soriano, F; Méndez, E

    1996-06-07

    Six distinct gamma- and omega-type secalins, together with two new low molecular mass glycoproteins, have been identified as the major coeliac immunoreactive proteins from a chloroform/methanol soluble extract from rye endosperm. These components were characterized by a combination of reverse-phase high-performance liquid chromatography, immunoblotting using a coeliac serum and microsequencing analysis. This allowed the identification of a group of secalins with different molecular masses according to their N-terminal amino-acid sequence: one omega-type secalin of 40 kDa (omega 1-40); three gamma-type secalins, one of 70 kDa (gamma-70) and two of 35 kDa (gamma-35); as well as two low molecular mass glycoproteins of 15 and 18 kDa, all exhibiting coeliac serum antigenicity. Moreover, four additional rye components, including two low molecular mass proteins, which did not react with coeliac sera, have also been identified. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of the three main purified coeliac immunogenic secalins, gamma-70, gamma-35 and omega 1-40, indicated molecular masses of 71457, 32240 and 39117 Da, respectively. The omega 1-40 secalin displays a significant absorption in the visible region which could be related to its peculiar low capacity to bind both coeliac sera antibodies and Coomassie brilliant blue dye.

  5. Mutational analysis of the major coat protein of M13 identifies residues that control protein display.

    PubMed Central

    Weiss, G. A.; Wells, J. A.; Sidhu, S. S.

    2000-01-01

    We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487-495). Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus. Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities. PMID:10794407

  6. Sperm Competition, Sperm Numbers and Sperm Quality in Muroid Rodents

    PubMed Central

    Gómez Montoto, Laura; Magaña, Concepción; Tourmente, Maximiliano; Martín-Coello, Juan; Crespo, Cristina; Luque-Larena, Juan José

    2011-01-01

    Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An “overall sperm quality” parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and

  7. Sperm competition and maternal effects differentially influence testis and sperm size in Callosobruchus maculatus.

    PubMed

    Gay, L; Hosken, D J; Vasudev, R; Tregenza, T; Eady, P E

    2009-05-01

    The evolutionary factors affecting testis size are well documented, with sperm competition being of major importance. However, the factors affecting sperm length are not well understood; there are no clear theoretical predictions and the empirical evidence is inconsistent. Recently, maternal effects have been implicated in sperm length variation, a finding that may offer insights into its evolution. We investigated potential proximate and microevolutionary factors influencing testis and sperm size in the bruchid beetle Callosobruchus maculatus using a combined approach of an artificial evolution experiment over 90 generations and an environmental effects study. We found that while polyandry seems to select for larger testes, it had no detectable effect on sperm length. Furthermore, population density, a proximate indicator of sperm competition risk, was not significantly associated with sperm length or testis size variation. However, there were strong maternal effects influencing sperm length.

  8. A high-fat, high-protein diet attenuates the negative impact of casein-induced chronic inflammation on testicular steroidogenesis and sperm parameters in adult mice.

    PubMed

    Zhao, Jing-Lu; Zhao, Yu-Yun; Zhu, Wei-Jie

    2017-10-01

    The interaction between obesity and chronic inflammation has been studied. Diet-induced obesity or chronic inflammation could reduce the testicular functions of males. However, the mechanism underlying the reproductive effects of fattening foods in males with or without chronic inflammation still needs further discussion. This study was aimed to investigate the effects of high-fat, high-protein diet on testicular steroidogenesis and sperm parameters in adult mice under physiological and chronic inflammatory conditions. Because casein can trigger a non-infectious systemic inflammatory response, we used casein injection to induce chronic inflammation in male adult Kunming mice. Twenty-four mice were randomly and equally divided into four groups: (i) normal diet+saline (Control); (ii) normal diet+casein (ND+CS); (iii) high-fat, high-protein diet+saline (HFPD+SI); (iv) high-fat, high-protein diet+casein (HFPD+CS). After 8weeks, there was a significant increase in body weight for groups HFPD+SI and HFPD+CS and a decrease in group ND+CS compared with the control. The serum levels of tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10) and lipid profiles were increased markedly in groups ND+CS, HFPD+SI and HFPD+CS compared with the control. A remarkable reduction of serum adiponectin level occurred in group HFPD+CS compared with group ND+CS. Sperm parameters (sperm count, viability and abnormality) were also adversely affected in groups ND+CS and HFPD+SI. Groups ND+CS and HFPD+SI showed severe pathological changes in testicular tissues. Semiquantitative RT-PCR, Western blot and immunohistochemical staining also showed significant reductions in both testicular mRNA and protein levels of steroidogenic acute regulatory (StAR) and cytochrome P450scc (CYP11A1) in groups HFPD+SI and HFPD+CS compared with the control, whereas testicular mRNA and protein levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) in groups HFPD+SI and HFPD+CS significantly increased. The m

  9. Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein

    PubMed Central

    Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C.; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

    2005-01-01

    We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561–565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an α-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38α or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa–egg interaction. PMID:15693750

  10. A pH Switch Regulates the Inverse Relationship between Membranolytic and Chaperone-like Activities of HSP-1/2, a Major Protein of Horse Seminal Plasma.

    PubMed

    Kumar, C Sudheer; Swamy, Musti J

    2016-07-05

    HSP-1/2, a major protein of horse seminal plasma binds to choline phospholipids present on the sperm plasma membrane and perturbs its structure by intercalating into the hydrophobic core, which results in an efflux of choline phospholipids and cholesterol, an important event in sperm capacitation. HSP-1/2 also exhibits chaperone-like activity (CLA) in vitro and protects target proteins against various kinds of stress. In the present study we show that HSP-1/2 exhibits destabilizing activity toward model supported and cell membranes. The membranolytic activity of HSP-1/2 is found to be pH dependent, with lytic activity being high at mildly acidic pH (6.0-6.5) and low at mildly basic pH (8.0-8.5). Interestingly, the CLA is also found to be pH dependent, with high activity at mildly basic pH and low activity at mildly acidic pH. Taken together the present studies demonstrate that the membranolytic and chaperone-like activities of HSP-1/2 have an inverse relationship and are regulated via a pH switch, which is reversible. The higher CLA observed at mildly basic pH could be correlated to an increase in surface hydrophobicity of the protein. To the best of our knowledge, this is the first study reporting regulation of two different activities of a chaperone protein by a pH switch.

  11. Comparative sperm ultrastructure in Nemertea.

    PubMed

    von Döhren, J; Beckers, P; Vogeler, R; Bartolomaeus, T

    2010-07-01

    Although the monophyly of Nemertea is strongly supported by unique morphological characters and results of molecular phylogenetic studies, their ingroup relationships are largely unresolved. To contribute solving this problem we studied sperm ultrastructure of 12 nemertean species that belong to different subtaxa representing the commonly recognized major monophyletic groups. The study yielded a set of 26 characters with an unexpected variation among species of the same genus (Tubulanus and Procephalothrix species), whereas other species varied in metric values or only one character state (Ramphogordius). In some species, the sperm nucleus has grooves (Zygonemertes virescens, Amphiporus imparispinosus) that may be twisted and give a spiral shape to the sperm head (Paranemertes peregrina, Emplectonema gracile). To make the characters from sperm ultrastructure accessible for further phylogenetic analyses, they were coded in a character matrix. Published data for eight species turned out to be sufficiently detailed to be included. Comparative evaluation of available information on the sperm ultrastructure suggests that subtaxa of Heteronemertea and Hoplonemertea are supported as monophyletic by sperm morphology. However, the data do not provide information on the existing contradictions regarding the internal relationships of "Palaeonemertea." Nevertheless, our study provides evidence that sperm ultrastructure yields numerous potentially informative characters that will be included in upcoming phylogenetic analyses.

  12. Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin)-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

    PubMed

    Mbanefo, Evaristus Chibunna; Kikuchi, Mihoko; Huy, Nguyen Tien; Shuaibu, Mohammed Nasir; Cherif, Mahamoud Sama; Yu, Chuanxin; Wakao, Masahiro; Suda, Yasuo; Hirayama, Kenji

    2014-01-01

    We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin)-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6) M) and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation), and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

  13. Major Urinary Protein Regulation of Chemical Communication and Nutrient Metabolism

    PubMed Central

    Zhou, Yingjiang; Rui, Liangyou

    2014-01-01

    Summary The major urinary protein (MUP) family members contain a conserved β-barrel structure with a characteristic central hydrophobic pocket. They are secreted by the liver and excreted into the urine. MUPs bind via their central pockets to volatile pheromones or other lipophilic molecules, and regulate pheromone transportation in the circulation, excretion in the kidney, and release into the air from urine marks. MUPs are highly polymorphic, and the MUP profiles in urine function as individual identity signatures of the owners. The MUP signatures are detected by the main and accessory olfactory systems and trigger adaptive behavioral responses and/or developmental processes. Circulating MUPs serve as a metabolic signal to regulate glucose and lipid metabolism. Recombinant MUP1 markedly ameliorates hyperglycemia and glucose intolerance in mice with type 2 diabetes. MUP1 suppresses hepatic gluconeogenesis and promotes energy expenditure in skeletal muscle by stimulating mitochondrial biogenesis and function. MUPs are unique members of the lipocalin super-family that mediate both chemical and metabolic signaling. PMID:20831945

  14. The quest for the sea urchin egg receptor for sperm.

    PubMed

    Vacquier, Victor D

    2012-08-31

    This review discusses identification, isolation and characterization of proteins mediating species-selective sperm-to-egg adhesion during sea urchin fertilization. Bindin is the only sea urchin sperm protein known to mediate species-selective sperm attachment to eggs. Two completely different egg surface proteins, 350-kDa and EBR1, have affinity for bindin and each one meets all the criteria to be a species-selective sperm receptor. Experiments suggest that sperm bindin recognizes both the sulfated O-linked oligosaccharides on the egg 350-kDa glycoprotein, and also the repeated protein sequence modules of EBR1.

  15. Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs.

    PubMed

    Yeste, Marc

    2016-01-01

    Cryopreservation is the most efficient method for long-term preservation of mammalian sperm. However, freeze-thawing procedures may strongly impair the sperm function and survival and thus decrease the reproductive performance. In addition, the sperm resilience to withstand cryopreservation, also known as freezability, presents a high individual variability. The present work summarizes the principles of cryoinjury and the relevance of permeating and nonpermeating cryoprotective agents. Descriptions about sperm cryodamage are mainly focused on boar sperm, but reference to other mammalian species is also made when relevant. Main cryoinjuries not only regard to sperm motility and membrane integrity, but also to the degradation effect exerted by freeze-thawing on other important components for sperm fertilizing ability, such as mRNAs. After delving into the main differences between good and poor freezability boar ejaculates, those protein markers predicting the sperm ability to sustain cryopreservation are also mentioned. Moreover, factors that may influence sperm freezability, such as season, diet, breed, or ejaculate fractions are discussed, together with the effects of different additives, like seminal plasma and antioxidants. After briefly referring to the effects of long-term sperm preservation in frozen state and the reproductive performance of frozen-thawed boar sperm, this work speculates with new research horizons on the preservation of boar sperm, such as vitrification and freeze-drying.

  16. Signaling in Sperm: More Different than Similar.

    PubMed

    Kaupp, U B; Strünker, T

    2017-02-01

    For a given sensory cell type, signaling motifs are rather uniform across phyla. By contrast, sperm from different species use diverse repertoires of sperm-specific signaling molecules and even closely related protein isoforms feature different properties and serve different functions. This surprising diversity has consequences for strategies in fertilization research and it will take some time to get the big picture. We discuss the function of receptors, ion channels, and exchangers embedded in cellular pathways from different sperm species.

  17. Identification of a Novel Hypocholesterolemic Protein, Major Royal Jelly Protein 1, Derived from Royal Jelly

    PubMed Central

    Asai, Saori; Kusada, Mio; Watanabe, Suzuyo; Kawashima, Takuji; Nakamura, Tadashi; Shimada, Masaya; Goto, Tsuyoshi; Nagaoka, Satoshi

    2014-01-01

    Royal jelly (RJ) intake lowers serum cholesterol levels in animals and humans, but the active component in RJ that lowers serum cholesterol level and its molecular mechanism are unclear. In this study, we set out to identify the bile acid-binding protein contained in RJ, because dietary bile acid-binding proteins including soybean protein and its peptide are effective in ameliorating hypercholesterolemia. Using a cholic acid-conjugated column, we separated some bile acid-binding proteins from RJ and identified the major RJ protein 1 (MRJP1), MRJP2, and MRJP3 as novel bile acid-binding proteins from RJ, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified MRJP1, which is the most abundant protein of the bile acid-binding proteins in RJ, exhibited taurocholate-binding activity in vitro. The micellar solubility of cholesterol was significantly decreased in the presence of MRJP1 compared with casein in vitro. Liver bile acids levels were significantly increased, and cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein tended to increase by MRJP1 feeding compared with the control. CYP7A1 mRNA and protein levels were significantly increased by MRJP1 tryptic hydrolysate treatment compared with that of casein tryptic hydrolysate in hepatocytes. MRJP1 hypocholesterolemic effect has been investigated in rats. The cholesterol-lowering action induced by MRJP1 occurs because MRJP1 interacts with bile acids induces a significant increase in fecal bile acids excretion and a tendency to increase in fecal cholesterol excretion and also enhances the hepatic cholesterol catabolism. We have identified, for the first time, a novel hypocholesterolemic protein, MRJP1, in RJ. Interestingly, MRJP1 exhibits greater hypocholesterolemic activity than the medicine β-sitosterol in rats. PMID:25144734

  18. Influence of macrophage migration inhibitory factor (MIF) on the zinc content and redox state of protein-bound sulphydryl groups in rat sperm: indications for a new role of MIF in sperm maturation.

    PubMed

    Eickhoff, Regina; Baldauf, Christina; Koyro, Hans-Werner; Wennemuth, Gunther; Suga, Yasushi; Seitz, Jürgen; Henkel, Ralf; Meinhardt, Andreas

    2004-08-01

    The function of macrophage migration inhibitory factor (MIF) in sperm maturation was studied by investigating its role in the biochemical maturation of the outer dense fibres. Rat sperm obtained from the caput and cauda epididymis were stimulated overnight with either recombinant MIF or MIF-containing vesicles originating from epididymal fluid at various concentrations. The zinc content of both the sperm and the medium was determined by means of atomic absorption spectrometry. Incubation in both recombinant MIF and vesicular MIF resulted in a statistically significant decrease of the zinc content in stimulated caput sperm of approximately 50%. In parallel, the conditioned media showed a clear increase in the concentration of this trace metal. The effect of MIF was less marked in cauda sperm. In addition, we demonstrated a statistically significant increase of detectable free thiol groups in the sperm mid- and principle piece in isolated rat sperm after stimulation with MIF at concentrations of 25 and 50 ng/ml. Our data suggest that MIF plays an important role in the maturation process of rat sperm during epididymal transit by inducing the elimination of zinc and affecting the amount of free sulphydryl groups in the sperm flagella.

  19. The Molecular Chaperone HSPA2 Plays a Key Role in Regulating the Expression of Sperm Surface Receptors That Mediate Sperm-Egg Recognition

    PubMed Central

    Redgrove, Kate A.; Nixon, Brett; Baker, Mark A.; Hetherington, Louise; Baker, Gordon; Liu, De-Yi; Aitken, R. John

    2012-01-01

    A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility. PMID:23209833

  20. Sperm motility initiation by egg jelly of the anuran, Discoglossus pictus may be mediated by sperm motility-initiating substance of the internally-fertilizing newt, Cynops pyrrhogaster.

    PubMed

    Takayama-Watanabe, Eriko; Campanella, Chiara; Kubo, Hideo; Watanabe, Akihiko

    2012-11-01

    The egg jelly of Discoglossus pictus contains sperm motility-activating activity, the molecular basis of which has not been studied. Discoglossus pictus sperm initiated motility immediately after immersion in egg-jelly extract, as well as after immersion in hyposmotic solution, which initiates sperm motility in the external fertilization of anuran amphibians. Sequential treatment of the D. pictus sperm with these two solutions revealed the predominant effect of hyposmolality in initiation of motility. The motility initiation induced by jelly extract was suppressed by a monoclonal antibody (mAb) that is specific for the 34 kDa sperm motility-initiating substance (SMIS) in the egg jelly of the newt, Cynops pyrrhogaster. Immunoblotting using the anti-SMIS mAb revealed several antigenic proteins that included major ones with sizes of 18- and 34-kDa in D. pictus jelly extract. Scanning electron microscopic observation revealed that granules of jelly matrix, in which SMIS localizes and which have a critical role in the internal fertilization of C. pyrrhogaster, were not observed near the surface of the D. pictus egg jelly. These results suggest that sperm motility-activating activity in egg jelly of D. pictus may be mediated by SMIS homologous proteins that act through a mechanism that is partially different from that of C. pyrrhogaster.

  1. An ARID domain-containing protein within nuclear bodies is required for sperm cell formation in Arabidopsis thaliana

    USDA-ARS?s Scientific Manuscript database

    In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells. Several genes have been found to act in this process, and DUO1 (DUO POLLEN 1), a transcription factor, p...

  2. Junctate, an inositol 1,4,5-triphosphate receptor associated protein, is present in rodent sperm and binds TRPC2 and TRPC5 but not TRPC1 channels.

    PubMed

    Stamboulian, Séverine; Moutin, Marie-Jo; Treves, Susan; Pochon, Nathalie; Grunwald, Didier; Zorzato, Francesco; De Waard, Michel; Ronjat, Michel; Arnoult, Christophe

    2005-10-01

    The acrosome reaction, the first step of the fertilization, is induced by calcium influx through Canonical Transient Receptor Potential channels (TRPC). The molecular nature of TRPC involved is still a debated question. In mouse, TRPC2 plays the most important role and is responsible for the calcium plateau. However, TRPC1 and TRPC5 are also localized in the acrosomal crescent of the sperm head and may participate in calcium signaling, especially in TRPC2-deficient mice. Activation of TRPC channels is an unresolved question in germ and somatic cells as well. In particular, in sperm, little is known concerning the molecular events leading to TRPC2 activation. From the discovery of IP3R binding domains on TRPC2, it has been suggested that TRPC channel activation may be due to a conformational coupling between IP3R and TRPC channels. Moreover, recent data demonstrate that junctate, an IP3R associated protein, participates also in the gating of some TRPC. In this study, we demonstrate that junctate is expressed in sperm and co-localizes with the IP3R in the acrosomal crescent of the anterior head of rodent sperm. Consistent with its specific localization, we show by pull-down experiments that junctate interacts with TRPC2 and TRPC5 but not with TRPC1. We focused on the interaction between TRPC2 and junctate, and we show that the N-terminus of junctate interacts with the C-terminus of TRPC2, both in vitro and in a heterologous expression system. We show that junctate binds to TRPC2 independently of the calcium concentration and that the junctate binding site does not overlap with the common IP3R/calmodulin binding sites. TRPC2 gating is downstream phospholipase C activation, which is a key and necessary step during the acrosome reaction. TRPC2 may then be activated directly by diacylglycerol (DAG), as in neurons of the vomeronasal organ. In the present study, we investigated whether DAG could promote the acrosome reaction. We found that 100 microM OAG, a permeant DAG

  3. Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family.

    PubMed

    Foster, J A; Gerton, G L

    1996-06-01

    We have cloned and sequenced cDNAs encoding autoantigen 1 (AA1), a testis-specific protein and the major autoantigen of the guinea pig sperm acrosome. The cDNA predicts a precursor protein of 244 amino acids including a 21 amino acid hydrophobic, secretory signal sequence. The mature polypeptide is predicted to have a molecular mass of 24,891 Daltons which agrees with the experimentally determined molecular weight of 25,000. Consistent with previous studies demonstrating that AA1 is not a glycoprotein, the predicted amino acid sequence contained no canonical sites for N-linked glycosylation. Comparison with other sequences showed that AA1 is the guinea pig homologue of the testis-specific protein Tpx-1 in mice and TPX1 in humans. AA1 also showed significant amino acid sequence homology with other cysteine-rich secretory proteins (CRISP's): rat and mouse acidic epididymal glycoproteins (AEG; also known as proteins D/E in rats) and helothermine, a toxin from the Mexican beaded lizard. In addition, AA1 had a lesser degree of homology with antigen 5 (vespid wasp venom), PR-1 (a plant pathogenesis related protein), and GliPR (a protein identified in human gliomas). Northern analysis of RNA from purified guinea pig spermatogenic cells showed that a 1.5 kb message was first detected in pachytene spermatocytes, was strongest in round spermatids, and was detected at a low level in condensing spermatids. Immunoblot analysis and metabolic labeling data of AA1 in spermatogenic cells showed that the protein was synthesized as early as the pachytene spermatocyte stage of spermatogenesis. Thus, the patterns of AA1 mRNA and protein expression during spermatogenesis are similar to the expression of other acrosomal mRNAs and proteins that are first detected meiotically.

  4. Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation.

    PubMed

    Cohen, Debora J; Da Ros, Vanina G; Busso, Dolores; Ellerman, Diego A; Maldera, Julieta A; Goldweic, Nadia; Cuasnicú, Patricia S

    2007-07-01

    Rat protein DE is an androgen-dependent cysteine-rich secretory protein (CRISP) synthesized by proximal epididymal regions. DE, also known as CRISP-1, is localized on the equatorial segment of acrosome-reacted spermatozoa and participates in gamete fusion through binding to egg complementary sites. Immunization of rats with DE inhibits fertility and sperm fusion ability, suggesting that DE represents a good epididymal contraceptive target. Recombinant DE fragments and synthetic peptides revealed that DE binds to the egg via a 12-amino acid region of an evolutionarily conserved motif, Signature 2 (S2). The ability of other CRISP to bind to the rat egg was correlated with their S2 amino acid sequences. Although testicular protein Tpx-1 (CRISP-2) was capable of binding to rodent eggs, human epididymal AEG-related protein (ARP) and helothermine (from lizard saliva) were not. The S2 region presented only two substitutions in Tpx-1 and four in ARP and helothermine, compared with the DE S2, suggesting that this amino acid sequence was relevant for egg interaction. Studies with Tpx-1 and anti-Tpx-1 revealed the participation of this protein in gamete fusion through binding to complementary sites in the egg. In competition studies, DE reduced binding of Tpx-1 dose-dependently, indicating that both CRISP share the egg complementary sites. That anti-DE and anti-Tpx-1 inhibit sperm-egg fusion while recognizing only the corresponding proteins, suggests functional cooperation between these homologous CRISP to ensure fertilization success. These results increase our understanding of the molecular mechanisms of gamete fusion and contribute to the development of new and safer fertility regulating methods.

  5. Disruption of Ttll5/Stamp Gene (Tubulin Tyrosine Ligase-like Protein 5/SRC-1 and TIF2-associated Modulatory Protein Gene) in Male Mice Causes Sperm Malformation and Infertility*

    PubMed Central

    Lee, Geun-Shik; He, Yuanzheng; Dougherty, Edward J.; Jimenez-Movilla, Maria; Avella, Matteo; Grullon, Sean; Sharlin, David S.; Guo, Chunhua; Blackford, John A.; Awasthi, Smita; Zhang, Zhenhuan; Armstrong, Stephen P.; London, Edra C.; Chen, Weiping; Dean, Jurrien; Simons, S. Stoney

    2013-01-01

    TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamptm/tm) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamptm/tm sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamptm/tm males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility. PMID:23558686

  6. Panning for sperm gold: Isolation and purification of apyrene and eupyrene sperm from lepidopterans.

    PubMed

    Karr, Timothy L; Walters, James R

    2015-08-01

    We describe a simple and straightforward procedure for the purification and separation of apyrene and eupyrene forms of lepidopteran sperm. The procedure is generally applicable to both butterfly and moth species with results varying according to the relative amounts of sperm produced and size of sperm storage organs. The technique relies upon inherent differences between eupyene sperm bundles and free apyrene sperm morphology. These differences allow for separation of the sperm morphs by repeated "panning" of sperm bundles into the center of a plastic dish. The purified eupyrene sperm bundles can then be removed and apyrene sperm collected from the supernatant by centrifugation. Efficacy of the purification process was confirmed by light microscopy and gel electrophoresis of the resulting fractions. Both one- and two-dimensional gel electrophoresis identified significant protein differences between the fractions further suggesting that the panning procedure effectively separated eurpyrene from apyrene sperm. The panning procedure should provide a convenient and accessible technique for further studies of sperm biology in lepidopterans.

  7. LOCALIZATION OF SP22 ON HUMAN SPERM OF DIFFERING QUALITY

    EPA Science Inventory

    LOCALIZATION OF SP22 ON HUMAN SPERM OF DIFFERING QUALITY. AE Lavers*1, GR Klinefelter2, DW Hamilton1, KP Roberts1, 1University of Minnesota, Minneapolis, MN and 2US EPA, Research Triangle Park, NC.
    SP22 is a sperm membrane protein that has been implicated in sperm function d...

  8. LOCALIZATION OF SP22 ON HUMAN SPERM OF DIFFERING QUALITY

    EPA Science Inventory

    LOCALIZATION OF SP22 ON HUMAN SPERM OF DIFFERING QUALITY. AE Lavers*1, GR Klinefelter2, DW Hamilton1, KP Roberts1, 1University of Minnesota, Minneapolis, MN and 2US EPA, Research Triangle Park, NC.
    SP22 is a sperm membrane protein that has been implicated in sperm function d...

  9. Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

    SciTech Connect

    Rana, Ritu; Jagadish, Nirmala; Garg, Manoj; Mishra, Deepshikha; Dahiya, Neetu; Chaurasiya, Dipak; Suri, Anil . E-mail: anil@nii.res.in

    2006-02-03

    Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.

  10. Evaluation of silica nanoparticle binding to major human blood proteins

    NASA Astrophysics Data System (ADS)

    Hata, Katsutomo; Higashisaka, Kazuma; Nagano, Kazuya; Mukai, Yohei; Kamada, Haruhiko; Tsunoda, Shin-ichi; Yoshioka, Yasuo; Tsutsumi, Yasuo

    2014-12-01

    Nanomaterials are used for various biomedical applications because they are often more effective than conventional materials. Recently, however, it has become clear that the protein corona that forms on the surface of nanomaterials when they make contact with biological fluids, such as blood, influences the pharmacokinetics and biological responses induced by the nanomaterials. Therefore, when evaluating nanomaterial safety and efficacy, it is important to analyze the interaction between nanomaterials and proteins in biological fluids and to evaluate the effects of the protein corona. Here, we evaluated the interaction of silica nanoparticles, a commonly used nanomaterial, with the human blood proteins albumin, transferrin, fibrinogen, and IgG. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the amount of albumin, transferrin, and IgG binding to the silica particles increased as the particle size decreased under conditions where the silica particle mass remained the same. However, under conditions in which the specific surface area remained constant, there were no differences in the binding of human plasma proteins to the silica particles tested, suggesting that the binding of silica particles with human plasma proteins is dependent on the specific surface area of the silica particles. Furthermore, the amount of albumin, transferrin, and IgG binding to silica nanoparticles with a diameter of 70 nm (nSP70) and a functional amino group was lower than that with unmodified nSP70, although there was no difference in the binding between nSP70 with the surface modification of a carboxyl functional group and nSP70. These results suggest that the characteristics of nanomaterials are important for binding with human blood proteins; this information may contribute to the development of safe and effective nanomaterials.

  11. Involvement of Protein cAMP-dependent Kinase, Phospholipase A2 and Phospholipase C in Sperm Acrosome Reaction of Chinchilla lanigera.

    PubMed

    Gramajo-Bühler, M C; Zelarayán, L; Sánchez-Toranzo, G

    2016-02-01

    The mechanisms involved in fertilization are the centre of attention in order to determine the conditions required to reproduce in vitro the events that take place in vivo, with special interest in endangered species. Previous data from mouse sperm, where acrosome reaction (AR) occurs more often in the interstitium of the cumulus oophorus, contribute to strengthen the use of progesterone as a physiological inducer of this process. We studied the participation of protein kinase A (PKA), phospholipases A2 and C (PLA2 , PLC) in the AR induced by progesterone from Chinchilla epididymal spermatozoa. The addition of db-cAMP to the incubation medium caused an increase of 58% in the AR, while the use of H89 (30 μm), a PKA inhibitor, reflected a decrease of 40% in the percentage of reacted gametes. The assays conducted with arachidonic acid showed a maximum increase of 23% in the AR. When gametes were pre-incubated with PLA2 inhibitors, a dose-dependent inhibitory effect was observed. The addition of phorbol12-myristate13-acetate (10 μm) revealed higher percentages of AR induction (60%). When PLC was inhibited with neomycin and U73122, a dose-dependent decrease in AR percentages was observed. Combined inhibition of PKA, PLA2 and PLC, AR values similar to control were obtained. This work shows evidence, for the first time in Chinchilla, that progesterone activates the AC/cAMP/PKA system as well as sperm phospholipases and that these signalling pathways participate jointly and cooperatively in AR. These results contribute to the understanding of the complex regulation that is triggered in sperm after the effect of progesterone.

  12. Efficient utilization of very dilute aquatic sperm: sperm competition may be more likely than sperm limitation when eggs are retained.

    PubMed Central

    Pemberton, Andrew J; Hughes, Roger N; Manríquez, Patricio H; Bishop, John D D

    2003-01-01

    Fertilization success may be severely limited in marine invertebrates that spawn both male and female gametes. In a diverse group of aquatic organisms only sperm are released, with sperm-egg fusion occurring at the mother. Here, we report fertilization kinetics data for two such 'brooding' or 'spermcast' species--representing each major clade of the animal kingdom. High levels of fertilization were achieved at sperm concentrations of two or three orders of magnitude lower than is common with broadcast spawning species. At a concentration of 100 sperm ml(-1), fertilization rates of a bryozoan and colonial ascidian were near maximum, whereas most broadcast spawners would have displayed near complete reproductive failure. A further experiment looked at the rate of uptake of sperm under natural conditions. Results suggested that sperm released at ca. 0.9 m from an acting female could be collected at a rate of 3-12 times greater than the minimum required simply to avoid sperm limitation. Thus, evolutionary pressures on gametic and other reproductive characteristics of many species that release sperm but retain eggs may be quite different from those of broadcast spawners and may confer on the former an enhanced scope for sperm competition and female choice. PMID:14667389

  13. Eosinophil cationic protein stimulates and major basic protein inhibits airway mucus secretion.

    PubMed

    Lundgren, J D; Davey, R T; Lundgren, B; Mullol, J; Marom, Z; Logun, C; Baraniuk, J; Kaliner, M A; Shelhamer, J H

    1991-03-01

    Possible roles of eosinophil (EO) products in modulating the release of mucus from airway explants were investigated. Cell- and membrane-free lysates from purified human EOs (1 to 20 x 10(5)) caused a dose-dependent release of respiratory glycoconjugates (RGC) from cultured feline tracheal explants. Crude extracts from isolated EO granules also stimulated RGC release, suggesting that a granular protein might be responsible. Three proteins derived from EO granules, EO-derived neurotoxin, EO cationic protein (ECP), and major basic protein (MBP) were separated by sequential sizing and affinity chromatography. ECP (0.025 to 25 micrograms/ml) caused a dose-dependent increase in RGC release from both feline and human airway explants and also stimulated the release of the serous cell-marker, lactoferrin, from human bronchial explants. EO-derived neurotoxin (0.025 to 50 micrograms/ml) failed to affect RGC release, whereas MBP (50 micrograms/ml) significantly inhibited RGC release from feline explants. Thus, ECP stimulates RGC and lactoferrin release from airway explants, whereas MBP inhibits RGC release.

  14. Cofilin is correlated with sperm quality and influences sperm fertilizing capacity in humans.

    PubMed

    Chen, S M; Chen, X M; Lu, Y L; Liu, B; Jiang, M; Ma, Y X

    2016-11-01

    Spermatozoa should undergo a series of biochemical modifications in female reproduction tract, which is collectively called sperm capacitation. The capacitated spermatozoa can bind to the egg zona pellucida, resulting in the occurrence of acrosome reaction which enabled spermatozoa penetrate into the egg. The formation of actin plays an important role in these processes. Actin polymerized during sperm capacitation, but the polymers dispersed before acrosome reaction. In this study, we take our focus on actin-binding protein, cofilin. Our results showed that the % and intensity of sperm expressing cofilin in normal sperm were significantly higher than in abnormal sperm, and the sperm expressing cofilin was correlated with sperm quality. Furthermore, treatment with anti-cofilin antibody increased the percentage of sperm capacitation and inhibited progesterone- or A23187- induced acrosome reaction in a dose-dependent manner. The presence of 100 ng/mL anti-cofilin antibodies markedly blocked the sperm penetration of zona-free hamster eggs. Besides, immunofluorescence results revealed that cofilin was colocalized with F-actin in the midpiece of spermatozoa; however, phospho-cofilin was expressed in the tail rather than in the midpiece of spermatozoa, which was not colocalized with F-actin in spermatozoa. Moreover, western blot revealed that phospho-cofilin increased in sperm capacitation, and the total cofilin and cofilin in insoluble fraction increased in acrosome reaction; immunofluorescence results showed that the amount of cofilin in acrosome increased in sperm capacitation. In conclusion, our study revealed that cofilin expression in human sperm is correlated with sperm quality and the alterations of cofilin and phospho-cofilin in fertilization affects sperm capacitation, acrosome reaction, and spermatozoa-oocyte fusion.

  15. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.

    PubMed

    Fernández, Jose Luis; Muriel, Lourdes; Rivero, Maria Teresa; Goyanes, Vicente; Vazquez, Rosana; Alvarez, Juan G

    2003-01-01

    Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test

  16. Mass-Specific Metabolic Rate and Sperm Competition Determine Sperm Size in Marsupial Mammals

    PubMed Central

    Tourmente, Maximiliano; Gomendio, Montserrat; Roldan, Eduardo R. S.

    2011-01-01

    Two complementary hypotheses have been proposed to explain variation in sperm size. The first proposes that post-copulatory sexual selection favors an increase in sperm size because it enhances sperm swimming speed, which is an important determinant of fertilization success in competitive contexts. The second hypothesis proposes that mass-specific metabolic rate acts as a constraint, because large animals with low mass-specific metabolic rates will not be able to process resources at the rates needed to produce large sperm. This constraint is expected to be particularly pronounced among mammals, given that this group contains some of the largest species on Earth. We tested these hypotheses among marsupials, a group in which mass-specific metabolic rates are roughly 30% lower than those of eutherian mammals of similar size, leading to the expectation that metabolic rate should be a major constraint. Our findings support both hypotheses because levels of sperm competition are associated with increases in sperm size, but low mass-specific metabolic rate constrains sperm size among large species. We also found that the relationship between sperm size and mass-specific metabolic rate is steeper among marsupials and shallower among eutherian mammals. This finding has two implications: marsupials respond to changes in mass-specific metabolic rate by modifying sperm length to a greater extent, suggesting that they are more constrained by metabolic rate. In addition, for any given mass-specific metabolic rate, marsupials produce longer sperm. We suggest that this is the consequence of marsupials diverting resources away from sperm numbers and into sperm size, due to their efficient sperm transport along the female tract and the existence of mechanisms to protect sperm. PMID:21731682

  17. Molecular Cloning of Spergen-4, Encoding a Spermatogenic Cell-Specific Protein Associated with Sperm Flagella and the Acrosome Region in Rat Spermatozoa.

    PubMed

    Howida, Ali; Salaheldeen, Elsaid; Iida, Hiroshi

    2016-04-01

    We used a differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene LOC690919 with an open reading frame of 1227-length nucleotides encoding a protein of 409 amino acids. This gene was designated as Spergen-4 (a spermatogenic cell-specific gene-4). Spergen-4 mRNA was highly expressed in testis, and its expression was detected in rat testis starting at three weeks of postnatal development and persisting up to adulthood. Mouse and human orthologs, which lack N-terminal 77 amino acid residues of rat Spegen-4, were found in the database. Immunofluorescence microscopy and immunoblot analysis demonstrated that Spergen-4 was not expressed in spermatogonia, spermatocytes, and round spermatids, but was restrictedly detected at sperm head, cytoplasm, and developing flagella of elongated spermatids in rat testis. In mature spermatozoa, Spergen-4 was detected at the acrosome region as well as the principal piece of flagella. Spergen-4 immunosignal disappeared from sperm heads on acrosome reaction induced by progesterone. These data suggest that Spergen-4 integrated into elongated spermatids during spermiogenesis serves as a constituent for acrosome region and flagella of rat spermatozoa.

  18. Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation.

    PubMed

    Intasqui, Paula; Camargo, Mariana; Del Giudice, Paula T; Spaine, Deborah M; Carvalho, Valdemir M; Cardozo, Karina H M; Cedenho, Agnaldo P; Bertolla, Ricardo P

    2013-09-01

    Sperm DNA fragmentation has been suggested as a marker for infertility diagnosis and prognosis. Hence, understanding its impact on male physiology and post-genomic pathways would be clinically important. We performed the proteomics and functional enrichment analyses of viable spermatozoa from ejaculates with low and high sperm DNA fragmentation to identify protein expression and pathways altered in association with sperm DNA fragmentation. Sperm DNA fragmentation using the Comet assay and the Komet 6.0.1 software was assessed in raw samples from 89 subjects from a human reproduction service. The Low and High sperm DNA fragmentation groups were formed according to the Olive Tail Moment variable. Spermatozoa proteins from these groups were pooled and analyzed by a shotgun proteomic approach (2D nanoUPLC-ESI-MS(E)). Differentially expressed proteins were used for a functional enrichment study. Two hundred and fifty-seven proteins were identified or quantified in sperm from the Low and High sperm DNA fragmentation groups. Of these, seventy-one proteins were exclusively or overexpressed in the Low group, whereas twenty-three proteins were exclusively or overexpressed in the High group. One hundred and sixty-three proteins were conserved between these groups. We also functionally related the differentially expressed proteins in viable spermatozoa from the groups. Processes such as triacylglycerol metabolism, energy production, protein folding, response to unfolded proteins, and cellular detoxification were found to be altered in these cells. Sperm DNA fragmentation is associated with differential protein expression in viable spermatozoa. These proteins may potentially be used as biomarkers for sperm DNA integrity.

  19. Circumvention of Immunity to the Adenovirus Major Coat Protein Hexon

    PubMed Central

    Roy, Soumitra; Shirley, Pamela S.; McClelland, Alan; Kaleko, Michael

    1998-01-01

    Immunity to adenoviruses is an important hurdle to be overcome for successful gene therapy. The presence of antibodies to the capsid proteins prevents efficacious adenovirus vector administration in vivo. We tested whether immunity to a particular serotype of adenovirus (Ad5) may be overcome with a vector that encodes the hexon sequences from a different adenovirus serotype (Ad12). We successfully constructed an adenovirus vector with a chimeric Ad5-Ad12 hexon which was not neutralized by plasma from C57BL/6 mice immunized with Ad5. The vector was also capable of transducing the livers of C57BL/6 mice previously immunized with Ad5. PMID:9658137

  20. Sugar‐coated sperm: Unraveling the functions of the mammalian sperm glycocalyx

    PubMed Central

    Tecle, Eillen

    2015-01-01

    SUMMARY Mammalian spermatozoa are coated with a thick glycocalyx that is assembled during sperm development, maturation, and upon contact with seminal fluid. The sperm glycocalyx is critical for sperm survival in the female reproductive tract and is modified during capacitation. The complex interplay among the various glycoconjugates generates numerous signaling motifs that may regulate sperm function and, as a result, fertility. Nascent spermatozoa assemble their own glycans while the cells still possess a functional endoplasmic reticulum and Golgi in the seminiferous tubule, but once spermatogenesis is complete, they lose the capacity to produce glycoconjugates de novo. Sperm glycans continue to be modified, during epididymal transit by extracellular glycosidases and glycosyltransferases. Furthermore, epididymal cells secrete glycoconjugates (glycophosphatidylinositol‐anchored glycoproteins and glycolipids) and glycan‐rich microvesicles that can fuse with the maturing sperm membrane. The sperm glycocalyx mediates numerous functions in the female reproductive tract, including the following: inhibition of premature capacitation; passage through the cervical mucus; protection from innate and adaptive female immunity; formation of the sperm reservoir; and masking sperm proteins involved in fertilization. The immense diversity in sperm‐associated glycans within and between species forms a remarkable challenge to our understanding of essential sperm glycan functions. Mol. Reprod. Dev. 82: 635–650, 2015. © 2015 The Authors. Molecular Reproduction and Development published by Wiley Periodicals, Inc. PMID:26061344

  1. Accurate sperm morphology assessment predicts sperm function.

    PubMed

    Abu Hassan Abu, D; Franken, D R; Hoffman, B; Henkel, R

    2012-05-01

    Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.

  2. Geometric Morphometrics of Rodent Sperm Head Shape

    PubMed Central

    Varea Sánchez, María; Bastir, Markus; Roldan, Eduardo R. S.

    2013-01-01

    Mammalian spermatozoa, particularly those of rodent species, are extremely complex cells and differ greatly in form and dimensions. Thus, characterization of sperm size and, particularly, sperm shape represents a major challenge. No consensus exists on a method to objectively assess size and shape of spermatozoa. In this study we apply the principles of geometric morphometrics to analyze rodent sperm head morphology and compare them with two traditional morphometry methods, that is, measurements of linear dimensions and dimensions-derived parameters calculated using formulae employed in sperm morphometry assessments. Our results show that geometric morphometrics clearly identifies shape differences among rodent spermatozoa. It is also capable of discriminating between size and shape and to analyze these two variables separately. Thus, it provides an accurate method to assess sperm head shape. Furthermore, it can identify which sperm morphology traits differ between species, such as the protrusion or retraction of the base of the head, the orientation and relative position of the site of flagellum insertion, the degree of curvature of the hook, and other distinct anatomical features and appendices. We envisage that the use of geometric morphometrics may have a major impact on future studies focused on the characterization of sperm head formation, diversity of sperm head shape among species (and underlying evolutionary forces), the effects of reprotoxicants on changes in cell shape, and phenotyping of genetically-modified individuals. PMID:24312234

  3. The Rhesus macaque (Macaca mulatta) sperm proteome.

    PubMed

    Skerget, Sheri; Rosenow, Matthew; Polpitiya, Ashoka; Petritis, Konstantinos; Dorus, Steve; Karr, Timothy L

    2013-11-01

    Mass spectrometry based proteomics has facilitated sperm composition studies in several mammalian species but no studies have been undertaken in non-human primate species. Here we report the analysis of the 1247 proteins that comprise the Rhesus macaque (Macaca mulatta) sperm proteome (termed the MacSP). Comparative analysis with previously characterized mouse and human sperm proteomes reveals substantial levels of orthology (47% and 40% respectively) and widespread overlap of functional categories based on Gene Ontology analyses. Approximately 10% of macaque sperm genes (113/1247) are significantly under-expressed in the testis as compared with other tissues, which may reflect proteins specifically acquired during epididymal maturation. Phylogenetic and genomic analyses of three MacSP ADAMs (A-Disintegrin and Metalloprotease proteins), ADAM18-, 20- and 21-like, provides empirical support for sperm genes functioning in non-human primate taxa which have been subsequently lost in the lineages leading to humans. The MacSP contains proteasome proteins of the 20S core subunit, the 19S proteasome activator complex and an alternate proteasome activator PA200, raising the possibility that proteasome activity is present in mature sperm. Robust empirical characterization of the Rhesus sperm proteome should greatly expand the possibility for targeted molecular studies of spermatogenesis and fertilization in a commonly used model species for human infertility.

  4. The Rhesus Macaque (Macaca mulatta) Sperm Proteome*

    PubMed Central

    Skerget, Sheri; Rosenow, Matthew; Polpitiya, Ashoka; Petritis, Konstantinos; Dorus, Steve; Karr, Timothy L.

    2013-01-01

    Mass spectrometry based proteomics has facilitated sperm composition studies in several mammalian species but no studies have been undertaken in non-human primate species. Here we report the analysis of the 1247 proteins that comprise the Rhesus macaque (Macaca mulatta) sperm proteome (termed the MacSP). Comparative analysis with previously characterized mouse and human sperm proteomes reveals substantial levels of orthology (47% and 40% respectively) and widespread overlap of functional categories based on Gene Ontology analyses. Approximately 10% of macaque sperm genes (113/1247) are significantly under-expressed in the testis as compared with other tissues, which may reflect proteins specifically acquired during epididymal maturation. Phylogenetic and genomic analyses of three MacSP ADAMs (A-Disintegrin and Metalloprotease proteins), ADAM18-, 20- and 21-like, provides empirical support for sperm genes functioning in non-human primate taxa which have been subsequently lost in the lineages leading to humans. The MacSP contains proteasome proteins of the 20S core subunit, the 19S proteasome activator complex and an alternate proteasome activator PA200, raising the possibility that proteasome activity is present in mature sperm. Robust empirical characterization of the Rhesus sperm proteome should greatly expand the possibility for targeted molecular studies of spermatogenesis and fertilization in a commonly used model species for human infertility. PMID:23816990

  5. Upregulation of breast cancer resistance protein and major vault protein in drug resistant epilepsy.

    PubMed

    Banerjee Dixit, Aparna; Sharma, Devina; Srivastava, Arpna; Banerjee, Jyotirmoy; Tripathi, Manjari; Prakash, Deepak; Sarat Chandra, P

    2017-04-01

    Identifying factors involved in the development of drug resistant epilepsy (DRE) remains a challenge. Candidate gene studies have shown modulation of resistance to drugs by various multidrug resistance proteins in DRE. However the resistance to drugs in DRE could be more complex and multifactorial involving molecules in different pharmacokinetic processes. In this study for the first time we have analyzed the relative expression of four molecules with different drug resistance mechanisms in two most common DRE pathologies, mesial temporal lobe epilepsy (MTLE) and focal cortical dysplasia (FCD) with respect to each other and also with different non-epileptic controls. Brain tissues resected from MTLE (n=16) and FCD type I and II (n=12) patients who had undergone surgery were analysed for mRNA levels of multidrug resistance-associated protein 1(MRP1), major vault protein (MVP), breast cancer resistance protein (BCRP), and one drug metabolising enzyme (UGT1A4) as compared to non-epileptic controls which were tissues resected from tumor periphery (n=6) and autopsy tissues (n=4) by quantitative PCR. We found significant upregulation of MVP and BCRP whereas MRP1 and UGT1A4 were unaltered in both pathologies. While upregulation of BCRP was significantly higher in MTLE (9.34±0.45; p<0.05), upregulation of MVP was significantly higher in FCD (2.94±0.65; p<0.01). We propose that upregulation of BCRP and MVP is associated with MTLE and FCD and these molecules not only may have the potential to predict pathology specific phenotypes but may also have therapeutic potential as adjunct treatment in these pathologies. Copyright © 2017 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

  6. The proline-rich region of glyceraldehyde-3-phosphate dehydrogenase from human sperm may bind SH3 domains, as revealed by a bioinformatic study of low-complexity protein segments.

    PubMed

    Tatjewski, Marcin; Gruca, Aleksandra; Plewczynski, Dariusz; Grynberg, Marcin

    2016-02-01

    Glyceraldehyde-3-phosphate dehydrogenase from human sperm (GAPDHS) provides energy to the sperm flagellum, and is therefore essential for sperm motility and male fertility. This isoform is distinct from somatic GAPDH, not only in being specific for the testis but also because it contains an additional amino-terminal region that encodes a proline-rich motif that is known to bind to the fibrous sheath of the sperm tail. By conducting a large-scale sequence comparison on low-complexity sequences available in databases, we identified a strong similarity between the proline-rich motif from GAPDHS and the proline-rich sequence from Ena/vasodilator-stimulated phosphoprotein-like (EVL), which is known to bind an SH3 domain of dynamin-binding protein (DNMBP). The putative binding partners of the proline-rich GAPDHS motif include SH3 domain-binding protein 4 (SH3BP4) and the IL2-inducible T-cell kinase/tyrosine-protein kinase ITK/TSK (ITK). This result implies that GAPDHS participates in specific signal-transduction pathways. Gene Ontology category-enrichment analysis showed several functional classes shared by both proteins, of which the most interesting ones are related to signal transduction and regulation of hydrolysis. Furthermore, a mutation of one EVL proline to leucine is known to cause colorectal cancer, suggesting that mutation of homologous amino acid residue in the GAPDHS motif may be functionally deleterious.

  7. Basic Endochitinases Are Major Proteins in Castanea sativa Cotyledons 1

    PubMed Central

    Collada, Carmen; Casado, Rosa; Fraile, Aurora; Aragoncillo, Cipriano

    1992-01-01

    Basic endochitinases are abundant proteins in Castanea sativa Mill. cotyledons. Three basic chitinases were purified with molecular masses of 25, 26, and 32 kD (Ch1, Ch2, and Ch3) and with isoelectric points between 8 and 9.5. Antibodies raised against Ch1 cross-reacted with Ch2 and Ch3. However, Ch3 showed differences when compared with the other two enzymes, especially in its higher cysteine content. The size, amino acid composition, and N-terminal sequence of Ch1 indicate that it is a class II endochitinase and, therefore, has no cysteine-rich hevein domain. Ch1 inhibits the growth of the fungus Trichoderma viride. The biological role of these endochitinases is discussed. Images Figure 2 Figure 4 Figure 5 Figure 6 PMID:16653058

  8. c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways link capacitation with apoptosis and seminal plasma proteins protect sperm by interfering with both routes†.

    PubMed

    Luna, Carolina; Mendoza, Noelia; Casao, Adriana; Pérez-Pé, Rosaura; Cebrián-Pérez, José A; Muiño-Blanco, Teresa

    2017-04-01

    The mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38) signaling cascades are involved in triggering apoptosis in somatic cells. Given that spermatozoa are able to undergo apoptosis, we tested the hypothesis that these pathways might be functional in ram spermatozoa as two signal transduction mechanisms that contribute to the modulation of capacitation and apoptosis. Indirect immunofluorescence and western blot analysis evidenced the presence of JNK and p38 in ram spermatozoa. To verify the involvement of these enzymes in sperm physiology, we determined the effect of specific inhibitors of JNK or p38 on in vitro capacitation induced with either cAMP-elevating agents or epidermal growth factor (EGF). Both inhibitions reduced the EGF-induced capacitation with a decrease in the chlortetracycline capacitated-sperm pattern, protein tyrosine phosphorylation, phosphatidylserine externalization, caspase-3 and -7 activation, and the proportion of DNA-damaged spermatozoa. No significant changes were found in the high-cAMP capacitated samples. The addition of 3.4 mg/ml seminal plasma proteins (SPPs) to the EGF-containing samples, either alone or together with each inhibitor, resulted in a decreased proportion of capacitated sperm pattern, protein tyrosine phosphorylation, loss of plasma membrane integrity, and apoptotic alterations. Furthermore, SPPs significantly reduced the phosphorylation level of JNK and p38 MAPK (active forms). These findings show a relationship between capacitation and apoptosis, and represent a step forward in the knowledge of the SPP protective mechanism in spermatozoa. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com.

  9. Molecular mechanisms involved in gamete interaction: evidence for the participation of cysteine-rich secretory proteins (CRISP) in sperm-egg fusion.

    PubMed

    Da Ros, V; Busso, D; Cohen, D J; Maldera, J; Goldweic, N; Cuasnicu, P S

    2007-01-01

    Epididymal protein DE and testicular protein Tpx-1 are two cysteine-rich secretory proteins also known as CRISP-1 and CRISP-2, respectively. DE/ CRISP-1 is localised on the equatorial segment of acrosome-reacted sperm and participates in rat gamete fusion through its binding to egg-complementary sites. Recent results using bacterially-expressed recombinant fragments of DE as well as synthetic peptides revealed that the ability of DE to bind to the egg surface and inhibit gamete fusion resides in a region of 12 amino acids corresponding to an evolutionary conserved motif of the CRISP family (Signature 2). Given the high degree of homology between DE/CRISP-1 and Tpx-1/CRISP-2, we also explored the potential participation of the testicular intra-acrosomal protein in gamete fusion. Results showing the ability of recombinant Tpx-1 to bind to the surface of rat eggs (evaluated by indirect immunofluorescence) and to significantly inhibit zona-free egg penetration, support the participation of this protein in gamete fusion through its interaction with egg-binding sites. Interestingly, rat Tpx-1 exhibits only two substitutions in Signature 2 when compared to this region in DE. Together, these results provide evidence for the involvement of both epididymal DE/CRISP-1 and testicular Tpx-1/CRISP-2 in gamete fusion suggesting the existence of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilisation.

  10. Insights into the lysine acetylproteome of human sperm.

    PubMed

    Sun, Guohai; Jiang, Min; Zhou, Tao; Guo, Yueshuai; Cui, Yiqiang; Guo, Xuejiang; Sha, Jiahao

    2014-09-23

    Protein lysine acetylation is a dynamic and reversible post-modification that is known to play diverse functions in eukaryotes. Nevertheless, the composition and function of non-histone lysine acetylation in gametes remain unknown. In humans, only capacitated sperm have the capacity to fertilize an egg. In the present study, we found complex composition of lysine acetylated proteins in capacitated human sperm. In vitro fertilization inhibition assay by anti-acetyllysine antibody showed essential roles of lysine acetylation in fertilization. And inhibition of lysine deacetylases, the histone deacetylases, by trichostatin A and nicotinamide, could significantly suppress sperm motility. After immunopurification enrichment of acetylpeptides with anti-acetyllysine antibody and high-throughput liquid chromatography-tandem mass spectrometry identification, we characterized 1206 lysine acetylated sites, corresponding to 576 lysine acetylated proteins in human capacitated sperm. Bioinformatics analysis showed that these proteins are associated with sperm functions, including motility, capacitation, acrosome reaction and sperm-egg interaction. Thus, lysine acetylation is expected to be an important regulatory mechanism for sperm functions. And our characterization of lysine acetylproteome could be a rich resource for the study of male fertility. Mature sperm are almost transcriptionally and translationally silent, thus post-translational modifications play important roles in sperm functions. Till now, only two types of PTMs, phosphorylation and glycosylation, are well studied in normal human sperm based on large scale proteomics. In the present study, we established the acetylproteome of capacitated human sperm. Over 1000 lysine acetylated sites were identified. Bioinformatics analysis shows that lysine acetylated proteins participate in many biological events of sperm functions. We further provided functional data that the lysine acetylation is essential for sperm motility

  11. Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction

    PubMed Central

    1989-01-01

    In the mouse, considerable evidence indicates that initial sperm binding to the zona pellucida (ZP) is mediated by ZP3. In addition, this same glycoprotein is also responsible for inducing the acrosome reaction (AR). Whereas the O-linked oligosaccharides of ZP3 appear to mediate sperm-ZP binding, the portion of ZP3 bearing AR activity has not been defined. To try to understand the bifunctional role of ZP3 (binding and AR inducing activities), we have examined the hypothesis that ZP3 aggregates sperm receptor molecules. By analogy with findings in a variety of other extracellular signal transducing systems, including receptors for growth factors and insulin, this aggregation event could initiate the cascade resulting in the AR. To test this hypothesis, we have generated monospecific polyclonal antibodies against ZP2 and against ZP3, and examined the effects of these probes on capacitated sperm incubated in the absence or presence of various ZP protein preparations. For some experiments, we have used proteolytic fragments of ZP3, a preparation known to retain specific binding, but not AR-inducing, activity. We show here that capacitated mouse sperm, incubated with ZP glycopeptides, displayed ARs when incubated subsequently with anti-ZP3 IgG; ARs did not occur when parallel sperm samples were incubated with anti-ZP2 IgG or with anti-ZP3 Fab fragments. When capacitated sperm were treated successively, with (a) ZP3 glycopeptides, (b) anti-ZP3 Fab fragments, and (c) goat anti-rabbit IgG, ARs occurred in the majority of sperm. An alternative approach to examine this hypothesis used ZP proteins obtained from tubal eggs treated previously with bioactive phorbol diester (12-O-tetradecanoyl phorbol-13-acetate [TPA]). This preparation arrests capacitated sperm in an intermediate state of the AR. We demonstrate here that these sperm can be induced to undergo a complete AR by subsequent treatment with anti-ZP3 IgG. Together, these findings are consistent with the hypothesis

  12. Bioenergetics of Mammalian Sperm Capacitation

    PubMed Central

    Ferramosca, Alessandra; Zara, Vincenzo

    2014-01-01

    After ejaculation, the mammalian male gamete must undergo the capacitation process, which is a prerequisite for egg fertilization. The bioenergetics of sperm capacitation is poorly understood despite its fundamental role in sustaining the biochemical and molecular events occurring during gamete activation. Glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) are the two major metabolic pathways producing ATP which is the primary source of energy for spermatozoa. Since recent data suggest that spermatozoa have the ability to use different metabolic substrates, the main aim of this work is to present a broad overview of the current knowledge on the energy-producing metabolic pathways operating inside sperm mitochondria during capacitation in different mammalian species. Metabolism of glucose and of other energetic substrates, such as pyruvate, lactate, and citrate, is critically analyzed. Such knowledge, besides its obvious importance for basic science, could eventually translate into the development of novel strategies for treatment of male infertility, artificial reproduction, and sperm selection methods. PMID:24791005

  13. Two types of assays for detecting frog sperm chemoattraction.

    PubMed

    Burnett, Lindsey A; Tholl, Nathan; Chandler, Douglas E

    2011-12-27

    Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm. Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg. Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 μm diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed

  14. Mammalian sperm nuclear organization: resiliencies and vulnerabilities.

    PubMed

    Champroux, A; Torres-Carreira, J; Gharagozloo, P; Drevet, J R; Kocer, A

    2016-01-01

    Sperm cells are remarkably complex and highly specialized compared to somatic cells. Their function is to deliver to the oocyte the paternal genomic blueprint along with a pool of proteins and RNAs so a new generation can begin. Reproductive success, including optimal embryonic development and healthy offspring, greatly depends on the integrity of the sperm chromatin structure. It is now well documented that DNA damage in sperm is linked to reproductive failures both in natural and assisted conception (Assisted Reproductive Technologies [ART]). This manuscript reviews recent important findings concerning - the unusual organization of mammalian sperm chromatin and its impact on reproductive success when modified. This review is focused on sperm chromatin damage and their impact on embryonic development and transgenerational inheritance.

  15. Sulfur dioxide inhalation lowers sperm quality and alters testicular histology via increasing expression of CREM and ACT proteins in rat testes.

    PubMed

    Zhang, Jianhai; Zheng, Fei; Liang, Chen; Zhu, Yuchen; Shi, Yan; Han, Yongli; Wang, Jundong

    2016-10-01

    Sulfur dioxide (SO2) is one of the main atmospheric pollutants worldwide, and is reported to be responsible for the formation of severe haze in China. Some studies have demonstrated a potential harmful effect of SO2 on the male reproductive system; however the underlying mechanism is still unknown. The purpose of this study is to investigate the roles of cytochrome P450 (P450), cAMP-responsive element modulator (CREM), and activator of CREM (ACT) in SO2-induced toxicity. Forty-eight male Wistar rats were randomly divided into an experimental and control group. The experiment group was exposed to SO2 in ambient air (10ppm, 4h/day), and the control group was treated with filtered air in the same conditions. After 2 weeks, the results showed a significant decrease in body weight and sperm motility, and an increase in the testis weight-to-body weight ratio as compared to the control group. Histological investigation suggested that SO2 exposure led to loose arrangement of the spermatogenic cells and local structural damage in the seminiferous tubules. Moreover, the expressions of P450, CREM and ACT proteins increased in the testes by 0.22%, 47.26% and 23.38%, respectively. Taken together, SO2 inhalation lowered sperm quality, altered testicular histology, and increased expressions of CREM and ACT proteins in the testes of rats. Overall, these results could contribute to a better understanding of SO2-induced male reproductive toxicity. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Two Distinct Ca2+ Signaling Pathways Modulate Sperm Flagellar Beating Patterns in Mice1

    PubMed Central

    Chang, Haixin; Suarez, Susan S.

    2011-01-01

    Hyperactivation, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization. It is characterized by asymmetrical flagellar beating and an increase of cytoplasmic Ca2+. We observed that some mouse sperm swimming in the oviduct produce high-amplitude pro-hook bends (bends in the direction of the hook on the head), whereas other sperm produce high-amplitude anti-hook bends. Switching direction of the major bends could serve to redirect sperm toward oocytes. We hypothesized that different Ca2+ signaling pathways produce high-amplitude pro-hook and anti-hook bends. In vitro, sperm that hyperactivated during capacitation (because of activation of CATSPER plasma membrane Ca2+ channels) developed high-amplitude pro-hook bends. The CATSPER activators procaine and 4-aminopyridine (4-AP) also induced high-amplitude pro-hook bends. Thimerosal, which triggers a Ca2+ release from internal stores, induced high-amplitude anti-hook bends. Activation of CATSPER channels is facilitated by a pH rise, so both Ca2+ and pH responses to treatments with 4-AP and thimerosal were monitored. Thimerosal triggered a Ca2+ increase that initiated at the base of the flagellum, whereas 4-AP initiated a rise in the proximal principal piece. Only 4-AP triggered a flagellar pH rise. Proteins were extracted from sperm for examination of phosphorylation patterns induced by Ca2+ signaling. Procaine and 4-AP induced phosphorylation of proteins on threonine and serine, whereas thimerosal primarily induced dephosphorylation of proteins. Tyrosine phosphorylation was unaffected. We concluded that hyperactivation, which is associated with capacitation, can be modulated by release of Ca2+ from intracellular stores to reverse the direction of the dominant flagellar bend and, thus, redirect sperm. PMID:21389347

  17. Comparative profiling of the sperm proteome.

    PubMed

    Holland, Ashling; Ohlendieck, Kay

    2015-02-01

    The highly complex and species-selective mechanism of fertilization is a central theme of developmental biology. Gametogenesis, sperm activation, and egg-sperm recognition are fundamental biological processes, warranting detailed studies into the molecular composition of gametes. Biological MS has been instrumental for the comprehensive itemizing of gamete proteomes. The protein constellation of sperm cells and its subcellular structures has been established for a variety of animal species. Spermatogenesis and the crucial activation of sperm cells as a prerequisite of successful fertilization and physiological adaptations to external stressors was investigated using proteomics, as well as the underlying mechanisms of male infertility with respect to proteome-wide alterations. This review outlines recent achievements of sperm proteomics and exemplifies the usefulness of gel-based surveys by outlining the comparative analysis of abnormal spermatozoa in globozoospermia. Besides label-free MS techniques and cell-based labeling methodology, high-resolution fluorescence 2DE has been shown to be highly suitable as a proteomic biomarker discovery tool in sperm protein research. The appropriateness of novel protein markers for improving our understanding of normal spermatogenesis and sperm activation versus the molecular pathogenesis of male infertility will be discussed. New biomarker candidates might be useful to improve diagnostic, prognostic, and therapeutic aspects of infertility.

  18. Redox regulation of mammalian sperm capacitation

    PubMed Central

    O’Flaherty, Cristian

    2015-01-01

    Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility. PMID:25926608

  19. Spata6 is required for normal assembly of the sperm connecting piece and tight head-tail conjunction.

    PubMed

    Yuan, Shuiqiao; Stratton, Clifford J; Bao, Jianqiang; Zheng, Huili; Bhetwal, Bhupal P; Yanagimachi, Ryuzo; Yan, Wei

    2015-02-03

    "Pinhead sperm," or "acephalic sperm," a type of human teratozoospermia, refers to the condition in which ejaculate contains mostly sperm flagella without heads. Family clustering and homogeneity of this syndrome suggests a genetic basis, but the causative genes remain largely unknown. Here we report that Spata6, an evolutionarily conserved testis-specific gene, encodes a protein required for formation of the segmented columns and the capitulum, two major structures of the sperm connecting piece essential for linking the developing flagellum to the head during late spermiogenesis. Inactivation of Spata6 in mice leads to acephalic spermatozoa and male sterility. Our proteomic analyses reveal that SPATA6 is involved in myosin-based microfilament transport through interaction with myosin subunits (e.g., MYL6).

  20. Sperm chromatin structure assay (SCSA®).

    PubMed

    Evenson, Donald P

    2013-01-01

    The SCSA(®) is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA(®) is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 × 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA(®) lab. The samples are individually thawed, prepared, and analyzed in ∼10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 × 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1-3%) and the same between fresh and frozen samples. SCSAsoft(®) software transforms the X-Y data to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose-response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA(®) simultaneously determines the % of sperm with high DNA stainability (%HDS

  1. Expression of the male reproduction-related gene in spermatic ducts of the blue swimming crab, Portunus pelagicus, and transfer of modified protein to the sperm acrosome.

    PubMed

    Sroyraya, Morakot; Hanna, Peter J; Changklungmoa, Narin; Senarai, Thanyaporn; Siangcham, Tanapan; Tinikul, Yotsawan; Sobhon, Prasert

    2013-01-01

    Expression of a sex-specific gene in Macrobrachium rosenbergii (Mr-Mrr), encoding a male reproduction-related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp-Mrr) and Scylla serrata (Ss-Mrr). We then investigated expression of Pp-Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp-Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp-Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti-Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp-Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs.

  2. The Drosophila don juan (dj) gene encodes a novel sperm specific protein component characterized by an unusual domain of a repetitive amino acid motif.

    PubMed

    Santel, A; Winhauer, T; Blümer, N; Renkawitz-Pohl, R

    1997-06-01

    We identified and characterized the don juan gene (dj) of Drosophila melanogaster. The don juan gene codes for a sperm specific protein component with an unusual repetitive six amino acid motif (DPCKKK) in the carboxy-terminal part of the protein. The expression of Don Juan is limited to male germ cells where transcription of the dj gene is initiated during meiotic prophase. But Western blot experiments indicate that DJ protein occurs just postmeiotically. Examination of transgenic flies bearing a dj-promoter-lacZ reporter construct revealed lacZ mRNA distribution resembling the expression pattern of the endogenous dj mRNA in the adult testes, whereas beta-galactosidase expression is exclusively present in postmeiotic germ cells. Thus, these observations strongly suggest that dj transcripts are under translational repression until in spermiogenesis. To study the function and subcellular distribution of DJ in spermiogenesis we expressed a chimaeric dj-GFP fusion gene in the male germline exhibiting strong GFP fluorescence in the liver testes, where only elongated spermatids are decorated. With regard to the characteristic expression pattern of DJ protein and its conspicuous repeat units possible functional roles are discussed.

  3. Ability of sulfated glycoconjugates and disulfide-reductants to release bovine epididymal sperm bound to the oviductal epithelium in vitro.

    PubMed

    Gualtieri, R; Mollo, V; Barbato, V; Talevi, R

    2010-05-01

    In Bos taurus, at ejaculation, epididymal sperm acquire a number of proteins secreted in the seminal plasma that increase their ability to interact with the female reproductive tract. Sperm-oviduct interaction comprises a transient sperm adhesion to the isthmus, the lower portion of the oviduct, followed by sperm release around ovulation. Oviductal fluid molecules, such as sulfated glycoconjugates and disulfide-reductants, are able to release bovine ejaculated sperm bound to the oviductal epithelium in vitro through the reduction of sperm surface protein disulfides to sulfhydryls. To understand whether the sperm molecules sensitive to releasing signals are already exposed on the surface of epididymal sperm, we studied the ability of cauda epididymal sperm to adhere to the oviductal epithelium and to be released by sulfated glycoconjugates and the disulfide-reductant penicillamine. Surface protein sulfhydryls in cauda epididymal sperm were analyzed in the initial suspension, in sperm bound to the in vitro-cultured oviductal epithelium, and in released sperm. Results showed that epididymal sperm are able to bind the oviductal epithelium in vitro, although at a lower extent than frozen-thawed ejaculated sperm; the interaction is mediated by oviductal cell microvilli that closely bind to the plasma membrane of the sperm head rostral region, as previously shown for ejaculated sperm. The sulfated glycoconjugates heparin, fucoidan, and dextran sulfate, as well as the disulfide-reductant penicillamine, are all powerful inducers of sperm release. The level of sulfhydryls in sperm surface proteins was (1) high in the initial sperm suspension; (2) low in bound sperm; (3) markedly increased in sperm released by heparin or by penicillamine. In conclusion, epididymal sperm are already able to bind the oviductal epithelium and to respond to the inducers of release through the reduction of sperm surface protein disulfides to sulfhydryls. (c) 2010 Elsevier Inc. All rights reserved.

  4. Major outer membrane proteins unique to reproductive cells of Hyphomonas jannaschiana.

    PubMed Central

    Shen, N; Dagasan, L; Sledjeski, D; Weiner, R M

    1989-01-01

    Separation on the basis of molecular weight resolved three proteins specific to the swarmer cell of Hyphomonas jannaschiana. In the reproductive cell, 4 major proteins were identified as cytoplasmic and 10 were identified as envelope. Of these envelope proteins, one was common to both the inner and outer membranes, four were common to the inner membrane, and five were common to the outer membrane. Four of these outer membrane proteins were specific to the reproductive cell, and two of these proteins, with apparent molecular weights of 116,000 and 29,000, constituted 19% of the total cell protein and 54% of the outer membrane protein. Images PMID:2703471

  5. Drosophila Sperm Motility in the Reproductive Tract1

    PubMed Central

    Yang, Yong; Lu, Xiangyi

    2011-01-01

    Motile cilia and flagella exhibit many waveforms as outputs of dynein activation sequences on the highly conserved axoneme. Motility change of sperm in the reproductive tract is difficult to study and remains an important area of investigation. Sperm typically execute a sinusoidal waveform. Increased viscosity in the medium induces somewhat unusual arc-line and helical waveforms in some sperm. However, whether the latter two waveforms occur in vivo is not known. Using green fluorescence protein imaging, we show that Drosophila sperm in the uterus move in circular foci via arc-line waves, predominantly in a tail-leading orientation. From the uterus, a small fraction of the sperm enters the seminal receptacle (SR) in parallel formations. After sperm storage and coincident with fertilization of the egg, the sperm exit the SR via head-leading helical waves. Consistent with the observed bidirectional movements, the sperm show the ability to propagate both base-to-tip and tip-to-base flagellar waves. Numerous studies have shown that sperm motility is regulated by intraflagellar calcium concentrations; in particular, the Pkd2 calcium channel has been shown to affect sperm storage. Our analyses here suggest that Pkd2 is required for the sperm to adopt the correct waveform and movement orientation during SR entry. A working model for the sperm's SR entry movement is proposed. PMID:21293028

  6. Reproductive development of Santa Inês rams during the first year of life: body and testis growth, testosterone concentrations, sperm parameters, age at puberty and seminal plasma proteins.

    PubMed

    Souza, C E A; Araújo, A A; Oliveira, J T A; Lima Souza, A C; Neiva, J N M; Moura, A A

    2010-08-01

    We have investigated the reproductive development of the tropically adapted Santa Inês ram, the most common hair sheep in Brazil. From 8 to 48 weeks of age, 16 animals were evaluated for body and testis growth, semen parameters, testosterone concentrations and seminal plasma proteins, using two-dimensional SDS-PAGE. Animals were weaned at 30 days and kept in feedlots thereafter, receiving hay, concentrate (18% of crude protein) and mineral supplement. Body weight increased from 12.3 +/- 0.7 to 54.3 +/- 1.6 kg between 8 and 48 weeks (p < 0.05), but changes in thoracic perimeter and scrotal circumference were non-significant after 36 weeks (p > 0.05). The percentage of motile sperm increased slowly until 23 weeks and more rapidly after that age, but significant changes in progressive motility occurred after 25 weeks. Presence of abnormal sperm related inversely to age. Most significant changes in sperm concentration occurred between 38 and 44 weeks (0.38 +/- 0.05 to 1.14 +/- 0.24 x 10(9) cells/ml, p < 0.05) and testosterone reached its highest concentrations at 42 weeks, decreasing afterwards. Rams reached puberty at 28.2 +/- 0.8 weeks. The number of protein spots on seminal plasma gels was similar from 15 to 18 weeks (45 and 47 spots; p > 0.05), increased until 24 weeks (141 spots) and 28 weeks (170 spots; p < 0.05) and remained without significant (p > 0.05) changes from 28 to 48 weeks (186 +/- 10 spots). Furthermore, the intensity of selected spots on 2D maps increased (p < 0.05) between 15 and 28 weeks, which preceded or coincided with the main developmental changes in sperm motility and percentage of defective sperm in the ejaculates. These results will support future studies designed to characterize specific seminal plasma proteins whose expression relate to the development of testis, epididymis and accessory sex glands.

  7. Major vault protein regulates cell growth/survival signaling through oxidative modifications.

    PubMed

    Das, Dividutta; Wang, Yi-Hsuan; Hsieh, Cheng-Ying; Suzuki, Yuichiro J

    2016-01-01

    Major vault protein forms a hollow, barrel-like structure in the cell called the vault, whose functions and regulation are not well understood. The present study reports that major vault protein regulates growth/survival signaling in human airway smooth muscle cells through oxidative modifications. The promotion of protein S-glutathionylation by asthma mediators such as interleukin-22 and platelet-derived growth factor or by knocking down glutaredoxin-1 or thioredoxin activated cell growth signaling. Mass spectrometry identified that major vault protein is glutathionylated. Major vault protein knockdown enhanced cell death and inhibited STAT3 and Akt signaling. We identified a protein partner of major vault protein that is regulated by glutaredoxin-1, namely myosin-9, which was found to serve as a cell death factor. Knocking down myosin-9 or promoting protein S-glutathionylation by knocking down glutaredoxin-1 inhibited the death of airway smooth muscle cells by heating to simulate bronchial thermoplasty, a clinically successful procedure for the treatment of severe asthma. These results establish a novel signaling pathway in which ligand/receptor-mediated oxidation promotes the S-glutathionylation of major vault protein, which in turn binds to myosin-9 to suppress the heating-induced death of airway smooth muscle cells.

  8. Sperm release pathway

    MedlinePlus

    ... vas deferens, a muscular tube, which propels sperm forward through smooth muscle contractions. The sperm arrive first ... added. From the ampulla, seminal fluid is propelled forward through the ejaculatory ducts toward the urethra, passing ...

  9. RAB-Like 2 Has an Essential Role in Male Fertility, Sperm Intra-Flagellar Transport, and Tail Assembly

    PubMed Central

    Lo, Jennifer C. Y.; Jamsai, Duangporn; O'Connor, Anne E.; Borg, Claire; Clark, Brett J.; Whisstock, James C.; Field, Mark C.; Adams, Vicki; Ishikawa, Tomomoto; Aitken, R. John; Whittle, Belinda; Goodnow, Christopher C.; Ormandy, Christopher J.; O'Bryan, Moira K.

    2012-01-01

    A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein–protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum. PMID:23055941

  10. Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane

    PubMed Central

    Du, Jian; Shen, Jian; Wang, Yuanxian; Pan, Chuanying; Pang, Weijun; Diao, Hua; Dong, Wuzi

    2016-01-01

    Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity (p < 0.05), increased total antioxidant capacity (T-AOC) activity and decreased malondialdehyde (MDA) content. The diluent containing four times concentration of exosomes compared to original semen was determined to inhibit premature capacitation, but not to influence capacitation induced in vitro. Inhibition of premature capacitation is likely related to the concentration of exosomes which had been demonstrated to transfer proteins including AWN and PSP-1 into sperm. In addition, using fluorescence microscopy and scanning electron microscopy analysis, it was demonstrated that exosomes in diluent were directly binding to the membrane of sperm head which could improve sperm plasma membrane integrity. PMID:27542209

  11. Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane.

    PubMed

    Du, Jian; Shen, Jian; Wang, Yuanxian; Pan, Chuanying; Pang, Weijun; Diao, Hua; Dong, Wuzi

    2016-09-13

    Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity (p < 0.05), increased total antioxidant capacity (T-AOC) activity and decreased malondialdehyde (MDA) content. The diluent containing four times concentration of exosomes compared to original semen was determined to inhibit premature capacitation, but not to influence capacitation induced in vitro. Inhibition of premature capacitation is likely related to the concentration of exosomes which had been demonstrated to transfer proteins including AWN and PSP-1 into sperm. In addition, using fluorescence microscopy and scanning electron microscopy analysis, it was demonstrated that exosomes in diluent were directly binding to the membrane of sperm head which could improve sperm plasma membrane integrity.

  12. Sperm donation in Israel.

    PubMed

    Mor-Yosef, S; Schenker, J G

    1995-04-01

    Science and technology in the field of human reproduction present new legal, ethical and religious questions which do not always have immediate answers. The first step in the rapidly developed field of reproductive technology was the use of sperm donation (artificial insemination by donor, AID) and the establishment of sperm banks. The state of Israel faced these problems when the regulations for sperm donation were discussed. The fact that the main holy places for the three monotheistic religions are in Israel directly influences the make-up of the population constituents. Therefore, besides a majority of secular people, a high percentage of the population of Israel is very religious: Jews, Moslems and Christians. Thus any resolution relating to AID should take this demographic combination into account. The practice of AID is opposed by the different monotheistic religions. To avoid the conflict between secular and religious people, and between the different religions' perspectives, the legal problem of AID in Israel was solved not by laws but by regulations which were published by the Ministry of Health. The main idea behind this attitude is that the state and its authorities should not and do not deal with ethical or religious questions. Thus, the decision was left to the couples and to the donors. The regulations address technical requirements, health problems and confidential issues concerning the couple, the donor and the child. In this paper we present the different views relating to these problems as perceived by the different religions, and describe the solution that was accepted by the Israeli Ministry of Health.

  13. In silico identification of the genes for sperm-egg interaction in the internal fertilization of the newt Cynops pyrrhogaster.

    PubMed

    Watanabe, Akihiko; Takayama-Watanabe, Eriko

    2014-01-01

    A specific sperm-egg interaction in the oviductal matrix is crucial for internal fertilization of the red-bellied newt, Cynops pyrrhogaster. An understanding of the molecular basis of this interaction is expected to elucidate the evolutionary history of internal fertilization in amphibians. Recently, deep sequencing technology has provided global gene information even in nonmodel animals, allowing us to understand specific features of the molecular mechanisms underlying fertilization in C. pyrrhogaster. In the present study, we screened de novo assembled RNAseq from ovary, testis, and oviduct samples in C. pyrrhogaster and identified the base sequences encoding zona pellucida (ZP) proteins, voltage-dependent Ca(2+) channels, and cysteine-rich secretory proteins (CRISPs), which respectively are sperm receptors for egg envelopes, major mediators of sperm intracellular signaling, and expected extracellular modulators for sperm function in the female reproductive tract. In the ovary, ZP homologues of all six subgroups were found, including a ZP1 homologue that was newly found in amphibians, a ZP4 homologue, and six ZPC homologues. The unique combination of ZP proteins suggests a new mechanism for sperm binding to egg envelopes in the internal fertilization of C. pyrrhogaster. In the testis, CaV1.1, 1.2, and 3.2, which are L- and T-type voltage-dependent Ca(2+) channels, were found as potential mediators for the internal fertilization-specific sperm-egg interaction. We also found CRISP 2 in the oviduct, which is speculated to participate in the sperm-egg interaction. These results indicate that the de novo assembled RNAseq is a powerful tool allowing analysis of the specific sperm-egg interactions in C. pyrrhogaster.

  14. Flow cytometry of sperm

    SciTech Connect

    Gledhill, B.L.

    1987-09-21

    This brief paper summarizes automated flow cytometric determination of sperm morphology and flow cytometry/sorting of sperm with application to sex preselection. In the latter context, mention is made of results of karyotypic determination of sex chromosome ratios in albumin-processed human sperm. 23 refs., 1 fig., 1 tab.

  15. Cryoprotective effect of resveratrol on DNA damage and crucial human sperm messenger RNAs, possibly through 5' AMP-activated protein kinase activation.

    PubMed

    Shabani Nashtaei, Maryam; Nekoonam, Saeid; Naji, Mohammad; Bakhshalizadeh, Shabnam; Amidi, Fardin

    2017-07-26

    This work aimed at investigating the effect of resveratrol on (1) DNA integrity and (2) fertilizing capacity of sperm by quantifying the presence of key paternal transcripts considered as markers for male fertility (protamine 1 [PRM1] and protamine 2 [PRM2]) and pregnancy success (adducin 1 alpha [ADD1]) in cryopreserved human spermatozoa through modulation of AMP-activated protein kinase (AMPK). The study populations was drawn from 22 normozoospermic healthy volunteers which were incubated with or without AMPK activator (resveratrol [RSV], 15 µM) or inhibitor (Compound C [CC], 30 µM) for 1 h and were then cryopreserved. Untreated frozen-thawed spermatozoa served as controls. The RSV-induced AMPK activation decreased the level of DNA fragmentation in comparison with the control (21.18 ± 0.92 vs. 22.50 ± 0.40; p < 0.01). The relative mRNA expression levels of protamines (1 and 2) and ADD1 in RSV pretreated frozen-thawed human spermatozoa were also improved significantly compared to the control (p < 0.05). Conversely, the inhibitory effect of CC on AMPK activity deteriorated the deleterious effects of cryopreservation on these parameters (p < 0.01). In conclusion, these results demonstrated the cryoprotective effect of the RSV-induced increase in AMPK activity on DNA integrity and key paternal transcripts of cryopreserved human spermatozoa. These findings are of great importance for improving the available cryopreservation protocols in terms of the number of lesions that produced over key genes and the dramatic effects on sperm DNA fragmentation.

  16. 2D-DIGE proteome analysis on the platelet proteins of patients with major depression

    PubMed Central

    2014-01-01

    Introduction Platelet activation is related to the psychopathology of major depression. We attempted to search and identify protein biomarkers from the platelets of patients with major depression. High resolution two-dimensional Differential Gel Electrophoresis (2D-DIGE), the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), Western blot, and bioinformatic tools were applied to examine the platelet proteins of 10 patients with major depression and 10 healthy controls. Results The levels of 8 proteins were significantly different between the patients with major depression in the acute phase and healthy controls. The levels of protein disulfide-isomerase A3 (PDIA3) and F-actin-capping protein subunit beta (CAPZB) were higher in patients with major depression than in healthy controls. The levels of fibrinogen beta chain (FIBB), fibrinogen gamma chain (FIBG), retinoic acid receptor beta (RARB), glutathione peroxidase 1 (GPX1), SH3 domain-containing protein 19 (SH319), and T-complex protein 1 subunit beta (TCPB) were lower in patients with major depression than in healthy controls. Conclusions Platelet provided valuable information about the pathways and processes of inflammation/immunity, oxidative stress, and neurogenesis, related to major depression. PMID:24383611

  17. The creation of transgenic pigs expressing human proteins using BAC-derived, full-length genes and intracytoplasmic sperm injection-mediated gene transfer.

    PubMed

    Watanabe, Masahito; Kurome, Mayuko; Matsunari, Hitomi; Nakano, Kazuaki; Umeyema, Kazuhiro; Shiota, Akira; Nakauchi, Hiromitsu; Nagashima, Hiroshi

    2012-06-01

    In most transgenic (Tg) animals created to date, a transgene consisting of the minimum promoter region linked to a cDNA has been used. However, transgenes on small plasmids are susceptible to the position effect, increasing the difficulty of controlling transgene expression. In this study, we attempted to create Tg pigs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) using two large genomic transgenes derived from a bacterial artificial chromosome (BAC) containing the full genomic region encoding two human proteins, type I collagen and albumin. The production efficiencies (Tg piglets/live offspring) of type I collagen and albumin Tg pigs were 11.8% (6/51) and 18.2% (2/11), respectively. In all of the Tg pigs examined by real-time PCR analysis, tissue-specific expression of the transgene was confirmed (type I collagen: skin, tendon, vessels, genitalia; albumin: liver). The production of human proteins derived from BAC transgenes was also confirmed. Fluorescence in situ hybridization analysis indicated that the BAC transgenes transferred into porcine oocytes by ICSI-MGT were integrated into single or multiple sites on the host chromosomes. These data demonstrate that Tg pigs expressing human proteins in a tissue-specific manner can be created using a BAC transgenic construct and the ICSI-MGT method.

  18. Assessment of sperm DNA fragmentation in stallion (Equus caballus) and donkey (Equus asinus) using the sperm chromatin dispersion test.

    PubMed

    Cortés-Gutiérrez, E I; Crespo, F; Serres-Dalmau, C; Gutiérrez de las Rozas, A L; Dávila-Rodríguez, M I; López-Fernández, C; Gósalvez, J

    2009-10-01

    Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys.

  19. Comparative Sperm Proteomics in Mouse Species with Divergent Mating Systems

    PubMed Central

    Vicens, Alberto; Borziak, Kirill; Karr, Timothy L.; Roldan, Eduardo R.S.

    2017-01-01

    Abstract Sexual selection is the pervasive force underlying the dramatic divergence of sperm form and function. Although it has been demonstrated that testis gene expression evolves rapidly, exploration of the proteomic basis of sperm diversity is in its infancy. We have employed a whole-cell proteomics approach to characterize sperm divergence among closely related Mus species that experience different sperm competition regimes and exhibit pronounced variation in sperm energetics, motility and fertilization capacity. Interspecific comparisons revealed significant abundance differences amongst proteins involved in fertilization capacity, including those that govern sperm-zona pellucida interactions, axoneme components and metabolic proteins. Ancestral reconstruction of relative testis size suggests that the reduction of zona pellucida binding proteins and heavy-chain dyneins was associated with a relaxation in sperm competition in the M. musculus lineage. Additionally, the decreased reliance on ATP derived from glycolysis in high sperm competition species was reflected in abundance decreases in glycolytic proteins of the principle piece in M. spretus and M. spicilegus. Comparison of protein abundance and stage-specific testis expression revealed a significant correlation during spermatid development when dynamic morphological changes occur. Proteins underlying sperm diversification were also more likely to be subject to translational repression, suggesting that sperm composition is influenced by the evolution of translation control mechanisms. The identification of functionally coherent classes of proteins relating to sperm competition highlights the utility of evolutionary proteomic analyses and reveals that both intensified and relaxed sperm competition can have a pronounced impact on the molecular composition of the male gamete. PMID:28333336

  20. Molecular architecture of the human sperm IZUMO1 and egg JUNO fertilization complex.

    PubMed

    Aydin, Halil; Sultana, Azmiri; Li, Sheng; Thavalingam, Annoj; Lee, Jeffrey E

    2016-06-23

    Fertilization is an essential biological process in sexual reproduction and comprises a series of molecular interactions between the sperm and egg. The fusion of the haploid spermatozoon and oocyte is the culminating event in mammalian fertilization, enabling the creation of a new, genetically distinct diploid organism. The merger of two gametes is achieved through a two-step mechanism in which the sperm protein IZUMO1 on the equatorial segment of the acrosome-reacted sperm recognizes its receptor, JUNO, on the egg surface. This recognition is followed by the fusion of the two plasma membranes. IZUMO1 and JUNO proteins are indispensable for fertilization, as constitutive knockdown of either protein results in mice that are healthy but infertile. Despite their central importance in reproductive medicine, the molecular architectures of these proteins and the details of their functional roles in fertilization are not known. Here we present the crystal structures of human IZUMO1 and JUNO in unbound and bound conformations. The human IZUMO1 structure exhibits a distinct boomerang shape and provides structural insights into the IZUMO family of proteins. Human IZUMO1 forms a high-affinity complex with JUNO and undergoes a major conformational change within its N-terminal domain upon binding to the egg-surface receptor. Our results provide insights into the molecular basis of sperm-egg recognition, cross-species fertilization, and the barrier to polyspermy, thereby promising benefits for the rational development of non-hormonal contraceptives and fertility treatments for humans and other mammals.

  1. Human sperm tail proteome suggests new endogenous metabolic pathways.

    PubMed

    Amaral, Alexandra; Castillo, Judit; Estanyol, Josep Maria; Ballescà, José Luís; Ramalho-Santos, João; Oliva, Rafael

    2013-02-01

    Proteomic studies are contributing greatly to our understanding of the sperm cell, and more detailed descriptions are expected to clarify additional cellular and molecular sperm attributes. The aim of this study was to characterize the subcellular proteome of the human sperm tail and, hopefully, identify less concentrated proteins (not found in whole cell proteome studies). Specifically, we were interested in characterizing the sperm metabolic proteome and gaining new insights into the sperm metabolism issue. Sperm were isolated from normozoospermic semen samples and depleted of any contaminating leukocytes. Tail fractions were obtained by means of sonication followed by sucrose-gradient ultracentrifugation, and their purity was confirmed via various techniques. Liquid chromatography and tandem mass spectrometry of isolated sperm tail peptides resulted in the identification of 1049 proteins, more than half of which had not been previously described in human sperm. The categorization of proteins according to their function revealed two main groups: proteins related to metabolism and energy production (26%), and proteins related to sperm tail structure and motility (11%). Interestingly, a great proportion of the metabolic proteome (24%) comprised enzymes involved in lipid metabolism, including enzymes for mitochondrial beta-oxidation. Unexpectedly, we also identified various peroxisomal proteins, some of which are known to be involved in the oxidation of very long chain fatty acids. Analysis of our data using Reactome suggests that both mitochondrial and peroxisomal pathways might indeed be active in sperm, and that the use of fatty acids as fuel might be more preponderant than previously thought. In addition, incubation of sperm with the fatty acid oxidation inhibitor etomoxir resulted in a significant decrease in sperm motility. Contradicting a common concept in the literature, we suggest that the male gamete might have the capacity to obtain energy from endogenous

  2. Gamete evolution and sperm numbers: sperm competition versus sperm limitation.

    PubMed

    Parker, Geoff A; Lehtonen, Jussi

    2014-09-22

    Both gamete competition and gamete limitation can generate anisogamy from ancestral isogamy, and both sperm competition (SC) and sperm limitation (SL) can increase sperm numbers. Here, we compare the marginal benefits due to these two components at any given population level of sperm production using the risk and intensity models in sperm economics. We show quite generally for the intensity model (where N males compete for each set of eggs) that however severe the degree of SL, if there is at least one competitor for fertilization (N - 1 ≥ 1), the marginal gains through SC exceed those for SL, provided that the relationship between the probability of fertilization (F) and increasing sperm numbers (x) is a concave function. In the risk model, as fertility F increases from 0 to 1.0, the threshold SC risk (the probability q that two males compete for fertilization) for SC to be the dominant force drops from 1.0 to 0. The gamete competition and gamete limitation theories for the evolution of anisogamy rely on very similar considerations: our results imply that gamete limitation could dominate only if ancestral reproduction took place in highly isolated, small spawning groups. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  3. Characterization of the major hnRNP proteins from Drosophila melanogaster

    PubMed Central

    1992-01-01

    To better understand the role(s) of hnRNP proteins in the process of mRNA formation, we have identified and characterized the major nuclear proteins that interact with hnRNAs in Drosophila melanogaster. cDNA clones of several D. melanogaster hnRNP proteins have been isolated and sequenced, and the genes encoding these proteins have been mapped cytologically on polytene chromosomes. These include the hnRNP proteins hrp36, hrp40, and hrp48, which together account for the major proteins of hnRNP complexes in D. melanogaster (Matunis et al., 1992, accompanying paper). All of the proteins described here contain two amino-terminal RNP consensus sequence RNA-binding domains and a carboxyl-terminal glycine-rich domain. We refer to this configuration, which is also found in the hnRNP A/B proteins of vertebrates, as 2 x RBD-Gly. The sequences of the D. melanogaster hnRNP proteins help define both highly conserved and variable amino acids within each RBD and support the suggestion that each RBD in multiple RBD-containing proteins has been conserved independently and has a different function. Although 2 x RBD-Gly proteins from evolutionarily distant organisms are conserved in their general structure, we find a surprising diversity among the members of this family of proteins. A mAb to the hrp40 proteins crossreacts with the human A/B and G hnRNP proteins and detects immunologically related proteins in divergent organisms from yeast to man. These data establish 2 x RBD-Gly as a prevalent hnRNP protein structure across eukaryotes. This information about the composition of hnRNP complexes and about the structure of hnRNA-binding proteins will facilitate studies of the functions of these proteins. PMID:1730754

  4. Differential extraction and protein sequencing reveals major differences in patterns of primary cell wall proteins from plants.

    PubMed

    Robertson, D; Mitchell, G P; Gilroy, J S; Gerrish, C; Bolwell, G P; Slabas, A R

    1997-06-20

    The proteins of the primary cell walls of suspension cultured cells of five plant species, Arabidopsis, carrot, French bean, tomato, and tobacco, have been compared. The approach that has been adopted is differential extraction followed by SDS-polyacrylamide gel electrophoresis (PAGE), rather than two-dimensional gel analysis, to facilitate protein sequencing. Whole cells were washed sequentially with the following aqueous solutions, CaCl2, CDTA (cyclohexane diaminotetraacetic acid, DTT (dithiothreitol), NaCl, and borate. SDS-PAGE analysis showed consistent differences between species. From the 233 proteins that were selected for sequencing, 63% gave N-terminal data. This analysis shows that (i) patterns of proteins revealed by SDS-PAGE are strikingly different for all five species, (ii) a large number of these proteins cannot be identified by data base searches indicating that a significant proportion of wall proteins have not been previously described, (iii) the major proteins that can be identified belong to very different classes of proteins, (iv) the majority of proteins found in the extracellular growth media are absent from their respective cell wall extracts, and (v) the results of the extraction process are indicative of higher order structure. It appears that aspects of speciation reside in the complement of extracellular wall proteins. The data represent a protein resource for cell wall studies complementary to EST (expressed sequence tag) and DNA sequencing strategies.

  5. Expression and Purification of Sperm Whale Myoglobin

    ERIC Educational Resources Information Center

    Miller, Stephen; Indivero, Virginia; Burkhard, Caroline

    2010-01-01

    We present a multiweek laboratory exercise that exposes students to the fundamental techniques of bacterial expression and protein purification through the preparation of sperm whale myoglobin. Myoglobin, a robust oxygen-binding protein, contains a single heme that gives the protein a reddish color, making it an ideal subject for the teaching…

  6. Expression and Purification of Sperm Whale Myoglobin

    ERIC Educational Resources Information Center

    Miller, Stephen; Indivero, Virginia; Burkhard, Caroline

    2010-01-01

    We present a multiweek laboratory exercise that exposes students to the fundamental techniques of bacterial expression and protein purification through the preparation of sperm whale myoglobin. Myoglobin, a robust oxygen-binding protein, contains a single heme that gives the protein a reddish color, making it an ideal subject for the teaching…

  7. Involvement of homocysteine, homocysteine thiolactone, and paraoxonase type 1 (PON-1) in the etiology of defective human sperm function.

    PubMed

    Aitken, R J; Flanagan, H M; Connaughton, H; Whiting, S; Hedges, A; Baker, M A

    2016-03-01

    This study reports, for the first time, the significant (p ≤ 0.01) accumulation of homocysteine residues in low density, defective sperm suspensions isolated from patients attending an infertility clinic. This overabundance of homocysteine was not related to a deficiency in folate availability but may have been a reflection of the oxidative stress that characterizes such defective sperm populations. Direct addition of the homocysteine cyclic congener, homocysteine thiolactone, to human spermatozoa resulted in the rapid induction of mitochondrial reactive oxygen species (ROS) generation (p < 0.001), the stimulation of lipid peroxidation (p < 0.01), the promotion of tyrosine phosphorylation (p < 0.001), and the suppression of sperm motility (p < 0.001) in the absence of any significant impact on DNA integrity. The parent homocysteine molecule was less active and took 24 h to stimulate mitochondrial ROS production possibly because of the need to convert this compound to the corresponding thiolactone before it could exert a measureable biological effect. Thiolactone was also effective in suppressing the carboxymethylation of key proteins in the sperm tail, which are thought to be involved in the regulation of sperm movement. The major enzyme responsible for removing thiolactone from proteins, paraoxonase (PON-1), was shown to be a major target for alkylation by lipid aldehydes, such as 4-hydroxynonenal, generated as a consequence of oxidative stress. Exposure of human spermatozoa to such aldehydes resulted in a dose-dependent accumulation of homocysteine in spermatozoa (p < 0.03). These results suggest that one of the consequences of oxidative stress in mammalian spermatozoa is the inhibition of PON-1, which then enhances the availability of homocysteine thiolactone to interact with the epsilon-amino group of lysine residues on sperm proteins, triggering a raft of significant biological changes in these cells that ultimately compromise sperm function.

  8. Increased sperm ubiquitination correlates with abnormal chromatin integrity.

    PubMed

    Hodjat, M; Akhondi, M A; Al-Hasani, S; Mobaraki, M; Sadeghi, M R

    2008-09-01

    Ubiquitin, a 8.5 kDa peptide that marks other proteins for proteasomal degradation, tags defective spermatozoa during epididymal passage and is proposed as a biomarker for sperm quality. The present study was designed to evaluate the relationships between sperm ubiquitination, sperm chromatin integrity and semen parameters. Semen samples from 63 couples were collected and analysed according to World Health Organization criteria. Each sample was evaluated for sperm ubiquitination by the direct immunofluorescence method, using anti-ubiquitin antibodies. Chromatin integrity of the same samples was analysed using acridine orange (AO) and toluidine blue (TB) tests. A positive correlation was found between ubiquitinated spermatozoa and the percentage of spermatozoa with abnormal chromatin (AO: r = 0.58, P < 0.001 and TB: r = 0.48, P < 0.001). Negative correlations were obtained between sperm ubiquitination and: sperm count (r = -0.2, P = 0.048), sperm morphology (r = -0.36, P = 0.003), rapidly progressive motility (r = -0.25, P = 0.044) and slow progressive motility (r = -0.28, P = 0.022). Sperm ubiquitination was positively correlated with the percentage of immotile spermatozoa. These results show that among semen parameters, chromatin abnormality is more closely associated with sperm ubiquitination and further validate sperm ubiquitination as a suitable marker for sperm quality.

  9. NMR studies of the membrane bound form of filamentous bacteriophage fd and Pfl major coat proteins

    SciTech Connect

    Schiksnis, R.A.; Bogusky, M.J.; Opella, S.J.

    1987-05-01

    The major coat proteins of the fd (M13) and Pf1 filamentous bacteriophage exist as integral membrane proteins during the viral life cycle. These proteins adopt their membrane bound conformations when solubilized by a variety of detergents, and the protein-detergent micelle complexes can be studied using solution NMR techniques. Determination of the structure of the coat proteins in their membrane bound form has been accomplished by qualitative interpretation of 2-dimensional /sup 1/H-/sup 1/H NOE spectra (NOESY). The critical amide proton resonance assignments were made through biosynthetic /sup 15/N labeling and /sup 1/H//sup 15/N heteronuclear chemical shift correlation techniques. The data indicate that both proteins adopt helical conformations within the micelle. The /sup 15/N//sup 1/H heteronuclear NOE has been used to characterize the backbone dynamics of both proteins in micelles. The lipid associated residues of the proteins are rigid on the nanosecond timescale, while the hydrophilic solvent associated N- and C-termini are high mobile. These results complement previously reported protein dynamics studies of membrane bound coat proteins conducted using solid state NMR methods. Solid state NMR studies reported in the literature have also investigated the structure and dynamics of the fd and Pf1 major coat proteins when bound to intact phage. Therefore, structure/dynamics comparisons of the proteins in their structural versus membrane bound forms can be made.

  10. Identification and characterization of the major cell envelope proteins of oral strains of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Di Rienzo, J M; Spieler, E L

    1983-01-01

    The major cell envelope protein compositions of seven Actinobacillus actinomycetemcomitans strains of human origin were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major envelope polypeptides were homogeneous, in relation to molecular weight, in all of the strains that were examined. The characterization of the five major proteins, designated Env1 through Env5, in the leukotoxic strain Y4 revealed that proteins Env2 to -5 may reside in the outer membrane as suggested by differential detergent extractions and 125I-labeling experiments. The proteins did not demonstrate covalent or ionic interactions with the peptidoglycan; however, one protein, Env2, displayed heat-modifiable properties, having apparent molecular weights of 32,000 and 45,000 when heated in sodium dodecyl sulfate at 50 and 100 degrees C, respectively. The protein composition of the extracellular "bleb" material, normally released by strain Y4, was determined, and proteins Env1 to -4 were the predominant protein species found. A comparison of the cell envelope proteins of strain Y4 with those of other members of the human oral flora, including species within the genera Capnocytophaga, Bacteroides, and Fusobacterium, revealed distinct differences on the basis of molecular size and heat-modifiable properties. However, the membrane proteins of Haemophilus aphrophilus showed a remarkable degree of homology with those of A. actinomycetemcomitans. Images PMID:6401694

  11. Identification of the major lipoproteins in crayfish hemolymph as proteins involved in immune recognition and clotting.

    PubMed

    Hall, M; van Heusden, M C; Söderhäll, K

    1995-11-22

    Lipid-containing hemolymph proteins from males of the crayfish Pacifastacus leniusculus were isolated by density gradient ultracentrifugation. Two major lipoproteins, one high density lipoprotein (HDL) and one very high density lipoprotein (VHDL), were characterized. The HDL and the VHDL were found to be identical to two proteins previously studied for their roles in immune recognition and hemolymph clotting, namely the beta-1,3-glucan binding protein and the clotting protein. These results imply that crayfish lipoproteins have dual functions, and that they are involved in immunity, hemolymph clotting, and lipid transport in these animals. Also, the oxygen-transporting protein hemocyanin was found to have a small lipid content.

  12. Boymaw, overexpressed in brains with major psychiatric disorders, may encode a small protein to inhibit mitochondrial function and protein translation.

    PubMed

    Ji, Baohu; Kim, Minjung; Higa, Kerin K; Zhou, Xianjin

    2015-06-01

    The t(1,11) chromosome translocation co-segregates with major psychiatric disorders in a large Scottish family. The translocation disrupts the DISC1and Boymaw (DISC1FP1) genes on chromosomes 1 and 11, respectively. After translocation, two fusion genes are generated. Our recent studies found that the DISC1-Boymaw fusion protein is localized in mitochondria and inhibits oxidoreductase activity, rRNA expression, and protein translation. Mice carrying the DISC1-Boymaw fusion genes display intermediate behavioral phenotypes related to major psychiatric disorders. Here, we report that the Boymaw gene may encode a small protein predominantly localized in mitochondria. The Boymaw protein inhibits oxidoreductase activity, rRNA expression, and protein translation in the same way as the DISC1-Boymaw fusion protein. Interestingly, Boymaw expression is up-regulated by different stressors at RNA and/or protein translational levels. In addition, we found that Boymaw RNA expression is significantly increased in the postmortem brains of patients with major psychiatric disorders. Our studies therefore suggest that the Boymaw gene could potentially be a susceptibility gene for major psychiatric disorders in both the Scottish t(1,11) family and the general population of patients.

  13. Purification and properties of hyaluronidase from bull sperm.

    PubMed

    Yang, C H; Srivastava, P N

    1975-01-10

    Hyaluronidase from bull sperm was fractionated by ammonium sulfate and further purified by DEAE-cellulose and Sephadex chromatography. The highly purified hyaluronidase preparation showed 2,370 units per mg of protein (68,730 N.F. units per mg of protein), i.e. 182-fold purification. Disc gel electrophoresis showed one major component. The molecular weight of bull sperm hyaluronidase was 62,000 by sodium dodecyl sulfate gel electrophoresis. Hyaluronidase from bull sperm has optimum activity at pH 3.8 and an absolute requirement for cations. Kplus and Naplus have a greater effect than Ca2plus, Mg2plus, and Mn2plus, whereas Co2plus, Cu2plus, and Zn2plus do not affect the enzyme activity. Purified preparations are less stable than crude extracts stored frozen at minus 15 degrees. Km of hyaluronidase with hyaluronic acid as substrate is 3.7 mg per ml and Vmax is 2.4 mumol per min by Hofstee plot.

  14. Identification, Purification and Characterization of Major Antigenic Proteins of Campylobacter jejuni

    DTIC Science & Technology

    1991-01-01

    ELISA -We next examined the potential application of antibodies to C. jejuni proteins for identification and diagnosis of Campylobacter and/or Helico...EXTRACT ANTI-PEBI Fio;. 5. Recognition of Campylobacter and Helicobacter t)ISCtTSSION cells by antisera to C. jejuni proteins by ELISA . Whoile...AD-A271 905 5 April 1991 Reprint Identification, Purification, and Characterization Army Project Order of Major Antigenic Proteins of Campylobacter

  15. Observation of sperm-head vacuoles and sperm morphology under light microscope.

    PubMed

    Park, Yong-Seog; Park, Sol; Ko, Duck Sung; Park, Dong Wook; Seo, Ju Tae; Yang, Kwang Moon

    2014-09-01

    The presence of sperm-head vacuoles has been suspected to be deleterious to the outcomes of assisted reproductive technology (ART). It is difficult to accurately distinguish morphologically abnormal sperm with vacuoles under a light microscope. This study was performed to analyze the result of the observation of sperm-head vacuoles using Papanicolaou staining under a light microscope and whether the male partner's age affects these vacuoles. Sperm morphology with vacuoles was evaluated using Papanicolaou staining and observed under a light microscope (400×) in 980 men. The normal morphology was divided into three categories (group A, <4% of normal morphology; group B, 4%-14% of normal morphology; and group C, >14% of normal morphology). The criteria for the sperm-head vacuoles were those given in the World Health Organization manual. For the analysis of the age factor, the participants were divided into the following groups: 26-30 years, 31-35 years, 36-40 years, 41-45 years, and 46-50 years. The percentage of sperm-head vacuoles increased with normal sperm morphology (group A vs. groups B, C) (p<0.05). In the case of the age factor, a statistically significant difference was not observed across any of the age groups. A majority of the sperm-head vacuoles showed a statistically significant difference among normal morphology groups. Therefore, we should consider the probability of the percentage of sperm-head vacuoles not increasing with age but with abnormal sperm morphology. A further study is required to clarify the effect of the sperm-head vacuoles on ART outcomes.

  16. Sperm navigation in complex environments

    NASA Astrophysics Data System (ADS)

    Olson, Sarah

    2016-11-01

    Sperm can swim in a variety of environments, interacting with chemicals and other proteins in the fluid. Some of these extra proteins or cells may act as friction, possibly preventing or enhancing forward progression of swimmers. The homogenized fluid flow is assumed to be governed by the incompressible Brinkman equation, where a friction term with a resistance parameter represents a sparse array of obstacles. Representing the swimmers with a centerline approximation, we employ regularized fundamental solutions to investigate swimming speeds, trajectories, and interactions of swimmers. Asymmetric waveforms due to an increase in flagellar calcium is known to be important for sperm to reach and fertilize the egg. The trajectories of hyperactivated swimmers are found to have a decreased path curvature. Although attraction of two swimmers is more efficient in the Stokes regime, we find that attraction does not occur for larger resistance. Additionally, we study interactions of swimmers in a channel. NSF DMS 1413110.

  17. CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline.

    PubMed

    Campbell, Anne C; Updike, Dustin L

    2015-05-15

    Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription.

  18. Methods of sperm DNA extraction for genetic and epigenetic studies.

    PubMed

    Griffin, Jeanine

    2013-01-01

    High quality DNA extractions developed for mammalian somatic cells are ineffective for sperm, due mainly to the high degree of nuclear compaction in sperm. The highly specialized nuclear proteins in sperm create a chromatin structure that is at least six times denser than histone bound DNA. Unlike somatic cells, sperm DNA is highly compacted by the replacement of histones with sperm-specific low molecular weight proteins called protamines. Both the protamines and the disulfide bridges formed within and between protamines inhibit the extraction of sperm DNA by standard techniques used for somatic cells. Here we describe the guanidine thiocyanate method reported by Hossain with additional modifications resulting in high molecular weight DNA of high quality with an A260/280 ratio ranging between 1.8 and 2.0 and an A260/230 ratio of 2.0 and greater. The DNA is efficiently digested with restriction enzymes and amplified by PCR.

  19. Ultrastructure of sperm, spermiogenesis, and sperm-egg interactions in selected invertebrates and lower vertebrates which use external fertilization.

    PubMed

    Koch, R A; Lambert, C C

    1990-10-01

    This review discusses the ultrastructure of sperm with reference to their development, the surface morphology of the egg, and the processes of sperm binding and penetration during fertilization. These topics are treated for selected invertebrates and lower vertebrates which live in aquatic environments and fertilize their eggs externally. Specifically, sperm eggs from cnidarians, echinoderms, decapod crustaceans, ascidians, lampreys, bony fishes, and amphibians are discussed. Sperm from the majority of these groups exhibit the classical head-midregion-tail configuration characteristic of primitive sperm. Specific variations within this general morphology have been described. The notable exceptions to the primitive-sperm paradigm are the sperm of decapod crustaceans and amphibians. Eggs from all of the animals considered are covered by complex vitelline envelopes except those of cnidarians. In general, the ultrastructural analysis of these egg envelopes shows that they are composed of fibrous subunits. Sperm bind to the vitelline envelope and then penetrate through it to fertilize the egg in all groups reviewed except fishes. In sperm ultrastructure which occur during penetration of the egg envelopes in both flagellated and non-flagellated sperm. These changes, which involve membrane fusion and reorganization as well as movement of membranous organelles, aid the sperm in reaching the actual site of gamete fusion.

  20. Mammalian sperm morphometry.

    PubMed Central

    Gage, M J

    1998-01-01

    Understanding the adaptive significance of sperm form and function has been a challenge to biologists because sperm are highly specialized cells operating at a microscopic level in a complex environment. A fruitful course of investigation has been to use the comparative approach. This comparative study attempts to address some fundamental questions of the evolution of mammalian sperm morphometry. Data on sperm morphometry for 445 mammalian species were collated from published sources. I use contemporary phylogenetic analysis to control for the inherent non-independence of species and explore relationships between the morphometric dimensions of the three essential spermatozoal components: head, mid-piece and flagellum. Energy for flagellar action is metabolized by the mitochondrial-dense mid-piece and these combine to propel the sperm head, carrying the male haplotype, to the ovum. I therefore search for evolutionary associations between sperm morphometry and body mass, karyotype and the duration of oestrus. In contrast to previous findings, there is no inverse correlation between body weight and sperm length. Sperm mid-piece and flagellum lengths are positively associated with both head length and area, and the slopes of these relationships are discussed. Flagellum length is positively associated with mid-piece length but, in contrast to previous research and after phylogenetic control, I find no relationship between flagellum length and the volume of the mitochondrial sheath. Sperm head dimensions are not related to either genome mass or chromosome number, and there are no relationships between sperm morphometry and the duration of oestrus. PMID:9474794

  1. Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli.

    PubMed Central

    Yenugu, Suresh; Hamil, Katherine G; Birse, Charles E; Ruben, Steven M; French, Frank S; Hall, Susan H

    2003-01-01

    During passage through the epididymis, sperm interact with secreted epididymal proteins that promote maturation, including the acquisition of motility and fertilization competence. Viewed previously as distinct from sperm maturation, host defence capabilities are now recognized functions of the human epididymis 2 (HE2) family of sperm-binding proteins. We analysed the potent dose and time-dependent bactericidal activity of recombinant HE2alpha, HE2beta1 and HE2beta2 and found that the full-length proteins (10 microg/ml or approximately 1 microM) caused more than a 50% decrease in Escherichia coli colony forming units within 15 min. By contrast, human beta-defensin-1, at a similar concentration, required more than 90 min to exhibit similar antibacterial activity. The epididymis-specific lipocalin, LCN6, failed to kill bacteria. Higher concentrations (25-100 microg/ml) of HE2 proteins and a longer duration of treatment resulted in near total inhibition of bacterial growth. The C-terminal peptides of HE2alpha, HEbeta1 and HEbeta2 proteins exhibited antibacterial activity similar to their full-length counterparts, indicating that the antibacterial activity of HE2 proteins resides in these C-terminal regions. Antibacterial activities of HE2 proteins and peptides were slightly inhibited by NaCl concentrations of up to 150 mM, while human beta-defensin-1 activity was nearly eliminated. Reduction and alkylation of disulphide bonds in HE2 proteins and their C-terminal peptides abolished their antibacterial activity. Consistent with the ability to kill bacteria, full-length HE2 proteins and C-terminal peptides caused rapid dose-dependent permeabilization of outer and cytoplasmic E. coli membranes. A much longer exposure time was required for human beta-defensin-1-mediated permeabilization of membranes, suggesting a possible difference in mode of action compared with the HE2 antibacterial peptides. PMID:12628001

  2. Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion.

    PubMed

    Coonrod, S A; Naaby-Hansen, S; Shetty, J; Shibahara, H; Chen, M; White, J M; Herr, J C

    1999-03-15

    The effect of phosphatidyinositol-specific phospholipase C (PI-PLC) on mouse sperm-egg interaction was investigated in this study to determine if glycosyl-phosphatidylinositol (GPI)-anchored proteins are involved in mammalian fertilization. When both sperm and zona-intact oocytes were pretreated with a highly purified preparation of PI-PLC and coincubated, there was no significant effect on sperm-zona pellucida binding; however, fertilization was reduced from 59.6% (control group) to 2.8% (treatment group). A similar reduction in fertilization rates was found when zona-intact oocytes were treated with PI-PLC and washed prior to incubation with untreated sperm. The effect of PI-PLC on sperm binding and fusion with zona-free oocytes was then investigated. Treatment of sperm with PI-PLC had no significant effect on sperm-egg binding or fusion. However, treatment of eggs with PI-PLC significantly reduced sperm-egg binding and fusion from 6.2 bound and 2.1 fused sperm per egg in the control group to 2.1 bound and 0.02 fused sperm per egg in the treatment group. This decrease in sperm-egg binding and fusion depended on the dose of PI-PLC employed, with a maximal inhibitory effect on binding and fusion at 5 and 1 U/ml, respectively. PI-PLC-treated oocytes could be artificially activated by calcium ionophore, demonstrating that the oocytes were functionally viable following treatment. Furthermore, treatment of oocytes with PI-PLC did not reduce the immunoreactivity of the non-GPI-anchored egg surface integrin, alpha6beta1. Taken together, these observations support the hypothesis that PI-PLC affects fertilization by specifically releasing GPI-anchored proteins from the oolemma. In order to identify the oolemmal GPI-anchored proteins involved in fertilization, egg surface proteins were labeled with sulfo-NHS biotin, treated with PI-PLC, and analyzed by two-dimensional gel electrophoresis followed by avidin blotting. A prominent high-molecular-weight protein cluster

  3. Characterization of the major proteins of tubers of yam bean (Pachyrhizus ahipa).

    PubMed

    Forsyth, Jane L; Shewry, Peter R

    2002-03-27

    Tubers of six accessions of ahipa (Pachyrhizus ahipa) contained between 0.77 and 1.34% nitrogen on a dry weight basis. This corresponds to 4.8 to 8.4% crude protein based on a nitrogen to protein conversion factor of 6.25; but detailed analysis of AC230 showed that although 93% of the total N was extracted with buffer containing 1.0 M NaCl, about a third of this was lost on dialysis. It was calculated, therefore, that salt-soluble proteins comprise about 60% of the total tuber nitrogen, with low-molecular-mass nitrogenous components comprising a further 30%. Electophoretic analysis of the salt-soluble proteins showed similar patterns of components in the six accessions, with none being present in amounts sufficiently high to suggest a role as storage proteins. Furthermore, light microscopy failed to show significant deposits of protein within the tuber cells. Five "major" protein bands, which together accounted for about 19% of the total salt-soluble protein fraction were purified and subjected to N-terminal amino acid sequencing. Comparison of these with sequences in protein databases revealed similarities to alpha-amylases, chitinases and chitin binding proteins, cysteine proteinases (including major components from P. erosus tubers), a tuberization-specific protein from potato, and proteins induced in soybean and pea by stress or the plant hormone abscisic acid, respectively. It was concluded that the primary roles of these proteins are probably in aspects of tuber metabolism and development and/or conferring protection to pests and pathogens, and that true storage proteins are not present. The absence of storage proteins is consistent with the biological role of the tubers as storage organs for carbohydrates (cf cassava tuberous roots) rather than as propagules (cf yam and potato tubers).

  4. Acetylproteomic analysis reveals functional implications of lysine acetylation in human spermatozoa (sperm).

    PubMed

    Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian

    2015-04-01

    Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed

  5. Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)*

    PubMed Central

    Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian

    2015-01-01

    Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed

  6. Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

    SciTech Connect

    Butler, C.A.; Hoffman, P.S. )

    1990-05-01

    A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled ((35S)cysteine or (35S)methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid per mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus.

  7. MSP Dynamics and Retraction in Nematode Sperm

    NASA Astrophysics Data System (ADS)

    Wolgemuth, Charles W.

    2005-03-01

    Most eukaryotic cells can crawl over surfaces. In general, this motility requires three distinct actions: polymerization at the leading edge, adhesion to the substrate, and retraction at the rear. Recent in vitro experiments with extracts from spermatozoa from the nematode Ascaris suum suggest that retraction forces are generated by depolymerization of the Major Sperm Protein (MSP) cytoskeleton. Combining polymer entropy with a simple kinetic model for disassembly I propose a model for disassembly-induced retraction that fit the in vitro experimental data. This model explains the mechanism by which deconstruction of the cytoskeleton produces the force necessary to pull the cell body forward and suggest further experiments that can test the validity of the model.

  8. MSP dynamics and retraction in nematode sperm

    NASA Astrophysics Data System (ADS)

    Wolgemuth, Charles; Miao, Long; Vanderlinde, Orion; Roberts, Tom; Oster, George

    2005-03-01

    Most eukaryotic cells can crawl over surfaces. In general, this motility requires three distinct actions: polymerization at the leading edge, adhesion to the substrate, and retraction at the rear. Recent in vitro experiments with extracts from spermatozoa from the nematode Ascaris suum suggest that retraction forces are generated by depolymerization of the Major Sperm Protein (MSP) cytoskeleton. Combining polymer entropy with a simple kinetic model for disassembly I propose a model for disassembly-induced retraction that fit the in vitro experimental data. This model explains the mechanism by which deconstruction of the cytoskeleton produces the force necessary to pull the cell body forward and suggest further experiments that can test the validity of the model.

  9. Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase

    PubMed Central

    Andrews, Rachel E.; Galileo, Deni S.; Martin-DeLeon, Patricia A.

    2015-01-01

    Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca2+ efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca2+, and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca2+ ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca2+-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca2+ concentration ([Ca2+]c) in capacitated sperm than at low [Ca2+]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca2+/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at high [Ca2+]c in

  10. Sperm Flagellum Volume Determines Freezability in Red Deer Spermatozoa

    PubMed Central

    Ros-Santaella, José Luis; Domínguez-Rebolledo, Álvaro Efrén; Garde, José Julián

    2014-01-01

    The factors affecting the inter-individual differences in sperm freezability is a major line of research in spermatology. Poor sperm freezability is mainly characterised by a low sperm velocity, which in turn is associated with low fertility rates in most animal species. Studies concerning the implications of sperm morphometry on freezability are quite limited, and most of them are based on sperm head size regardless of the structural parts of the flagellum, which provides sperm motility. Here, for the first time, we determined the volumes of the flagellum structures in fresh epididymal red deer spermatozoa using a stereological method under phase contrast microscopy. Sperm samples from thirty-three stags were frozen and classified as good freezers (GF) or bad freezers (BF) at two hours post-thawing using three sperm kinetic parameters which are strongly correlated with fertility in this species. Fourteen stags were clearly identified as GF, whereas nineteen were BF. No significant difference in sperm head size between the two groups was found. On the contrary, the GF exhibited a lower principal piece volume than the BF (6.13 µm3 vs 6.61 µm3, respectively, p = 0.006). The volume of the flagellum structures showed a strong negative relationship with post-thawing sperm velocity. For instance, the volume of the sperm principal piece was negatively correlated with sperm velocity at two hours post-thawing (r = −0.60; p<0.001). Our results clearly show that a higher volume of the sperm principal piece results in poor freezability, and highlights the key role of flagellum size in sperm cryopreservation success. PMID:25380133

  11. Sperm flagellum volume determines freezability in red deer spermatozoa.

    PubMed

    Ros-Santaella, José Luis; Domínguez-Rebolledo, Alvaro Efrén; Garde, José Julián

    2014-01-01

    The factors affecting the inter-individual differences in sperm freezability is a major line of research in spermatology. Poor sperm freezability is mainly characterised by a low sperm velocity, which in turn is associated with low fertility rates in most animal species. Studies concerning the implications of sperm morphometry on freezability are quite limited, and most of them are based on sperm head size regardless of the structural parts of the flagellum, which provides sperm motility. Here, for the first time, we determined the volumes of the flagellum structures in fresh epididymal red deer spermatozoa using a stereological method under phase contrast microscopy. Sperm samples from thirty-three stags were frozen and classified as good freezers (GF) or bad freezers (BF) at two hours post-thawing using three sperm kinetic parameters which are strongly correlated with fertility in this species. Fourteen stags were clearly identified as GF, whereas nineteen were BF. No significant difference in sperm head size between the two groups was found. On the contrary, the GF exhibited a lower principal piece volume than the BF (6.13 µm3 vs 6.61 µm3, respectively, p = 0.006). The volume of the flagellum structures showed a strong negative relationship with post-thawing sperm velocity. For instance, the volume of the sperm principal piece was negatively correlated with sperm velocity at two hours post-thawing (r = -0.60; p<0.001). Our results clearly show that a higher volume of the sperm principal piece results in poor freezability, and highlights the key role of flagellum size in sperm cryopreservation success.

  12. Association of seminal plasma motility inhibitors/semenogelins with sperm in asthenozoospermia-infertile men.

    PubMed

    Terai, K; Yoshida, K; Yoshiike, M; Fujime, M; Iwamoto, T

    2010-01-01

    Seminal plasma motility inhibitors (SPMIs) are proteinase-resistant fragments of semenogelin I and II (Sgs), which are the major proteins of semen coagulum. SPMIs inhibit the motility of spermatozoa, and Sgs are thought to be natural regulators of human sperm function. The mechanism underlying sperm motility regulation and its association with defective motility in infertile men remain unclear. The purpose of this study was to investigate the association between SPMIs and spermatozoa in infertile men with asthenozoospermia. Fifty-four semen samples from 37 asthenozoospermic patients and 17 samples from 9 normal healthy subjects were analyzed. Spermatozoa, washed by Percoll density gradients, were immunostained with anti-SPMI antibody and subjected to flow cytometric analysis. The proportion of spermatozoa labeled with the antibody and the average intensity of fluorescence labeling per spermatozoa were analyzed in relation to the parameters used for semen analysis. A significant negative correlation was found between sperm motility and the proportion (R = -0.68) and intensity (R = -0.38) of labeling. These results suggest that SPMIs remain on the sperm surface after liquefaction. This might account for some disorders of sperm motility observed in infertile men with asthenozoospermia.

  13. Sperm Proteome: What Is on the Horizon?

    PubMed

    Mohanty, Gayatri; Swain, Nirlipta; Samanta, Luna

    2015-06-01

    As the mammalian spermatozoa transcends from the testis to the end of the epididymal tubule, the functionally incompetent spermatozoa acquires its fertilizing capability. Molecular changes in the spermatozoa at the posttesticular level concern qualitative and quantitative modifications of proteins along with their sugar moieties and membranous lipids mostly associated with motility, egg binding, and penetration processes. Proteomic studies have identified numerous sperm-specific proteins, and recent reports have provided a further understanding of their function with respect to male fertility. High-throughput techniques such as mass spectrometry have shown drastic potential for the identification and study of sperm proteins. In fact, compelling evidence has provided that proteins are critically important in cellular remodeling event and that aberrant expression is associated with pronounced defects in sperm function. This review highlights the posttesticular functional transformation in the epididymis and female reproductive tract with due emphasis on proteomics.

  14. OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

    SciTech Connect

    Zhong, Wenbin; Zhou, You; Li, Jiwei; Mysore, Raghavendra; Luo, Wei; Li, Shiqian; Chang, Mau-Sun; Olkkonen, Vesa M.; Yan, Daoguang

    2014-04-01

    We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle. - Highlights: • The oxysterol-binding protein ORP8 was found to interact with the mitotic regulator SPAG5/Astrin. • Treatment of HepG2 cells with 25-hydroxycholesterol caused cell cycle retardation in G2/M. • ORP8 overexpression caused a similar G2/M accumulation, and ORP8 knock-down reversed the 25-hydroxycholesterol effect. • Reduction of cellular of SPAG5/Astrin reversed the cell cycle effects of both 25-hydroxycholesterol and ORP8 overexpression. • Our results suggest that ORP8 mediates via SPAG5/Astrin the oxysterol interference of HepG2 cell cycle.

  15. Sperm macromolecules associated with bull fertility.

    PubMed

    Kaya, Abdullah; Memili, Erdoğan

    2016-06-01

    Bull fertility, ability of the sperm to fertilize and activate the egg that sustain embryo development, is vitally important for effective and efficient production of cattle. Fertility is a complex trait with low heritability. Despite recent advances in genomic selection and possibility of enormous paternal benefits to profitable cattle production, there exist no reliable tests for evaluating semen quality and predicting bull fertility. This review focuses on sperm macromolecules such as transcripts, proteins and the epigenome, i.e., the functional genome that are associated with bull fertility. Generating new information in these systems is important beyond agriculture because such progress advances the fundamental science of the mammalian male gamete while at the same time introduces biotechnology into livestock production. Sperm macromolecules and epigenome markers associated with bull fertility can be used alone or in combination with the current SNP microarrays to determine sperm quality and to indicate bull fertility.

  16. Interaction of the major protein from bovine seminal plasma, PDC-109 with phospholipid membranes and soluble ligands investigated by fluorescence approaches.

    PubMed

    Anbazhagan, V; Damai, Rajani S; Paul, Aniruddha; Swamy, Musti J

    2008-06-01

    The major protein from bovine seminal plasma, PDC-109 binds selectively to choline phospholipids on the sperm plasma membrane and plays a crucial role in priming spermatozoa for fertilization. The microenvironment and accessibility of tryptophans of PDC-109 in the native state, in the presence of phosphorylcholine (PrC) and phospholipid membranes as well as upon denaturation have been investigated by fluorescence approaches. Quenching of the protein intrinsic fluorescence by different quenchers decreased in the order: acrylamide>succinimide>Cs(+)>I(-). Ligand binding afforded considerable protection from quenching, with shielding efficiencies following the order: dimyristoylphosphatidylcholine (DMPC)>lysophosphatidylcholine (Lyso-PC)>PrC. This has been attributed to a partial penetration of the protein into the DMPC membranes and Lyso-PC micelles, as well as a further stabilization of the binding due to the interaction of PDC-109 with lipid acyl chains and the resulting tightening of the protein structure, leading to a decreased accessibility of the tryptophan residues. Red-edge excitation shift (REES) studies yielded REES values of 4 nm for both native and denatured PDC-109, whereas reduced and denatured protein gave a REES of only 0.5 nm, clearly indicating that the structural and dynamic features of the microenvironment around the tryptophan residues are retained even after denaturation, presumably due to the constraints imposed on the protein structure by disulfide bonds. Upon binding of PDC-109 to DMPC membranes and Lyso-PC micelles the REES values were reduced to 2.5 and 1.0 nm, respectively, which could be due to the penetration of some parts of the protein, especially the segment containing Trp-90 into the membrane interior, where the red-edge effects are considerably reduced.

  17. Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs.

    PubMed

    Aagaard, Jan E; Yi, Xianhua; MacCoss, Michael J; Swanson, Willie J

    2006-11-14

    Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins.

  18. Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs

    PubMed Central

    Aagaard, Jan E.; Yi, Xianhua; MacCoss, Michael J.; Swanson, Willie J.

    2006-01-01

    Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins. PMID:17085584

  19. Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

    NASA Technical Reports Server (NTRS)

    Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

  20. Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

    NASA Technical Reports Server (NTRS)

    Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

  1. Nuclear magnetic resonance studies of amino acids and proteins. Side-chain mobility of methionine in the crystalline amonio acid and in crystallne sperm whale (Physeter catodon) myoglobin

    SciTech Connect

    Keniry, M.A.; Rothgeb, T.M.; Smith, R.L.; Gutowsky, H.S.; Oldfield, E.

    1983-04-12

    Deuterium (/sup 2/H) nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation times (T/sub 1/) were obtained of L-(epsilon-/sup 2/H/sub 3/)methionine, L-(epsilon-/sup 2/H/sub 3/)methionine in a D,L lattice, and (S-methyl-/sup 2/H/sub 3/)methionine in the crystalline solid state, as a function of temperature, in addition to obtaining /sup 2/H T/sub 1/ and line-width results as a function of temperature on (epsilon-/sup 2/H/sub 3/)methionine-labeled sperm whale (Physeter catodon) myoglobins by using the method of magnetic ordering. Also recorded were /sup 13/C cross-polarization ''magic-angle'' sample-spinning NMR spectra of (epsilon-/sup 13/C)methionine-labeled crystalline cyanoferrimyoglobin (at 37.7 MHz, corresponding to a magnetic field strength of 3.52 T) and of the same protein in aqueous solution. (JMT)

  2. The regulation of spermatogenesis and sperm function in nematodes

    PubMed Central

    Ellis, Ronald E.; Stanfield, Gillian M.

    2014-01-01

    In the nematode C. elegans, both males and self-fertile hermaphrodites produce sperm. As a result, researchers have been able to use a broad range of genetic and genomic techniques to dissect all aspects of sperm development and function. Their results show that the early stages of spermatogenesis are controlled by transcriptional and translational processes, but later stages are dominated by protein kinases and phosphatases. Once spermatids are produced, they participate in many interactions with other cells — signals from the somatic gonad determine when sperm activate and begin to crawl, signals from the female reproductive tissues guide the sperm, and signals from sperm stimulate oocytes to mature and be ovulated. The sperm also show strong competitive interactions with other sperm and oocytes. Some of the molecules that mediate these processes have conserved functions in animal sperm, others are conserved proteins that have been adapted for new roles in nematode sperm, and some are novel proteins that provide insights into evolutionary change. The advent of new techniques should keep this system on the cutting edge of research in cellular and reproductive biology. PMID:24718317

  3. Healthy Sperm: Improving Your Fertility

    MedlinePlus

    ... of fruits and vegetables, which are rich in antioxidants — and might help improve sperm health. Prevent sexually ... Moderate physical activity can increase levels of powerful antioxidant enzymes, which can help protect sperm. Sperm can ...

  4. Chemical definition, cloning, and expression of the major protein of the leprosy bacillus.

    PubMed

    Rivoire, B; Pessolani, M C; Bozic, C M; Hunter, S W; Hefta, S A; Mehra, V; Brennan, P J

    1994-06-01

    The decline in prevalence of leprosy is not necessarily matched by a fall in incidence, emphasizing the need for new antigens to measure disease transmission and reservoirs of infection. Mycobacterium leprae obtained from armadillo tissues was disrupted and subjected to differential centrifugation to arrive at preparations of cell wall, cytoplasmic membrane, and cytosol. By committing 0.3 g of M. leprae to the task, it was possible to isolate from the cytosol and fully define the major cytosolic protein. Amino-terminus sequencing and chemical and enzymatic cleavage, followed by more sequencing and fast atom bombardment-mass spectrometry of fragments, allowed description of the entire amino acid sequence of a protein of 10,675-Da molecular mass. The sequence derived by chemical means is identical to that deduced previously from DNA analysis of the gene of a 10-kDa protein, a GroES analog. The work represents the first complete chemical definition of an M. leprae protein. PCR amplification of the 10-kDa protein gene, when cloned into Escherichia coli with a pTRP expression vector, allowed production of the recombinant protein. Chemical analysis of the expressed protein demonstrated that it exactly reflected the native protein. The recombinant major cytosolic protein appears to be a promising reagent for skin testing, still probably the most appropriate and pragmatic means of measuring incidence of leprosy.

  5. Lipids and proteins--major targets of oxidative modifications in abiotic stressed plants.

    PubMed

    Anjum, Naser A; Sofo, Adriano; Scopa, Antonio; Roychoudhury, Aryadeep; Gill, Sarvajeet S; Iqbal, Muhammad; Lukatkin, Alexander S; Pereira, Eduarda; Duarte, Armando C; Ahmad, Iqbal

    2015-03-01

    Stress factors provoke enhanced production of reactive oxygen species (ROS) in plants. ROS that escape antioxidant-mediated scavenging/detoxification react with biomolecules such as cellular lipids and proteins and cause irreversible damage to the structure of these molecules, initiate their oxidation, and subsequently inactivate key cellular functions. The lipid- and protein-oxidation products are considered as the significant oxidative stress biomarkers in stressed plants. Also, there exists an abundance of information on the abiotic stress-mediated elevations in the generation of ROS, and the modulation of lipid and protein oxidation in abiotic stressed plants. However, the available literature reflects a wide information gap on the mechanisms underlying lipid- and protein-oxidation processes, major techniques for the determination of lipid- and protein-oxidation products, and on critical cross-talks among these aspects. Based on recent reports, this article (a) introduces ROS and highlights their relationship with abiotic stress-caused consequences in crop plants, (b) examines critically the various physiological/biochemical aspects of oxidative damage to lipids (membrane lipids) and proteins in stressed crop plants, (c) summarizes the principles of current technologies used to evaluate the extent of lipid and protein oxidation, (d) synthesizes major outcomes of studies on lipid and protein oxidation in plants under abiotic stress, and finally, (e) considers a brief cross-talk on the ROS-accrued lipid and protein oxidation, pointing to the aspects unexplored so far.

  6. Production and glycosylation of sperm constitutive proteins in the lizard Lacerta vivipara. Evolution during the reproductive period.

    PubMed

    Depeiges, A; Force, A; Dufaure, J P

    1987-01-01

    From epididymal fluid samples taken at three different times during the reproductive period (early April, late April, mid-May), the soluble proteins were separated with one dimensional electrophoresis on polyacrylamide gel. Their evolution was studied: firstly quantitatively, after staining with Coomassie blue, or, for one protein (the "L" protein), by immunodetection; secondly, according to their glycosylation after transfer to nitrocellulose and treatment with a set of labelled lectins: from Wheat germ, Ricinus communis, Lens culinaris, Asparagus pea or Canavalia ensiformis, with or without use of their specific inhibitor sugars. At least 15 proteins underwent a quantitative and/or qualitative evolution, mainly during the month of April. Protein "L" (19 kDa), which is androgen dependent and which fixates on to spermatozoa during their epididymal transit, appears to be little or not glycosylated. By contrast its accumulation in the epididymal canal increases considerably during the month of April. Five other proteins proved to be especially interesting because of their evolution during this same period, notably the MW 94, 67, 35, 29 and 25.5 kDa proteins. With the exception of the 67 kDa all the others increased quantitatively. All were decisively enriched in mannose or in methyl-mannoside residues. The proteins of MW 29 and 25.5 kDa were also enriched in galactose or N-acetyl galactosamine residues. These findings are of physiological significance since they are set up concomitantly with the acquisition of maximum motility of spermatozoa in the distal segment of the epididymis, and they coincide with a very great increase in testosteronemia.

  7. Genetic sperm defects.

    PubMed

    Chenoweth, Peter J

    2005-08-01

    Genetic sperm defects are specific sperm defects, which have been shown to have a genetic mode of transmission. Such genetic linkage, either direct or indirect, has been associated with a number of sperm defects in different species, with this number increasing with improved diagnostic capabilities. A number of sperm defects, which have proven or suspected genetic modes of transmission are discussed herein, with particular emphasis on cattle. These include: 1. Acrosome defects (knobbed, ruffled and incomplete); 2. Head defects (abnormal condensation, decapitated, round head, rolled head, nuclear crest); 3. Midpiece abnormalities ("Dag" defect, "corkscrew" defect, "pseudo-droplet" defect); 4. Tail defects ("tail stump" defect, primary ciliary dyskinesia).

  8. Protein-sparing therapy after major abdominal surgery: lack of clinical effects. Protein-Sparing Therapy Study Group.

    PubMed Central

    Doglietto, G B; Gallitelli, L; Pacelli, F; Bellantone, R; Malerba, M; Sgadari, A; Crucitti, F

    1996-01-01

    OBJECTIVE: A prospective multicenter randomized trial was designed to evaluate the clinical efficacy of postoperative protein-sparing therapy. SUMMARY BACKGROUND DATA: The metabolic effect of postoperative protein-sparing therapy has been shown by several studies, but the clinical utility of this treatment has not been investigated by large prospective trials. METHODS: Six hundred seventy-eight patients undergoing major elective abdominal surgery were randomly assigned to receive either protein-sparing therapy after surgery (protein-sparing therapy group) or conventional therapy (control group). The patients were monitored for postoperative complications and mortality. RESULTS: The rate of major postoperative complications was similar in both groups (protein-sparing therapy group, 19.5%; control group, 20.9%; p=0.66) as were the overall postoperative mortality rates (4.7% and 3.5%, respectively; p=0.43). CONCLUSIONS: The present study indicates that routine protein-sparing therapy for patients normonourished or mildly malnourished undergoing major abdominal surgery is not clinically justified. PMID:8633913

  9. Protein C levels in beta-thalassemia major patients in the east Nile delta of Egypt.

    PubMed

    Hassan, Tamer H; Elbehedy, Rabab M; Youssef, Doaa M; Amr, Ghada E

    2010-01-01

    Thalassemic patients have an increased risk for thromboembolic complications. To determine if this might be due to a deficiency in protein C, we investigated the status of the protein C anticoagulant pathway in thalassemia major patients and its relationship to the hypercoagulable state. Fifty patients with beta-thalassemia major (30 non-splenectomized and 20 splenectomized) and 20 healthy children as a control group were tested for levels of serum ferritin, liver enzymes, serum albumin, fibrinogen, protein C and protein S, thrombin antithrombin complex (TAT) and D-dimer. Thalassemic patients had lower levels of protein C and S and higher levels of D-dimer and TAT than the control group. These findings were more obvious in splenectomized patients and in those with infrequent blood transfusion. Protein C plays a major role in the hypercoagulable state in thalassemic patients. These findings raise the issue as to whether it would be cost-beneficial to recommend prophylactic antithrombotic therapy in high-risk thalassemic patients. A wider prospective study is necessary to delineate under which circumstances therapy might be needed, and at what level of protein C deficiency to start prophylactic antithrombotic therapy.

  10. Functional distribution of synapsin I in human sperm

    PubMed Central

    Coleman, William L.; Kulp, Adam C.; Venditti, Jennifer J.

    2015-01-01

    Proteins known to function during cell–cell communication and exocytosis in neurons and other secretory cells have recently been reported in human sperm. Synapsins are a group of proteins that have been very well characterized in neurons, but little is known about synapsin function in other cell types. Based upon previous findings and the known function of synapsin, we tested the hypothesis that synapsin I was present in human sperm. Washed, capacitated, and acrosome induced sperm preparations were used to evaluate the functional distribution of synapsin I using immunocytochemistry. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were used for protein blotting techniques. Immunolocalization revealed synapsin I was enriched in the sperm equatorial segment. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were positive for synapsin I using several different antibodies, and dot blot results were confirmed by Western blot analyses. Finally, treatment of capacitated and acrosome reaction induced samples with anti-synapsin antibodies significantly reduced sperm motility. Localization of synapsin I in human sperm is a novel finding. The association of synapsin I with the sperm equatorial segment and effects on motility are suggestive of a role associated with capacitation and/or acrosome reaction, processes that render sperm capable of fertilizing an oocyte. PMID:26566474

  11. Expression of a hydrophilic surface protein in infective stages of Leishmania major.

    PubMed

    Flinn, H M; Rangarajan, D; Smith, D F

    1994-06-01

    A family of differentially expressed genes from Leishmania major contains one sequence (Gene B) that encodes a novel, hydrophilic protein found on the surface of infective parasite stages. The 177-residue, acidic Gene B protein is characterised by an amino acid repetitive element, comprising 45% of the total molecule, that is related to the cell-wall binding domain of protein A from Staphylococcus aureus. No identifiable signal peptide, membrane-spanning domain or consensus for glycosylphosphatidylinositol anchor attachment to the cell surface is found elsewhere in the deduced protein sequence. In vitro, the Gene B protein fractionates with the parasite cell surface glycoconjugates, lipophosphoglycan and the glycoinositolphospholipids. This protein is the first characterised surface pep