Science.gov

Sample records for major sperm protein

  1. Role of major sperm protein (MSP) in the protrusion and retraction of Ascaris sperm.

    PubMed

    Roberts, Thomas M; Stewart, Murray

    2012-01-01

    Nematode sperm offer a unique perspective for investigating amoeboid cell motility. These cells display the hallmark features of amoeboid movement but power their locomotion with a cytoskeleton composed of major sperm protein (MSP) filaments in place of the familiar actin cytoskeleton found in other crawling cells. Thus, properties of sperm can be compared to those of actin-rich cells to identify the shared features that are essential to motility. Sperm are simple cells in which cytoskeletal dynamics are tightly coupled to protrusion of the leading edge and retraction of the cell body. These features have facilitated reconstitution of both protrusion and retraction in cell-free extracts and enabled identification of accessory components in the motility apparatus as well as elucidation of the mechanical basis of movement. Six MSP accessory proteins have been isolated including four components of the sperm cytoskeleton and two enzymes that play key roles in regulating cytoskeletal dynamics and locomotion. Analysis of this versatile in vitro motility system has identified motor-independent mechanisms for protrusion and retraction that are based on changes in filament-packing density. These changes result in expansion and contraction of the MSP-filament network that generate the forces for movement. We discuss how the mechanisms of motility that operate in nematode sperm may contribute generally to the movement of crawling cells. PMID:22608562

  2. Mammalian Sperm Fertility Related Proteins

    PubMed Central

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

    2013-01-01

    Infertility is an important aspect of human and animal reproduction and still presents with much etiological ambiguity. As fifty percent of infertility is related to the male partner, molecular investigations on sperm and seminal plasma can lead to new knowledge on male infertility. Several comparisons between fertile and infertile human and other species sperm proteome have shown the existence of potential fertility markers. These proteins have been categorized into energy related, structural and other functional proteins which play a major role in sperm motility, capacitation and sperm-oocyte binding. The data from these studies show the impact of sperm proteome studies on identifying different valuable markers for fertility screening. In this article, we review recent development in unraveling sperm fertility related proteins. PMID:24151436

  3. 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

    2015-03-01

    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

  4. 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

  5. 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. PMID:26386584

  6. 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.

  7. 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. PMID:25399599

  8. Sperm specific proteins-potential candidate molecules for fertility control

    PubMed Central

    Suri, Anil

    2004-01-01

    The increase in population growth rate warrants the development of additional contraceptive methods that are widely acceptable, free from side effects and less expensive. Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm egg binding and fertilization, offers an attractive approach to control fertility. The development of a contraceptive vaccine based on sperm antigen represents a promising approach to contraception. In mammals, fertilization is completed by the direct interaction of sperm and egg, a process mediated primarily by sperm surface proteins. Sperm have proteins that are unique, cell specific, immunogenic and accessible to antibodies. A few of the sperm specific proteins have been isolated and characterized. The antibodies raised against the sperm specific antigens have proved to be extremely effective at reducing sperm-egg interaction in vitro; fertility trials in sub-human primates would eventually prove the effectiveness of the sperm antigens in terms of contraceptive efficacy. PMID:15012833

  9. 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.

  10. Dynamics of heparin-binding proteins on boar sperm.

    PubMed

    Dapino, Dora G; Teijeiro, Juan M; Cabada, Marcelo O; Marini, Patricia E

    2009-12-01

    The presence, topology and dynamics of heparin-binding proteins (HBP) on boar sperm were evaluated. HBP distribution was analyzed by subcellular parting, using biotinylated heparin followed by colorimetric detection. HBP were detected as peripherical and integral periacrosomal membrane proteins. Indirect fluorescence microscopy of sperm incubated with biotinylated heparin was used to evidence heparin binding on sperm at different physiological stages. Two different fluorescent patterns (A and B) were found, which probably correspond to non-capacitated and capacitated sperm as assessed by the ability to undergo acrosome reaction with calcium ionophore A23187 and by the increase of p32 phosphorylated protein. In A pattern, corresponding to untreated sperm, fluorescence located mostly on the post-acrosomal region; in B pattern, corresponding to incubated sperm, on the acrosomal region. Upon incubation under capacitating conditions (TALP), sperm having the B pattern was augmented compared with non-incubated sperm (p<0.001). Differences in the HBP patterns (p<0.0001) were observed in sperm incubated under non-capacitating conditions in relation to sperm incubated in TALP, indicating that the modification of HBP patterns is probably related to capacitation. No difference was observed when untreated sperm were permeabilized prior to staining, suggesting that HBP are present on the sperm surface. The effect of heparin on capacitation dependent protein tyrosine phosphorylation was also analyzed, finding a decrease in p32 phosphorylation in the presence of heparin. This suggests that the capacitation enhancement mediated by this glycosaminoglycan involves an alternative intracellular pathway. The finding that heparin binds to sperm differently according to its physiological state, is a new evidence of the remodelling of sperm membrane surface upon capacitation and may provide a useful and relatively simple method to evaluate in vitro modification of boar sperm physiological

  11. 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. PMID:26223815

  12. 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. PMID:26631595

  13. Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

    PubMed Central

    Martin-DeLeon, Patricia A

    2015-01-01

    A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca2+-ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca2+ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion–competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca2+-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach. PMID:26112481

  14. Freezability prediction of boar ejaculates assessed by functional sperm parameters and sperm proteins.

    PubMed

    Casas, I; Sancho, S; Briz, M; Pinart, E; Bussalleu, E; Yeste, M; Bonet, S

    2009-10-15

    The objective of this work was to look for useful predictive indicators of the potentially "good" or "poor" ability of a boar ejaculate to sustain cryopreservation by assessing both the conventional sperm quality parameters (Study 1) and the immunolabeling of three proteins involved in the physiology of the sperm cell: GLUT3, HSP90AA1 and Cu/ZnSOD (Study 2). Study 1 was carried out in three different steps during the cryopreservation process of the sperm-rich fraction of 29 Piétrain boar ejaculates (17 degrees C, 5 degrees C, and 240min postthaw). These ejaculates were clustered based on sperm quality parameters analyzed at 240min postthaw, obtaining 16 good freezability ejaculates (GFEs) and 13 poor freezability ejaculates (PFEs). The sperm linearity (LIN) and the straightforward (STR) indexes at 5 degrees C showed higher hyperactivated movement in the PFEs than in the GFEs, which suggests that analyzing these sperm kinematic parameters could be a useful tool for predicting the potential freezability of an ejaculate. This statement was demonstrated by grouping the 29 ejaculates into two clusters (A and B) based on LIN and STR values assessed after 30 min at 5 degrees C, which resulted in around 72% of coincidence with the GFE and PFE groups. Study 2, performed at 17 degrees C and 240 min postthaw, revealed no differences between GFEs and PFEs in the immunolabeling of the three proteins within a same step, in terms of location and reactivity, although reactivity was generally weaker at 240 min postthaw in both groups. Additional studies on Western blot are currently being carried out with the objective to quantify the expression of the three proteins in GFEs and PFEs in the three steps of the cryopreservation process. PMID:19651432

  15. Sperm-egg recognition in the mouse: characterization of sp56, a sperm protein having specific affinity for ZP3

    PubMed Central

    1994-01-01

    Recognition between mammalian gametes occurs when the plasma membrane of the sperm head binds to the zona pellucida (ZP), an extracellular coat surrounding eggs. ZP3, one of three glycoproteins in the ZP, is the egg protein recognized by sperm. A mouse sperm surface protein, sp56 (M(r) = 56,000), has been identified on the basis of its specific affinity for ZP3 (Bleil, J. D., and P. M. Wassarman. 1990. Proc. Natl. Acad. Sci. USA. 87:5563-5567). Studies presented here were designed to characterize mouse sperm sp56 and to further test whether or not this protein specifically recognizes ZP3. sp56 was purified by both ZP3 affinity chromatography and by ion exchange chromatography followed by size-exclusion chromatography. The purified native protein eluted from size-exclusion columns as a homomultimer (M(r) approximately 110,000). Each monomer of the protein contains intramolecular disulfide bonds, consistent with its extracellular location. Immunohistochemical and immunoblotting studies, using monoclonal antibodies, demonstrated that sp56 is a peripheral membrane protein located on the outer surface of the sperm head plasma membrane, precisely where sperm bind ZP3. Results of crosslinking experiments demonstrated that the ZP3 oligosaccharide recognized by sperm has specific affinity for sp56. Collectively, these results suggest that sp56 may be the sperm protein responsible for sperm-egg recognition in the mouse. PMID:8188752

  16. Development of antifertility vaccine using sperm specific proteins.

    PubMed

    Bandivdekar, A H

    2014-11-01

    Sperm proteins are known to be associated with normal fertilization as auto- or iso-antibodies to these proteins may cause infertility. Therefore, sperm proteins have been considered to be the potential candidate for the development of antifertility vaccine. Some of the sperm proteins proved to be promising antigens for contraceptive vaccine includes lactate dehydrogenase (LDH-C4), protein hyaluronidase (PH-20), and Eppin. Immunization with LDH-C4 reduced fertility in female baboons but not in female cynomolgus macaques. Active immunization with PH-20 resulted in 100 per cent inhibition of fertility in male guinea pigs but it induced autoimmune orchitis. Immunization with Eppin elicited high antibody titres in 78 per cent of immunized monkeys and induced infertility but the immunopathological effect of immunization was not examined. Human sperm antigen (80 kDa HSA) is a sperm specific, highly immunogenic and conserved sperm protein. Active immunization with 80 kDa HSA induced immunological infertility in male and female rats. Partial N-terminal amino acid sequence of 80 kDa HSA (Peptide NT) and its peptides (Peptides 1, 2, 3 and 4) obtained by enzymatic digestion did not show homology with any of the known proteins in gene bank. Peptides NT, 1, 2 and 4 were found to mimic immunobiological activity of native protein. Passive administration of antibodies to peptides NT, 1, 2 and 4 induced infertility in male and female rats and peptide 1 was found to be most effective in suppressing fertility. Active immunization with keyhole limpet haemocynin (KLH) conjugated synthetic peptide 1 impaired fertility in all the male rabbits and six of the seven male marmosets. The fertility was restored following decline in antibody titre. All these findings on 80 kDA HAS suggest that the synthetic Peptide-1 of 80 kDa HSA is the promising candidate for development of male contraceptive vaccine. PMID:25673547

  17. Development of antifertility vaccine using sperm specific proteins

    PubMed Central

    Bandivdekar, A.H.

    2014-01-01

    Sperm proteins are known to be associated with normal fertilization as auto- or iso-antibodies to these proteins may cause infertility. Therefore, sperm proteins have been considered to be the potential candidate for the development of antifertility vaccine. Some of the sperm proteins proved to be promising antigens for contraceptive vaccine includes lactate dehydrogenase (LDH-C4), protein hyaluronidase (PH-20), and Eppin. Immunization with LDH-C4 reduced fertility in female baboons but not in female cynomolgus macaques. Active immunization with PH-20 resulted in 100 per cent inhibition of fertility in male guinea pigs but it induced autoimmune orchitis. Immunization with Eppin elicited high antibody titres in 78 per cent of immunized monkeys and induced infertility but the immunopathological effect of immunization was not examined. Human sperm antigen (80kDa HSA) is a sperm specific, highly immunogenic and conserved sperm protein. Active immunization with 80kDa HSA induced immunological infertility in male and female rats. Partial N-terminal amino acid sequence of 80kDa HSA (Peptide NT) and its peptides (Peptides 1, 2, 3 and 4) obtained by enzymatic digestion did not show homology with any of the known proteins in gene bank. Peptides NT, 1, 2 and 4 were found to mimic immunobiological activity of native protein. Passive administration of antibodies to peptides NT, 1, 2 and 4 induced infertility in male and female rats and peptide 1 was found to be most effective in suppressing fertility. Active immunization with keyhole limpet haemocynin (KLH) conjugated synthetic peptide 1 impaired fertility in all the male rabbits and six of the seven male marmosets. The fertility was restored following decline in antibody titre. All these findings on 80kDA HAS suggest that the synthetic Peptide-1 of 80kDa HSA is the promising candidate for development of male contraceptive vaccine. PMID:25673547

  18. Sperm Motility Requires Wnt/GSK3 Stabilization of Proteins.

    PubMed

    De Robertis, Edward M; Ploper, Diego

    2015-11-23

    Inhibition of GSK3 by Wnt signaling stabilizes many cellular proteins, but proof that this effect is independent of β-catenin-mediated transcription is lacking. Koch, Acebron, and colleagues (2015) now demonstrate that transcriptionally silent mammalian sperm require Wnt signaling via exosomes to prevent protein degradation during their lengthy travels through the epididymis. PMID:26609954

  19. Alteration of sperm protein profile induced by cigarette smoking.

    PubMed

    Chen, Xiaohui; Xu, Wangjie; Miao, Maohua; Zhu, Zijue; Dai, Jingbo; Chen, Zhong; Fang, Peng; Wu, Junqing; Nie, Dongsheng; Wang, Lianyun; Wang, Zhaoxia; Qiao, Zhongdong; Shi, Huijuan

    2015-07-01

    Cigarette smoking is associated with lower semen quality, but how cigarette smoking changes the semen quality remains unclear. The aim of this study was to screen the differentially expressed proteins in the sperm of mice with daily exposure to cigarette smoke. The 2D gel electrophoresis (2DE) and mass spectrometry (MS) analyses results showed that the mouse sperm protein profile was altered by cigarette smoking. And 22 of the most abundant proteins that correspond to differentially expressed spots in 2DE gels of the sperm samples were identified. These proteins were classified into different groups based on their functions, such as energy metabolism, reproduction, and structural molecules. Furthermore, the 2DE and MS results of five proteins (Aldoa, ATP5a1, Gpx4, Cs, and Spatc1) were validated by western blot analysis and reverse transcriptase-polymerase chain reaction. Results showed that except Spatc1 the other four proteins showed statistically significant different protein levels between the smoking group and the control group (P < 0.05). The expressions of three genes (Aldoa, Gpx4, and Spatc1) were significantly different (P < 0.05) at transcription level between the smoking group and the control group. In addition, five proteins (Aldoa, ATP5a1, Spatc1, Cs, and Gpx4) in human sperm samples from 30 male smokers and 30 non-smokers were detected by western blot analysis. Two proteins (Aldoa and Cs) that are associated with energy production were found to be significantly altered, suggesting that these proteins may be potential diagnostic markers for evaluation of smoking risk in sperm. Further study of these proteins may provide insight into the pathogenic mechanisms underlying infertility in smoking persons. PMID:26063603

  20. Seminal vesicle proteins SVS3 and SVS4 facilitate SVS2 effect on sperm capacitation.

    PubMed

    Araki, Naoya; Kawano, Natsuko; Kang, Woojin; Miyado, Kenji; Yoshida, Kaoru; Yoshida, Manabu

    2016-10-01

    Mammalian spermatozoa acquire their fertilizing ability in the female reproductive tract (sperm capacitation). On the other hand, seminal vesicle secretion, which is a major component of seminal plasma, inhibits the initiation of sperm capacitation (capacitation inhibition) and reduces the fertility of the capacitated spermatozoa (decapacitation). There are seven major proteins involved in murine seminal vesicle secretion (SVS1-7), and we have previously shown that SVS2 acts as both a capacitation inhibitor and a decapacitation factor, and is indispensable for in vivo fertilization. However, the effects of SVSs other than SVS2 on the sperm have not been elucidated. Since mouse Svs2-Svs6 genes evolved by gene duplication belong to the same gene family, it is possible that SVSs other than SVS2 also have some effects on sperm capacitation. In this study, we examined the effects of SVS3 and SVS4 on sperm capacitation. Our results showed that both SVS3 and SVS4 are able to bind to spermatozoa, but SVS3 alone showed no effects on sperm capacitation. On the other hand, SVS4 acted as a capacitation inhibitor, although it did not show decapacitation abilities. Interestingly, SVS3 showed an affinity for SVS2 and it facilitated the effects of SVS2. Interaction of SVS2 and spermatozoa is mediated by the ganglioside GM1 in the sperm membrane; however, both SVS3 and SVS4 had weaker affinities for GM1 than SVS2. Therefore, we suggest that separate processes may cause capacitation inhibition and decapacitation, and SVS3 and SVS4 act on sperm capacitation cooperatively with SVS2. PMID:27486266

  1. Quantitative analysis of flagellar proteins in Drosophila sperm tails.

    PubMed

    Mendes Maia, Teresa; Paul-Gilloteaux, Perrine; Basto, Renata

    2015-01-01

    The cilium has a well-defined structure, which can still accommodate some morphological and molecular composition diversity to suit the functional requirements of different cell types. The sperm flagellum of the fruit fly Drosophila melanogaster appears as a good model to study the genetic regulation of axoneme assembly and motility, due to the wealth of genetic tools publically available for this organism. In addition, the fruit fly's sperm flagellum displays quite a long axoneme (∼1.8mm), which may facilitate both histological and biochemical analyses. Here, we present a protocol for imaging and quantitatively analyze proteins, which associate with the fly differentiating, and mature sperm flagella. We will use as an example the quantification of tubulin polyglycylation in wild-type testes and in Bug22 mutant testes, which present defects in the deposition of this posttranslational modification. During sperm biogenesis, flagella appear tightly bundled, which makes it more challenging to get accurate measurements of protein levels from immunostained specimens. The method we present is based on the use of a novel semiautomated, macro installed in the image processing software ImageJ. It allows to measure fluorescence levels in closely associated sperm tails, through an exact distinction between positive and background signals, and provides background-corrected pixel intensity values that can directly be used for data analysis. PMID:25837396

  2. 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. PMID:27470200

  3. A role for the WH-30 protein in sperm-sperm adhesion during rouleaux formation in the guinea pig.

    PubMed

    Flaherty, S P; Swann, N J; Primakoff, P; Myles, D G

    1993-03-01

    Mammalian spermatozoa participate in specific cell adhesion phenomena during their development and functional lifespan; this includes interaction with Sertoli cells, the zona pellucida, and the oolemma. In some species such as the guinea pig, an additional sperm-sperm adhesion occurs during epididymal maturation which results in the formation of rouleaux in which the sperm heads are stacked one upon the other and the periacrosomal plasma membranes of adjacent sperm are linked by periodic cross-bridges. In this study, we have used a monoclonal antibody to investigate the role of the WH-30 protein on the sperm surface in the formation of the junctional zones between adjacent guinea pig sperm in rouleaux. WH-30 monoclonal antibodies caused a dose- and time-dependent dissociation of rouleaux and an increase in the percentage of single, acrosome-intact sperm; there were no effects on sperm motility (maintained at 80-90%) or ultrastructure during the 120-min incubations. The maximal effect of about 80% single sperm was obtained with a 1:4 dilution of the WH-30 hybridoma supernatant or 5-50 micrograms/ml of purified WH-30 IgG. In contrast, incubation of sperm in AH-20 IgG, myeloma cell supernatants, or purified, nonspecific mouse IgG1 had no effect on rouleaux. Treatment of sperm with a WH-30 Fab fragment resulted in almost complete dissociation of rouleaux without any observed effect on sperm motility or acrosomal status. Surface labeling of sperm followed by immunoprecipitation and SDS-PAGE revealed that the WH-30 antibody recognizes a single polypeptide of 43-45 kDa. Using immunofluorescence, the WH-30 protein was localized over the entire surface of the sperm head (whole-head pattern), and immunogold labeling showed that WH-30 is localized in the glycocalyx on both the dorsal and ventral surfaces of the periacrosomal and postacrosomal plasma membranes. These results indicate that the WH-30 protein on the sperm surface is a cell adhesion protein which is involved in

  4. SEMINAL PROTEINS BUT NOT SPERM INDUCE MORPHOLOGICAL CHANGES IN THE DROSOPHILA MELANOGASTER FEMALE REPRODUCTIVE TRACT DURING SPERM STORAGE

    PubMed Central

    Adams, Erika M.; Wolfner, Mariana F.

    2007-01-01

    In most insects, sperm transferred by the male to the female during mating are stored within the female reproductive tract for subsequent use in fertilization. In Drosophila melanogaster, male accessory gland proteins (Acps) within the seminal fluid are required for efficient transfer and subsequent accumulation of sperm in the female's sperm storage organs. To determine the events within the female reproductive tract that occur during sperm storage, and the role that Acps and sperm play in these events, we identified morphological changes that take place during sperm storage in females mated to wild-type, Acp-deficient or sperm-deficient males. A reproducible set of morphological changes occurs in a wild-type mating. These were categorized into 10 stereotypic stages. Sperm are not needed for progression through these stages in females, but receipt of Acps is essential for progression beyond the first few stages of morphological changes. Furthermore, females that received small quantities of Acps reached slightly later stages than females that received no Acps. Our results suggest that timely morphological changes in the female reproductive tract, possibly muscular in nature, may be needed for successful sperm storage, and that Acps from the male are needed in order for these changes to occur. PMID:17276455

  5. 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.

  6. Effects of milk proteins on sperm binding to the zona pellucida and intracellular Ca(2+) concentration in stallion sperm.

    PubMed

    Coutinho da Silva, Marco A; Seidel, George E; Squires, Edward L; Graham, James K; Carnevale, Elaine M

    2014-11-10

    Objectives were to determine the effects of extracellular Ca(2+) and milk proteins on intracellular Ca(2+) concentrations in stallion sperm; and to determine the effects of single caseins on sperm binding to the zona pellucida (ZP). In Experiment I, sperm were incubated in media containing 2 or 4mM Ca(2+) and intracellular Ca(2+) concentration was determined after ionomycin treatment and long-term incubation (3h). Extracellular Ca(2+) concentrations (2 compared with 4mM) did not affect baseline intracellular Ca(2+) concentration of sperm. However, incubating sperm in a medium containing 4 compared with 2mM Ca(2+) resulted in greater (P<0.05) influx of Ca(2+) into sperm. In Experiment II, sperm incubated in media containing 1mg/mL of native phosphocaseinate (NP) or sodium caseinate (SC) showed similar baseline intracellular Ca(2+) and influx of Ca(2+) than control (TALP). In Experiment III, sperm-ZP binding assays were performed in TALP medium containing: no additions (TALP); 1mg/mL SC; 1 or 3mg/mL of α-casein; 1 or 3mg/mL of β-casein; and 1 or 3mg/mL of κ-casein. The number of stallion sperm bound to bovine ZP was greatest (P<0.05) when SC was used. Co-incubation in media containing single caseins (α-, β- or κ-casein) resulted in similar results to TALP; however, a dose effect (P<0.05) was observed for β- and κ-caseins. In conclusion, extracellular Ca(2+) concentration and milk proteins did not affect baseline intracellular calcium in stallion sperm. It appears that β- and κ-caseins may be responsible for enhancing sperm binding to ZP, but the mechanism remains unknown. PMID:25213434

  7. Cigarette smoke affects posttranslational modifications and inhibits capacitation-induced changes in human sperm proteins.

    PubMed

    Shrivastava, Vibha; Marmor, Hannah; Chernyak, Sholom; Goldstein, Marc; Feliciano, Miriam; Vigodner, Margarita

    2014-01-01

    Sperm are highly dependent on posttranslational modifications of proteins. Massive phosphorylation on tyrosine residue is required for sperm capacitation. Sumoylation has also been recently implicated in spermatogenesis and sperm functions. Cigarette smoke is known to cause oxidative stress in different tissues, and several studies suggest that it causes oxidative stress in sperm. Whether tobacco affects posttranslational modifications in human sperm is currently unknown. In this study, we show that a short exposure of human sperm to physiological concentrations of cigarette smoke extract (CSE) causes the partial de-sumoylation of many sperm proteins. Furthermore, the presence of a low concentration of CSE in the human tubal fluid during an induction of in vitro capacitation inhibits the capacitation-associated increase in protein phosphorylation. Collectively, changes in posttranslational modifications may be one of the mechanisms through which exposure to tobacco can negatively affect sperm functions and cause fertility problems. PMID:24345728

  8. Sperm and Spermatids Contain Different Proteins and Bind Distinct Egg Factors

    PubMed Central

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

    2014-01-01

    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. PMID:25244019

  9. Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion.

    PubMed

    Primakoff, P; Hyatt, H; Tredick-Kline, J

    1987-01-01

    Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The mAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 micrograms/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75%. The average number of sperm fused per egg was also reduced by 75%. In contrast a control mAb, PH-1, preincubated with sperm at 400 micrograms/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two 125I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes. Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears. After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to approximately 49 kD and approximately 33 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44- and 60-kD bands. In the absence of

  10. Sperm flagella: comparative and phylogenetic perspectives of protein components.

    PubMed

    Inaba, Kazuo

    2011-08-01

    Sperm motility is necessary for the transport of male DNA to eggs in species with both external and internal fertilization. Flagella comprise several proteins for generating and regulating motility. Central cytoskeletal structures called axonemes have been well conserved through evolution. In mammalian sperm flagella, two accessory structures (outer dense fiber and the fibrous sheath) surround the axoneme. The axonemal bend movement is based on the active sliding of axonemal doublet microtubules by the molecular motor dynein, which is divided into outer and inner arm dyneins according to positioning on the doublet microtubule. Outer and inner arm dyneins play different roles in the production and regulation of flagellar motility. Several regulatory mechanisms are known for both dyneins, which are important in motility activation and chemotaxis at fertilization. Although dynein itself has certain properties that contribute to the formation and propagation of flagellar bending, other axonemal structures-specifically, the radial spoke/central pair apparatus-have essential roles in the regulation of flagellar bending. Recent genetic and proteomic studies have explored several new components of axonemes and shed light on the generation and regulation of sperm motility during fertilization. PMID:21586547

  11. Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster.

    PubMed Central

    Tram, U; Wolfner, M F

    1999-01-01

    The seminal fluid that is transferred along with sperm during mating acts in many ways to maximize a male's reproductive success. Here, we use transgenic Drosophila melanogaster males deficient in the seminal fluid proteins derived from the accessory gland (Acps) to investigate the role of these proteins in the fate of sperm transferred to females during mating. Competitive PCR assays were used to show that while Acps contribute to the efficiency of sperm transfer, they are not essential for the transfer of sperm to the female. In contrast, we found that Acps are essential for storage of sperm by females. Direct counts of stored sperm showed that 10% of normal levels are stored by females whose mates transfer little or no Acps along with sperm. PMID:10511561

  12. Fully effective contraception in male and female guinea pigs immunized with the sperm protein PH-20.

    PubMed

    Primakoff, P; Lathrop, W; Woolman, L; Cowan, A; Myles, D

    1988-10-01

    Immunization of male and female animals with extracts of whole sperm cells is known to cause infertility. Also, men and women who spontaneously produce antisperm antibodies are infertile but otherwise healthy. Although the critical sperm antigens are unknown, these observations have led to the proposal that sperm proteins might be useful in the development of a contraceptive vaccine. The guinea pig sperm surface protein PH-20 is essential in sperm adhesion to the extracellular coat (zona pellucida) of the egg, a necessary initial step in fertilization. Here, we report that 100% effective contraception was obtained in male and female guinea pigs immunized with PH-20. Antisera from immunized females had high titres, specifically recognized PH-20 in sperm extracts, and blocked sperm adhesion to the egg zona pellucida in vitro. The contraceptive effect was long-lasting and reversible: immunized females, mated at intervals of six to fifteen months after immunization, progressively regained fertility. PMID:3419530

  13. Telomere-telomere interactions and candidate telomere binding protein(s) in mammalian sperm cells.

    PubMed

    Zalensky, A O; Tomilin, N V; Zalenskaya, I A; Teplitz, R L; Bradbury, E M

    1997-04-10

    We have used fluorescent in situ hybridization to localize telomeres within the nuclei of sperm from six mammals (human, rat, mouse, stallion, boar, and bull). In minimally swollen sperm of mouse and rat, most of the telomeres are clustered within a limited area in the posterior part of nuclei. In sperm of other species, telomeres associate into tetrameres and dimers. On swelling of sperm cells with heparin/dithiotriethol, telomere associations disperse, and hybridization signals become smaller in size and their numbers approach or correspond to the number of chromosome ends in a haploid genome. Quantitation of telomere loci indicates that dimeric associations are prominent features of mammalian sperm nuclear architecture. Higher order telomere-telomere interactions and organization develop during meiotic stages of human spermatogenesis. At this stage, telomeres also become associated with the nuclear membrane. In an attempt to elucidate the molecular mechanisms underlying telomere interactions in sperm, we have identified a novel protein activity that binds to the double-stranded telomeric repeat (TTAGGG)n. Sperm telomere binding protein(s) (STBP) was extracted from human and bull sperm by 0.5 M NaCl. STBP does not bind single-stranded telomeric DNA and is highly specific for single base substitutions in a duplex DNA sequence. Depending on the conditions of binding, we observed the formation of several nucleoprotein complexes. We have shown that there is a transition between complexes, which indicates that the slower migrating complex is a multimer of the higher mobility one. We propose that STBP participates in association between the telomere domains which were microscopically observed in mammalian spermatozoa. PMID:9141618

  14. 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. PMID:24848520

  15. Characterization of maturation-dependent extrinsic proteins of the rat sperm surface

    SciTech Connect

    Rifkin, J.M.; Olson, G.E.

    1985-05-01

    Mammalian spermatozoa must mature in the epididymis before they can fertilize an egg. It is known that modification of the protein composition of the sperm surface is an important part of the maturation process. In this paper, the authors present data on two related glycoproteins that can be extracted from mature but not immature spermatozoa. Cell surface radioiodination has shown that these proteins are on the sperm surface, and immunofluorescence microscopy, by use of monospecific antibodies to the proteins, has indicated that their localization is restricted to the periacrosomal region of the sperm head. The authors have also shown that in vitro, these proteins will bind to the identical region of immature sperm. Immunohistochemical localization of the proteins in the epididymis shows that they are produced and secreted by the cauda region. The significance of the addition of these proteins to the sperm surface in both maturation and fertilization is discussed.

  16. 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. PMID:24004935

  17. 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

  18. Identification of guanylate cyclases and related signaling proteins in sperm tail from sea stars by mass spectrometry.

    PubMed

    Nakachi, Mia; Matsumoto, Midori; Terry, Philip M; Cerny, Ronald L; Moriyama, Hideaki

    2008-01-01

    Marine invertebrates employ external fertilization to take the advantages of sexual reproduction as one of excellent survival strategies. To prevent mismatching, successful fertilization can be made only after going though strictly defined steps in the fertilization. In sea stars, the fertilization process starts with the chemotaxis of sperm followed by hyperactivation of sperm upon arriving onto the egg coat, and then sperm penetrate to the egg coat before achieving the fusion. To investigate whether the initiation of chemotaxis and the following signaling has species specificity, we conducted comparative studies in the protein level among sea stars, Asterias amurensis, A. forbesi, and Asterina pectinifera. Since transcription of messenger ribonucleic acid (mRNA) has been suppressed in gamete, the roles of sperm proteins during the fertilization cannot be investigated by examining the mRNA profile. Therefore, proteomics analysis by mass spectrometry was used in this study. In sea stars, upon receiving asteroidal sperm-activating peptide (asterosap), the receptor membrane-bound guanylate cyclases in the sperm tail trigger sperm chemotaxis. We confirmed the presence of membrane-bound guanylate cyclases in the three sea star species, and they all had the same structural domains including the extracellular domain, kinase-like domain, and guanylate cyclase domain. The majority of peptides recovered were from alpha-helices distributed on the solvent side of the protein. More peptides were recovered from the intracellular domains. The transmembrane domain has not been recovered. The functions of the receptors seemed to be conserved among the species. Furthermore, we identified proteins that may be involved in the guanylate cyclase-triggered signaling pathway. PMID:18461395

  19. Phylogeny of major intrinsic proteins.

    PubMed

    Danielson, Jonas A H; Johanson, Urban

    2010-01-01

    Major intrinsic proteins (MIPs) form a large superfamily of proteins that can be divided into different subfamilies and groups according to phylogenetic analyses. Plants encode more MIPs than o ther organisms and se ven subfamilies have been defined, whereofthe Nodulin26-like major intrinsic proteins (NIPs) have been shown to permeate metalloids. In this chapter we review the phylogeny of MIPs in general and especially of the plant MIPs. We also identify bacterial NIP-like MIPs and discuss the evolutionary implications of this finding regarding the origin and ancestral transport specificity of the NIPs. PMID:20666221

  20. 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.

  1. Transmembrane adenylyl cyclase regulates amphibian sperm motility through Protein Kinase A activation

    PubMed Central

    O’Brien, Emma D.; Krapf, Darío; Cabada, Marcelo O.; Visconti, Pablo E.; Arranz, Silvia E.

    2014-01-01

    Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation. PMID:21126515

  2. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. 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. PMID:26046548

  5. 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).Reproduction (2016) 151 1-10. PMID:26860122

  6. 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. PMID:26810830

  7. Bisphenol A accelerates capacitation-associated protein tyrosine phosphorylation of rat sperm by activating protein kinase A.

    PubMed

    Wan, Xiaofeng; Ru, Yanfei; Chu, Chen; Ni, Zimei; Zhou, Yuchuan; Wang, Shoulin; Zhou, Zuomin; Zhang, Yonglian

    2016-06-01

    Bisphenol A (BPA) is a synthetic estrogen-mimic chemical. It has been shown to affect many reproductive endpoints. However, the effect of BPA on the mature sperm and the mechanism of its action are not clear yet. Here, our in vitro studies indicated that BPA could accelerate sperm capacitation-associated protein tyrosine phosphorylation in time- and dose-dependent manners. In vivo, the adult male rats exposed to a high dose of BPA could result in a significant increase in sperm activity. Further investigation demonstrated that BPA could accelerate capacitation-associated protein tyrosine phosphorylation even if sperm were incubated in medium devoid of BSA, HCO3 (-), and Ca(2+) However, this action of BPA stimulation could be blocked by H89, a highly selective blocker of protein kinase A (PKA), but not by KH7, a specific inhibitor of adenylyl cyclase. These data suggest that BPA may activate PKA to affect sperm functions and male fertility. PMID:27174873

  8. Equatorial Segment Protein (ESP) Is a Human Alloantigen Involved in Sperm-Egg Binding and Fusion

    PubMed Central

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

    2010-01-01

    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. PMID:17978344

  9. Cadmium inhibits mouse sperm motility through inducing tyrosine phosphorylation in a specific subset of proteins.

    PubMed

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

    2016-08-01

    Cadmium (Cd) has been reported to impair male fertility, primarily by disrupting sperm motility, but the underlying molecular mechanism remains unclear. Here we investigated the effects of Cd on sperm motility, tyrosine phosphorylation, AMP-activated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, and ATP levels in vitro. Our results demonstrated that Cd inhibited sperm motility, GAPDH activity, AMPK activity and ATP production, and induced tyrosine phosphorylation of 55-57KDa proteins. Importantly, all the parameters affected by Cd were restored to normal levels when incubated with 10μM Cd in the presence of 30μM ethylene diamine tetraacetic acid (EDTA). Interestingly, changes of tyrosine phosphorylation levels of 55-57KDa proteins are completely contrary to that of other parameters. These results suggest that Cd-induced tyrosine phosphorylation of 55-57KDa proteins might act as an engine to block intracellular energy metabolism and thus decrease sperm motility. PMID:27233480

  10. 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.

  11. Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation.

    PubMed

    Imam, S; Ansari, M R; Ahmed, N; Kumaresan, A

    2008-05-01

    The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS-PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen-thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P<0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P<0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity. PMID:18359071

  12. Sperm surface protein PH-20 is bifunctional: one activity is a hyaluronidase and a second, distinct activity is required in secondary sperm-zona binding.

    PubMed

    Hunnicutt, G R; Primakoff, P; Myles, D G

    1996-07-01

    In previous studies, we have found that the sperm membrane protein PH-20 acts during two different stages of fertilization. On acrosome-intact sperm, PH-20 has a hyaluronidase activity that is required for sperm penetration through the cumulus cell layer that surrounds the oocyte. On acrosome-reacted sperm, PH-20 has a required function in sperm-zona binding (secondary binding). Because hyaluronic acid (HA) has been detected in the zona pellucida, secondary sperm-zona adhesion could depend on repetitive binding and hydrolysis of HA by PH-20 acting as a hyaluronidase. Alternatively, PH-20 may be bifunctional and have a second, different activity required for secondary binding. To distinguish between these two possibilities, in this study we used reagents that inhibit either PH-20's function in sperm-zona binding or its hyaluronidase activity. We found that an anti-PH-20 monoclonal antibody that inhibited sperm-zona binding (approximately 90%) had no effect on hyaluronidase activity. Conversely, apigenin, a hyaluronidase inhibitor, blocked PH-20 hyaluronidase activity 93% without inhibiting sperm-zona binding. Similarly, another anti-PH-20 monoclonal antibody that inhibited hyaluronidase activity 95% only partially inhibited sperm-zona binding (approximately 45%). We also extensively pretreated oocytes with hyaluronidase to remove all accessible HA on or in the zona pellucida and found little or no effect on secondary sperm-zona binding. Our results suggest that PH-20 is bifunctional and has two activities: a hyaluronidase activity and a second, separate activity required for secondary sperm-zona binding. PMID:8793062

  13. Vaccine for human contraception targeting sperm Izumo protein and YLP12 dodecamer peptide

    PubMed Central

    Naz, Rajesh K

    2014-01-01

    There is an urgent need to develop a better method of contraception which is non-steroidal and reversible to control world population explosion and unintended pregnancies. Contraceptive vaccines (CV), especially targeting sperm-specific proteins, can provide an ideal contraceptive modality. Sperm-specific proteins can induce an immune response in women as well as men, thus can be used for CV development in both sexes. In this article, we will review two sperm-specific proteins, namely Izumo protein and YLP12 dodecamer peptide. Gene-knockout studies indicate that Izumo protein is essential for sperm–egg membrane fusion. Vaccination with Izumo protein or its cDNA causes a significant reduction in fertility of female mice. The antibodies to human Izumo inhibit human sperm penetration assay. Recently, our laboratory found that a significant percentage of infertile women have antibodies to Izumo protein. The second sperm-specific protein is YLP12, a peptide mimetic sequence present on human sperm involved in recognition and binding to the human oocyte zona pellucida. Vaccination with YLP12 or its cDNA causes long-term, reversible contraception, without side effects, in female mice. Infertile, but not fertile, men and women have antibodies to YLP12 peptide. Our laboratory has isolated, cloned, and sequenced cDNA encoding human single chain variable fragment (scFv) antibody from infertile men which reacts with YLP12 peptide. The human YLP12 scFv antibody may provide a novel passive immunocontraceptive, the first of its kind. In conclusion, sperm-specific Izumo protein and YLP12 peptide can provide exciting candidates for antisperm CV development. PMID:24723387

  14. Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

    PubMed Central

    Capkova, Jana; Kubatova, Alena; Ded, Lukas; Tepla, Olina; Peknicova, Jana

    2016-01-01

    Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins. PMID:25926605

  15. Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies.

    PubMed

    Capkova, Jana; Kubatova, Alena; Ded, Lukas; Tepla, Olina; Peknicova, Jana

    2016-01-01

    Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins. PMID:25926605

  16. 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.

  17. 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. PMID:26902467

  18. 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

  19. Epididymal protein Rnase10 is required for post-testicular sperm maturation and male fertility.

    PubMed

    Krutskikh, Anton; Poliandri, Ariel; Cabrera-Sharp, Victoria; Dacheux, Jean Louis; Poutanen, Matti; Huhtaniemi, Ilpo

    2012-10-01

    Eutherian spermatozoa are dependent on the environment of the proximal epididymis to complete their maturation; however, no specific epididymal factors that mediate this process have so far been identified. Here, we show that targeted disruption of the novel gene Rnase10 encoding a secreted proximal epididymal protein in the mouse results in a binding defect in spermatozoa and their inability to pass through the uterotubal junction in the female. The failure to gain the site of fertilization in the knockout spermatozoa is associated with a gradual loss of ADAM3 and ADAM6 proteins during epididymal transit. In the distal epididymis, these spermatozoa appear to lack calcium-dependent associations with the immobilizing glutinous extracellular material and are released as single, vigorously motile cells that display no tendency for head-to-head agglutination and lack affinity to the oviductal epithelium. In sperm-egg binding assay, they are unable to establish a tenacious association with the zona pellucida, yet they are capable of fertilization. Furthermore, these sperm show accelerated capacitation resulting in an overall in vitro fertilizing ability superior to that of wild-type sperm. We conclude that the physiological role of sperm adhesiveness is in the mechanism of restricted sperm entry into the oviduct rather than in sperm-egg interaction. PMID:22750516

  20. Reactive oxygen species and boar sperm function.

    PubMed

    Awda, Basim J; Mackenzie-Bell, Meghan; Buhr, Mary M

    2009-09-01

    Boar spermatozoa are very susceptible to reactive oxygen species (ROS), but ROS involvement in damage and/or capacitation is unclear. The impact of exposing fresh boar spermatozoa to an ROS-generating system (xanthine/xanthine oxidase; XA/XO) on sperm ROS content, membrane lipid peroxidation, phospholipase (PL) A activity, and motility, viability, and capacitation was contrasted to ROS content and sperm function after cryopreservation. Exposing boar sperm (n = 4-5 ejaculates) to the ROS-generating system for 30 min rapidly increased hydrogen peroxide (H2O2) and lipid peroxidation in all sperm, increased PLA in dead sperm, and did not affect intracellular O2- (flow cytometry of sperm labeled with 2',7'-dichlorodihydrofluorscein diacetate, BODIPY 581/591 C11, bis-BODIPY-FL C11, hydroethidine, respectively; counterstained for viability). Sperm viability remained high, but sperm became immotile. Cryopreservation decreased sperm motility, viability, and intracellular O2- significantly, but did not affect H2O2. As expected, more sperm incubated in capacitating media than Beltsville thawing solution buffer underwent acrosome reactions and protein tyrosine phosphorylation (four proteins, 58-174 kDa); which proteins were tyrosine phosphorylated was pH dependent. Pre-exposing sperm to the ROS-generating system increased the percentage of sperm that underwent acrosome reactions after incubation in capacitating conditions (P < 0.025), and decreased capacitation-dependent increases in two tyrosine-phosphorylated proteins (P < or = 0.035). In summary, H2O2 is the major free radical mediating direct ROS effects, but not cryopreservation changes, on boar sperm. Boar sperm motility, acrosome integrity, and lipid peroxidation are more sensitive indicators of oxidative stress than viability and PLA activity. ROS may stimulate the acrosome reaction in boar sperm through membrane lipid peroxidation and PLA activation. PMID:19357363

  1. 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

  2. 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.

  3. Mass Spectrometry and Next-Generation Sequencing Reveal an Abundant and Rapidly Evolving Abalone Sperm Protein

    PubMed Central

    Palmer, Melody R.; McDowall, Margo H.; Stewart, Lia; Ouaddi, Aleena; MacCoss, Michael J.; Swanson, Willie J.

    2014-01-01

    SUMMARY Abalone, a broadcast spawning marine mollusk, is an important model for molecular interactions and positive selection in fertilization, but the focus has previously been on only two sperm proteins, lysin and sp18.We used genomic and proteomic techniques to bring new insights to this model by characterizing the testis transcriptome and sperm proteome of the Red abalone Haliotis rufescens. One pair of homologous, testis-specific proteins contains a secretion signal and is small, abundant, and associated with the acrosome. Comparative analysis revealed that homologs are extremely divergent between species, and show strong evidence for positive selection. The acrosomal localization and rapid evolution of these proteins indicates that they play an important role in fertilization, and could be involved in the species-specificity of sperm-egg interactions in abalone. Our genomic and proteomic characterization of abalone fertilization resulted in the identification of interesting, novel peptides that have eluded detection in this important model system for 20 years. PMID:23585193

  4. β1 Integrin is an Adhesion Protein for Sperm Binding to Eggs

    PubMed Central

    Baessler, Keith A.; Lee, Younjoo; Sampson, Nicole S.

    2009-01-01

    We investigated the role of β1 integrin in mammalian fertilization and the mode of inhibition of fertilinβ-derived polymers. We determined that polymers displaying the Glu-Cys-Asp peptide from the fertilinβ disintegrin domain mediate inhibition of mammalian fertilization through a β1 integrin receptor on the egg surface. Inhibition of fertilization is a consequence of competition with sperm binding to the cell surface, not activation of an egg-signaling pathway. The presence of the β1 integrin on the egg surface increases the rate of sperm attachment, but does not alter the total number of sperm that can attach or fuse to the egg. We conclude that the presence of β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane and that subsequent attachment and fusion are mediated by additional egg and sperm proteins present in the β1 integrin complex. Therefore, the mechanisms by which sperm fertilize wild-type and β1 knockout eggs are different. PMID:19338281

  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. 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

  7. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. 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. PMID:16026541

  9. 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

  10. Effects of lactoferrin, a protein present in the female reproductive tract, on parameters of human sperm capacitation and gamete interaction.

    PubMed

    Zumoffen, C M; Massa, E; Caille, A M; Munuce, M J; Ghersevich, S A

    2015-11-01

    In a recent study, lactoferrin (LF) was detected in human oviductal secretion. The protein was able to bind to oocytes and sperm, and modulated gamete interaction. The aim of the present study was to investigate the effect of LF on parameters related to human sperm capacitation and sperm-zona pellucida interaction. Semen samples were obtained from healthy normozoospermic donors (n = 7). Human follicular fluids and oocytes were collected from patients undergoing in vitro fertilization. Motile sperm obtained by swim-up were incubated for 6 or 22 h under capacitating conditions with LF (0-100 μg/mL). After incubations, viability, motility, presence of α-d-mannose receptors (using a fluorescent probe on mannose coupled to bovine serum albumin), spontaneous and induced acrosome reaction (assessed with Pisum sativum agglutinin conjugated to fluorescein isothiocyanate), and tyrosine phosphorylation of sperm proteins were evaluated. Sperm-zona pellucida interaction in the presence of LF was investigated using the hemizone assay. The presence of LF did not affect sperm viability or motility, but caused a dose-dependent significant decrease in sperm α-d-mannose-binding sites, and the effect was already significant with the lowest concentration of the protein used after 22 h incubation. Dose-dependent significant increases in both induced acrosome reaction and tyrosine phosphorylation of sperm proteins were observed in the presence of LF. The present data indicate that LF modulates parameters of sperm function. The inhibition of gamete interaction by LF could be partially explained by the decrease in sperm d-mannose-binding sites. The presence of the LF promoted sperm capacitation in vitro. PMID:26445132

  11. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.

    PubMed

    González-Fernández, Lauro; Macías-García, Beatriz; Loux, Shavahn C; Varner, Dickson D; Hinrichs, Katrin

    2013-06-01

    Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways. PMID:23595906

  12. 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. PMID:24494601

  13. 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...

  14. 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

  15. 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.

  16. Association of heat shock protein 90 with motility of post-thawed sperm in bulls.

    PubMed

    Zhang, Xiao-Gang; Hu, Shan; Han, Cong; Zhu, Qing-Chao; Yan, Guan-Jie; Hu, Jian-Hong

    2015-04-01

    The correlation between the 90 kDa heat-shock protein (HSP90) and sperm quality following the process of freezing-thawing in bulls has not been studied clearly. Therefore, the objective of the present was to clarify the relationship between HSP90 level and semen parameters during the process of cryopreservation in bulls. Semen samples from 5 Holstein bulls were obtained by artificial vagina. Characteristics of these semen at three stages (fresh, after equilibration and frozen-thawed), including motility, plasma membrane integrity and acrosome integrity were evaluated. The mRNA expression level of HSP90 at the three stages was evaluated by using quantitative Real-Time PCR. Meanwhile, the protein level of HSP90 expression at the three stages was detected according to Western blot. The results showed that sperm parameters evaluated in fresh semen was the highest in the three groups. Sperm parameters in semen after equilibration were lower than those in fresh semen (P>0.05) and higher than those in post-thawed semen (P<0.05). Sperm parameters in frozen-thawed semen were the lowest among the three groups (P<0.05). This study indicated that HSP90 expression is proportional to sperm quality. HSP90 expression level in fresh semen was significantly higher than that in frozen-thawed semen (P<0.05). Although no significant differences in HSP90 expression were observed between fresh semen and semen after equilibration (P>0.05). Results in this study suggest that HSP90 level in bull spermatozoa was gradually declined following the process of freezing-thawing, and might be associated with sperm motility, plasma membrane integrity and acrosome integrity. PMID:25578982

  17. 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...

  18. Epididymal Binder of SPerm genes and proteins: what do we know a decade later?

    PubMed

    Plante, G; Manjunath, P

    2015-09-01

    Binder of SPerm (BSP) proteins from ungulates (more specifically bull, bison, buffalo, stallion, boar, goat, and ram) have been extensively studied in the past 30 years. These proteins secreted by seminal vesicles constitute between 1 and 60% of total seminal plasma proteins depending on the species. In addition to sharing many biochemical characteristics such as the ability to bind to gelatin, glycosaminoglycans, choline phospholipids, and lipoproteins, they also share a main function: promoting sperm capacitation. Over the last 10 years, new members of the BSP superfamily have been discovered. These proteins found in bulls, humans, mice, and rabbits are expressed in the epididymides rather than in seminal vesicles and constitute only a minute percentage of the seminal plasma proteins. However, they share many characteristics with BSPs expressed by accessory glands including their structure, their ability to bind to the aforementioned BSP ligands, as well as their ability to promote sperm capacitation. More investigations need to be done on epididymal BSP proteins, but studies described in this review constitute a solid foundation toward deciphering the significance of epididymal BSP proteins in male fertility. PMID:26236016

  19. Enkurin is a novel calmodulin and TRPC channel binding protein in sperm.

    PubMed

    Sutton, Keith A; Jungnickel, Melissa K; Wang, Yanli; Cullen, Kay; Lambert, Stephen; Florman, Harvey M

    2004-10-15

    The TRPC cation channel family has been implicated in receptor- or phospholipase C (PLC)-mediated Ca2+ entry into animal cells. These channels are present in mammalian sperm and are assigned a role in ZP3-evoked Ca2+ influx that drives acrosome reactions. However, the mechanisms controlling channel activity and coupling Ca2+ entry through these channels to cellular responses are not well understood. A yeast two-hybrid screen was carried out to identify TRPC-interacting proteins that would be candidate regulators or effectors. We identified a novel protein, enkurin, that is expressed at high levels in the testis and vomeronasal organ and at lower levels in selected other tissues. Enkurin interacts with several TRPC proteins (TRPC1, TRPC2, TRPC5, but not TRPC3) and colocalizes with these channels in sperm. Three protein-protein interaction domains were identified in enkurin: a C-terminal region is essential for channel interaction; an IQ motif binds the Ca2+ sensor, calmodulin, in a Ca2+-dependent manner; and a proline-rich N-terminal region contains predicted ligand sequences for SH3 domain proteins, including the SH3 domain of the p85 regulatory subunit of 1-phosphatidylinositol-3-kinase. We suggest that enkurin is an adaptor that functions to localize a Ca2+ sensitive signal transduction machinery in sperm to a Ca2+-permeable ion channel. PMID:15385169

  20. Preliminary study of sperm chromatin characteristics of the brachyuran crab Maja brachydactyla. Histones and nucleosome-like structures in decapod crustacean sperm nuclei previously described without SNBPs.

    PubMed

    Kurtz, K; Ausió, J; Chiva, M

    2009-10-01

    An interesting characteristic of decapod crustacean sperm nuclei is that they do not contain highly packaged chromatin. In the present study we re-examine the presence of DNA-interacting proteins in sperm nuclei of the brachyuran Maja brachydactyla. Although previous reports have indicated that, unlike the majority of sperm cells, DNA of decapod sperm is not organized by basic proteins, in this work we show that: (1) histones are present in sperm of M. brachydactyla; (2) histones are associated with sperm DNA; (3) histone H3 appears in lower proportions than the other core histones, while histone H2B appears in higher proportions; and (4) histone H3 in sperm nuclei is acetylated. This work complements a previous study of sperm histones of Cancer pagurus and supports the suggestion that decapod crustacean sperm chromatin deserves further attention. PMID:19324386

  1. Protein fraction isolated from epididymal fluid re-associates sperm in vitro: possible role of serpins in rat rosettes assembly.

    PubMed

    Monclus, María A; Andreina, Cesari; Cabrillana, María E; Lancellotti, Tania E Saez; Rensetti, Daniel E; Clementi, Marisa A; Boarelli, Paola V; Vincenti, Amanda E; Fornés, Miguel W

    2010-05-01

    In many mammalian species, sperm associate as a consequence of the epididymal transit. From the classic Rouleaux in guinea pig to the most recent work in mouse and echidna, authors have focused mainly on a detailed morphological description of this phenomenon. Some of these articles have also begun to describe the nature of the material present between sperm heads. Here, we try to better understand the factor/s involved in rat sperm association (Rosette). Based on previous work describing the appearance of Rosettes in the distal segments of the rat epididymis, we consider that sperm during their transit must be in contact with factor/s present in the caudal lumen in order to associate with each other. By an in vitro sperm re-associating assay, we try to determine the in vivo phenomenon observed in the lumen. The assay consists of co-incubating non-associated sperm with several protein fractions obtained from epididymal caudal fluid. After establishing the most active fraction, the proteins were characterized by MALDI-TOF mass spectrometry. Among the proteins we found two members of the serine protease inhibitors family; an alpha-1 antitrypsin and a new protein with an alpha-1 antitrypsin like domain which includes a sequence compatible with the serpins' reactive center loop. These serpins may play a role in the assembly/disassembly process of Rosettes by modulating lumenal protease activity. Finally, a biochemical-morphological model which explains the sperm-proteases interaction was proposed. PMID:20143401

  2. 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. PMID

  3. 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. PMID:25638741

  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.- PMID:24970390

  5. Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit.

    PubMed

    Nishijima, Kazutoshi; Tanaka, Mai; Sakai, Yusuke; Koshimoto, Chihiro; Morimoto, Masatoshi; Watanabe, Teruo; Fan, Jianglin; Kitajima, Shuji

    2014-08-01

    We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN2) and then preserved in the LN2. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN2. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos. PMID:24809634

  6. 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. PMID:21088232

  7. 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

  8. 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. PMID:27579691

  9. 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. PMID:27423488

  10. Analysis of the Sperm Head Protein Profiles in Fertile Men: Consistency across Time in the Levels of Expression of Heat Shock Proteins and Peroxiredoxins

    PubMed Central

    Kichine, Elsa; Di Falco, Marcos; Hales, Barbara F.; Robaire, Bernard; Chan, Peter

    2013-01-01

    We investigated the identity and quantitative variations of proteins extracted from human sperm heads using a label-free Gel-MS approach. Sperm samples were obtained from three men with high sperm counts at three different time points. This design allowed us to analyse intra-individual and inter-individual variations of the human sperm head proteome. Each time point was analyzed in triplicate to minimize any background artifactual effects of the methodology on the variation analyses. Intra-individual analysis using the spectral counting method revealed that the expression levels of 90% of the common proteins identified in three samples collected at various time-points, separated by several months, had a coefficient of variation of less than 0.5 for each man. Across individuals, the expression level of more than 80% of the proteins had a CV under 0.7. Interestingly, 83 common proteins were found within the core proteome as defined by the intra- and inter-variation analyses set criteria (CV<0.7). Some of these uniformly expressed proteins were chaperones, peroxiredoxins, isomerases, and cytoskeletal proteins. Although there is a significant level of inter-individual variation in the protein profiles of human sperm heads even in a well-defined group of men with high sperm counts, the consistent expression levels of a wide range of proteins points to their essential role during spermatogenesis. PMID:24204839

  11. 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.

  12. Sperm and seminal fluid proteomes of the field cricket Teleogryllus oceanicus: identification of novel proteins transferred to females at mating.

    PubMed

    Simmons, L W; Tan, Y-F; Millar, A H

    2013-02-01

    Reproductive proteins are amongst the most evolutionarily divergent proteins known, and research on genetically well-characterized species suggests that postcopulatory sexual selection might be important in their evolution; however, we lack the taxonomic breadth of information on reproductive proteins that is required to determine the general importance of sexual selection for their evolution. We used transcriptome sequencing and proteomics to characterize the sperm and seminal fluid proteins of a cricket, Teleogryllus oceanicus, that has been widely used in the study of postcopulatory sexual selection. We identified 57 proteins from the sperm of these crickets. Many of these had predicted function in glycolysis and metabolism, or were structural, and had sequence similarity to sperm proteins found across taxa ranging from flies to humans. We identified 21 seminal fluid proteins, some of which resemble those found to be involved in postmating changes to female reproduction in other species. Some 27% of sperm proteins and 48% of seminal fluid proteins were of unknown function. The characterization of seminal fluid proteins in this species will allow us to explore their adaptive significance, and to contribute comparative data that will facilitate a general appreciation of the evolution of reproductive proteins within and among animal taxa. PMID:23211034

  13. Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression

    PubMed Central

    Kouprina, Natalay; Noskov, Vladimir N.; Pavlicek, Adam; Collins, N. Keith; Schoppee Bortz, Pamela D.; Ottolenghi, Chris; Loukinov, Dmitri; Goldsmith, Paul; Risinger, John I.; Kim, Jung-Hyun; Westbrook, V. Anne; Solomon, Gregory; Sounders, Hanna; Herr, John C.; Jurka, Jerzy; Lobanenkov, Victor; Schlessinger, David; Larionov, Vladimir

    2007-01-01

    The sperm protein associated with nucleus in the X chromosome (SPANX) genes cluster at Xq27 in two subfamilies, SPANX-A/D and SPANX-N. SPANX-A/D is specific for hominoids and is fairly well characterized. The SPANX-N gave rise to SPANX-A/D in the hominoid lineage ∼7 MYA. Given the proposed role of SPANX genes in spermatogenesis, we have extended studies to SPANX-N gene evolution, variation, regulation of expression, and intra-sperm localization. By immunofluorescence analysis, SPANX-N proteins are localized in post-meiotic spermatids exclusively, like SPANX-A/D. But in contrast to SPANX-A/D, SPANX-N are found in all ejaculated spermatozoa rather than only in a subpopulation, are localized in the acrosome rather than in the nuclear envelope, and are expressed at a low level in several nongametogenic adult tissues as well as many cancers. Presence of a binding site for CTCF and its testis-specific paralogue BORIS in the SPANX promoters suggests, by analogy to MAGE-A1 and NY-ESO-1, that their activation in spermatogenesis is mediated by the programmed replacement of CTCF by BORIS. Based on the relative density of CpG, the more extended expression of SPANX-N compared to SPANX-A/D in nongametogenic tissues is likely attributed to differences in promoter methylation. Our findings suggest that the recent duplication of SPANX genes in hominoids was accompanied by different localization of SPANX-N proteins in post-meiotic sperm and additional expression in several nongonadal tissues. This suggests a corresponding functional diversification of SPANX gene families in hominoids. SPANX proteins thus provide unique targets to investigate their roles in the function of spermatozoa, selected malignancies, and for SPANX-N, in other tissues as well. PMID:17406683

  14. IGF1 stabilizes sperm membrane proteins to reduce cryoinjury and maintain post-thaw sperm motility in buffalo (Bubalus bubalis) spermatozoa.

    PubMed

    Selvaraju, Sellappan; Krishnan, Binsila B; Archana, Santhanahalli Siddalingappa; Ravindra, Janivara Parameshwaraiah

    2016-08-01

    Insulin like growth factor 1 (IGF1) in the seminal plasma is reported to improve sperm motility by reducing oxidative stress. The present study was conducted to assess the effect of addition of IGF1 on sperm function and protein composition during cryopreservation process. Semen samples were collected from six Murrah buffaloes (2 ejaculates from each animal) and diluted (80 million/ml) in tris egg yolk extender and divided into control, T1, T2 and T3, groups supplemented with 0, 50, 100 and 150 ng of IGF1/mL, respectively. The semen was filled in straws (250 μL) and straws from each group were divided into two batches. One batch was processed for freezing and another batch was incubated at 4 °C for 4 h. The sperm kinematic and functional parameters were studied in both the batches. A significant (P < 0.05) positive effect of IGF1 was observed on functional membrane integrity (%) during incubation at 4 °C for 4 h in T3 as compared to control group. The spermatozoa (%) positive for structural membrane integrity, mitochondrial membrane potential and the metabolic activity in post-thaw semen were significantly (P < 0.05) high in T3 than the control group. The acrosomal integrity was significantly (P < 0.05) higher in T2 group as compared to control. The proteins (kDa) of 17.3 with pI 4.2 (calmodulin), 11.3 with pI 6.5 (dermcidin) and 18.1 with pI 5.5 (sperm acrosome membrane associated protein3) were protected in IGF1 group. The study suggests that IGF1 can be added to the extender for improving cryosurvial of buffalo spermatozoa. PMID:27256665

  15. Minimization of apoptosis-like changes in cryopreserved buffalo bull sperm by supplementing extender with Bcl-2 protein

    PubMed Central

    Dalal, Jasmer; Kumar, Ajeet; Honparkhe, Mrigank; Deka, Dipak; Singh, Narinder

    2016-01-01

    Aim: This study was aimed at evaluating the anti-apoptotic effects of Bcl-2 protein in cryopreserved buffalo bull sperm. Materials and Methods: A total 10 ejaculates from two buffalo bulls (5 each) were collected using artificial vagina method, and semen was evaluated using a standard protocol. Semen was extended by Tris egg yolk extender supplemented with Bcl-2 protein at 5, 10, and 15 µM. Semen was cryopreserved at ultra-low temperature using traditional vapor freezing method. Pre-freeze and post-thaw semen samples were evaluated for percent motility, viability, hypo-osmotic swelling test (HOST) reactive sperms; status of mitochondrial membrane activity and status of sperm phospholipase A1 and phospholipase A2 activity. Results: There were no significant effects of Bcl-2 protein supplementation on pre-freeze sperm quality. Percent motility and active mitochondria in post-thaw Bcl-2 supplemented and control groups were also similar. However, viable sperms were significantly (p<0.05) higher (74.29±4.23%) in Bcl-2 supplemented group (5 µM) as compared to control (51.6±5.77%). The proportion of HOST reactive sperms was also higher (63.1±6.73%) in Bcl-2 supplemented (5 µM) group as compared to control (50.7±6.98%). The sperm with low PLA activity (non-apoptotic) was significantly (p<0.05) higher in all the supplemented doses of Bcl-2 protein, i.e., at 5 µM (73.42±5.79%), 10 µM (75.51±6.22%), and 15 µM (74.78±5.89%) as compared to control (60.23±4.45%). We found that Bcl-2 protein supplementation at 5 µM dose improved the post-thaw semen quality indicated by higher viability, HOST reactive sperms, and sperm with low PLA activity (non-apoptotic sperms). Conclusion: Bcl-2 protein supplementation exerts its protective effect on spermatozoa against apoptosis-like changes developed during cryopreservation. PMID:27284216

  16. An ARID Domain-Containing Protein within Nuclear Bodies Is Required for Sperm Cell Formation in Arabidopsis thaliana

    PubMed Central

    Zheng, Binglian; He, Hui; Zheng, Yanhua; Wu, Wenye; McCormick, Sheila

    2014-01-01

    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, plays a key role in sperm cell formation by activating expression of several germline genes. But how DUO1 itself is activated and how sperm cell formation is initiated remain unknown. To expand our understanding of sperm cell formation, we characterized an ARID (AT-Rich Interacting Domain)-containing protein, ARID1, that is specifically required for sperm cell formation in Arabidopsis. ARID1 localizes within nuclear bodies that are transiently present in the generative cell from which sperm cells arise, coincident with the timing of DUO1 activation. An arid1 mutant and antisense arid1 plants had an increased incidence of pollen with only a single sperm-like cell and exhibited reduced fertility as well as reduced expression of DUO1. In vitro and in vivo evidence showed that ARID1 binds to the DUO1 promoter. Lastly, we found that ARID1 physically associates with histone deacetylase 8 and that histone acetylation, which in wild type is evident only in sperm, expanded to the vegetative cell nucleus in the arid1 mutant. This study identifies a novel component required for sperm cell formation in plants and uncovers a direct positive regulatory role of ARID1 on DUO1 through association with histone acetylation. PMID:25057814

  17. Sperm nuclear proteome and its epigenetic potential.

    PubMed

    Castillo, J; Amaral, A; Oliva, R

    2014-05-01

    The main function of the sperm cell is to transmit the paternal genetic message and epigenetic information to the embryo. Importantly, the majority of the genes in the sperm chromatin are highly condensed by protamines, whereas genes potentially needed in the initial stages of development are associated with histones, representing a form of epigenetic marking. However, so far little attention has been devoted to other sperm chromatin-associated proteins that, in addition to histones and protamines, may also have an epigenetic role. Therefore, with the goal of contributing to cover this subject we have compiled, reviewed and report a list of 581 chromatin or nuclear proteins described in the human sperm cell. Furthermore, we have analysed their Gene Ontology Biological Process enriched terms and have grouped them into different functional categories. Remarkably, we show that 56% of the sperm nuclear proteins have a potential epigenetic activity, being involved in at least one of the following functions: chromosome organization, chromatin organization, protein-DNA complex assembly, DNA packaging, gene expression, transcription, chromatin modification and histone modification. In addition, we have also included and compared the sperm cell proteomes of different model species, demonstrating the existence of common trends in the chromatin composition in the mammalian mature male gamete. Taken together, our analyses suggest that the mammalian sperm cell delivers to the offspring a rich combination of histone variants, transcription factors, chromatin-associated and chromatin-modifying proteins which have the potential to encode and transmit an extremely complex epigenetic information. PMID:24327354

  18. A 130-kDa membrane protein of sperm flagella is the receptor for asterosaps, sperm-activating peptides of starfish Asterias amurensis.

    PubMed

    Nishigaki, T; Chiba, K; Hoshi, M

    2000-03-01

    Spermatozoa of the starfish, Asterias amurensis, have a specific receptor for asterosap, a sperm-activating peptide isolated from the jelly coat of homologous eggs. We characterized the receptor by using several asterosap derivatives. Analysis of equilibrium binding of radioactive di-iodinated Bolton-Hunter reagent-labeled asterosap ((125)I(2)-BHP15) to the spermatozoa indicated that the cell has 1.1 x 10(5) binding sites of high affinity (K(d) = 57 pM), and also the receptor showed positive cooperativity for asterosap binding. When spermatozoa were treated with fluorophore-labeled asterosap, the sperm flagella were labeled, indicating that the receptors are mostly localized in the sperm tail. When spermatozoa were reacted with radioactive asterosap prelabeled with photoaffinity cross-linkers, a single 130-kDa membrane protein of sperm flagella was specifically radiolabeled. This result was reproducible regardless of the length of spacer arm of cross-linkers so far studied. Therefore, the 130-kDa protein is likely to be the receptor for asterosaps. Modification of asterosap at the N-terminal region with bulky molecules such as carboxyfluorescein did not affect the activity of asterosap, suggesting that the N-terminus of asterosap is not involved in the ligand-receptor interaction. On the other hand, S-alkylated asterosaps did not compete with (125)I(2)-BHP15 for binding to the receptor, indicating that disulfide linkage of asterosap is essential for the ligand-receptor interaction. The properties of the receptor, high affinity and high concentration, enabled us to apply the fluorescence polarization technique to study the molecular interaction between asterosap and the receptor. Using this method, we performed binding experiments in almost real time and found that divalent cations are significantly involved in the interaction between asterosap and the receptor. PMID:10677262

  19. A role for sperm surface protein disulfide isomerase activity in gamete fusion: evidence for the participation of ERp57.

    PubMed

    Ellerman, Diego A; Myles, Diana G; Primakoff, Paul

    2006-06-01

    In mammals, sperm-egg interaction is based on molecular events either unique to gametes or also present in somatic cells. In gamete fusion, it is unknown which features are gamete specific and which are shared with other systems. Conformational changes mediated by thiol-disulfide exchange are involved in the activation of some virus membrane fusion proteins. Here we asked whether that mechanism is also operative in sperm-egg fusion. Different inhibitors of protein disulfide isomerase (PDI) activity were able to inhibit sperm-egg fusion in vitro. While pretreatment of oocytes had no effect, pretreatment of sperm reduced their fusion ability. Some members of the PDI family were detected on the sperm head, and use of specific antibodies and substrates suggested that the oxidoreductase ERp57 has a role in gamete fusion. The results support the idea that thiol-disulfide exchange is a mechanism that may act in gamete fusion to produce conformational changes in fusion-active proteins. PMID:16740484

  20. 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. PMID:9311151

  1. The structure of sperm Izumo1 reveals unexpected similarities with Plasmodium invasion proteins.

    PubMed

    Nishimura, Kaoru; Han, Ling; Bianchi, Enrica; Wright, Gavin J; de Sanctis, Daniele; Jovine, Luca

    2016-07-25

    Fertilization, the culminating event in sexual reproduction, occurs when haploid sperm and egg recognize each other and fuse to form a diploid zygote. In mammals this process critically depends on the interaction between Izumo1, a protein exposed on the equatorial segment of acrosome-reacted sperm, and the egg plasma-membrane-anchored receptor Juno [1,2]. The molecular mechanism triggering gamete fusion is unresolved because both Izumo1 and Juno lack sequence similarity to known membrane fusogens. Here we report the crystal structure of Izumo1, which reveals a membrane distal region composed of a four-helix bundle connected to a carboxy-terminal immunoglobulin (Ig)-like domain through a β-hairpin stabilized by disulfide bonds. Remarkably, different regions of Izumo1 display significant structural similarities to two proteins expressed by the invasive sporozoite stage of Plasmodium parasites: SPECT1, which is essential for host cell traversal and hepatocyte invasion [3]; and TRAP, which is necessary for gliding motility and invasion [4]. These observations suggest a link between the molecular mechanisms underlying host cell invasion by the malaria parasite and gamete membrane fusion at fertilization. PMID:27374339

  2. Data on endogenous chicken sperm peptides and small proteins obtained through Top-Down High Resolution Mass Spectrometry.

    PubMed

    Soler, L; Labas, V; Thélie, A; Teixeira-Gomes, A P; Grasseau, I; Bouguereau, L; Blesbois, E

    2016-09-01

    The endogenous peptides and small proteins present in chicken sperm were identified in the context of the characterization of a fertility-diagnostic method based on the use of ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). The interpretation and description of these data can be found in a research article, "Intact cell MALDI-TOF MS on sperm: a molecular test for male fertility diagnosis" (Soler et al., 2016) [1], and raw data derived from this analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002768. Here, we describe the inventory of all the molecular species identified, along with their biochemical features and functional analysis. This peptide/protein catalogue can be further employed as reference for other studies and reveal that the use of proteomics allows for a global evaluation of sperm cells functions. PMID:27617276

  3. Sperm protein 17 is an oncofetal antigen: a lesson from a murine model.

    PubMed

    Arnaboldi, F; Menon, A; Menegola, E; Di Renzo, F; Mirandola, L; Grizzi, F; Figueroa, J A; Cobos, E; Jenkins, M; Barajon, I; Chiriva-Internati, Maurizio

    2014-10-01

    Sperm protein 17 (Sp17) was originally identified in the flagellum of spermatozoa and subsequently included in the subfamily of tumor-associated antigens known as cancer-testes antigens (CTA). Sp17 has been associated with the motility and migratory capacity in tumor cells, representing a link between gene expression patterns in germinal and tumor cells of different histological origins. Here we review the relevance of Sp17 expression in the mouse embryo and cancerous tissues, and present additional data demonstrating Sp17 complex expression pattern in this murine model. The expression of Sp17 in embryonic as well as adult neoplastic cells, but not normal tissues, suggests this protein should be considered an "oncofetal antigen." Further investigations are necessary to elucidate the mechanisms and functional significance of Sp17 aberrant expression in human adult cells and its implication in the pathobiology of cancer. PMID:24811209

  4. 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. PMID:9166697

  5. Tctex2: a sperm tail surface protein mapping to the t-complex.

    PubMed

    Huw, L Y; Goldsborough, A S; Willison, K; Artzt, K

    1995-07-01

    Transmission ratio distortion (TRD) in mouse t-haplotypes remains the most significant example of meiotic drive in vertebrates. While the underlying mechanism that fuels it is still mysterious, TRD is clearly a complex multigene phenomenon. The characterization of Tctex2 (t-complex testis expressed 2) shows it to be one of several candidates for involvement in TRD. Tctex2 maps to the t-complex and encodes a membrane-associated protein found exclusively on the sperm tail. The t-haplotype form of Tctex2 is aberrant in both the level of its expression and its primary amino acid sequence, but is nonetheless translated and transported to its normal location. The multiple amino acid changes in the t-form make it extremely unlikely that it can function normally and, since it is found on sperm tails, suggest that it may actively interfere with the development of normal gamete function in males. The possible role of Tctex2 in t-complex transmission ratio distortion and sterility is discussed. PMID:7601308

  6. Loss of Zona Pellucida Binding Proteins in the Acrosomal Matrix Disrupts Acrosome Biogenesis and Sperm Morphogenesis▿ †

    PubMed Central

    Lin, Yi-Nan; Roy, Angshumoy; Yan, Wei; Burns, Kathleen H.; Matzuk, Martin M.

    2007-01-01

    Zona pellucida binding protein 1 (ZPBP1), a spermatid and spermatozoon protein that localizes to the acrosome, was originally identified in pigs and named for its binding to the oocyte zona pellucida. In an in silico search for germ cell-specific genes, Zpbp1 and its novel paralog, Zpbp2, were discovered and confirmed to be expressed only in the testes in both mice and humans. To study the in vivo functions of both ZPBP proteins, we disrupted Zpbp1 and Zpbp2 in mice. Males lacking ZPBP1 were sterile, with abnormal round-headed sperm morphology and no forward sperm motility. Ultrastructural studies demonstrated that absence of ZPBP1 prevents proper acrosome compaction, resulting in acrosome fragmentation and disruption of the Sertoli-spermatid junctions. Males null for ZPBP2 were subfertile, demonstrated aberrant acrosomal membrane invaginations, and produced dysmorphic sperm with reduced ability to penetrate zona pellucida. Molecular phylogenetic analysis of ZPBPs from amphibians, birds, and mammals suggests that these paralogous genes coevolved to play cooperative roles during spermiogenesis. Whereas ZPBP1 was discovered for an in vitro role in sperm-egg interactions, we have shown that both ZPBP proteins play an earlier structural role during spermiogenesis. PMID:17664285

  7. 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

  8. Requirement for nuclear autoantigenic sperm protein mRNA expression in bovine preimplantation development.

    PubMed

    Nagatomo, Hiroaki; Kohri, Nanami; Akizawa, Hiroki; Hoshino, Yumi; Yamauchi, Nobuhiko; Kono, Tomohiro; Takahashi, Masashi; Kawahara, Manabu

    2016-03-01

    Nuclear autoantigenic sperm protein (NASP) is associated with DNA replication, cell proliferation, and cell cycle progression through its specific binding to histones. The aim of this study was to examine the roles of NASP in bovine preimplantation embryonic development. Using NASP gene knockdown (KD), we confirmed the reduction of NASP messenger RNA (mRNA) expression during preimplantation development. NASP KD did not affect cleavage but significantly decreased development of embryos into the blastocyst stage. Furthermore, blastocyst hatching was significantly decreased in NASP KD embryos. Cell numbers in the inner cell mass of NASP KD blastocysts were also decreased compared to those of controls. These results suggest that NASP mRNA expression is required for preimplantation development into the blastocyst stage in cattle. PMID:26690724

  9. 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

  10. 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

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

    PubMed

    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 1.4×10(4) 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 8.61×10(3) 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. PMID:25909900

  12. 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.

  13. 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

  14. Identification and characterization of a bovine sperm protein that binds specifically to single-stranded telomeric deoxyribonucleic acid.

    PubMed

    Kozik, A; Bradbury, E M; Zalensky, A O

    2000-02-01

    Telomere DNA at the physical termini of chromosomes forms a single-stranded 3' overhang. In lower eukaryotes, e.g., ciliated protozoa, this DNA extension is capped by specific proteins that have been structurally and functionally characterized. Much less is known about single-stranded telomere DNA-binding proteins in vertebrates. Here we describe a new protein from bovine sperm designated bsSSTBP that specifically interacts with single-stranded (TTAGGG)(N) DNA. The bsSSTBP was extracted from nuclei by 0.6 M KCl. The native size of this protein, estimated by gel filtration, was 20-40 kDa. SDS-PAGE of the UV cross-linked complex between bsSSTBP and telomere DNA indicated that several polypeptides are involved in complex formation. Bovine sSSTB had high specificity toward nucleotide sequence, since single nucleotide substitutions in the (TTAGGG)(4) substrate suppressed binding. The minimal number of (TTAGGG) repeats required for binding of bsSSTBP was 3, and the protein recognized linear but not folded DNA structures. We propose that the bsSSTBP participates in telomere-telomere interactions and the telomere membrane localization observed in mature sperm. In mammals, somatic telomere-binding proteins are apparently substituted by sperm-specific ones that may lead to a structural reorganization of telomere domains to fulfill functions important during meiosis and fertilization. PMID:10642571

  15. Loss of R2D2 proteins ROPN1 and ROPN1L causes defects in murine sperm motility, phosphorylation, and fibrous sheath integrity.

    PubMed

    Fiedler, Sarah E; Dudiki, Tejasvi; Vijayaraghavan, Srinivasan; Carr, Daniel W

    2013-02-01

    The fibrous sheath (FS) is a flagellar cytoskeletal structure unique to sperm that surrounds the outer dense fibers and axoneme. Its primary components are A-kinase anchoring proteins (AKAPs) 3 and 4, which suggests that the FS affects flagellar beating via the scaffolding of signaling pathways necessary for motility. Sperm proteins ROPN1 and ROPN1L bind AKAP3. To determine the role of ROPN1 and ROPN1L in sperm function, we created mice deficient in ROPN1 (RKO), mice deficient in ROPN1L (RLKO), and double knockout mice (DKO). All three strains of mice had normal testicular morphology and spermatogenesis. Only the DKOs had obvious defects in sperm morphology (thinning and shredding of the principal piece), which was accompanied by a reduction in AKAP3 levels. RLKO mice had slightly reduced sperm motility and increased levels of ROPN1. RKO mice had moderately impaired motility and increased levels of ROPN1L. DKO sperm were immotile. We have previously determined that RKO male mice are subfertile, and DKO males are infertile. Together these data indicate that ROPN1L and ROPN1 compensate for each other in the absence of the opposing protein, possibly to maintain AKAP3 incorporation in the FS. Sperm from mice lacking ROPN1L exhibited reductions in both cAMP-dependent protein kinase (PKA) phosphorylation of a 270-kDa protein (perhaps FSCB), and in capacitation-induced tyrosine phosphorylation. Sperm from mice lacking ROPN1 had reduced levels of FSCB and increased tyrosine phosphorylation of noncapacitated sperm. These data demonstrate that mutations in ROPN1 and ROPN1L can cause defects in FS integrity, sperm motility, and PKA-dependent signaling processes, leading to male infertility. PMID:23303679

  16. 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. PMID:22486240

  17. Extracellular Regulation of Sperm Transmembrane Adenylyl Cyclase by a Forward Motility Stimulating Protein

    PubMed Central

    Dey, Souvik; Roy, Debarun; Majumder, Gopal C.; Bhattacharyya, Debdas

    2014-01-01

    Forward motility stimulating factor (FMSF), a glycoprotein isolated from buffalo serum, binds to the surface of the mature sperm cells to promote their progressive motility. This article reports the mode of signal transduction of this extracellular factor in goat sperm. The mechanism was investigated by assaying intracellular second messenger level and forward motility in presence of different pharmacological modulators. Mg++-dependent Forskolin responsive form of transmembrane adenylyl cyclase (tmAC) of goat spermatozoa was probed for its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of tmACs, was used to identify the role of this enzyme in the scheme of FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has been inspected using GTPγS. Participation of protein kinase A (PKA) and tyrosine kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC activity in a dose-dependent manner through receptor/G-protein activation to enhance intracellular cAMP and forward motility. Motility boosting effects of this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF displayed substantial motility promoting activity when movement of spermatozoa was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in mediating FMSF action was confirmed by the application of dibutyryl cAMP. Observed motility regulatory effects with IP20 and genistein indicate contribution of PKA and tyrosine kinase in FMSF activity; enhanced phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that augments intracellular cAMP, which through downstream crosstalk of phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus, this article for the first time describes conventional tmAC-dependent profound activation

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

    PubMed

    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

  19. 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

  20. 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

  1. 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

  2. Rheotaxis guides mammalian sperm

    PubMed Central

    Miki, Kiyoshi; Clapham, David E

    2013-01-01

    Background In sea urchins, spermatozoan motility is altered by chemotactic peptides, giving rise to the assumption that mammalian eggs also emit chemotactic agents that guide spermatozoa through the female reproductive tract to the mature oocyte. Mammalian spermatozoa indeed undergo complex adaptations within the female (the process of capacitation) that are initiated by agents ranging from pH to progesterone, but these factors are not necessarily taxic. Currently, chemotaxis, thermotaxis, and rheotaxis have not been definitively established in mammals. Results Here, we show that positive rheotaxis, the ability of organisms to orient and swim against the flow of surrounding fluid, is a major taxic factor for mouse and human sperm. This flow is generated within 4 hours of sexual stimulation and coitus in female mice; prolactin-triggered oviductal fluid secretion clears the oviduct of debris, lowers viscosity, and generates the stream that guides sperm migration in the oviduct. Rheotaxic movement is demonstrated in capacitated and uncapacitated spermatozoa in low and high viscosity medium. Finally, we show that a unique sperm motion we quantify using the sperm head's rolling rate reflects sperm rotation that generates essential force for positioning the sperm in the stream. Rotation requires CatSper channels, presumably by enabling Ca2+ influx. Conclusions We propose that rheotaxis is a major determinant of sperm guidance over long distances in the mammalian female reproductive tract. Coitus induces fluid flow to guide sperm in the oviduct. Sperm rheotaxis requires rotational motion during CatSper channel-dependent hyperactivated motility. PMID:23453951

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

    PubMed Central

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

    2011-01-01

    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. PMID:21571637

  4. 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

  5. Etiologies of sperm oxidative stress.

    PubMed

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

    2016-04-01

    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

  6. Sperm Proteome Maturation in the Mouse Epididymis

    PubMed Central

    Skerget, Sheri; Rosenow, Matthew A.; Petritis, Konstantinos; Karr, Timothy L.

    2015-01-01

    In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm. PMID:26556802

  7. 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. PMID:25660692

  8. 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

  9. Discovery, Primary, and Crystal Structures and Capacitation-related Properties of a Prostate-derived Heparin-binding Protein WGA16 from Boar Sperm*

    PubMed Central

    Garénaux, Estelle; Kanagawa, Mayumi; Tsuchiyama, Tomoyuki; Hori, Kazuki; Kanazawa, Takeru; Goshima, Ami; Chiba, Mitsuru; Yasue, Hiroshi; Ikeda, Akemi; Yamaguchi, Yoshiki; Sato, Chihiro; Kitajima, Ken

    2015-01-01

    Mammalian sperm acquire fertility through a functional maturation process called capacitation, where sperm membrane molecules are drastically remodeled. In this study, we found that a wheat germ agglutinin (WGA)-reactive protein on lipid rafts, named WGA16, is removed from the sperm surface on capacitation. WGA16 is a prostate-derived seminal plasma protein that has never been reported and is deposited on the sperm surface in the male reproductive tract. Based on protein and cDNA sequences for purified WGA16, it is a homologue of human zymogen granule protein 16 (ZG16) belonging to the Jacalin-related lectin (JRL) family in crystal and primary structures. A glycan array shows that WGA16 binds heparin through a basic patch containing Lys-53/Lys-73 residues but not the conventional lectin domain of the JRL family. WGA16 is glycosylated, contrary to other ZG16 members, and comparative mass spectrometry clearly shows its unique N-glycosylation profile among seminal plasma proteins. It has exposed GlcNAc and GalNAc residues without additional Gal residues. The GlcNAc/GalNAc residues can work as binding ligands for a sperm surface galactosyltransferase, which actually galactosylates WGA16 in situ in the presence of UDP-Gal. Interestingly, surface removal of WGA16 is experimentally induced by either UDP-Gal or heparin. In the crystal structure, N-glycosylated sites and a potential heparin-binding site face opposite sides. This geography of two functional sites suggest that WGA16 is deposited on the sperm surface through interaction between its N-glycans and the surface galactosyltransferase, whereas its heparin-binding domain may be involved in binding to sulfated glycosaminoglycans in the female tract, enabling removal of WGA16 from the sperm surface. PMID:25568322

  10. 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.

  11. Heat shock protein A8 stabilizes the bull sperm plasma membrane during cryopreservation: Effects of breed, protein concentration, and mode of use.

    PubMed

    Holt, W V; Del Valle, I; Fazeli, A

    2015-09-15

    Heat shock protein A8 (HSPA8) is a highly conserved member of the Hsp70 family, which is expressed in oviductal cells, translocated into oviductal fluid, and becomes attached to the sperm surface during sperm transport. Previous research has shown that HSPA8 supports mammalian sperm viability during in vitro incubation at both 5 °C and body temperature. The present series of experiments was designed to explore the possibility that bovine recombinant HSPA8 might therefore protect bull spermatozoa during cryopreservation through its beneficial effects on the sperm plasma membrane. Soy-based cryopreservation media were used in these experiments. The effects of HSPA8 addition before freezing were examined at concentrations ranging from 0.2 to 6.4 μg/mL, whereas the effects of postthaw HSPA8 addition were tested between 0.2 and 12.8 μg/mL. When bull spermatozoa (from beef and dairy breeds) were frozen in the presence of HSPA8, beneficial but complex effects on postthaw viability were observed. Low HSPA8 concentrations (0.2 and 0.4 μg/mL) resulted in significantly reduced postthaw sperm viability, but concentrations above 0.8 μg/mL improved plasma membrane integrity. If HSPA8 was added to spermatozoa after thawing, outcomes were also biphasic and beneficial effects on viability were only seen if the HSPA8 concentration exceeded 3.2 μg/mL. Beneficial effects were significantly more apparent with beef rather than dairy breeds. When HSPA8 was used in combination with cholesterol-loaded cyclodextrin, spermatozoa from the beef breeds showed significantly lower apoptotic effects. This was not observed with the dairy breeds. PMID:26047707

  12. Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa.

    PubMed

    Huang, S Y; Kuo, Y H; Lee, W C; Tsou, H L; Lee, Y P; Chang, H L; Wu, J J; Yang, P C

    1999-04-01

    The decline in boar semen quality after cryopreservation may be attributed to changes in intracellular proteins. Thus, the aim of the present study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually mature boars (mean age = 25.5+/-12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15 degrees C, after cooling to 5 degrees C, following thawing after freezing to -100 degrees C, and following thawing after 1 wk of cryopreservation at -196 degrees C. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5x10(6) spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was 90 kDa heat-shock protein (HSP90). Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5 degrees C. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5 degrees C. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100 degrees C. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The length of time between a decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa. PMID:10729022

  13. FRET analysis using sperm-activating peptides tagged with fluorescent proteins reveals that ligand-binding sites exist as clusters.

    PubMed

    Arcos-Hernández, César; Romero, Francisco; Sánchez-Guevara, Yoloxochitl; Beltrán, Carmen; Nishigaki, Takuya

    2016-02-01

    Long-range cellular communication between the sperm and egg is critical for external fertilization. Sperm-activating peptides (SAPs) are diffusible components of the outer layer of eggs in echinoderms, and function as chemoattractants for spermatozoa. The decapeptide named speract is the best-characterized sea urchin SAP. Biochemical and physiological actions of speract have been studied with purified or chemically synthesized peptides. In this work, we prepared recombinant speract fused to a fluorescent protein (FP; FP-speract) using three color variants: a cyan (eCFP), a yellow (mVenus) and a large Stokes shift yellow (mAmetrine) FP. Although these fluorescence tags are 20 times larger than speract, competitive binding experiments using mAmetrine-speract revealed that this FP-speract has binding affinity to the receptor that is comparable (7.6-fold less) to that of non-labeled speract. Indeed, 10 nmol l(-1) eCFP-speract induces physiological sperm responses such as membrane potential changes and increases in intracellular pH and Ca(2+) concentrations similar to those triggered by 10 nmol l(-1) speract. Furthermore, FP-speract maintains its fluorescence upon binding to its receptor. Using this property, we performed fluorescence resonance energy transfer (FRET) measurements with eCFP-speract and mVenus-speract as probes and obtained a positive FRET signal upon binding to the receptor, which suggests that the speract receptor exists as an oligomer, at least as a dimer, or alternatively that a single speract receptor protein possesses multiple binding sites. This property could partially account for the positive and/or negative cooperative binding of speract to the receptor. PMID:26889001

  14. Study of sperm cell phosphorylating systems using nucleotide photoaffinity probes

    SciTech Connect

    Khatoon, S.

    1983-01-01

    The major thrust of the research presented in this thesis was to identify specific nucleotide binding proteins and phosphoproteins of rat caput and cauda sperm. Also, the differences in these proteins between caput and cauda sperm were investigated as well as determination of the membrane sidedness of the proteins and their location in either the head or tail/mid-piece region. In addition, the effects of small molecular weight modifers such as cGMP, cAMP and Ca/sup 2 +/ on the detection of binding proteins and phosphorylated proteins was studied. The technique used to identify and locate nucleotide binding proteins was photoaffinity labeling using the proven 8-azidopurine nucleotide analogs of cAMP, ATP and GTP in radioactive form. The first study presented involved the use of (/sup 32/P)8-N /sub 3/cAMP which showed that both caput and cauda sperm contained both type I and type II regulatory subunits (R/sub I/ and R/sub II/, respectively) of the cAMP dependent kinases and that the great majority of the regulatory subunits were located in the tail/mid-piece section and not in the sperm head. The second phase of this study involved the use of (..gamma../sup 32/P)8-azidoadensosine triphosphate ((..gamma../sup 32/P)8-N/sub 3/ATP) and (..gamma../sup 32/P)8-azidoguanosine triphosphate ((..gamma../sup 32/P)8-N/sub 3/GTP) to photolable specific ATP and GTP binding proteins and to phosphorylate specific phosphoproteins. Again, this was done on caput versus cauda sperm and the location of the majority of the photolabeled or phosphorylated proteins was shown to be in the tail/mid-piece fraction. In addition, considerable differences were found in both the phosphorylated and photolabeled proteins of caput versus cauda sperm.

  15. CHARACTERIZATION OF MAJOR ALLERGEN PROTEINS OF SOYBEAN BY PROTEOMIC TOOLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We investigated profiles of three major allergen proteins (Gly m Bd 60K, Gly m Bd 30K and Gly m Bd 28K) in sixteen different soybean genotypes that included four groups; wild, cultivated, modern (elite), and ancestor. Four genotypes were studied within each group. All allergen proteins were well s...

  16. Identification of sea urchin sperm adenylate cyclase

    PubMed Central

    1990-01-01

    Calmodulin (CaM) affinity chromatography of a detergent extract of sea urchin sperm yielded approximately 20 major proteins. One of these proteins, of Mr 190,000, was purified and used to immunize rabbits. After absorption with living sperm, the serum reacted monospecifically on one- and two-dimensional Western immunoblots with the Mr 190,000 protein. The anti-190-kD serum inhibited 94% of the adenylate cyclase (AC) activity of the CaM eluate. An immunoaffinity column removed 95% of the AC activity, and the purified (but inactive) Mr 190,000 protein was eluted from the column. The antiserum also inhibited 23% of the activity of bovine brain CaM-sensitive AC and 90% of the activity of horse sperm CaM-sensitive AC. These data support the hypothesis that the Mr 190,000 protein is sea urchin sperm AC. Although this AC bound to CaM, it was not possible to demonstrate directly a Ca2+ or CaM sensitivity. However, two CaM antagonists, calmidazolium and chlorpromazine, both inhibited AC activity, and the inhibition was released by added CaM, suggesting the possibility of regulation of this AC by CaM. Indirect immunofluorescence showed the Mr 190,000 protein to be highly concentrated on only the proximal half of the sea urchin sperm flagellum. This asymmetric localization of AC may be important to its function in flagellar motility. This is the first report of the identification of an AC from animal spermatozoa. PMID:2121742

  17. 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.

  18. 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

  19. Modulation of bovine sperm signalling pathways: correlation between intracellular parameters and sperm capacitation and acrosome exocytosis.

    PubMed

    Pons-Rejraji, Hanae; Bailey, Janice L; Leclerc, Pierre

    2009-01-01

    In the present study, the viability, intracellular pH (pHi), cAMP ([cAMP]i), calcium concentration and protein phosphotyrosine content were evaluated in relation to the acrosomal and capacitation status of freshly ejaculated bull spermatozoa. These parameters were evaluated before and after incubation with the capacitation inducer heparin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), the phosphotyrosyl-protein phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate, and hydrogen peroxide. The results obtained were integrated to address the physiological interactions between the different signalling events affecting sperm capacitation and acrosome reaction. As expected, heparin promoted the expression of the 'B' pattern of chlortetracycline binding, increased pHi, [cAMP]i and the phosphotyrosine content of sperm proteins. The effects of heparin were enhanced by IBMX. Both PAO and sodium orthovanadate stimulated protein phosphotyrosine content and acrosomal exocytosis, although only PAO affected pH, Ca2+ and cAMP levels. Intracellular pH was increased while both Ca2+ and [cAMP]i were decreased. Physiological concentrations of H2O2 increased [cAMP]i and promoted acrosomal exocytosis. A significant positive correlation was found between sperm capacitation, protein phosphotyrosine content and stored Ca2+ concentration, whereas the acrosome reaction was correlated with pHi and Ca2+ concentration. This study presents the first global analysis of the major elements individually described during sperm capacitation and acrosome reaction signalling pathways, supported by statistical correlations. PMID:19383258

  20. 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

  1. Potent protein glycation inhibition of plantagoside in Plantago major seeds.

    PubMed

    Matsuura, Nobuyasu; Aradate, Tadashi; Kurosaka, Chihiro; Ubukata, Makoto; Kittaka, Shiho; Nakaminami, Yuri; Gamo, Kanae; Kojima, Hiroyuki; Ohara, Mitsuharu

    2014-01-01

    Plantagoside (5,7,4',5'-tetrahydroxyflavanone-3'-O-glucoside) and its aglycone (5,7,3',4',5'-pentahydroxyflavanone), isolated from a 50% ethanol extract of Plantago major seeds (Plantaginaceae), were established to be potent inhibitors of the Maillard reaction. These compounds also inhibited the formation of advanced glycation end products in proteins in physiological conditions and inhibited protein cross-linking glycation. These results indicate that P. major seeds have potential therapeutic applications in the prevention of diabetic complications. PMID:24895551

  2. 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

  3. Multi-transgenic pigs expressing three fluorescent proteins produced with high efficiency by sperm mediated gene transfer.

    PubMed

    Webster, Nicole L; Forni, Monica; Bacci, Maria Laura; Giovannoni, Roberto; Razzini, Riccardo; Fantinati, Paolo; Zannoni, Augusta; Fusetti, Lisa; Dalprà, Leda; Bianco, Maria Rosaria; Papa, Michele; Seren, Eraldo; Sandrin, Mauro S; Mc Kenzie, Ian F C; Lavitrano, Marialuisa

    2005-09-01

    Multi-gene transgenic pigs would be of benefit for large animal models in medical, agricultural, and pharmaceutical applications; in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We used the sperm mediated gene transfer (SMGT) method to produce with high efficiency multi-gene transgenic pigs using three genes coding for fluorescent proteins: enhanced blue (EBFP), green (EGFP), and red (DsRed2). All three fluorescent proteins were expressed in 171 out of 195 normally developed morula/blastocysts examined at day 6 post insemination (88%). Genomic DNA of 18 piglets born from two litters was screened by PCR, showing that all piglets were transgenic with at least one gene, 7/18 piglets were triple transgenic, 7/18 double transgenic, and 4/18 single transgenic. Fluorescence in situ hybridization (FISH) analysis revealed multiple sites of integration of the transgenes. RNA and protein expression was found in muscle, heart, liver, hair, and peripheral blood mononuclear cells (PBMCs). These results show that SMGT is an effective method for introducing multiple genes into pigs as shown by the simultaneous expression of three fluorescent proteins. PMID:15906394

  4. 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. PMID:27161017

  5. 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. PMID:25277530

  6. Novel SNPs in heat shock protein 70 gene and their association with sperm quality traits of Boer goats and Boer crosses.

    PubMed

    Nikbin, S; Panandam, J M; Yaakub, H; Murugaiyah, M; Sazili, A Q

    2014-05-01

    The semen quality of bucks affects the reproduction performance of the herd and is influenced by genetic and non-genetic factors. Heat shock protein 70 (HSP70) is considered as an important gene affecting semen quality traits. The objectives of this study are to find single nucleotide polymorphisms in HSP70 coding region and their association with semen quality traits on Boer and Boer cross bucks. DNA isolated from 53 goats (36 pure South African Boer and 17 Boer crosses) was subjected to PCR amplification of the exon 1 region of the caprine HSP70 gene. Single-strand conformation polymorphism (SSCP) was used to detect polymorphisms and the variant DNA fragments were sequenced. Two synonymous SNPs (74A>C (ss836187517) and 191C>G (ss836187518)) were detected. Qualities of fresh and post-thaw semen were evaluated for sperm concentration, semen volume, sperm motility and velocity traits, live sperm percentage, and abnormal sperm rate. The C allele of ss836187517 and G allele of ss836187518 were at higher frequencies in both the breeds. The C allele of ss836187517 appeared to be the favorable allele for semen concentration, progressive motility of fresh semen, and motility and sperm lateral head displacement of post-thaw semen. A negative overdominance was observed for ss836187517 alleles on velocity traits of post-thaw semen. The C allele of ss836187518 was favorable for sperm concentration and progressive motility. Results herein suggest that the SNPs in HSP70 may affect on semen quality in tropical regions and specially on the potential of semen for freezing. PMID:24674824

  7. Species-specific sequences of abalone lysin, the sperm protein that creates a hole in the egg envelope.

    PubMed Central

    Vacquier, V D; Carner, K R; Stout, C D

    1990-01-01

    Abalone eggs are contained within a rigid, elevated vitelline envelope through which the sperm must pass before reaching the egg cell membrane. Abalone spermatozoa possess an acrosomal protein called lysin that creates a hole in the egg vitelline envelope by a nonenzymatic mechanism. Lysins from two species of abalone, termed pink and red, which share the same habitat, exhibit species specificity in the dissolution of isolated egg envelopes. Cloning and sequencing the cDNAs for pink and red abalone lysins reveal transcript lengths of approximately 660 nucleotides. The open reading frames of 465 (pink) and 462 (red) nucleotides show a 13% difference. The 3' untranslated regions before the poly(A) tails are 170 (pink) and 165 (red) nucleotides long and differ from each other by about 7%. The protein sequences show nearly identical signal sequences of 18 amino acids for both lysins. The mature protein is 137 amino acids in the pink abalone and 136 in the red abalone; the two mature lysins differ in 29 of 137 amino acids (21%). The most variable region, which may account for lysin's species specificity, is at the NH2 terminus, where 11 of the 15 amino acids differ between the two species. Predictions of secondary structure indicate that both lysins contain four homologous amphiphilic alpha-helices. Images PMID:2377618

  8. 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. PMID:27335066

  9. Evidence of 5-HT components in human sperm: implications for protein tyrosine phosphorylation and the physiology of motility

    PubMed Central

    Jiménez-Trejo, Francisco; Tapia-Rodríguez, Miguel; Cerbón, Marco; Kuhn, Donald M; Manjarrez-Gutiérrez, Gabriel; Mendoza-Rodríguez, C Adriana; Picazo, Ofir

    2016-01-01

    Serotonin (5-hydroxytryptamine; C10H12N2O (5-HT)) is produced in the CNS and in some cells of peripheral tissues. In the mammalian male reproductive system, both 5-HT and tryptophan hydroxylase (TPH) have been described in Leydig cells of the testis and in principal cells of the caput epididymis. In capacitated hamster sperm, it has been shown that 5-HT promotes the acrosomal reaction. The aim of this work was to explore the existence of components of the serotoninergic system and their relevance in human sperm physiology. We used both immunocytochemistry and western blot to detect serotoninergic markers such as 5-HT, TPH1, MAOA, 5-HT1B, 5-HT3, and 5HTT; HPLC for TPH enzymatic activity; Computer Assisted Semen Analysis assays to measure sperm motility parameters and pharmacological approaches to show the effect of 5-HT in sperm motility and tyrosine phosphorylation was assessed by western blot. We found the presence of serotoninergic markers (5-HT, TPH1, MAOA, 5-HT1B, 5-HT2A, 5-HT3, 5-HTT, and TPH enzymatic activity) in human sperm. In addition, we observed a significant increase in tyrosine phosphorylation and changes in sperm motility after 5-HT treatment. In conclusion, our data demonstrate the existence of components of a serotoninergic system in human sperm and support the notion for a functional role of 5-HT in mammalian sperm physiology, which can be modulated pharmacologically. PMID:23028123

  10. Identification of major immunogenic proteins of Mycoplasma synoviae isolates.

    PubMed

    Bercic, Rebeka Lucijana; Slavec, Brigita; Lavric, Miha; Narat, Mojca; Bidovec, Andrej; Dovc, Peter; Bencina, Dusan

    2008-02-01

    Mycoplasma synoviae isolates differ in patterns of immunogenic proteins, but most of them have not been identified yet. The main aim of this study was their identification in two closely related M. synoviae isolates, ULB 02/P4 and ULB 02/OV6, recovered recently from chickens in Slovenia. N-terminal sequencing identified 17 M. synoviae proteins. Amongst them were 14 major, highly expressed but previously unidentified proteins, including enzymes, chaperones and putative lipoproteins. ULB 02/P4 proteins with increasing molecular weight (M(w)) in the region above the lipoprotein MSPB (approximately 40 kDa) were elongation factor EF-Tu, enolase, NADH oxidase, haemagglutinin MSPA, ATP synthase beta chain, trigger factor, pyruvate kinase and chaperone DnaK. Enolase (approximately 47 kDa) seemed to be immunogenic for chickens infected with M. synoviae, whereas EF-Tu, which might cross-react with antibodies to the P1 adhesin of Mycoplasma pneumoniae, was not. ULB 02/OV6 synthesized several immunogenic proteins and those with M(w) of approximately 70, 78, 82, 90, 110 and 160 kDa, cross-reacted with antibodies to Mycoplasma gallisepticum. They remain to be identified, because besides putative lipoproteins, protein bands of 78, 82, 85 and 110 kDa contained also dehydrogenase PdhD, elongation factor EF-G, enzyme PtsG and putative neuraminidase, respectively. PMID:17720337

  11. Actin related protein complex subunit 1b controls sperm release, barrier integrity and cell division during adult rat spermatogenesis.

    PubMed

    Kumar, Anita; Dumasia, Kushaan; Deshpande, Sharvari; Gaonkar, Reshma; Balasinor, N H

    2016-08-01

    Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Knockdown also compromised BTB integrity and caused polarity defects of mature spermatids. Apart from these effects pertaining to Sertoli cells, Arpc1b reduction perturbed ability of germ cells to enter G2/M phase thus hindering cell division. In summary, Arpc1b, an estrogen responsive gene, is a regulator of spermiation, mature spermatid polarity, BTB integrity and cell division during adult spermatogenesis. PMID:27113856

  12. 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

  13. 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. PMID:17562090

  14. 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

  15. Functional role of feline zona pellucida protein 4 trefoil domain: a sperm receptor or structural component of the domestic cat zona pellucida?

    PubMed

    Braun, B C; Ringleb, J; Waurich, R; Viertel, D; Jewgenow, K

    2009-07-01

    The trefoil domain (TFD) is a protein structure characterized by six cysteines, which form a typical three-loop structure by three disulphide bridges. It is assumed that two of these loops generate a hydrophobic groove, which could be a binding site for carbohydrate residues or proteins. The zona pellucida (ZP) protein, ZP4, contains such a TFD. The carbohydrate-/protein-binding property of TFD allows us to assume a potential sperm receptor function of this domain. Additionally, gastrointestinal trefoil peptides are stable against proteases; therefore, a structural role of TFD within the ZP might also be possible. We were able to show that the synthesized and natural folded feline TFD (fTFD) expresses the typical protease resistance that vanished under reducing conditions and after substitution of cysteine residues within the peptide. Furthermore, an antibody directed against the first loop of fTFD was almost unable to bind to intact in vitro mature cat oocytes. Pre-incubation of oocytes in the reducing agent (DDT), however, improved antibody binding substantially. Therefore, we suggest structural masking of the fTFD domain within the intact ZP. An interaction between fTFD and feline sperm cells was examined using several methods, including immunocytochemistry, immunoelectron microscopy, co-immunoprecipitation and far western blot, but we found no indication for an involvement of TFD in the primary sperm binding to the ZP. To summarize, there is increasing evidence that the TFD of fZP4 has a structural rather than a sperm-binding function. PMID:19754576

  16. Drosophila sperm motility in the reproductive tract.

    PubMed

    Yang, Yong; Lu, Xiangyi

    2011-05-01

    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

  17. 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.

  18. Mice lacking FABP9/PERF15 develop sperm head abnormalities but are fertile

    PubMed Central

    Selvaraj, Vimal; Asano, Atsushi; Page, Jennifer L.; Nelson, Jacquelyn L.; Kothapalli, Kumar S. D.; Foster, James A.; Brenna, J. Thomas; Weiss, Robert S.; Travis, Alexander J.

    2010-01-01

    The male germ cell-specific fatty acid binding protein 9 (FABP9/PERF15) is the major component of the murine sperm perforatorium and perinuclear theca. Based on its cytoskeletal association and sequence homology to myelin P2 (FABP8), it has been suggested that FABP9 tethers sperm membranes to the underlying cytoskeleton. Furthermore, its upregulation in apoptotic testicular germ cells and its increased phosphorylation status during capacitation suggested multiple important functions for FABP9. Therefore, we investigated specific functions for FABP9 by means of targeted gene disruption in mice. FABP9−/− mice were viable and fertile. Phenotypic analysis showed that FABP9−/− mice had significant increases in sperm head abnormalities (~8% greater than their WT cohorts); in particular, we observed the reduction or absence of the characteristic structural element known as the “ventral spur” in ~10% of FABP9−/− sperm. However, deficiency of FABP9 neither affected membrane tethering to the perinuclear theca nor the fatty acid composition of sperm. Moreover, epididymal sperm numbers were not affected in FABP9−/− mice. Therefore, we conclude that FABP9 plays only a minor role in providing the murine sperm head its characteristic shape and is not absolutely required for spermatogenesis or sperm function. PMID:20920498

  19. 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. PMID:22541550

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  1. 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.

  2. 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

  3. 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. PMID:26417650

  4. 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

  5. ZP-binding peptides identified via phage display stimulate production of sperm antibodies in dogs.

    PubMed

    Samoylova, Tatiana I; Cox, Nancy R; Cochran, Anna M; Samoylov, Alexandre M; Griffin, Brenda; Baker, Henry J

    2010-07-01

    Zona pellucida (ZP) glycoproteins play a central role in sperm-oocyte binding and fertilization. Sperm protein sequences that are involved in sperm-ZP recognition and have an important role in fertilization represent attractive targets for development of contraceptive vaccines, yet are currently unknown. To identify peptide sequences that recognize and bind to ZP proteins, we developed a novel selection procedure from phage display libraries that utilizes intact oocytes surrounded by ZP proteins. The major advantage of this procedure is that ZP proteins remain in their native conformation unlike a selection protocol previously published that utilized solubilized ZP on artificial solid support. Several peptides of 7 and 12 amino acids with binding specificity to canine ZP proteins were identified. Four of them (LNSFLRS, SSWYRGA, YLPIYTIPSMVY, and NNQSPILKLSIH) plus a control ZP-binding peptide (YLPVGGLRRIGG) from the literature were synthesized and tested for antigenic properties in dogs. NNQSPILKLSIH peptide stimulated production of anti-peptide antibodies. These antibodies bind to the acrosomal region of the canine sperm cell, demonstrating ability to act as sperm antibodies. The identified ZP-binding peptides (mimicking sperm cell surface antigens) may be useful in the design of immunocontraceptive agents for dogs. PMID:20434854

  6. Characterization of major lipid droplet proteins from Dunaliella.

    PubMed

    Davidi, Lital; Katz, Adriana; Pick, Uri

    2012-07-01

    Many green algal species can accumulate large amounts of triacylglycerides (TAG) under nutrient deprivation, making them a potential source for production of biodiesel. TAG are organized in cytoplasmic lipid bodies, which contain a major lipid droplet protein termed MLDP. Green algae MLDP differ in sequence from plant oleosins and from animal perilipins, and their structure and function are not clear. In this study, we describe the isolation of MLDP from three species of the extreme halotolerant green algae Dunaliella. Sequence alignment with other green algae MLDP proteins identified a conserved 4-proline domain that may be considered as a signature domain of Volvocales green algae MLDP. Gold immunolabeling localized MLDP at the surface of lipid droplets in D. salina. The induction of MLDP by nitrogen deprivation is kinetically correlated with TAG accumulation, and inhibition of TAG biosynthesis impairs MLDP accumulation suggesting that MLDP induction is co-regulated with TAG accumulation. These results can lead to a better understanding of the structure and function of Volvocales green algae MLDP proteins. PMID:22231009

  7. Evolution of myelin ultrastructure and the major structural myelin proteins.

    PubMed

    Inouye, Hideyo; Kirschner, Daniel A

    2016-06-15

    Myelin sheaths, as the specialized tissue wrapping the nerve fibers in the central and peripheral nervous systems (CNS and PNS), are responsible for rapid conduction of electrical signals in these fibers. We compare the nerve myelin sheaths of different phylogenetic origins-including mammal, rodent, bird, reptile, amphibian, lungfish, teleost, and elasmobranch-with respect to periodicities and inter-membrane separations at their cytoplasmic and extracellular appositions, and correlate these structural parameters with biochemical composition. P0 glycoprotein and P0-like proteins are present in PNS of terrestrial species or land vertebrates (Tetrapod) and in CNS and PNS of aquatic species. Proteolipid protein (PLP) is a major component only in the CNS myelin of terrestrial species and is involved in compaction of the extracellular apposition. The myelin structures of aquatic garfish and lungfish, which contain P0-like protein both in CNS and PNS, are similar to those of terrestrial species, indicating that they may be transitional organisms between water and land species. This article is part of a Special Issue entitled SI: Myelin Evolution. PMID:26519753

  8. Binding patterns of bovine seminal plasma proteins A1/A2, 30 kDa and osteopontin on ejaculated sperm before and after incubation with isthmic and ampullary oviductal fluid.

    PubMed

    Souza, Carlos Eduardo A; Moura, Arlindo A; Monaco, Elisa; Killian, Gary J

    2008-04-01

    Previous studies from our laboratory have reported empirical associations between bovine seminal plasma protein(s) (BSP) A1/A2 and 30 kDa and osteopontin (OPN) in accessory sex gland fluid and bull fertility. These BSP and OPN are believed to bind to sperm at ejaculation and to remain bound until sperm reach the oviduct. The objective of the present study was to evaluate the topographical distribution of BSP A1/A2, 30 kDa and OPN binding on: (1) bovine ejaculated sperm; (2) ejaculated sperm incubated with isthmic oviductal fluid (ODF); (3) ejaculated sperm+isthmic ODF incubated in ampullary ODF. From each of these media, aliquots of sperm for BSP and OPN were processed for immunocytochemistry and analysis by laser scanning confocal microscopy. Isthmic and ampullary ODF was collected from indwelling catheters and used as pools from three cows in the non-luteal phase of the estrous cycle. Anti-BSP A1/A2 was detected bound to the midpiece, post-equatorial and equatorial segments and acrosome of sperm after ejaculation and after incubation with isthmic and ampullary ODF. The BSP A1/A2 fluorescence was more concentrated on the midpiece and increased as acrosome-intact sperm came in contact with ODF. As compared with acrosome-intact sperm, non-intact acrosome intact sperm had 39 and 68% reductions of acrosome fluorescence and 36% and 90% increases of post-equatorial fluorescence after contact with isthmic and ampullary ODF (P<0.05). Anti-BSP 30 kDa was more intense on the midpiece than on post-equatorial, equatorial and acrosome regions of sperm after ejaculation and contact with ODF. However, equatorial fluorescence was 141% and 89% more intense and acrosome stainning was 80% and 76% less (P<0.05) in non-intact acrosome sperm than in acrosome intact cells, during all ODF incubations. Anti-OPN was identified on the acrosome of ejaculated sperm, but with less fluorescence (P<0.05) on the post-equatorial segment and midpiece. Incubation of sperm with isthmic ODF increased

  9. 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. PMID:20544873

  10. Soluble sperm extract specifically recapitulates the initial phase of the Ca2+ response in the fertilized oocyte of P. occelata following a G-protein/ PLCβ signaling pathway.

    PubMed

    Nakano, Takeshi; Kyozuka, Keiichiro

    2015-12-01

    Matured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte. To study how sperm trigger the Ca2+ increases during fertilization, we prepared soluble sperm extract (SE) and examined its ability to induce Ca2+ increases in the oocyte. The SE could evoke a Ca2+ increase in the oocyte when it was added to the medium, but not when it was delivered by microinjection. However, the second-step Ca2+ increase leading to the resumption of meiosis did not follow in these eggs. Local application of SE induced a non-propagating Ca2+ increase and formed a cytoplasmic protrusion that was similar to that created by the fertilizing sperm at the first stage of the Ca2+ response, important for sperm incorporation into the oocyte. Our results suggest that the fertilizing spermatozoon may trigger the first-step Ca2+ increase before it fuses with the oocyte in a pathway that involves the G-protein-coupled receptor and phospholipase C. Thus, the first phase of the Ca2+ response in the fertilized egg of this species is independent of the second phase of the Ca2+ increase for egg activation. PMID:25318389

  11. 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

    2016-07-01

    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. PMID:27292547

  12. 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. PMID:22805164

  13. Relationship of sperm small heat-shock protein 10 and voltage-dependent anion channel 2 with semen freezability in boars.

    PubMed

    Vilagran, Ingrid; Yeste, Marc; Sancho, Sílvia; Casas, Isabel; Rivera del Álamo, Maria M; Bonet, Sergi

    2014-08-01

    Freezability differences between boar ejaculates exist, but there is no useful method to predict the ejaculate freezability before sperm cryopreservation takes place. In this context, the present study sought to determine whether the amounts of small heat-shock protein 10 (also known as outer dense fiber protein 1) (ODF1/HSPB10) and voltage-dependent anion channel 2 (VDAC2) may be used as boar sperm freezability markers. With this aim, 26 boar ejaculates were split into two fractions: one for protein extraction and the other for cryopreservation purposes. Ejaculates were subsequently classified into two groups (good freezability ejaculates [GFE] and poor freezability ejaculates [PFE]) based on viability and sperm motility assessments after 30 and 240 minutes of after thawing. Although the VDAC2 amounts, analyzed through Western blot, were significantly higher (P < 0.01) in GFE (1.15 ± 0.18 density mm(2)) than in PFE (0.16 ± 0.03 density mm(2)), no significant differences were observed in ODF1/HSPB10 between both groups (i.e., 1.97 ± 0.38 density mm(2) in GFE vs. 1.87 ± 1.54 density mm(2) in PFE). In addition, principal component and multiple regression analyses indicated that the component explaining most of the variance (78.41%) in ejaculate freezability at 240 minutes after thawing resulted to be significantly (P < 0.05) correlated with VDAC2 content. This result revealed that the amounts of VDAC2 but not those of ODF1/HSPB10 may be used to predict the freezability of a given boar ejaculate before starting cryopreservation procedures. PMID:24933094

  14. Assembly of nuclear pore complexes mediated by major vault protein.

    PubMed

    Vollmar, Friederike; Hacker, Christian; Zahedi, René-Peiman; Sickmann, Albert; Ewald, Andrea; Scheer, Ulrich; Dabauvalle, Marie-Christine

    2009-03-15

    During interphase growth of eukaryotic cells, nuclear pore complexes (NPCs) are continuously incorporated into the intact nuclear envelope (NE) by mechanisms that are largely unknown. De novo formation of NPCs involves local fusion events between the inner and outer nuclear membrane, formation of a transcisternal membranous channel of defined diameter and the coordinated assembly of hundreds of nucleoporins into the characteristic NPC structure. Here we have used a cell-free system based on Xenopus egg extract, which allows the experimental separation of nuclear-membrane assembly and NPC formation. Nuclei surrounded by a closed double nuclear membrane, but devoid of NPCs, were first reconstituted from chromatin and a specific membrane fraction. Insertion of NPCs into the preformed pore-free nuclei required cytosol containing soluble nucleoporins or nucleoporin subcomplexes and, quite unexpectedly, major vault protein (MVP). MVP is the main component of vaults, which are ubiquitous barrel-shaped particles of enigmatic function. Our results implicate MVP, and thus also vaults, in NPC biogenesis and provide a functional explanation for the association of a fraction of vaults with the NE and specifically with NPCs in intact cells. PMID:19240118

  15. An ARID domain-containing protein within nuclear bodies is required for sperm cell formation in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. Quantification of Protein-Lipid Selectivity using FRET: Application to the M13 Major Coat Protein

    PubMed Central

    Fernandes, Fábio; Loura, Luís M. S.; Koehorst, Rob; Spruijt, Ruud B.; Hemminga, Marcus A.; Fedorov, Alexander; Prieto, Manuel

    2004-01-01

    Quantification of lipid selectivity by membrane proteins has been previously addressed mainly from electron spin resonance studies. We present here a new methodology for quantification of protein-lipid selectivity based on fluorescence resonance energy transfer. A mutant of M13 major coat protein was labeled with 7-diethylamino-3((4′iodoacetyl)amino)phenyl-4-methylcoumarin to be used as the donor in energy transfer studies. Phospholipids labeled with N-(7-nitro-2-1,3-benzoxadiazol-4-yl) were selected as the acceptors. The dependence of protein-lipid selectivity on both hydrophobic mismatch and headgroup family was determined. M13 major coat protein exhibited larger selectivity toward phospholipids which allow for a better hydrophobic matching. Increased selectivity was also observed for anionic phospholipids and the relative association constants agreed with the ones already presented in the literature and obtained through electron spin resonance studies. This result led us to conclude that fluorescence resonance energy transfer is a promising methodology in protein-lipid selectivity studies. PMID:15240469

  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. Use of Differential Isotopic Labeling and Mass Spectrometry To Analyze Capacitation-Associated Changes in the Phosphorylation Status of Mouse Sperm Proteins

    PubMed Central

    Platt, Mark D.; Salicioni, Ana M.; Hunt, Donald F.; Visconti, Pablo E.

    2010-01-01

    Mammalian sperm need to reside in the female reproductive tract for a finite period of time before acquiring fertilizing competence. The biochemical changes associated with this process are collectively known as “capacitation”. With the use of the mouse as an experimental model, we have previously demonstrated that capacitation is associated with a cAMP-dependent increase in protein tyrosine phosphorylation. However, little is known about the identity and function of the protein targets of this phosphorylation cascade. In the present work, we have used differential isotopic labeling coupled with immobilized metal affinity chromatography (IMAC)-based phosphopeptide enrichment and analysis on a hybrid linear ion trap/FT-ICR mass spectrometer to measure the changes in protein phosphorylation resulting from the capacitation process. As no kinase activators and/or phosphatase inhibitors were used in the preparation of the sperm samples, phosphorylated residues identified in this study represent in vivo sites of phosphorylation. Also, in contrast to other methods which rely on the incorporation of isotopically labeled amino acids at the protein level (e.g., SILAC), the present technique is based on the Fisher esterification of protein digests, allowing for the comparison of phosphorylation status in the absence of protein synthesis. This approach resulted in the identification of 55 unique, in vivo sites of phosphorylation and permitted the relative extent of phosphorylation, as a consequence of capacitation, to be calculated for 42 different phosphopeptides. This work represents the first effort to determine which specific protein phosphorylation sites change their phosphorylation status in vivo as a result of the mammalian capacitation process. PMID:19186949

  19. Cryopreservation of Fish Sperm

    NASA Astrophysics Data System (ADS)

    Kurokura, Hisashi

    Present status of research activities in cryopreservation of fish gamete in aquaculture field was introduced. More than 59 fish species have been reported in the research histories and nearly half of them were studied during recent 10 years. This means that the research activities are increasing, though commercial profit have not obtained yet. Fish species of which sperm can successfully cryopreserved is still limited comparing to numerous species in telost. One of the major obstacle for improvement of the technique is existence of wide specie specific variance in the freezing tolerance of fish sperm. The varianc can possibly be explaind thorugh the informations obtained by the studies in comparative spermatology, which is recently activated field in fish biology.

  20. Sugar-coated sperm: Unraveling the functions of the mammalian sperm glycocalyx.

    PubMed

    Tecle, Eillen; Gagneux, Pascal

    2015-09-01

    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. PMID:26061344

  1. 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...

  2. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.

    PubMed

    Stanger, Simone J; Law, Estelle A; Jamsai, Duangporn; O'Bryan, Moira K; Nixon, Brett; McLaughlin, Eileen A; Aitken, R John; Roman, Shaun D

    2016-08-01

    Spermatozoa require the process of capacitation to enable them to fertilize an egg. PKA is crucial to capacitation and the development of hyperactivated motility. Sperm PKA is activated by cAMP generated by the germ cell-enriched adenylyl cyclase encoded by Adcy10 Male mice lacking Adcy10 are sterile, because their spermatozoa are immotile. The current study was designed to identify binding partners of the sperm-specific (Cα2) catalytic subunit of PKA (PRKACA) by using it as the "bait" in a yeast 2-hybrid system. This approach was used to identify a novel germ cell-enriched protein, sperm PKA interacting factor (SPIF), in 25% of the positive clones. Homozygous Spif-null mice were embryonically lethal. SPIF was coexpressed and coregulated with PRKACA and with t-complex protein (TCP)-11, a protein associated with PKA signaling. We established that these 3 proteins form part of a novel complex in mouse spermatozoa. Upon capacitation, the SPIF protein becomes tyrosine phosphorylated in >95% of sperm. An apparent molecular rearrangement in the complex occurs, bringing PRKACA and TCP11 into proximity. Taken together, these results suggest a role for the novel complex of SPIF, PRKACA, and TCP11 during sperm capacitation, fertilization, and embryogenesis.-Stanger, S. J., Law, E. A., Jamsai, D., O'Bryan, M. K., Nixon, B., McLaughlin, E. A., Aitken, R. J., Roman, S. D. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation. PMID:27105888

  3. Sperm viability - Determination of sperm viability using fluorescence microscopy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To determine the percentage of viable sperm in a semen sample using stains that differentiates viable (live) sperm from nonviable (dead) sperm. Viable sperm are detected by SYBR-14, which stains the sperm nuclei green. Nonviable sperm are detected by propidium iodide (PI), which stains the sperm red...

  4. Transcript levels of the soluble sperm factor protein phospholipase C zeta 1 (PLCζ1) increase through induced spermatogenesis in European eel.

    PubMed

    Morini, Marina; Peñaranda, David S; Vílchez, María C; Gallego, Víctor; Nourizadeh-Lillabadi, Rasoul; Asturiano, Juan F; Weltzien, Finn-Arne; Pérez, Luz

    2015-09-01

    Activation at fertilization of the vertebrate egg is triggered by Ca(2+) waves. Recent studies suggest the phospholipase C zeta (PLCζ), a sperm-specific protein, triggers egg activation by an IP3-mediated Ca(2+) release and allow Ca(2+) waves at fertilization. In the present study we cloned, characterized, and phylogenetically positioned the European eel PLCζ (PLCζ1). It is 1521 bp long, with 10 exons encoding an open reading frame of 506 amino acids. The amino acid sequence contains an EF-hand domain, X and Y catalytic domains, and a carboxy-terminal C2 domain, all typical of other PLCζ orthologous. The tissue distribution was studied, and the gene expression was determined in testis during induced sexual maturation at three different thermal regimes. Also, brain and pituitary expression was studied through sex maturation at constant temperature. plcζ1 was expressed in brain of male and female, in testis but not in ovaries. By first time in vertebrates, it is reported plcζ1 expression in the pituitary gland. Testis plcζ1 expression increased through spermatogenesis under all the thermal regimes, but being significantly elevated at lower temperatures. It was very low when testis contained only spermatogonia or spermatocytes, while maximum expression was found during spermiogenesis. These results support the hypothesis for an eel sperm-specific PLCζ1 inducing egg activation, similarly to mammals and some teleosts, but different from some other teleost species, which express this protein in ovaries, but not in testes. PMID:26051612

  5. 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.

  6. Porcine oviduct sperm binding glycoprotein and its deleterious effect on sperm: a mechanism for negative selection of sperm?

    PubMed

    Teijeiro, Juan M; Dapino, Dora G; Marini, Patricia E

    2011-01-01

    In their journey through the oviduct some subpopulations of sperm are preserved in a reservoir, while others are negatively selected. Sperm binding glycoprotein (SBG) is a pig oviductal epithelial cell glycoprotein that produces, under capacitating conditions, acrosome alteration, p97 tyrosine-phosphorylation and reduction of the motility of sperm. In this paper, we show that SBG is accessible at the extracellular surface of the oviductal epithelial cells, supporting a sperm interaction biological role in situ. We analyze the possible dependence of the tyrosine-phosphorylation of p97 on the PKA mechanism, finding that apparently it is not PKA dependent. Also, after SBG treatment the phosphorylated proteins locate mainly at the detached periacrosomal region and at the tail of sperm; the latter may be related to SBG's motility reduction effect. The study of the time course effect of SBG on sperm as detected by chlortetracycline (CTC) staining and of its binding to sperm by immunodetection in conjunction with CTC, shows results in agreement with the hypothesis that this glycoprotein is involved in the alteration of acrosomes in a specific sperm subpopulation. The results suggest that SBG may be part of a mechanism for negative selection of sperm. PMID:22446595

  7. 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. PMID:26699205

  8. The role of protein intrinsic disorder in major psychiatric disorders.

    PubMed

    Tovo-Rodrigues, Luciana; Recamonde-Mendoza, Mariana; Paixão-Côrtes, Vanessa Rodrigues; Bruxel, Estela M; Schuch, Jaqueline B; Friedrich, Deise C; Rohde, Luis A; Hutz, Mara H

    2016-09-01

    Although new candidate genes for Autism Spectrum Disorder (ASD), Schizophrenia (SCZ), Attention-Deficit/Hyperactivity Disorder (ADHD), and Bipolar Disorder (BD) emerged from genome-wide association studies (GWAS), their underlying molecular mechanisms remain poorly understood. Evidences of the involvement of intrinsically disordered proteins in diseases have grown in the last decade. These proteins lack tridimensional structure under physiological conditions and are involved in important cellular functions such as signaling, recognition and regulation. The aim of the present study was to identify the role and abundance of intrinsically disordered proteins in a set of psychiatric diseases and to test whether diseases are different regarding protein intrinsic disorder. Our hypothesis is that differences across psychiatric illnesses phenotypes and symptoms may arise from differences in intrinsic protein disorder content and properties of each group. A bioinformatics prediction of intrinsic disorder was performed in proteins retrieved based on top findings from GWAS, Copy Number Variation and candidate gene investigations for each disease. This approach revealed that about 80% of studied proteins presented long stretches of disorder. This amount was significantly higher than that observed in general eukaryotic proteins, and those involved in cardiovascular diseases. These results suggest that proteins with intrinsic disorder are a common feature of neurodevelopment and synaptic transmission processes which are potentially involved in the etiology of psychiatric diseases. Moreover, we identified differences between ADHD and ASD when the binary prediction of structure and putative binding sites were compared. These differences may be related to variation in symptom complexity between both diseases. © 2016 Wiley Periodicals, Inc. PMID:27184105

  9. 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

  10. Fluorescence investigations on choline phospholipid binding and chemical unfolding of HSP-1/2, a major protein of horse seminal plasma.

    PubMed

    Kumar, C Sudheer; Sivaramakrishna, D; Ravi, Sanjay K; Swamy, Musti J

    2016-05-01

    Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigated in the native state, in the presence of PrC and phosphatidylcholines (PCs) with short (valeryl, C-5) and long (myristoyl, C-14) chains, and upon denaturation using fluorescence quenching, time-resolved fluorescence and red-edge excitation shift (REES) measurements. The results obtained show that the environment of tryptophan residues in HSP-1/2 is more heterogeneous as compared to that in PDC-109. Binding of choline containing ligands afforded a protection to the tryptophan residues with the shielding order being: PrC≤divalaroyl PCprotein. In the presence of PrC the transition midpoints shifted to higher concentrations of the denaturant together with a broadening of the sigmoidal transitions, indicating that ligand binding as well as polydispersity modulate the unfolding process. PMID:26963430

  11. 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.

  12. 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

  13. 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. PMID:27032685

  14. Protein Phosphorylation: A Major Switch Mechanism for Metabolic Regulation.

    PubMed

    Humphrey, Sean J; James, David E; Mann, Matthias

    2015-12-01

    Metabolism research is undergoing a renaissance because many diseases are increasingly recognized as being characterized by perturbations in intracellular metabolic regulation. Metabolic changes can be conferred through changes to the expression of metabolic enzymes, the concentrations of substrates or products that govern reaction kinetics, or post-translational modification (PTM) of the proteins that facilitate these reactions. On the 60th anniversary since its discovery, reversible protein phosphorylation is widely appreciated as an essential PTM regulating metabolism. With the ability to quantitatively measure dynamic changes in protein phosphorylation on a global scale - hereafter referred to as phosphoproteomics - we are now entering a new era in metabolism research, with mass spectrometry (MS)-based proteomics at the helm. PMID:26498855

  15. Sperm phosphoproteomics: historical perspectives and current methodologies

    PubMed Central

    Porambo, James R; Salicioni, Ana M; Visconti, Pablo E; Platt, Mark D

    2013-01-01

    Mammalian sperm are differentiated germ cells that transfer genetic material from the male to the female. Owing to this essential role in the reproductive process, an understanding of the complex mechanisms that underlie sperm function has implications ranging from the development of novel contraceptives to the treatment of male infertility. While the importance of phosphorylation in sperm differentiation, maturation and fertilization has been well established, the ability to directly determine the sites of phosphorylation within sperm proteins and to quantitate the extent of phosphorylation at these sites is a recent development that has relied almost exclusively on advances in the field of proteomics. This review will summarize the work that has been carried out to date on sperm phosphoproteomics and discuss how the resulting qualitative and quantitative information has been used to provide insight into the manner in which protein phosphorylation events modulate sperm function. The authors also present the proteomics process as it is most often utilized for the elucidation of protein expression, with a particular emphasis on the way in which the process has been modified for the analysis of protein phosphorylation in sperm. PMID:23194270

  16. 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

  17. Ultrastructure of bovine sperm chromatin.

    PubMed

    Filho, Romualdo Morandi; Beletti, Marcelo Emilio; de Oliveira, Fabio

    2015-12-01

    Mammalian semen chromatin comprises DNA, protamine, and, at lower levels, other proteins. This constitution confers intense compaction to the chromatin, helping to protect the DNA and causing the head of the sperm to be very small, facilitating the safe transport of its genetic contents. It is known that changes in the sperm chromatin compaction lead to fertility problems in bulls, justifying studies of this structure. Although there are theoretical models of sperm chromatin because of its high compaction, there is no morphological evidence of such models. The aim of this study was to demonstrate the ultrastructure of bovine sperm chromatin in an attempt to corroborate the theoretical chromatin models existing today. The isolated bull sperm heads had their chromatin partially unpacked by chemical treatment using sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) and were then embedded in Epon resin. Using an ultramicrotome, ultrathin sections were obtained, which were contrasted with uranyl acetate and lead citrate, and then viewed under transmission electron microscopy. The methodology used allowed the visualization of toroidal structures interconnected by a filamentous nuclear matrix, which is entirely consistent with the most current theoretical models. PMID:26515508

  18. Phosphorylation of Cysteine String Protein Triggers a Major Conformational Switch.

    PubMed

    Patel, Pryank; Prescott, Gerald R; Burgoyne, Robert D; Lian, Lu-Yun; Morgan, Alan

    2016-08-01

    Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 chaperone family that localizes to neuronal synaptic vesicles. Impaired CSP function leads to neurodegeneration in humans and model organisms as a result of misfolding of client proteins involved in neurotransmission. Mammalian CSP is phosphorylated in vivo on Ser10, and this modulates its protein interactions and effects on neurotransmitter release. However, there are no data on the structural consequences of CSP phosphorylation to explain these functional effects. We show that Ser10 phosphorylation causes an order-to-disorder transition that disrupts CSP's extreme N-terminal α helix. This triggers the concomitant formation of a hairpin loop stabilized by ionic interactions between phosphoSer10 and the highly conserved J-domain residue, Lys58. These phosphorylation-induced effects result in significant changes to CSP conformation and surface charge distribution. The phospho-switch revealed here provides structural insight into how Ser10 phosphorylation modulates CSP function and also has potential implications for other DnaJ phosphoproteins. PMID:27452402

  19. Two Types of Assays for Detecting Frog Sperm Chemoattraction

    PubMed Central

    Burnett, Lindsey A.; Tholl, Nathan; Chandler, Douglas E.

    2011-01-01

    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 pipette1. 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

  20. 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

  1. 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

  2. G Protein-Coupled Receptors in Major Psychiatric Disorders

    PubMed Central

    Catapano, Lisa A.; Manji, Husseini K.

    2007-01-01

    Although the molecular mechanisms underlying psychiatric illnesses such as depression, bipolar disorder and schizophrenia remain incompletely understood, there is increasing clinical, pharmacologic, and genetic evidence that G protein-coupled receptors (GPCRs) play critical roles in these disorders and their treatments. This perspectives paper reviews and synthesizes the available data. Dysfunction of multiple neurotransmitter and neuropeptide GPCRs in frontal cortex and limbic-related regions, such as the hippocampus, hypothalamus and brainstem, likely underlies the complex clinical picture that includes cognitive, perceptual, affective and motoric symptoms. The future development of novel agents targeting GPCR signaling cascades remains an exciting prospect for patients refractory to existing therapeutics. PMID:17078926

  3. Spata6 is required for normal assembly of the sperm connecting piece and tight head–tail conjunction

    PubMed Central

    Yuan, Shuiqiao; Stratton, Clifford J.; Bao, Jianqiang; Zheng, Huili; Bhetwal, Bhupal P.; Yanagimachi, Ryuzo; Yan, Wei

    2015-01-01

    “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). PMID:25605924

  4. 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

    2015-02-01

    "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). PMID:25605924

  5. Regulation of axonemal motility in demembranated equine sperm.

    PubMed

    Loux, Shavahn C; Macías-Garcia, Beatríz; González-Fernández, Lauro; Canesin, Heloisa DeSiqueira; Varner, Dickson D; Hinrichs, Katrin

    2014-12-01

    Equine in vitro fertilization is not yet successful because equine sperm do not effectively capacitate in vitro. Results of previous studies suggest that this may be due to failure of induction of hyperactivated motility in equine sperm under standard capacitating conditions. To evaluate factors directly affecting axonemal motility in equine sperm, we developed a demembranated sperm model and analyzed motility parameters in this model under different conditions using computer-assisted sperm analysis. Treatment of ejaculated equine sperm with 0.02% Triton X-100 for 30 sec maximized both permeabilization and total motility after reactivation. The presence of ATP was required for motility of demembranated sperm after reactivation, but cAMP was not. The calculated intracellular pH of intact equine sperm was 7.14 ± 0.07. Demembranated sperm showed maximal total motility at pH 7. Neither increasing pH nor increasing calcium levels, nor any interaction of the two, induced hyperactivated motility in demembranated equine sperm. Motility of demembranated sperm was maintained at free calcium concentrations as low as 27 pM, and calcium arrested sperm motility at much lower concentrations than those reported in other species. Calcium arrest of sperm motility was not accompanied by flagellar curvature, suggesting a failure of calcium to induce the tonic bend seen in other species and thought to support hyperactivated motility. This indicated an absence, or difference in calcium sensitivity, of the related asymmetric doublet-sliding proteins. These studies show a difference in response to calcium of the equine sperm axoneme to that reported in other species that may be related to the failure of equine sperm to penetrate oocytes in vitro under standard capacitating conditions. Further work is needed to determine the factors that stimulate hyperactivated motility at the axonemal level in equine sperm. PMID:25339104

  6. 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

  7. Intracytoplasmic Sperm Injection (ICSI)

    MedlinePlus

    ... male partner produces too few sperm to do artificial insemination (intrauterine insemination [IUI]) or IVF. • The sperm may ... birth defects may actually be due to the infertility and not the treatments used to overcome the ...

  8. 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. PMID:7650152

  9. 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.

  10. The major vault protein is related to the toxic anion resistance protein (TelA) family.

    PubMed

    Suprenant, Kathy A; Bloom, Nathan; Fang, Jianwen; Lushington, Gerald

    2007-03-01

    Vaults are barrel-shaped ribonucleoprotein particles that are abundant in certain tumors and multidrug resistant cancer cells. Prokaryotic relatives of the major vault protein, MVP, have not been identified. We used sequence analysis and molecular modeling to show that MVP and the toxic anion resistance protein, TelA of Rhodobacter sphaeroides strain 2.4.1, share a novel fold that consists of a three-stranded antiparallel beta-sheet. Because of this strong structural correspondence, we examined whether mammalian cell vaults respond to tellurite treatment. In the presence of the oxyanion tellurite, large vault aggregates, or vaultosomes, appear at the cell periphery in 15 min or less. Vaultosome formation is temperature-dependent, reversible, and occurs in normal human umbilical vein endothelial cells as well as transformed HeLa cervical cancer cells. Vaultosome formation is not restricted to tellurite and occurs in the presence of other toxic oxyanions (selenate, selinite, arsenate, arsenite, vanadate). In addition, vaultosomes form independently from other stress-induced ribonucleoprotein complexes, stress granules and aggresomes. Vaultosome formation is therefore a unique cellular response to an environmental toxin. PMID:17337707

  11. 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. PMID:27309818

  12. Effects of ethanol ingestion on sperm monosaccharides and fertility.

    PubMed

    Srikanth, V; Aruldhas, M M; Srinivasan, N; Govindarajulu, P; Balasubramanian, K

    1999-01-01

    Chronic alcohol abuse is often associated with reproductive disorders. Sperm monosaccharides play an indispensable role in sperm-egg interactions and fertilization. Ethanol (3 g/kg body weight as 25%, v/v) was given by gastric intubation twice daily for 30 days while in another group, rats which had been treated with ethanol were withdrawn from treatment for a further period of 30 days, in order to assess the reversibility of the ethanol-induced effects. Epididymal ethanol content, sperm monosaccharides and the fertility of ethanol treated and ethanol withdrawn rats were assessed. Ethanol ingestion caused a significant decrease in sperm monosaccharides suggesting defective glycosylation of sperm surface proteins. Sperm monosaccharides and fertility were returned to normal following the withdrawal of ethanol. Ethanol-induced changes in sperm monosaccharides may be one of the reasons for the reduced fertility of ethanol treated rats. PMID:10092946

  13. Exogenous neurotensin modulates sperm function in Japanese Black cattle.

    PubMed

    Umezu, Kohei; Hiradate, Yuuki; Oikawa, Toshinori; Ishiguro, Hirotoshi; Numabe, Takashi; Hara, Kenshiro; Tanemura, Kentaro

    2016-08-25

    Recently, the conception rates after artificial insemination have been pointed out to decline continuously. To overcome this problem, the control of frozen and thawed sperm quality is required. However, the mechanism of bovine sperm functional regulation is still largely unknown. In mammals, the ejaculated sperm are capable of showing fertilizing ability during migration in the female reproductive organs. It is well known that these female organs secrete several factors contributing to sperm capacitation. We previously reported that neurotensin (NT) secreted from the oviduct and cumulus cells enhanced sperm capacitation and acrosome reaction in mice. In this study, we confirmed the expression of the NT receptor (NTR1) in the bovine sperm neck region and the secretion of NT in the bovine uterus and oviduct. The similar expression patterns of NT and NTR1 suggests a conserved mechanism of sperm functional regulation between mouse and cattle. Thus, we examined the effects of exogenous NT on the bovine sperm functions. First, we showed that NT induced sperm protein tyrosine phosphorylation in a dose-dependent manner, suggesting that NT enhances sperm capacitation. Second, we showed that NT induced acrosome reactions of capacitated sperm in a dose-dependent manner, suggesting that NT facilitates acrosome reaction. Finally, we used a computer-aided sperm analysis system to show that NT did not have a great effect on sperm motility. These results suggest that NT acts as a facilitator of sperm capacitation and acrosome reaction in the female reproductive tracts in cattle, highlighting the importance of NT-mediated signaling to regulate sperm functions. PMID:27210588

  14. Exogenous neurotensin modulates sperm function in Japanese Black cattle

    PubMed Central

    UMEZU, Kohei; HIRADATE, Yuuki; OIKAWA, Toshinori; ISHIGURO, Hirotoshi; NUMABE, Takashi; HARA, Kenshiro; TANEMURA, Kentaro

    2016-01-01

    Recently, the conception rates after artificial insemination have been pointed out to decline continuously. To overcome this problem, the control of frozen and thawed sperm quality is required. However, the mechanism of bovine sperm functional regulation is still largely unknown. In mammals, the ejaculated sperm are capable of showing fertilizing ability during migration in the female reproductive organs. It is well known that these female organs secrete several factors contributing to sperm capacitation. We previously reported that neurotensin (NT) secreted from the oviduct and cumulus cells enhanced sperm capacitation and acrosome reaction in mice. In this study, we confirmed the expression of the NT receptor (NTR1) in the bovine sperm neck region and the secretion of NT in the bovine uterus and oviduct. The similar expression patterns of NT and NTR1 suggests a conserved mechanism of sperm functional regulation between mouse and cattle. Thus, we examined the effects of exogenous NT on the bovine sperm functions. First, we showed that NT induced sperm protein tyrosine phosphorylation in a dose-dependent manner, suggesting that NT enhances sperm capacitation. Second, we showed that NT induced acrosome reactions of capacitated sperm in a dose-dependent manner, suggesting that NT facilitates acrosome reaction. Finally, we used a computer-aided sperm analysis system to show that NT did not have a great effect on sperm motility. These results suggest that NT acts as a facilitator of sperm capacitation and acrosome reaction in the female reproductive tracts in cattle, highlighting the importance of NT-mediated signaling to regulate sperm functions. PMID:27210588

  15. Major basic protein, but not eosinophil cationic protein or eosinophil protein X, is related to atopy in cystic fibrosis.

    PubMed

    Koller, D Y; Halmerbauer, G; Müller, J; Frischer, T; Schierl, M

    1999-10-01

    Increased eosinophil granule proteins have been described in serum and sputum samples of patients with cystic fibrosis (CF). It has been assumed that eosinophil degranulation is enhanced in atopic subjects - as in asthmatics. Since in CF no differences in eosinophil cationic protein (ECP), eosinophil protein X (EPX), and eosinophil peroxidase between atopic and nonatopic subjects have been detected, we investigated whether major basic protein (MBP) is increased in serum and sputum samples derived from atopic (n = 14) compared with nonatopic CF subjects (n = 26). In CF patients, high mean serum (sputum) levels of ECP 29.7 microg/l (2.7 mg/l), EPX 53.7 microg/l (7.9 mg/l), and MBP 984.6 microg/l but low sputum MBP levels (57.4 microg/l) were measured. In addition, in serum and in sputum samples, a significant correlation between MBP and ECP (P<0.03 and P<0.0001, respectively) or EPX (P<0.05 and P<0.0004, respectively) was detected. By subdivision of the patients into allergic and nonallergic subjects, significant differences were found for serum MBP values only(mean 1382.2 microg/l vs. 770.5 microg/l; P<0.0001), but not for ECP or EPX serum levels or for eosinophil proteins in sputum. Although no differences between atopic and nonatopic CF patients in ECP and EPX were found, serum MBP levels were higher in patients sensitized to inhalant allergens than in nonsensitized subjects. These results indicate differential release of eosinophil granule proteins in peripheral blood from eosinophils, and they also indicate that MBP in serum likely is to be a better discriminator of atopy in CF. PMID:10536888

  16. 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…

  17. 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. PMID:25100694

  18. 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. PMID:26825875

  19. The Complete Set of Genes Encoding Major Intrinsic Proteins in Arabidopsis Provides a Framework for a New Nomenclature for Major Intrinsic Proteins in Plants1

    PubMed Central

    Johanson, Urban; Karlsson, Maria; Johansson, Ingela; Gustavsson, Sofia; Sjövall, Sara; Fraysse, Laure; Weig, Alfons R.; Kjellbom, Per

    2001-01-01

    Major intrinsic proteins (MIPs) facilitate the passive transport of small polar molecules across membranes. MIPs constitute a very old family of proteins and different forms have been found in all kinds of living organisms, including bacteria, fungi, animals, and plants. In the genomic sequence of Arabidopsis, we have identified 35 different MIP-encoding genes. Based on sequence similarity, these 35 proteins are divided into four different subfamilies: plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins also called NOD26-like MIPs, and the recently discovered small basic intrinsic proteins. In Arabidopsis, there are 13 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, nine NOD26-like intrinsic proteins, and three small basic intrinsic proteins. The gene structure in general is conserved within each subfamily, although there is a tendency to lose introns. Based on phylogenetic comparisons of maize (Zea mays) and Arabidopsis MIPs (AtMIPs), it is argued that the general intron patterns in the subfamilies were formed before the split of monocotyledons and dicotyledons. Although the gene structure is unique for each subfamily, there is a common pattern in how transmembrane helices are encoded on the exons in three of the subfamilies. The nomenclature for plant MIPs varies widely between different species but also between subfamilies in the same species. Based on the phylogeny of all AtMIPs, a new and more consistent nomenclature is proposed. The complete set of AtMIPs, together with the new nomenclature, will facilitate the isolation, classification, and labeling of plant MIPs from other species. PMID:11500536

  20. Korean red ginseng extract rejuvenates testicular ineffectiveness and sperm maturation process in aged rats by regulating redox proteins and oxidative defense mechanisms.

    PubMed

    Kopalli, Spandana Rajendra; Hwang, Seock-Yeon; Won, Yu-Jin; Kim, Sung-Won; Cha, Kyu-Min; Han, Chang-Kyun; Hong, Jae-Yup; Kim, Si-Kwan

    2015-09-01

    Distortion of intracellular oxidant and antioxidant balances appears to be a common feature that underlies in age-related male sexual impairment. Therefore regulating oxidative defense mechanisms might be an ideal approach in improving male sexual dysfunctions. In the present study, the effect of Korean red ginseng aqueous extract (KRG) on age-induced testicular dysfunction in rats was investigated. KRG (200mg/kg) mixed with regular pellet diet was administered orally for six months and the morphological, spermatogenic and antioxidant enzyme status in testis of aged rats (18months) were evaluated. Data indicated a significant change in morphology and decrease in spermatogenesis-related parameters in aged rats (AC) compared with young rats (YC). Sperm number, germ cell count, Sertoli cell count and Sertoli cell index were significantly (p<0.05) restored in KRG-treated aged rat groups (G-AC). Further the increased lipid peroxidation as measured by malondialdehyde (p<0.05), and altered enzymatic (superoxide dismutase, glutathione peroxidase, glutathione S-transferase, glutathione reductase and catalase) and non-enzymatic (reduced glutathione, ascorbic acid and α-tocopherol) antioxidants (p<0.05) were attenuated by KRG treatment in aged rats to near normal levels as in YC groups. Furthermore, proteomic analysis demonstrated differential expression of selected proteins such as phosphatidylinositol transfer protein, fatty acid binding protein-9, triosephosphate isomerase-1 and aldehyde (aldose) reductase-1in aged rats was significantly (p<0.05) protected by KRG treatment. In conclusion, long-term administration of KRG restored aging-induced testicular ineffectiveness in rats by modulating redox proteins and oxidative defense mechanisms. PMID:25980653

  1. 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.

  2. 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.

  3. 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

  4. 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. PMID:25376881

  5. 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. PMID:7488215

  6. Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase.

    PubMed

    Andrews, Rachel E; Galileo, Deni S; Martin-DeLeon, Patricia A

    2015-11-01

    Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) 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 Ca(2+), 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 Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-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 Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]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 Ca(2+)/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

  7. Virus-specific interaction between the human cytomegalovirus major capsid protein and the C terminus of the assembly protein precursor.

    PubMed Central

    Beaudet-Miller, M; Zhang, R; Durkin, J; Gibson, W; Kwong, A D; Hong, Z

    1996-01-01

    We previously identified a minimal 12-amino-acid domain in the C terminus of the herpes simplex virus type 1 (HSV-1) scaffolding protein which is required for interaction with the HSV-1 major capsid protein. An alpha-helical structure which maximizes the hydropathicity of the minimal domain is required for the interaction. To address whether cytomegalovirus (CMV) utilizes the same strategy for capsid assembly, several glutathione S-transferase fusion proteins to the C terminus of the CMV assembly protein precursor were produced and purified from bacterial cells. The study showed that the glutathione S-transferase fusion containing 16 amino acids near the C-terminal end was sufficient to interact with the major capsid protein. Interestingly, no cross-interaction between HSV-1 and CMV could be detected. Mutation analysis revealed that a three-amino-acid region at the N-terminal side of the central Phe residue of the CMV interaction domain played a role in determining the viral specificity of the interaction. When this region was converted so as to correspond to that of HSV-1, the CMV assembly protein domain lost its ability to interact with the CMV major capsid protein but gained full interaction with the HSV-1 major capsid protein. To address whether the minimal interaction domain of the CMV assembly protein forms an alpha-helical structure similar to that in HSV-1, peptide competition experiments were carried out. The results showed that a cyclic peptide derived from the interaction domain with a constrained (alpha-helical structure competed for interaction with the major capsid protein much more efficiently than the unconstrained linear peptide. In contrast, a cyclic peptide containing an Ala substitution for the critical Phe residue did not compete for the interaction at all. The results of this study suggest that (i) CMV may have developed a strategy similar to that of HSV-1 for capsid assembly; (ii) the minimal interaction motif in the CMV assembly protein

  8. 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. PMID:26925808

  9. 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

  10. Induction of the major heat-stress protein in purified rat glial cells

    SciTech Connect

    Nishimura, R.N.; Dwyer, B.E.; Welch, W.; Cole, R.; de Vellis, J.; Liotta, K.

    1988-05-01

    Cultured purified oligodendroglia and astroglia exposed to heat stress (45 degrees C, 10 or 20 min) synthesized a 68-kDa heat-stress protein, which migrates on two-dimensional gel electrophoresis and reacts with a specific monoclonal antibody suggesting it is similar to a major 72-kDa heat-shock protein previously reported in other cell types. This protein was not detected in control glial cultures. Actinomycin D prevented synthesis of this protein demonstrating an absolute requirement for newly synthesized mRNA. The response was prolonged by increasing the period of heat stress from 10 to 20 min. In addition to the 68-kDa HSP protein, the incorporation of radioactivity into 70-, 89-, and 97-kDa proteins was also increased after heating, but in contrast to the 68 kDa protein these proteins appeared to be made in control glial cultures.

  11. 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. PMID:25943690

  12. 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.

  13. Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization.

    PubMed

    Chalbi, Myriam; Barraud-Lange, Virginie; Ravaux, Benjamin; Howan, Kevin; Rodriguez, Nicolas; Soule, Pierre; Ndzoudi, Arnaud; Boucheix, Claude; Rubinstein, Eric; Wolf, Jean Philippe; Ziyyat, Ahmed; Perez, Eric; Pincet, Frédéric; Gourier, Christine

    2014-10-01

    Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery. PMID:25209248

  14. 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)

  15. 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). PMID:11902937

  16. Functional Amyloids in the Mouse Sperm Acrosome

    PubMed Central

    Guyonnet, Benoit; Egge, Nathan

    2014-01-01

    The acrosomal matrix (AM) is an insoluble structure within the sperm acrosome that serves as a scaffold controlling the release of AM-associated proteins during the sperm acrosome reaction. The AM also interacts with the zona pellucida (ZP) that surrounds the oocyte, suggesting a remarkable stability that allows its survival despite being surrounded by proteolytic and hydrolytic enzymes released during the acrosome reaction. To date, the mechanism responsible for the stability of the AM is not known. Our studies demonstrate that amyloids are present within the sperm AM and contribute to the formation of an SDS- and formic-acid-resistant core. The AM core contained several known amyloidogenic proteins, as well as many proteins predicted to form amyloid, including several ZP binding proteins, suggesting a functional role for the amyloid core in sperm-ZP interactions. While stable at pH 3, at pH 7, the sperm AM rapidly destabilized. The pH-dependent dispersion of the AM correlated with a change in amyloid structure leading to a loss of mature forms and a gain of immature forms, suggesting that the reversal of amyloid is integral to AM dispersion. PMID:24797071

  17. 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.

  18. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

    PubMed

    Zimmerman, Shawn W; Manandhar, Gaurishankar; Yi, Young-Joo; Gupta, Satish K; Sutovsky, Miriam; Odhiambo, John F; Powell, Michael D; Miller, David J; Sutovsky, Peter

    2011-01-01

    Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced

  19. Polyspermy in birds: sperm numbers and embryo survival

    PubMed Central

    Hemmings, N.; Birkhead, T. R.

    2015-01-01

    Polyspermy is a major puzzle in reproductive biology. In some taxa, multiple sperm enter the ovum as part of the normal fertilization process, whereas in others, penetration of the ovum by more than one sperm is lethal. In birds, several sperm typically enter the germinal disc, yet only one fuses with the female pronucleus. It is unclear whether supernumerary sperm play an essential role in the avian fertilization process and, if they do, how females regulate the progression of sperm through the oviduct to ensure an appropriate number reach the ovum. Here, we show that when very few sperm penetrate the avian ovum, embryos are unlikely to survive beyond the earliest stages of development. We also show that when the number of inseminated sperm is limited, a greater proportion than expected reach and penetrate the ovum, indicating that females compensate for low sperm numbers in the oviduct. Our results suggest a functional role for supernumerary sperm in the processes of fertilization and early embryogenesis, providing an exciting expansion of our understanding of sperm function in birds. PMID:26511048

  20. Polyspermy in birds: sperm numbers and embryo survival.

    PubMed

    Hemmings, N; Birkhead, T R

    2015-11-01

    Polyspermy is a major puzzle in reproductive biology. In some taxa, multiple sperm enter the ovum as part of the normal fertilization process, whereas in others, penetration of the ovum by more than one sperm is lethal. In birds, several sperm typically enter the germinal disc, yet only one fuses with the female pronucleus. It is unclear whether supernumerary sperm play an essential role in the avian fertilization process and, if they do, how females regulate the progression of sperm through the oviduct to ensure an appropriate number reach the ovum. Here, we show that when very few sperm penetrate the avian ovum, embryos are unlikely to survive beyond the earliest stages of development. We also show that when the number of inseminated sperm is limited, a greater proportion than expected reach and penetrate the ovum, indicating that females compensate for low sperm numbers in the oviduct. Our results suggest a functional role for supernumerary sperm in the processes of fertilization and early embryogenesis, providing an exciting expansion of our understanding of sperm function in birds. PMID:26511048

  1. 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.

  2. Characterization of the major proteins in gamma particles, cytoplasmic organelles in Blastocladiella emersonii zoospores.

    PubMed

    Hohn, T M; Lovett, J S; Bracker, C E

    1984-04-01

    The gamma particles of Blastocladiella emersonii are 0.5-micron (diameter), electron dense, membrane-enclosed organelles in the cytoplasm of zoospores that have been reported (E.C. Cantino and G.L. Mills, in P. Lemke, ed., Viruses and Plasmids in Fungi, 1979, and R.B. Myers and E.C. Cantino, in A. Wolsky (ed.), Monographs in Developmental Biology, 1974) to store the enzyme chitin synthetase. These particles were isolated from zoospores, and the two major proteins were purified for an analysis of their composition and function. The lower-molecular-weight protein (apparent molecular weight, 41,000) was insoluble in aqueous buffers, had an unusual, very basic amino acid composition, and comprised the characteristic electron-dense inclusions seen in micrographs of sections of fixed and stained gamma particles. After dispersal of the gamma particle membranes with detergent, the higher-molecular-weight protein (apparent molecular weight, 43,000) and a third minor protein (apparent molecular weight, 45,000) sedimented through sucrose cushions with the 41 kilodalton inclusion body protein but were dissociated from it by sonication in buffer containing 7 M urea. Together, the two major proteins represent 60 to 70% of the total protein in the gamma particle and 2.9% of the total protein in zoospores. Tests with specific antisera showed that the two major proteins were not antigenically related, a result consistent with the differences in amino acid composition. When zoospore lysates were centrifuged in sucrose density gradients, the major gamma particle proteins and chitin synthetase activity migrated to regions of different density. Proteins from sporulating thalli and germinating zoospores were separated by gel electrophoresis, and the two major gamma particle proteins were detected by reaction with specific antisera after electrophoretic transfer to nitrocellulose filters. Neither protein could be found in growth phase cells; the appearance and disappearances of both

  3. PUB1: a major yeast poly(A)+ RNA-binding protein.

    PubMed Central

    Matunis, M J; Matunis, E L; Dreyfuss, G

    1993-01-01

    The expression of RNA polymerase II transcripts can be regulated at the posttranscriptional level by RNA-binding proteins. Although extensively characterized in metazoans, relatively few RNA-binding proteins have been characterized in the yeast Saccharomyces cerevisiae. Three major proteins are cross-linked by UV light to poly(A)+ RNA in living S. cerevisiae cells. These are the 72-kDa poly(A)-binding protein and proteins of 60 and 50 kDa (S.A. Adam, T.Y. Nakagawa, M.S. Swanson, T. Woodruff, and G. Dreyfuss, Mol. Cell. Biol. 6:2932-2943, 1986). Here, we describe the 60-kDa protein, one of the major poly(A)+ RNA-binding proteins in S. cerevisiae. This protein, PUB1 [for poly(U)-binding protein 1], was purified by affinity chromatography on immobilized poly(rU), and specific monoclonal antibodies to it were produced. UV cross-linking demonstrated that PUB1 is bound to poly(A)+ RNA (mRNA or pre-mRNA) in living cells, and it was detected primarily in the cytoplasm by indirect immunofluorescence. The gene for PUB1 was cloned and sequenced, and the sequence was found to predict a 51-kDa protein with three ribonucleoprotein consensus RNA-binding domains and three glutamine- and asparagine-rich auxiliary domains. This overall structure is remarkably similar to the structures of the Drosophila melanogaster elav gene product, the human neuronal antigen HuD, and the cytolytic lymphocyte protein TIA-1. Each of these proteins has an important role in development and differentiation, potentially by affecting RNA processing. PUB1 was found to be nonessential in S. cerevisiae by gene replacement; however, further genetic analysis should reveal important features of this class of RNA-binding proteins. Images PMID:8413213

  4. Evaluation of Lasting Effects of Heat Stress on Sperm Profile and Oxidative Status of Ram Semen and Epididymal Sperm

    PubMed Central

    Hamilton, Thais Rose dos Santos; Mendes, Camilla Mota; de Castro, Letícia Signori; de Assis, Patrícia Monken; Siqueira, Adriano Felipe Perez; Delgado, Juliana de Carvalho; Goissis, Marcelo Demarchi; Muiño-Blanco, Teresa; Cebrián-Pérez, José Álvaro; Nichi, Marcílio; Visintin, José Antonio; Assumpção, Mayra Elena Ortiz D'Ávila

    2016-01-01

    Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long-term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm. PMID:26881013

  5. Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

    PubMed Central

    Díaz, Rommy; Boguen, Rodrigo; Martins, Simone Maria Massami Kitamura; Ravagnani, Gisele Mouro; Leal, Diego Feitosa; Oliveira, Melissa de Lima; Muro, Bruno Bracco Donatelli; Parra, Beatriz Martins; Meirelles, Flávio Vieira; Papa, Frederico Ozanan; Dell’Aqua, José Antônio; Alvarenga, Marco Antônio; Moretti, Aníbal de Sant’Anna; Sepúlveda, Néstor

    2016-01-01

    Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility. PMID:27529819

  6. Chromatin and extracellular vesicle associated sperm RNAs

    PubMed Central

    Johnson, Graham D.; Mackie, Paula; Jodar, Meritxell; Moskovtsev, Sergey; Krawetz, Stephen A.

    2015-01-01

    A diverse pool of RNAs remain encapsulated within the transcriptionally silent spermatozoon despite the dramatic reduction in cellular and nuclear volume following cytoplasm/nucleoplasm expulsion. The impact of this pronounced restructuring on the distribution of transcripts inside the sperm essentially remains unknown. To define their compartmentalization, total RNA >100 nt was extracted from sonicated (SS) mouse spermatozoa and detergent demembranated sucrose gradient fractionated (Cs/Tx) sperm heads. Sperm RNAs predominately localized toward the periphery. The corresponding distribution of transcripts and thus localization and complexity were then inferred by RNA-seq. Interestingly, the number of annotated RNAs in the CsTx sperm heads exhibiting reduced peripheral enrichment was restricted. However this included Cabyr, the calcium-binding tyrosine phosphorylation-regulated protein encoded transcript. It is present in murine zygotes prior to the maternal to the zygotic transition yet absent in oocytes, consistent with the delivery of internally positioned sperm-borne RNAs to the embryo. In comparison, transcripts enriched in sonicated sperm contributed to the mitochondria and exosomes along with several nuclear transcripts including the metastasis associated lung adenocarcinoma transcript 1 (Malat1) and several small nucleolar RNAs. Their preferential peripheral localization suggests that chromatin remodeling during spermiogenesis is not limited to nucleoproteins as part of the nucleoprotein exchange. PMID:26071953

  7. Sperm Patch-Clamp

    PubMed Central

    Lishko, Polina; Clapham, David E.; Navarro, Betsy; Kirichok, Yuriy

    2014-01-01

    Sperm intracellular pH and calcium concentration ([Ca2+]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca2+] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels. PMID:23522465

  8. Presence and Function of Dopamine Transporter (DAT) in Stallion Sperm: Dopamine Modulates Sperm Motility and Acrosomal Integrity

    PubMed Central

    Covarrubias, Alejandra A.; Rodríguez-Gil, Joan Enric; Ramírez-Reveco, Alfredo; Concha, Ilona I.

    2014-01-01

    Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP+), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility. PMID:25402186

  9. A sperm-activating peptide controls a cGMP-signaling pathway in starfish sperm.

    PubMed

    Matsumoto, Midori; Solzin, Johannes; Helbig, Annika; Hagen, Volker; Ueno, Sei-ichi; Kawase, Osamu; Maruyama, Yoshinori; Ogiso, Manabu; Godde, Matthias; Minakata, Hiroyuki; Kaupp, U Benjamin; Hoshi, Motonori; Weyand, Ingo

    2003-08-15

    Peptides released from eggs of marine invertebrates play a central role in fertilization. About 80 different peptides from various phyla have been isolated, however, with one exception, their respective receptors on the sperm surface have not been unequivocally identified and the pertinent signaling pathways remain ill defined. Using rapid mixing techniques and novel membrane-permeable caged compounds of cyclic nucleotides, we show that the sperm-activating peptide asterosap evokes a fast and transient increase of the cGMP concentration in sperm of the starfish Asterias amurensis, followed by a transient cGMP-stimulated increase in the Ca(2+) concentration. In contrast, cAMP levels did not change significantly and the Ca(2+) response evoked by photolysis of caged cAMP was significantly smaller than that using caged cGMP. By cloning of cDNA and chemical crosslinking, we identified a receptor-type guanylyl cyclase in the sperm flagellum as the asterosap-binding protein. Sperm respond exquisitely sensitive to picomolar concentrations of asterosap, suggesting that the peptide serves a chemosensory function like resact, a peptide involved in chemotaxis of sperm of the sea urchin Arbacia punctulata. A unifying principle emerges that chemosensory transduction in sperm of marine invertebrates uses cGMP as the primary messenger, although there may be variations in the detail. PMID:12921734

  10. Flow Cytometry Analysis Reveals That Only a Subpopulation of Mouse Sperm Undergoes Hyperpolarization During Capacitation1

    PubMed Central

    Escoffier, Jessica; Navarrete, Felipe; Haddad, Doug; Santi, Celia M.; Darszon, Alberto; Visconti, Pablo E.

    2015-01-01

    To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K+, by PKA inhibitors, and by SLO3 inhibitors in CD1 mouse sperm, and undetectable in Slo3 knockout mouse sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3, and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream of a cAMP-dependent pathway and is mediated by the activation of SLO3 K+ channels. PMID:25855261

  11. Arterivirus Minor Envelope Proteins Are a Major Determinant of Viral Tropism in Cell Culture

    PubMed Central

    Tian, Debin; Wei, Zuzhang; Zevenhoven-Dobbe, Jessika C.; Liu, Runxia; Tong, Guangzhi

    2012-01-01

    Arteriviruses are enveloped positive-strand RNA viruses for which the attachment proteins and cellular receptors have remained largely controversial. Arterivirus particles contain at least eight envelope proteins, an unusually large number among RNA viruses. These appear to segregate into three groups: major structural components (major glycoprotein GP5 and membrane protein [M]), minor glycoproteins (GP2a, GP3, and GP4), and small hydrophobic proteins (E and the recently discovered ORF5a protein). Biochemical studies previously suggested that the GP5-M heterodimer of porcine reproductive and respiratory syndrome virus (PRRSV) interacts with porcine sialoadhesin (pSn) in porcine alveolar macrophages (PAM). However, another study proposed that minor protein GP4, along with GP2a, interacts with CD163, another reported cellular receptor for PRRSV. In this study, we provide genetic evidence that the minor envelope proteins are the major determinant of arterivirus entry into cultured cells. A PRRSV infectious cDNA clone was equipped with open reading frames (ORFs) encoding minor envelope and E proteins of equine arteritis virus (EAV), the only known arterivirus displaying a broad tropism in cultured cells. Although PRRSV and EAV are only distantly related and utilize diversified transcription-regulating sequences (TRSs), a viable chimeric progeny virus was rescued. Strikingly, this chimeric virus (vAPRRS-EAV2ab34) acquired the broad in vitro cell tropism of EAV, demonstrating that the minor envelope proteins play a critical role as viral attachment proteins. We believe that chimeric arteriviruses of this kind will be a powerful tool for further dissection of the arterivirus replicative cycle, including virus entry, subgenomic RNA synthesis, and virion assembly. PMID:22258262

  12. Elemental composition of human semen is associated with motility and genomic sperm defects among older men

    PubMed Central

    Schmid, Thomas E.; Grant, Patrick G.; Marchetti, Francesco; Weldon, Rosana H.; Eskenazi, Brenda; Wyrobek, Andrew J.

    2013-01-01

    BACKGROUND Older men tend to have poorer semen quality and are generally at higher risks for infertility and abnormal reproductive outcomes. METHODS We employed proton-induced X-ray emission (PIXE, 3 MeV proton beam) to investigate the concentrations of zinc, copper, calcium, sulfur, chlorine, potassium, titanium, iron and nickel in washed sperm and seminal plasma from non-smoking groups of 10 older men (65–80 years old) and 10 younger men (22–28 years old) who were concurrently assayed for sperm function and genomicly defective sperm. RESULTS The older group showed elevated zinc, copper and calcium in sperm and elevated sulfur in seminal plasma compared with the younger men. The older group also showed reduced motility as well as increased sperm DNA fragmentation, achondroplasia mutations, DNA strand breaks and chromosomal aberrations. Sperm calcium and copper were positively associated with sperm DNA fragmentation (P < 0.03). Seminal sulfur was positively associated with sperm DNA fragmentation and chromosomal aberrations (P < 0.04), and negatively associated with sperm motility (P < 0.05). Sperm calcium was negatively associated with sperm motility, independent of male age (P = 0.01). CONCLUSIONS We identified major differences in elemental concentrations between sperm and seminal plasma and that higher sperm copper, sulfur and calcium are quantitatively associated with poorer semen quality and increased frequencies of genomic sperm defects. PMID:23042799

  13. Sperm Pretreatment with Dithiothreitol Increases Male Pronucleus Formation Rates After Intracytoplasmic Sperm Injection (ICSI) in Swamp Buffalo Oocytes

    PubMed Central

    CHANKITISAKUL, Vibuntita; AM-IN, Nutthee; THARASANIT, Theerawat; SOMFAI, Tamas; NAGAI, Takashi; TECHAKUMPHU, Mongkol

    2012-01-01

    Abstract Failure of male pronucleus formation has hampered the success of intracytoplasmic sperm injection (ICSI) in swamp buffalo. The aim of the present study was to improve male pronucleus formation by pretreating sperm with various chemicals before ICSI. In Experiments1 and 2, sperm were treated according to one of the following protocols: (1) 0.1% Triton-X 100 (TX) for 1 min, (2) 10 µM calcium ionophore (CaI) for 20 min, (3) freezing and thawing (FT) without any cryoprotectant, or (4) no treatment (control). These sperm treatment groups then either did or did not receive additional sperm treatment with 5 mM dithiothreitol (DTT) for 20 min. Acrosomal integrity (Experiment 1) and DNA fragmentation (Experiment 2) were evaluated in the sperm before ICSI. In Experiment 3, oocytes matured in vitro were subjected to ICSI using pretreated sperm as described above and then were cultured either with or without activation. The TX- and CaI-treated sperm caused an increase in the number of acrosome-loss sperm, whereas the FT treatment and control increased the proportion of acrosome-reacted sperm (P<0.05). The DNA fragmentation did not differ among treatments (P>0.05). At 18 h post-ICSI, pronucleus (PN) formation was found only in activated oocytes. The majority of the activated ICSI oocytes contained intact sperm heads. Normal fertilization was observed in the CaI and FT treatment groups and control group when sperm were treated with DTT before ICSI. In conclusion, DTT treatment of sperm with reacted acrosomes before ICSI together with activation of the ICSI oocytes is important for successful male pronucleus formation. PMID:23132520

  14. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein.

    PubMed

    Nawagitgul, P; Morozov, I; Bolin, S R; Harms, P A; Sorden, S D; Paul, P S

    2000-09-01

    Porcine circovirus 2 (PCV2), a single-stranded DNA virus associated with post-weaning multisystemic wasting syndrome of swine, has two potential open reading frames, ORF1 and ORF2, greater than 600 nucleotides in length. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA, while ORF2 contains a conserved basic amino acid sequence at the N terminus resembling that of the major structural protein of chicken anaemia virus. Thus far, the structural protein(s) of PCV2 have not been identified. In this study, a viral structural protein of 30 kDa was identified in purified PCV2 particles. ORF2 of PCV2 was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. The expressed ORF2 gene product had a molecular mass of 30 kDa, similar to that detected in purified virus particles. The recombinant ORF2 protein self-assembled to form capsid-like particles when viewed by electron microscopy. Antibodies against the ORF2 protein were detected in samples of sera obtained from pigs as early as 3 weeks after experimental infection with PCV2. These results show that the major structural protein of PCV2 is encoded by ORF2 and has a molecular mass of 30 kDa. PMID:10950986

  15. Bromodomain and extra-terminal (BET) family proteins: New therapeutic targets in major diseases.

    PubMed

    Padmanabhan, Balasundaram; Mathur, Shruti; Manjula, Ramu; Tripathi, Shailesh

    2016-06-01

    The bromodomains and extra-terminal domain (BET) family proteins recognize acetylated chromatin through their bromodomains (BDs) and help in regulating gene expression. BDs are chromatin 'readers': by interacting with acetylated lysines on the histone tails, they recruit chromatin-regulating proteins on the promoter region to regulate gene expression and repression. Extensive efforts have been employed by scientific communities worldwide to identify and develop potential inhibitors of BET family BDs to regulate protein expression by inhibiting acetylated histone (H3/H4) interactions. Several small molecule inhibitors have been reported, which not only have high affinity but also have high specificity to BET BDs. These developments make BET family proteins an important therapeutic targets for major diseases such as cancer, neurological disorders, obesity and inflammation. Here, we review and discuss the structural biology of BET family BDs and their applications in major diseases. PMID:27240990

  16. Eosinophil granule-derived major basic protein induces IL-8 expression in human intestinal myofibroblasts.

    PubMed

    Furuta, G T; Ackerman, S J; Varga, J; Spiess, A M; Wang, M Y; Wershil, B K

    2000-10-01

    Eosinophil infiltration occurs in a variety of allergic and inflammatory diseases. The release of preformed mediators from eosinophils may contribute to inflammatory responses. We investigated the ability of eosinophil-derived major basic protein and eosinophil-derived neurotoxin to stimulate production of IL-8 from intestinal myofibroblasts. Intestinal myofibroblasts (18-Co cells) were incubated with major basic protein, eosinophil-derived neurotoxin, or a synthetic analogue of major basic protein, poly-L-arginine. Immunoreactive IL-8 was measured by ELISA and IL-8 mRNA levels were analysed by Northern blot or reverse transcription-polymerase chain assay. Major basic protein induced IL-8 mRNA production and release of significant levels of IL-8 immunoreactive protein. By contrast, eosinophil-derived neurotoxin stimulated little IL-8 release. The induction of IL-8 mRNA by poly-L-arginine was significantly inhibited by actinomycin D. These findings demonstrate a novel interaction between eosinophils and intestinal fibroblasts that may be involved in the pathogenesis of diseases associated with tissue eosinophilia. PMID:11012615

  17. Diisopropyl fluorophosphate labeling of sperm-associated proteinases

    SciTech Connect

    Odem, R.R.; Willand, J.L.; Polakoski, K.L. )

    1990-02-01

    Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.

  18. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis

    SciTech Connect

    Caldwell, H.D.; Kromhout, J.; Schachter, J.

    1981-03-01

    Elementary bodies (EB) of Chlamydia trachomatis serotypes C, E, and L2 were extrinsically radioiodinated, and whole-cell lysates of these serotypes were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of the polypeptide profiles identified a major surface protein with an apparent subunit molecular weight of 39,500 that was common to each C. trachomatis serotype. The abilities of nonionic (Triton X-100), dipolar ionic (Zwittergent TM-314), mild (sodium deoxycholate and sodium N-lauroyl sarcosine), and strongly anionic (SDS) detergents to extract this protein from intact EB of the L2 serotype were investigated by SDS-PAGE analysis of the soluble and insoluble fractions obtained after each detergent treatment. Only SDS readily extracted this protein from intact EB. Sarkosyl treatment selectively solubilized the majority of other EB proteins, leaving the 39,500-dalton protein associated with the Sarkosyl-insoluble fraction. Ultrastructural studies of the Sarkosyl-insoluble EB pellet showed it to consist of empty EB particles possessing an apparently intact outer membrane. No structural evidence for a peptidoglycan-like cell wall was found. Morphologically these chlamydial outer membrane complexes (COMC) resembled intact chlamydial EB outer membranes. The 39,500-dalton outer membrane protein was quantitatively extracted from COMC by treating them with 2% SDS at 60 degrees C. This protein accounted for 61% of the total COMC-associated protein, and its extraction resulted in a concomitant loss of the COMC membrane structure and morphology. The 39,500-dalton major outer membrane protein is a serogroup antigen of C. trachomatis organisms.

  19. Extraction, purification, and characterization of major outer membrane proteins from Wolinella recta ATCC 33238.

    PubMed Central

    Kennell, W L; Holt, S C

    1991-01-01

    The outer membrane of Wolinella recta ATCC 33238 was isolated by French pressure cell disruption and differential centrifugation. Outer membrane proteins (OMPs) were solubilized by Zwittergent 3.14 extraction and separated by DEAE-Sephacel ion-exchange chromatography. The major OMPs that were found in W. recta ATCC 33238 and in several other Wolinella spp. consisted of proteins with apparent molecular masses of 51, 45, and 43 kDa. These three conserved proteins were purified to essential homogeneity by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized chemically. Heating at between 75 and 100 degrees C revealed both the 43- and 51-kDa proteins to be heat modified from apparent molecular masses of 32 and 38 kDa, respectively. The 45-kDa protein was unmodified at all temperatures tested. Two-dimensional isoelectric focusing-SDS-PAGE revealed the 51-kDa protein to be composed of multiple pIs between a pH of 5.0 and greater than 8.0 while the 43- and 45-kDa proteins had a pI of approximately 6.0. N'-terminal amino acid sequence analysis of the first 30 to 40 amino acids and search of the Protein Identification Resource data base for similar proteins only revealed the 43-kDa protein to be similar to the P.69 OMP of Bordetella pertussis; however, the homology was weak (33%). Amino acid analysis revealed the 43-kDa protein to be noncharged and the 45- and 51-kDa proteins to be hydrophilic, containing between 38 to 42% polar residues but no cysteine. This study reports the purification and partial characterization of three conserved proteins in W. recta ATCC 33238. Images PMID:1894372

  20. Annexins V and VI: major calcium-dependent atrial secretory granule-binding proteins.

    PubMed

    Doubell, A F; Bester, A J; Thibault, G

    1991-11-01

    Atrial natriuretic peptide is stored by atrial myocytes in secretory granules, known as atrial specific granules, and is released from these granules by exocytosis. We have isolated a group of atrial proteins by affinity chromatography that bind to atrial specific granules in a calcium-dependent manner. The two major proteins isolated (32.5 kd and 67 kd) are calcium-binding proteins and have been identified as annexins V and VI by immunoblotting with specific antisera. The calcium dependence of their binding to atrial specific granules has been characterized in vitro and indicates that this interaction takes place at micromolar levels of calcium. In addition, the group of proteins isolated includes another calcium-binding protein of 20 kd, as well as GTP-binding proteins of 22 to 26 kd. Membrane interactions during exocytosis are presumably mediated by the interaction of specific proteins with the granule membrane. The properties of the proteins described here, and their ability to bind to atrial specific granules in a calcium-dependent manner, make them likely candidates in the search for regulatory proteins mediating atrial natriuretic peptide secretion. PMID:1834552

  1. The Spectrum of Major Seed Storage Genes and Proteins in Oats (Avena sativa)

    PubMed Central

    Anderson, Olin D.

    2014-01-01

    Background The oat seed storage proteins are mainly composed of two classes: the globulins and avenins. Among the major cereals, the globulins are the major seed protein class in rice and oats, and along with the higher protein content of oats is the basis for the relative higher nutrition content in oats compared to the other cereals. The second major class of oat seed proteins is the avenins; also classified as prolamins – seed proteins high in proline and glutamine amino acids. The prolamins are associated with celiac disease, an autoimmune disorder of the gastrointestinal tract. In spite of their importance, neither the oat globulins nor the avenins have been completely analyzed and described for any single germplasm. Results Using available EST resources for a single hexaploid oat cultivar, the spectrum of avenin and globulin sequences are described for the gene coding regions and the derived protein sequences. The nine unique avenin sequences are suggested to be divided into 3–4 distinct subclasses distributed in the hexaploid genome. The globulins from the same germplasm include 24 distinct sequences. Variation in globulin size results mainly from a glutamine-rich domain, similar to as in the avenins, and to variation in the C-terminal sequence domain. Two globulin genes have premature stop codons that shorten the resulting polypeptides by 9 and 17 amino acids, and eight of the globulin sequences form a branch of the globulins not previously reported. Conclusions A more complete description of the major oat seed proteins should allow a more thorough analysis of their contributions to those oat seed characteristics related to nutritional value, evolutionary history, and celiac disease association. PMID:25054628

  2. Primary structure of a sperm cell anion exchanger and its messenger ribonucleic acid expression during spermatogenesis.

    PubMed

    Holappa, K; Mustonen, M; Parvinen, M; Vihko, P; Rajaniemi, H; Kellokumpu, S

    1999-10-01

    Chloride/bicarbonate (Cl-/HCO(3)-) exchangers are a family of proteins (anion exchanger [AE] gene family) that regulate many vital cellular processes such as intracellular pH, cell volume, and Cl- concentration. They may also be involved in the regulation of sperm cell motility and acrosome reaction during fertilization, as these two phenomena are bicarbonate dependent, and we have previously shown that a polypeptide immunologically related to erythrocyte band 3 is expressed in mammalian sperm cells. We have now identified this putative sperm cell anion exchanger as the AE2 isoform of this gene family. First, we determined its complete primary structure from the human testis lambda gt 11 cDNA library. The cloned sequence was found to consist of 3896 base pairs (bp) with an open reading frame of 3726 bp, and to be almost identical to the previously published human genomic AE2 sequence. Only four amino acid disparities were found between these two sequences. Second, our in situ hybridization analyses showed that AE2 mRNA is expressed in developing sperm cells, indicating that the cloned sequence corresponds to the sperm cell AE. Our reverse transcription-polymerase chain reaction analyses suggested further that the expression of AE2 mRNA was variable to some extent during the epithelial cell cycle. Strongest expression was observed at stages VII-XIV except for stage X, i.e., when major structural and morphological changes take place. These results suggest that the full-length AE2 isoform regulates HCO(3)- transport in mature sperm cells and thus their motility in vivo. PMID:10491633

  3. Kinesin-1 Prevents Capture of the Oocyte Meiotic Spindle by the Sperm Aster

    PubMed Central

    McNally, Karen L.P.; Fabritius, Amy S.; Ellefson, Marina L.; Flynn, Jonathan R.; Milan, Jennifer A.; McNally, Francis J.

    2012-01-01

    Centrioles are lost during oogenesis and inherited from the sperm at fertilization. In the zygote, the centrioles recruit pericentriolar proteins from the egg to form a mature centrosome that nucleates a sperm aster. The sperm aster then captures the female pronucleus to join the maternal and paternal genomes. Because fertilization occurs before completion of female meiosis, some mechanism must prevent capture of the meiotic spindle by the sperm aster. Here we show that in wild-type Caenorhabditis elegans zygotes, maternal pericentriolar proteins are not recruited to the sperm centrioles until after completion of meiosis. Depletion of kinesin-1 heavy chain or its binding partner resulted in premature centrosome maturation during meiosis and growth of a sperm aster that could capture the oocyte meiotic spindle. Kinesin prevents recruitment of pericentriolar proteins by coating the sperm DNA and centrioles and thus prevents triploidy by a non-motor mechanism. PMID:22465668

  4. Structural characterization of dioscorin, the major tuber protein of yams, by near infrared Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Liao, Yu-Hsiu; Tseng, Chi-Yin; Chen, Wenlung

    2006-01-01

    As very little is known about the molecular structure of dioscorin, the major storage protein of yam tuber, we report here FT-Raman spectroscopic investigation of this yam protein isolated from D. alata L., for the first time. According to a series of purification and identification by ion-exchange chromatography, gel chromatography, SDS-PAGE, and MALDI-TOF-MS, it shows that the major storage protein is made up of dioscorin A (M.W. ~33 kDa) and dioscorin B (M.W. ~31 kDa). Raman spectral results indicate that the secondary structure of dioscorin A is major in α-helix, while dioscorin B belongs to anti-parallel β- sheet. It also shows that the microenvironment of major amino acids including tyrosine, phenylalanine, tryptophan, and methionine, and cysteine exhibit explicit differences between these two components. The conformation of disulfide bonding in dioscorin A predominates in Gauche-Gauche-Trans form, while Gauche-Gauche-Gauche and Trans-Gauche-Trans share the conformation in dioscorin B. Structural resemblance between dioscorin A and crude yam proteins implies that dioscorin A exhibits structural preference even though its content is lower than dioscorin B.

  5. Supplementation with major royal jelly proteins increases lifespan, feeding and fecundity in Drosophila

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The major royal-jelly proteins (MRJPs) are the main constituents responsible for the specific physiological role of royal jelly (RJ) in honeybees. Male and female Drosophila flies were fed diets containing either no MRJPs (A) or casein (B) at 1.25% (w/w) of diet or MRJPs at 1.25% (C), 2.50% (D), or ...

  6. Production and Characterization of Monoclonal Antibodies Against a Major Membrane Protein of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Mycobacterium avium subsp. paratuberculosis 35-kDa major membrane protein (MMP) encoded by MAP2121c has been shown to play a role in invasion of epithelial cells and is an important membrane antigen recognized by cattle with Johne’s disease. In this study, purified recombinant MMP was used to p...

  7. Role of posttranslational modifications in C. elegans and ascaris spermatogenesis and sperm function.

    PubMed

    Miao, Long; L'Hernault, Steven W

    2014-01-01

    Generally, spermatogenesis and sperm function involve widespread posttranslational modification of regulatory proteins in many different species. Nematode spermatogenesis has been studied in detail, mostly by genetic/molecular genetic techniques in the free-living Caenorhabditis elegans and by biochemistry/cell biology in the pig parasite Ascaris suum. Like other nematodes, both of these species produce sperm that use a form of amoeboid motility termed crawling, and many aspects of spermatogenesis are likely to be similar in both species. Consequently, work in these two nematode species has been largely complementary. Work in C. elegans has identified a number of spermatogenesis-defective genes and, so far, 12 encode enzymes that are implicated as catalysts of posttranslational protein modification. Crawling motility involves extension of a single pseudopod and this process is powered by a unique cytoskeleton composed of Major Sperm Protein (MSP) and accessory proteins, instead of the more widely observed actin. In Ascaris, pseudopod extension and crawling motility can be reconstituted in vitro, and biochemical studies have begun to reveal how posttranslational protein modifications, including phosphorylation, dephosphorylation and proteolysis, participate in these processes. PMID:25030766

  8. Supporters of sperm

    PubMed Central

    Løvlie, Hanne

    2014-01-01

    The Biology of Spermatozoa (BoS) meetings have run on a biannual basis since the early 1990s. They are dedicated to the fascinating research topic of sperm and their complicated route to fertilization. The BoS meetings focus on sperm, but they also explore additional supporting factors important in fertilization, such as those present in seminal and ovarian fluid, as well as the genomic bases of sperm biology. Here, I present a report of the recent BoS meeting, and showcase some of the highlights of this year’s meeting. PMID:25225623

  9. Long-lived sperm in the geothermal bryophyte Pohlia nutans

    PubMed Central

    Rosenstiel, Todd N.; Eppley, Sarah M.

    2009-01-01

    Non-vascular plants rely on sperm to cross the distance between male and female reproductive organs for fertilization and sexual reproduction to occur. The majority of non-vascular plants have separate sexes, and thus, this distance may be a few millimetres to many metres. Because sperm need water for transport, it has been assumed that sperm lifespans are short and that this type of sexual reproduction limits the expansion of non-vascular plants in terrestrial environments. However, little data is available on the lifespan of sperm in non-vascular plants, and none is available for bryophytes, the group thought to have first colonized terrestrial habitats. Here, we documented the lifespan of sperm of Pohlia nutans, collected from a geothermal spring's area, and tested the effects of variation under environmental conditions on this lifespan. Surprisingly, 20 per cent of the sperm were still motile after 100 h, and sperm lifespan was not significantly affected by temperature variation between 22 and 60°C. Lifespan was significantly affected by sperm dilution and temperatures above 75°C. These results suggest the need to reconsider the importance of sperm motility in bryophyte fertilization. PMID:19640871

  10. Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction*

    PubMed Central

    De La Vega-Beltran, Jose Luis; Sánchez-Cárdenas, Claudia; Krapf, Darío; Hernandez-González, Enrique O.; Wertheimer, Eva; Treviño, Claudia L.; Visconti, Pablo E.; Darszon, Alberto

    2012-01-01

    Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pHi) and Ca2+ ([Ca2+]i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K+ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K+ or by addition of solubilized zona pellucida (sZP). Moreover, K+ and sZP were also able to increase [Ca2+]i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K+ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca2+]i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction. PMID:23095755

  11. Oviductosome-Sperm Membrane Interaction in Cargo Delivery

    PubMed Central

    Al-Dossary, Amal A.; Bathala, Pradeepthi; Caplan, Jeffrey L.; Martin-DeLeon, Patricia A.

    2015-01-01

    Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca2+ efflux pump, plasma membrane Ca2+ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability (Al-Dossary, A. A., Strehler, E. E., and Martin-DeLeon, P. A. (2013) PloS one 8, e80181). It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Here we define a novel experimental approach for the assessment of the interaction of OVS with sperm at a nanoscale level, using a lipophilic dye (FM4–64FX) and three-dimensional SR/SIM, which has an 8-fold increase in volumetric resolution, compared with conventional confocal microscopy. Coincubation assays detected fusion of prelabeled OVS with sperm, primarily over the head and midpiece. Immunofluorescence revealed oviductosomal delivery of PMCA4a to WT and Pmca4 KO sperm, and also endogenous PMCA4a on the inner acrosomal membrane. Fusion was confirmed by transmission immunoelectron microscopy, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Immunofluorescence colocalized OVS with the αv integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. In capacitated and acrosome reacted sperm, fusion was significantly (p < 0.001) inhibited by blocking integrin/ligand interactions via antibodies, exogenous ligands (vitronectin and fibronectin), and their RGD recognition motif. Our results provide evidence that receptor/ligand interactions, involving αvβ3 and α5β1integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be also involved in cargo delivery of prostasomes, epididymosomes, and uterosomes. PMID:26023236

  12. Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia.

    PubMed Central

    Kropinski, A M; Parr, T R; Angus, B L; Hancock, R E; Ghiorse, W C; Greenberg, E P

    1987-01-01

    The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl. Images PMID:3025168

  13. Crystal structure of CspA, the major cold shock protein of Escherichia coli.

    PubMed

    Schindelin, H; Jiang, W; Inouye, M; Heinemann, U

    1994-05-24

    The major cold shock protein of Escherichia coli, CspA, produced upon a rapid downshift in growth temperature, is involved in the transcriptional regulation of at least two genes. The protein shares high homology with the nucleic acid-binding domain of the Y-box factors, a family of eukaryotic proteins involved in transcriptional and translational regulation. The crystal structure of CspA has been determined at 2-A resolution and refined to R = 0.187. CspA is composed of five antiparallel beta-strands forming a closed five-stranded beta-barrel. The three-dimensional structure of CspA is similar to that of the major cold shock protein of Bacillus subtilis, CspB, which has recently been determined at 2.45-A resolution. However, in contrast to CspB, no dimer is formed in the crystal. The surface of CspA is characteristic for a protein interacting with single-stranded nucleic acids. Due to the high homology of the bacterial cold shock proteins with the Y-box factors, E. coli CspA and B. subtilis CspB define a structural framework for the common cold shock domain. PMID:8197194

  14. Rapid separation and quantification of major caseins and whey proteins of bovine milk by capillary electrophoresis.

    PubMed

    Vallejo-Cordoba, B

    1997-01-01

    A rapid capillary zone electrophoresis (CZE) method was established for separating and quantifying major casein and whey proteins in milk. Optimum sample preparation and electrophoretic conditions in a coated capillary maintained at 40 degrees C allowed accurate and reproducible quantification of milk proteins in a single analysis. Sample and run buffer allowed caseins to be maintained in solution by using a combination of urea and a nonionic detergent in phosphate buffer at pH 2.5. Quantitative CZE protein data were derived by calculating percentages and concentrations (mg/mL) of alpha-casein, beta-casein, alpha-lactalbumin, and beta-lactoglobulin. Calibration curves followed linear relationships with highly significant (p < 0.1) correlation coefficients. Relative standard deviations of less than 0.82 (%) for migration times and 2.18 (%) for percent protein indicated that the technique was reproducible. Electrophoretic protein profiles of fresh bovine milk and rehydrated dry milk showed marked quantitative differences in whey protein concentrations. Whey protein represented 12.37 +/- 0.07% beta-lactoglobulin and 3.05 +/- 0.08% alpha-lactalbumin of total protein in typical fresh milk, while only 1.90 +/- 0.16% beta-lactoglobulin and 0.86 +/- 0.04% alpha-lactalbumin of total protein were detected in a commercial rehydrated milk powder. By quantifying these differences, the established technique may allow the detection of substitution of fresh milk with rehydrated milk powder. The accuracy and reproducibility of the technique permitted the quantitation of individual protein concentrations in milk samples, which agreed with ranges reported in the literature. CZE may be well suited for routine use by dairies and regulatory agencies, since it allows the determination of milk proteins in less than 60 min. PMID:9725120

  15. Lead chloride affects sperm motility and acrosome reaction in mice: lead affects mice sperm motility and acrosome reaction.

    PubMed

    Oliveira, Helena; Spanò, Marcello; Santos, Conceição; Pereira, Maria de Lourdes

    2009-08-01

    Lead is highly toxic and persistent in the environment and, thus, a major concern for public health. In this study, the effects of lead chloride (PbCl2) on mouse epididymal sperm were evaluated. Male mice were subcutaneously injected with 74 and 100 mg PbCl2/kg body weight for four consecutive days. Sperm was collected from the epididymis and several parameters of sperm function, such as sperm density, motility, viability, mitochondrial function, acrosome integrity and morphology, were evaluated. Furthermore, DNA fragmentation was assessed by the terminal deoxylnucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay and chromatin integrity was evaluated by sperm chromatin structure assay (SCSA). In order to assess direct effects on existing sperm population, we sacrificed one group for each condition at day 5. The effects of lead upon one entire spermatogenic cycle were evaluated on day 35. Both lead concentrations used in this work affected sperm motility, although no significant differences were observed in sperm viability, mitochondrial function and DNA/chromatin integrity. However, a decrease in the percentage of intact acrosomes was also observed, mirroring a lead-induced premature acrosome reaction. Thus, the results obtained indicate that, together with impaired motility, the effect of lead toxicity on acrosome integrity, leading to premature reaction, may compromise the ability of sperm to fertilize the oocyte. PMID:18594995

  16. The Molecules of Sperm Exocytosis.

    PubMed

    Belmonte, Silvia A; Mayorga, Luis S; Tomes, Claudia N

    2016-01-01

    Exocytosis is a fundamental process used by eukaryotic cells to release biological compounds and to insert lipids and proteins in the plasma membrane. Specialized secretory cells undergo regulated exocytosis in response to physiological signals. Sperm exocytosis or acrosome reaction (AR) is essentially a regulated secretion with special characteristics. We will focus here on some of these unique features, covering the topology, kinetics, and molecular mechanisms that prepare, drive, and regulate membrane fusion during the AR. Last, we will compare acrosomal release with exocytosis in other model systems. PMID:27194350

  17. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid.

    PubMed Central

    Wise, K S; Kim, M F

    1987-01-01

    -modified hydrophobic membrane proteins are major surface antigens of M. hyopneumoniae and that structural variants of surface antigens occur within this species. Images PMID:3680170

  18. The solubilisation of boar sperm membranes by different detergents - a microscopic, MALDI-TOF MS, 31P NMR and PAGE study on membrane lysis, extraction efficiency, lipid and protein composition

    PubMed Central

    2009-01-01

    Background Detergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs) from cells. Different detergents affect different membrane structures according to their physico-chemical properties. However, the effects of different detergents on membrane lysis of boar spermatozoa and the lipid composition of DRMs prepared from the affected sperm membranes have not been investigated so far. Results Spermatozoa were treated with the selected detergents Pluronic F-127, sodium cholate, CHAPS, Tween 20, Triton X-100 and Brij 96V. Different patterns of membrane disintegration were observed by light and electron microscopy. In accordance with microscopic data, different amounts of lipids and proteins were released from the cells by the different detergents. The biochemical methods to assay the phosphorus and cholesterol contents as well as 31P NMR to determine the phospholipids were not influenced by the presence of detergents since comparable amounts of lipids were detected in the organic extracts from whole cell suspensions after exposure to each detergent. However, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry applied to identify phospholipids was essentially disturbed by the presence of detergents which exerted particular suppression effects on signal intensities. After separation of the membrane fractions released by detergents on a sucrose gradient only Triton X-100 and sodium cholate produced sharp turbid DRM bands. Only membrane solubilisation by Triton X-100 leads to an enrichment of cholesterol, sphingomyelin, phosphatidylinositol and phosphatidylethanolamine in a visible DRM band accompanied by a selective accumulation of proteins. Conclusion The boar sperm membranes are solubilised to a different extent by the used detergents. Particularly, the very unique DRMs isolated after Triton X-100 exposure are interesting candidates for further studies regarding the architecture of sperm. PMID

  19. Pore-forming ability of major outer membrane proteins from Wolinella recta ATCC 33238.

    PubMed Central

    Kennell, W L; Egli, C; Hancock, R E; Holt, S C

    1992-01-01

    Three major outer membrane proteins with apparent molecular masses of 43, 45, and 51 kDa were purified from Wolinella recta ATCC 33238, and their pore-forming abilities were determined by the black lipid bilayer method. The non-heat-modifiable 45-kDa protein (Omp 45) showed no pore-forming activity even at high KCl concentrations. The single-channel conductances in 1 M KCl of the heat-modifiable proteins with apparent molecular masses of 43 kDa (Omp 43) and 51 kDa (Omp 51) were 0.49 and 0.60 nS, respectively. The proteins formed nonselective channels and, as determined by experiments of ion selectivity and zero-current potential, were weakly anion selective. Images PMID:1370429

  20. Structural characterization of the major ampullate silk spidroin-2 protein produced by the spider Nephila clavipes.

    PubMed

    Santos-Pinto, José Roberto Aparecido Dos; Arcuri, Helen Andrade; Lubec, Gert; Palma, Mario Sergio

    2016-10-01

    Major ampullate spidroin-2 (MaSp2) is one of the most important spider silk protein, but up to now no information is available regarding the post-translational modifications (PTMs) of this protein. A gel-based mass spectrometry strategy using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods was used to sequence Nephila clavipes MaSp2 (including the N- and C-terminal non-repetitive domains, and the great part of the central core), and to assign a series of post-translational modifications (PTMs) on to the MaSp2 sequence. Two forms of this protein were identified, with different levels of phosphorylation along their sequences. These findings provide a basis for understanding mechanoelastic properties and can support the future design of recombinant spider silk proteins for biotechnological applications. PMID:27208434

  1. Sperm release pathway

    MedlinePlus Videos and Cool Tools

    ... top of the seminiferous tubules in the testes is the epididymis. The sperm migrate from of the ... vesicle are added. From the ampulla, seminal fluid is propelled forward through the ejaculatory ducts toward the ...

  2. Sperm release pathway

    MedlinePlus Videos and Cool Tools

    ... stored in this structure. The ejaculation process begins as the penis fills with blood and becomes erect. ... travel from the epididymis through the vas deferens, a muscular tube, which propels sperm forward through smooth ...

  3. Identification of Peroxiredoxin-5 in Bovine Cauda Epididymal Sperm

    PubMed Central

    Nagdas, Subir K; Buchanan, Teresa; Raychoudhury, Samir

    2013-01-01

    Developing spermatozoa require a series of post-testicular modifications within the luminal environment of the epididymis to achieve maturation; this involves several surface modifications including changes in plasma membrane lipids, proteins, carbohydrates, and alterations in the outer acrosomal membrane. Epididymal maturation can therefore allow sperm to gain forward motility and fertilization capabilities. The objective of this study was to identify maturation dependent protein(s) and to investigate their role with the production of functionally competent spermatozoa. Lectin blot analyses of caput and cauda sperm plasma membrane fractions identified a 17.5kDa Wheat Germ Agglutinin (WGA) binding polypeptide present in the cauda sperm plasma membrane not in the caput sperm plasma membrane. Among the several WGA stained bands, the presence of a 17.5kDa WGA binding polypeptide band was detected only in cauda epididymal fluid not in caput epididymal fluid suggesting that the 17.5kDa WGA-binding polypeptide is secreted from the cauda epididymis and binds to the cauda sperm plasma membrane during epididymal transit. Proteomic identification of the 17.5kDa polypeptide yielded 13 peptides that matched the sequence of peroxiredoxin-5 (PRDX5) protein (Bos Taurus). We propose that bovine cauda sperm PRDX5 acts as an antioxidant enzyme in the epididymal environment, which is crucial in protecting the viable sperm population against the damage caused by endogeneous or exogeneous peroxide. PMID:24186847

  4. Structures of an all-α protein running along the DNA major groove.

    PubMed

    Yu, Li-Yan; Cheng, Wang; Zhou, Kang; Li, Wei-Fang; Yu, Hong-Mei; Gao, Xinlei; Shen, Xudong; Wu, Qingfa; Chen, Yuxing; Zhou, Cong-Zhao

    2016-05-01

    Despite over 3300 protein-DNA complex structures have been reported in the past decades, there remain some unknown recognition patterns between protein and target DNA. The silkgland-specific transcription factor FMBP-1 from the silkworm Bombyx mori contains a unique DNA-binding domain of four tandem STPRs, namely the score and three amino acid peptide repeats. Here we report three structures of this STPR domain (termed BmSTPR) in complex with DNA of various lengths. In the presence of target DNA, BmSTPR adopts a zig-zag structure of three or four tandem α-helices that run along the major groove of DNA. Structural analyses combined with binding assays indicate BmSTPR prefers the AT-rich sequences, with each α-helix covering a DNA sequence of 4 bp. The successive AT-rich DNAs adopt a wider major groove, which is in complementary in shape and size to the tandem α-helices of BmSTPR. Substitutions of DNA sequences and affinity comparison further prove that BmSTPR recognizes the major groove mainly via shape readout. Multiple-sequence alignment suggests this unique DNA-binding pattern should be highly conserved for the STPR domain containing proteins which are widespread in animals. Together, our findings provide structural insights into the specific interactions between a novel DNA-binding protein and a unique deformed B-DNA. PMID:26939889

  5. Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

    PubMed Central

    Chang, C P; Jacobs, Y; Nakamura, T; Jenkins, N A; Copeland, N G; Cleary, M L

    1997-01-01

    The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1. PMID:9315626

  6. Molecular cloning and characterization of the structural gene for protein I, the major outer membrane protein of Neisseria gonorrhoeae.

    PubMed Central

    Carbonetti, N H; Sparling, P F

    1987-01-01

    Protein I (P.I) is the major outer membrane protein of Neisseria gonorrhoeae and serves as a porin. By using oligonucleotide probes derived from the known amino-terminal sequence of the mature protein, we have cloned the gene encoding the P.I of gonococcal strain FA19 in three overlapping fragments and determined the DNA sequence. The gene sequence predicts a protein with characteristics typical of the porins of other Gram-negative bacteria. A clone expressing P.I in Escherichia coli was obtained by removing a portion of the P.I gene promoter and reconstructing the entire P.I gene in a position just downstream from a phage T7 promoter. Expression of P.I was then achieved by introducing this recombinant plasmid into an E. coli strain containing an inducible T7 polymerase gene. The clone produced a protein that was identical in size to native P.I and reacted with anti-P.I monoclonal antibodies. Prolonged expression of the protein apparently was lethal for E. coli, possibly explaining failures to clone an intact P.I gene with its own promoter. Images PMID:3122212

  7. With or without you - Proteomics with or without major plasma/serum proteins.

    PubMed

    Gianazza, Elisabetta; Miller, Ingrid; Palazzolo, Luca; Parravicini, Chiara; Eberini, Ivano

    2016-05-17

    The first sections of this review compile and discuss strategies and protocols for managing plasma/serum as a source of biomarkers relevant to human disease. In many such cases, depletion of abundant protein(s) is a crucial preliminary step to the procedure; specific conceptual and technical approaches, however, make it possible to effectively use to this purpose whole plasma/serum. The final sections focus instead on the complexity associated with each of the major serum/plasma proteins in terms of both, multiple molecular structures (existence of a number of protein species) and of multiple molecular functions (behavior as multifunctional/multitasking/moonlighting proteins). Reviewing evidence in these and some related fields (regulation of the synthetic pattern by proteins and non-protein compounds and its connection with health and disease) prompts the suggestion/recommendation that information on the abundant components of plasma/serum proteome is routinely obtained and processed/mined as a valuable contribution to the characterization of any non-physiological condition and to the understanding of its mechanisms and of its implications/sequels. PMID:27072114

  8. Sperm studies in anesthesiologists

    SciTech Connect

    Wyrobek, A.J.; Brodsky, J.; Gordon, l.; Moore, D.H., II; Watchmaker, G.; Cohen, E.N.

    1981-11-01

    Semen samples were collected from 46 anesthesiologists each of whom had worked a minimum of one year in hospital operating rooms ventilated with modern gas-scavenging devices. Samples collected from 26 beginning residents in anesthesiology served as controls. Concentrations of sperm and percentage of sperm having abnormal head shapes were determined for each sample. No significant differences were found between anesthesiologists and beginning residents. Limiting the analyses to men having no confounding factors (varicocele, recent illness, medications, heavy smoking, frequent sauna use) did not change the results. The sperm concentration and morphology in 13 men did not change signficantly after one year of exposure to anesthetic gases. However, the group of men who had one or more confounding factors (excluding exposure to anesthetic gases) showed significantly higher percentages of sperm abnormalities than did the group of men without such factors. These results suggest that limited exposure to anesthetic gases does not significantly affect sperm production as judged by changes in sperm concentration and morphology. These data are reassuring, but since the hospitals surveyed used modern gas-scavenging devices, men who are occupationally exposed to anesthetic gases without this protection should be studied for fuller assessment of the possible human spermatotoxic effects.

  9. Major peptides from amaranth (Amaranthus cruentus) protein inhibit HMG-CoA reductase activity.

    PubMed

    Soares, Rosana Aparecida Manólio; Mendonça, Simone; de Castro, Luíla Ívini Andrade; Menezes, Amanda Caroline Cardoso Corrêa Carlos; Arêas, José Alfredo Gomes

    2015-01-01

    The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. PMID:25690031

  10. Crystal Structure of Major Envelope Protein VP24 from White Spot Syndrome Virus

    PubMed Central

    Sun, Lifang; Su, Yintao; Zhao, Yanhe; Fu, Zheng-qing; Wu, Yunkun

    2016-01-01

    White spot syndrome virus (WSSV) is one of the major and most serious pathogen in the shrimp industry. As one of the most abundant envelope protein, VP24 acts as a core protein interacting with other structure proteins and plays an important role in virus assembly and infection. Here, we have presented the crystal structure of VP24 from WSSV. In the structure, VP24 consists of a nine-stranded β–barrel fold with mostly antiparallel β-strands, and the loops extending out the β–barrel at both N-terminus and C-terminus, which is distinct to those of the other two major envelope proteins VP28 and VP26. Structural comparison of VP24 with VP26 and VP28 reveals opposite electrostatic surface potential properties of them. These structural differences could provide insight into their differential functional mechanisms and roles for virus assembly and infection. Moreover, the structure reveals a trimeric assembly, suggesting a likely natural conformation of VP24 in viral envelope. Therefore, in addition to confirming the evolutionary relationship among the three abundant envelope proteins of WSSV, our structural studies also facilitate a better understanding of the molecular mechanism underlying special roles of VP24 in WSSV assembly and infection. PMID:27572278

  11. Major Peptides from Amaranth (Amaranthus cruentus) Protein Inhibit HMG-CoA Reductase Activity

    PubMed Central

    Soares, Rosana Aparecida Manólio; Mendonça, Simone; de Castro, Luíla Ívini Andrade; Menezes, Amanda Caroline Cardoso Corrêa Carlos; Arêas, José Alfredo Gomes

    2015-01-01

    The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. PMID:25690031

  12. Identification of major Toxoneuron nigriceps venom proteins using an integrated transcriptomic/proteomic approach.

    PubMed

    Laurino, Simona; Grossi, Gerarda; Pucci, Pietro; Flagiello, Angela; Bufo, Sabino Aurelio; Bianco, Giuliana; Salvia, Rosanna; Vinson, S Bradleigh; Vogel, Heiko; Falabella, Patrizia

    2016-09-01

    Endoparasitoids in the order Hymenoptera are natural enemies of several herbivorous insect pest species. During oviposition they inject a mixture of factors, which include venom, into the host, ensuring the successful parasitism and the development of their progeny. Although these parasitoid factors are known to be responsible for host manipulation, such as immune system suppression, little is known about both identity and function of the majority of their venom components. To identify the major proteins of Toxoneuron nigriceps (Hymenoptera: Braconidae) venom, we used an integrated transcriptomic and proteomic approach. The tandem-mass spectrometric (LC-MS/MS) data combined with T. nigriceps venom gland transcriptome used as a reference database resulted in the identification of a total of thirty one different proteins. While some of the identified proteins have been described in venom from several parasitoids, others were identified for the first time. Among the identified proteins, hydrolases constituted the most abundant family followed by transferases, oxidoreductases, ligases, lyases and isomerases. The hydrolases identified in the T. nigriceps venom glands included proteases, peptidases and glycosidases, reported as common components of venom from several parasitoid species. Taken together, the identified proteins included factors that could potentially inhibit the host immune system, manipulate host physiological processes and host development, as well as provide nutrients to the parasitoid progeny, degrading host tissues by specific hydrolytic enzymes. The venom decoding provides us with information about the identity of candidate venom factors which could contribute to the success of parasitism, together with other maternal and embryonic factors. PMID:27388778

  13. Complex architecture of major histocompatibility complex class II promoters: reiterated motifs and conserved protein-protein interactions.

    PubMed Central

    Jabrane-Ferrat, N; Fontes, J D; Boss, J M; Peterlin, B M

    1996-01-01

    The S box (also known as at the H, W, or Z box) is the 5'-most element of the conserved upstream sequences in promoters of major histocompatibility complex class II genes. It is important for their B-cell-specific and interferon gamma-inducible expression. In this study, we demonstrate that the S box represents a duplication of the downstream X box. First, RFX, which is composed of the RFX5-p36 heterodimer that binds to the X box, also binds to the S box and its 5'-flanking sequence. Second, NF-Y, which binds to the Y box and increases interactions between RFX and the X box, also increases the binding of RFX to the S box. Third, RFXs bound to S and X boxes interact with each other in a spatially constrained manner. Finally, we confirmed these protein-protein and protein-DNA interactions by expressing a hybrid RFX5-VP16 protein in cells. We conclude that RFX binds to S and X boxes and that complex interactions between RFX and NF-Y direct B-cell-specific and interferon gamma-inducible expression or major histocompatibility complex class II genes. PMID:8756625

  14. Leishmania major MPK7 Protein Kinase Activity Inhibits Intracellular Growth of the Pathogenic Amastigote Stage ▿

    PubMed Central

    Morales, Miguel A.; Pescher, Pascale; Späth, Gerald F.

    2010-01-01

    During the infectious cycle, protozoan parasites of the genus Leishmania undergo several adaptive differentiation steps that are induced by environmental factors and crucial for parasite infectivity. Genetic analyses of signaling proteins underlying Leishmania stage differentiation are often rendered difficult due to lethal null mutant phenotypes. Here we used a transgenic strategy to gain insight into the functions of the mitogen-activated Leishmania major protein kinases LmaMPK7 and LmaMPK10 in parasite virulence. We established L. major and Leishmania donovani lines expressing episomal green fluorescent protein (GFP)-LmaMPK7 and GFP-LmaMPK10 fusion proteins. The transgenic lines were normal in promastigote morphology, growth, and the ability to differentiate into metacyclic and amastigote stages. While parasites expressing GFP-LmaMPK10 showed normal infectivity by mouse footpad analysis and macrophage infection assays, GFP-LmaMPK7 transgenic parasites displayed a strong delay in lesion formation and reduced intracellular parasite growth. Significantly, the effects of GFP-LmaMPK7 on virulence and proliferation were due exclusively to protein kinase activity, as the overexpression of two kinase-dead mutants had no effect on parasite infectivity. GFP-LmaMPK7 transgenic L. donovani cells revealed a reversible, stage-specific growth defect in axenic amastigotes that was independent of cell death but linked to nonsynchronous growth arrest and a significant reduction of de novo protein biosynthesis. Our data suggest that LmaMPK7 protein kinase activity may be implicated in parasite growth control and thus relevant for the development of nonproliferating stages during the infectious cycle. PMID:19801421

  15. Ubiquitination and its influence in boar sperm physiology and cryopreservation.

    PubMed

    Purdy, P H

    2008-09-15

    Recent reports document the potential use of the ubiquitin protein as an indicator of mammalian sperm quality or fertility, based on poor morphology, sperm count, and other cellular qualities. However, its influence on cellular physiologic mechanisms and boar sperm cryopreservation are unknown. The objective of this research was to determine the influence of boar sperm ubiquitination (n=12 boars) on motility (using CASA), and flow cytometry and fluorescent probes (in parentheses) to evaluate mitochondrial activity (JC-1), plasma and acrosomal membrane integrity (PI and FITC-PNA), membrane fluidity (M540), and chromatin stability (TUNEL) for fresh and frozen-thawed samples. The effects of ubiquitination (determined flow cytometrically) on the ability of frozen-thawed boar sperm to capacitate (FLUO-3AM) and acrosome react (FITC-PNA) were also investigated using flow cytometry. Cryopreservation induced a decrease in the percentage of sperm that were ubiquitinated from 29 to 20% (P<0.0001), but no significant effects of ubiquitin on sperm quality (motility, membrane integrities and organization) were detected. The ability of sperm to capacitate and acrosome react was influenced by ubiquitination. Samples with more ubiquitinated boar sperm were able to maintain plasma membrane integrity (PMI) better and have fewer live acrosome-reacted cells over 120 min of induced capacitation (P<0.05). In conclusion, frozen-thawed ubiquitinated boar sperm were better able to survive the physical stresses of induced capacitation, yet were still capable of capacitating and acrosome reacting, which may enable use of this assay for in the vitro evaluation of the quality of boar sperm. PMID:18579194

  16. Proteomic identification of sperm antigens using serum samples from individuals with and without antisperm antibodies.

    PubMed

    Nowicka-Bauer, K; Kamieniczna, M; Cibulka, J; Ulcova-Gallova, Z; Kurpisz, M

    2016-08-01

    The aim of the study was to identify human sperm antigens reacting with polyclonal antisperm antibodies. Protein sperm extracts were subjected to electrofocusing, and next immune reactions (immunoblotting) were carried out with positive for antisperm antibodies and control (not containing antisperm antibodies) serum samples. Proteomic analysis of human sperm proteins resulted in identification of 80 sperm antigens that could be divided into three groups: antigens specific for patients with antisperm antibodies (32), antigens recognised by both infertile patients and control sera (35) and antigens detected by control serum samples only (13). Among antigens specific for infertile patients, there were 12 sperm entities known to be involved in fertilisation process. We have also characterised three protein entities identified only by sera of infertile women. Altogether, the proteomic analysis resulted in identification of 27 sperm entities not reported previously in human sperm proteome. Identified proteins are sperm antigens that could be potentially responsible for immunological infertility. The study also sheds new light on the sperm antigens in aspect of gender specificity. The investigation of human sperm proteome by the use of antisperm antibodies-containing sera of infertile individuals not only may indicate new proteins but also can draft their immunological nature. PMID:26659478

  17. A Systematic Analysis of a Deep Mouse Epididymal Sperm Proteome

    SciTech Connect

    Chauvin, Theodore; Xie, Fang; Liu, Tao; Nicora, Carrie D.; Yang, Feng; Camp, David G.; Smith, Richard D.; Roberts, Kenneth P.

    2012-12-21

    Spermatozoa are highly specialized cells that, when mature, are capable of navigating the female reproductive tract and fertilizing an oocyte. The sperm cell is thought to be largely quiescent in terms of transcriptional and translational activity. As a result, once it has left the male reproductive tract, the sperm cell is essentially operating with a static population of proteins. It is therefore theoretically possible to understand the protein networks contained in a sperm cell and to deduce its cellular function capabilities. To this end we have performed a proteomic analysis of mouse sperm isolated from the cauda epididymis and have confidently identified 2,850 proteins, which is the most comprehensive sperm proteome for any species reported to date. These proteins comprise many complete cellular pathways, including those for energy production via glycolysis, β-oxidation and oxidative phosphorylation, protein folding and transport, and cell signaling systems. This proteome should prove a useful tool for assembly and testing of protein networks important for sperm function.

  18. Sperm Proteomics: Road to Male Fertility and Contraception

    PubMed Central

    Rahman, Md Saidur; Lee, June-Sub

    2013-01-01

    Spermatozoa are highly specialized cells that can be easily obtained and purified. Mature spermatozoa are transcriptionally and translationally inactive and incapable of protein synthesis. In addition, spermatozoa contain relatively higher amounts of membrane proteins compared to other cells; therefore, they are very suitable for proteomic studies. Recently, the application of proteomic approaches such as the two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential in-gel electrophoresis has identified several sperm-specific proteins. These findings have provided a further understanding of protein functions involved in different sperm processes as well as of the differentiation of normal state from an abnormal one. In addition, studies on the sperm proteome have demonstrated the importance of spermatozoal posttranslational modifications and their ability to induce physiological changes responsible for fertilization. Large-scale proteomic studies to identify hundreds to thousands of sperm proteins will ultimately result in the development of novel biomarkers that may help to detect fertility, the state of complete contraception, and beyond. Eventually, these protein biomarkers will allow for a better diagnosis of sperm dysfunctions and aid in drug development. This paper reviews the recent scientific publications available from the PubMed database to address sperm proteomics and its potential application to characterize male fertility and contraception. PMID:24363670

  19. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.

  20. Identification of vitronectin as a major plasma protein adsorbed on polymer surfaces of different copolymer composition.

    PubMed

    Bale, M D; Wohlfahrt, L A; Mosher, D F; Tomasini, B; Sutton, R C

    1989-12-01

    The arrays of proteins adsorbed from plasma onto a series of polystyrene copolymeric latexes were analyzed by enzyme-linked immunosorbent assay (ELISA) of washed beads and immunoblotting of proteins desorbed from the beads and separated by polyacrylamide gel electrophoresis (PAGE). Beads were prepared by continuous emulsion polymerization in the absence of surfactant. Coomassie brilliant blue staining of gel electropherograms of desorbed proteins indicated that the presence of small amounts of comonomers (1 to 10 mole %) significantly influenced the composition of the adsorbed protein layer. Immunoblotting revealed that fibrinogen, fibronectin, and vitronectin were adsorbed by all surfaces investigated. C3 and Clq adsorption varied significantly with copolymer composition. The ELISAs revealed that although the concentrations of vitronectin and fibronectin in plasma are similar, the extent of vitronectin adsorption from 70% to 85% plasma was greater by two orders of magnitude than fibronectin adsorption. Vitronectin adsorbed on carboxylic acid-containing copolymers reacted more strongly with a conformationally sensitive antivitronectin monoclonal antibody (MoAb) than vitronectin adsorbed to polystyrene and was more susceptible to cleavage by plasma proteases(s). The results show that vitronectin is a major protein adsorbed from concentrated plasma and that small changes in the chemical composition of a copolymer profoundly affects the extent and nature of protein adsorption from complex mixtures such as plasma. PMID:2479428

  1. A Novel Plant Major Intrinsic Protein in Physcomitrella patens Most Similar to Bacterial Glycerol Channels1

    PubMed Central

    Gustavsson, Sofia; Lebrun, Anne-Sophie; Nordén, Kristina; Chaumont, François; Johanson, Urban

    2005-01-01

    A gene encoding a novel fifth type of major intrinsic protein (MIP) in plants has been identified in the moss Physcomitrella patens. Phylogenetic analyses show that this protein, GlpF-like intrinsic protein (GIP1;1), is closely related to a subclass of glycerol transporters in bacteria that in addition to glycerol are highly permeable to water. A likely explanation of the occurrence of this bacterial-like MIP in P. patens is horizontal gene transfer. The expressed P. patens GIP1;1 gene contains five introns and encodes a unique C-loop extension of approximately 110 amino acid residues that has no obvious similarity with any other known protein. Based on alignments and structural comparisons with other MIPs, GIP1;1 is suggested to have retained the permeability for glycerol but not for water. Studies on heterologously expressed GIP1;1 in Xenopus laevis oocytes confirm the predicted substrate specificity. Interestingly, proteins of one of the plant-specific subgroups of MIPs, the NOD26-like intrinsic proteins, are also facilitating the transport of glycerol and have previously been suggested to have evolved from a horizontally transferred bacterial gene. Further studies on localization and searches for GIP1;1 homologs in other plants will clarify the function and significance of this new plant MIP. PMID:16113222

  2. Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins

    SciTech Connect

    Tsukamotot, A.S.; Ponticelli, A.; Berk, A.J.; Gaynor, R.B.

    1986-07-01

    Adenovirus early region 1A (E1A) encodes two acidic phosphoproteins which are required for transactivation of viral transcription, efficient viral DNA replication in phase G/sub 0/-arrested human cells, and oncogenic transformation of rodent cells. Biochemical analysis of in vivo /sup 32/P-labeled adenovirus type 2 E1A proteins purified with monoclonal antibodies demonstrated that these proteins were phosphorylated at multiple serine residues. Two-dimensional phosphotryptic peptide maps of wild-type and mutant E1A proteins were used to locate a major site of E1A protein phosphorylation at serine-219 of the large E1A protein. Although this serine fell within a consensus sequence for phosphorylation by the cyclic AMP-dependent protein kinases, experiments with mutant CHO cells defective in these enzymes indicated that it was not. Oligonucleotide-directed mutagenesis was used to substitute an alanine for serine-219. This mutation prevented phosphorylation at this site. Nonetheless, the mutant was indistinguishable from the wild type for early gene transactivation, replication on G/sub 0/-arrested WI-38 cells, and transformation of cloned rat embryo fibroblast cells.

  3. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

    PubMed Central

    Haynes, J I; Consigli, R A

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein. Images PMID:1318417

  4. Outer membrane protein DsrA is the major fibronectin-binding determinant of Haemophilus ducreyi.

    PubMed

    Leduc, Isabelle; White, C Dinitra; Nepluev, Igor; Throm, Robert E; Spinola, Stanley M; Elkins, Christopher

    2008-04-01

    The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains. PMID:18212073

  5. Structures of an all-α protein running along the DNA major groove

    PubMed Central

    Yu, Li-Yan; Cheng, Wang; Zhou, Kang; Li, Wei-Fang; Yu, Hong-Mei; Gao, Xinlei; Shen, Xudong; Wu, Qingfa; Chen, Yuxing; Zhou, Cong-Zhao

    2016-01-01

    Despite over 3300 protein–DNA complex structures have been reported in the past decades, there remain some unknown recognition patterns between protein and target DNA. The silkgland-specific transcription factor FMBP-1 from the silkworm Bombyx mori contains a unique DNA-binding domain of four tandem STPRs, namely the score and three amino acid peptide repeats. Here we report three structures of this STPR domain (termed BmSTPR) in complex with DNA of various lengths. In the presence of target DNA, BmSTPR adopts a zig-zag structure of three or four tandem α-helices that run along the major groove of DNA. Structural analyses combined with binding assays indicate BmSTPR prefers the AT-rich sequences, with each α-helix covering a DNA sequence of 4 bp. The successive AT-rich DNAs adopt a wider major groove, which is in complementary in shape and size to the tandem α-helices of BmSTPR. Substitutions of DNA sequences and affinity comparison further prove that BmSTPR recognizes the major groove mainly via shape readout. Multiple-sequence alignment suggests this unique DNA-binding pattern should be highly conserved for the STPR domain containing proteins which are widespread in animals. Together, our findings provide structural insights into the specific interactions between a novel DNA-binding protein and a unique deformed B-DNA. PMID:26939889

  6. Serine proteinase of Renibacterium salmoninarum digests a major autologous extracellular and cell-surface protein.

    PubMed

    Rockey, D D; Turaga, P S; Wiens, G D; Cook, B A; Kaattari, S L

    1991-10-01

    Renibacterium salmoninarum is a pathogen of salmonid fish that produces large amounts of extracellular protein (ECP) during growth. A proteolytic activity present in ECP at elevated temperatures digested the majority of the proteins in ECP. This digestion was also associated with the loss of ECP immunosuppressive function. In vitro activity of the proteinase in ECP was temperature dependent: it was not detected in an 18-h digest at 4 and 17 degrees C but became readily apparent at 37 degrees C. Proteinase activity was detected at bacterial physiological temperatures (17 degrees C) in reactions incubated for several days. Under these conditions, digestion of partially purified p57, a major constituent of ECP and a major cell-surface protein, yielded a spectrum of breakdown products similar in molecular weight and antigenicity to those in ECP. This pattern of digestion suggests that most of the immunologically related constituents of ECP are p57 and its breakdown products. The proteolytic activity was sensitive to phenylmethylsulfonyl fluoride, methanol, and ethanol and to 10-min incubation at temperatures above 65 degrees C. Electrophoretic analysis of the proteinase on polyacrylamide gels containing proteinase substrates indicated the native form to be 100 kDa or greater. The enzyme was active against selected unrelated substrates only when coincubated with a denaturant (0.1% lauryl sulfate) and (or) a reducing agent (20 mM dithiothreitol). PMID:1777853

  7. Vaults and the major vault protein: novel roles in signal pathway regulation and immunity.

    PubMed

    Berger, W; Steiner, E; Grusch, M; Elbling, L; Micksche, M

    2009-01-01

    The unique and evolutionary highly conserved major vault protein (MVP) is the main component of ubiquitous, large cellular ribonucleoparticles termed vaults. The 100 kDa MVP represents more than 70% of the vault mass which contains two additional proteins, the vault poly (ADP-ribose) polymerase (vPARP) and the telomerase-associated protein 1 (TEP1), as well as several short untranslated RNAs (vRNA). Vaults are almost ubiquitously expressed and, besides chemotherapy resistance, have been implicated in the regulation of several cellular processes including transport mechanisms, signal transmissions and immune responses. Despite a growing amount of data from diverse species and systems, the definition of precise vault functions is still highly complex and challenging. Here we review the current knowledge on MVP and vaults with focus on regulatory functions in intracellular signal transduction and immune defence. PMID:18759128

  8. Shape and shear guide sperm cells spiraling upstream

    NASA Astrophysics Data System (ADS)

    Kantsler, Vasily; Dunkel, Jorn; Goldstein, Raymond E.

    2014-11-01

    A major puzzle in biology is how mammalian sperm determine and maintain the correct swimming direction during the various phases of the sexual reproduction process. Currently debated mechanisms for sperm long range travel vary from peristaltic pumping to temperature sensing (thermotaxis) and direct response to fluid flow (rheotaxis), but little is known quantitatively about their relative importance. Here, we report the first quantitative experimental study of mammalian sperm rheotaxis. Using microfluidic devices, we investigate systematically the swimming behavior of human and bull sperm over a wide range of physiologically relevant shear rates and viscosities. Our measurements show that the interplay of fluid shear, steric surface-interactions and chirality of the flagellar beat leads to a stable upstream spiraling motion of sperm cells, thus providing a generic and robust rectification mechanism to support mammalian fertilization. To rationalize these findings, we identify a minimal mathematical model that is capable of describing quantitatively the experimental observations.

  9. Major royal jelly proteins as markers of authenticity and quality of honey.

    PubMed

    Bilikova, Katarina; Kristof Krakova, Tatiana; Yamaguchi, Kikuji; Yamaguchi, Yoshihisa

    2015-12-01

    Until now, the properties of honey have been defined based exclusively on the content of plant components in the nectar of given plant. We showed that apalbumin1, the major royal jelly (RJ) protein, is an authentic and regular component of honey. Apalbumin1 and other RJ proteins and peptides are responsible for the immunostimulatory properties and antibiotic activity of honey. For the quantification of apalbumin1, an enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal anti-apalbumin1 antibody. The method is suitable for honey authenticity determination; moreover it is useful for detection of the honey, honeybee pollen and RJ in products of medicine, pharmacy, cosmetics, and food industry, where presences of these honeybee products are declared. Results from the analysis for presence and amount of apalbumin1 in honeys will be used for high-throughput screening of honey samples over the world. On the basis of our experiments which show that royal jelly proteins are regular and physiologically active components of honey we propose to change the definition of honey (according to the EU Honey Directive 2001/110/EC) as follows: Honey is a natural sweet substance produced by honey bees from nectar of plants or from secretions of plants, or excretions of plant sucking insects, which honey bees collect, transform by combining with major royal jelly proteins and other specific substances of their own, deposit, dehydrate, store and leave in the honey comb to ripen and mature. PMID:26751857

  10. Peroxidase as the Major Protein Constituent in Areca Nut and Identification of Its Natural Substrates

    PubMed Central

    Liu, Yu-Ching; Chen, Chao-Jung; Lee, Miau-Rong; Li, Mi; Hsieh, Wen-Tsong; Chung, Jing-Gung; Ho, Heng-Chien

    2013-01-01

    Numerous reports illustrate the diverse effects of chewing the areca nut, most of which are harmful and have been shown to be associated with oral cancer. Nearly all of the studies are focused on the extract and/or low molecular weight ingredients in the areca nut. The purpose of this report is to identify the major protein component in the areca nut. After ammonium sulfate fractionation, the concentrated areca nut extract is subjected to DEAE-cellulose chromatography. A colored protein is eluted at low NaCl concentration and the apparently homogeneous eluent represents the major protein component compared to the areca nut extract. The colored protein shares partial sequence identity with the royal palm tree peroxidase and its peroxidase activity is confirmed using an established assay. In the study, the natural substrates of areca nut peroxidase are identified as catechin, epicatechin, and procyanidin B1. The two former substrates are similarly oxidized to form a 576 Da product with concomitant removal of four hydrogen atoms. Interestingly, oxidation of procyanidin B1 occurs only in the presence of catechin or epicatechin and an additional product with an 864 Da molecular mass. In addition, procyanidin B1 is identified as a peroxidase substrate for the first time. PMID:24250715

  11. RubisCO is not a major fraction of total protein in marine phytoplankton

    NASA Astrophysics Data System (ADS)

    Losh, J.; Young, J. N.; Morel, F. M.

    2012-12-01

    Ribulose 1,5 bisphosphate carboxylase oxygnenase (Rubisco) concentrations were quantified as a proportion of total protein in diatoms to determine whether Rubisco is as abundant in phytoplankton as previously thought. This enzyme has been assumed to be a major fraction of total protein in phytoplankton, as has been demonstrated in plants, potentially constituting a large sink for cellular nitrogen (N). Marine diatoms were grown in batch cultures, and in N-limited continuous cultures at various carbon dioxide (CO2) levels. Quantitative western blots were performed using commercially available global antibodies and protein standards. Field incubations with natural populations of organisms from the coast of California were conducted under both N-limited and N-replete incubations with varying CO2. In all experiments, Rubisco represented less than 5% of total protein. Within exponentially growing batch cultures, concentrations ranged from 2-4%, while in N-limited laboratory and field cultures, concentrations were less than 1%. In some experiments under N-limiting conditions, Rubisco concentrations decreased with decreasing growth rates or with increasing CO2. These results were used as a basis of a theoretical calculation of maximum Rubisco activity and suggest that phytoplankton contain the minimum amount of Rubisco necessary to operate. Unlike plants, Rubisco is not a major sink of cellular N in phytoplankton. This has implications for phytoplankton's response to increasing CO2 under N-limitation.

  12. The Mg2+ transporter CNNM4 regulates sperm Ca2+ homeostasis and is essential for reproduction.

    PubMed

    Yamazaki, Daisuke; Miyata, Haruhiko; Funato, Yosuke; Fujihara, Yoshitaka; Ikawa, Masahito; Miki, Hiroaki

    2016-05-01

    Ca(2+) influx triggers sperm capacitation; however, the underlying regulatory mechanisms remain incompletely understood. Here, we show that CNNM4, a Mg(2+) transporter, is required for Ca(2+) influx during capacitation. We find that Cnnm4-deficient male mice are almost infertile because of sperm dysfunction. Motion analyses show that hyperactivation, a qualitative change in the mode of sperm motility during capacitation, is abrogated in Cnnm4-deficient sperm. In contrast, tyrosine phosphorylation of flagellar proteins, a hallmark of capacitation, is excessively augmented. These seemingly paradoxical phenotypes of Cnnm4-deficient sperm are very similar to those of sperm lacking a functional cation channel of sperm (CatSper) channel, which plays an essential role in Ca(2+) influx during sperm capacitation. Ca(2+) imaging analyses demonstrate that Ca(2+) influx is perturbed in Cnnm4-deficient sperm, and forced Ca(2+) entry into these sperm normalizes the level of tyrosine phosphorylation. Furthermore, we confirm the importance of CNNM4 in sperm by generating germ-cell-specific Cnnm4-deficient mice. These results suggest a new role of CNNM4 in sperm Ca(2+) homeostasis. PMID:27006114

  13. Changes of sperm quality and hormone receptors in the rat testis after exposure to methamphetamine.

    PubMed

    Nudmamud-Thanoi, Sutisa; Sueudom, Wanvipa; Tangsrisakda, Nareelak; Thanoi, Samur

    2016-10-01

    Methamphetamine (METH) is known to damage neurons and induce psychosis. It can also induce apoptosis in seminiferous tubules and affect sperm quality. The present study was carried out to investigate the effect of a rat model of METH addiction on sperm quality and expression of progesterone receptors (PR) and estrogen receptors (ER) in the testis. Sperm quality parameters including sperm motility, sperm morphology and sperm concentration were examined. Protein and gene expressions PR, ERα and ERβ were studied using immunohistochemistry and reverse transcriptase-polymerase chain reaction, respectively. The percentages of normal sperm motility and normal sperm morphology were significantly decreased in animals receiving METH, especially in escalating dose (ED METH) and escalating dose-binge (ED-binge METH) groups when compared with control. In addition, sperm concentrations in ED METH and ED-binge METH groups were numerically decreased. PR, ERα and ERβ immunoreactive cells were significantly decreased in spermatogonia, spermatogenic cells and especially in Sertoli cells in all METH-treated groups. Furthermore, messenger RNA expression of PR, ERα and ERβ were also significantly decreased in all METH-treated animals. These results indicate that METH can induce abnormal sperm quality. These changes of sperm quality may relate to the reduction of PR, ERα and ERβ expressions in male germ cells and Sertoli cells which are essential for spermatogenesis and development of sperm. PMID:26864947

  14. Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

    SciTech Connect

    Huser, T; Orme, C; Hollars, C; Corzett, M; Balhorn, R

    2009-03-09

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

  15. Male sperm storage compromises sperm motility in guppies

    PubMed Central

    Gasparini, Clelia; Kelley, Jennifer L.; Evans, Jonathan P.

    2014-01-01

    Sperm senescence can have important evolutionary implications due to its deleterious effects on sperm quality and offspring performance. Consequently, it has been argued that polyandry (female multiple mating) may facilitate the selection of younger, and therefore competitively superior, sperm when ejaculates from multiple males compete for fertilization. Surprisingly, however, unequivocal evidence that sperm ageing influences traits that underlie sperm competitiveness is lacking. Here, we used a paired experimental design that compares sperm quality between ‘old’ and ‘young’ ejaculates from individual male guppies (Poecilia reticulata). We show that older sperm exhibit significant reductions in sperm velocity compared with younger sperm from the same males. We found no evidence that the brightness of the male's orange (carotenoid) spots, which are thought to signal resistance to oxidative stress (and thus age-related declines in sperm fitness), signals a male's ability to withstand the deleterious effects of sperm ageing. Instead, polyandry may be a more effective strategy for females to minimize the likelihood of being fertilized by aged sperm. PMID:25392314

  16. Male sperm storage compromises sperm motility in guppies.

    PubMed

    Gasparini, Clelia; Kelley, Jennifer L; Evans, Jonathan P

    2014-11-01

    Sperm senescence can have important evolutionary implications due to its deleterious effects on sperm quality and offspring performance. Consequently, it has been argued that polyandry (female multiple mating) may facilitate the selection of younger, and therefore competitively superior, sperm when ejaculates from multiple males compete for fertilization. Surprisingly, however, unequivocal evidence that sperm ageing influences traits that underlie sperm competitiveness is lacking. Here, we used a paired experimental design that compares sperm quality between 'old' and 'young' ejaculates from individual male guppies (Poecilia reticulata). We show that older sperm exhibit significant reductions in sperm velocity compared with younger sperm from the same males. We found no evidence that the brightness of the male's orange (carotenoid) spots, which are thought to signal resistance to oxidative stress (and thus age-related declines in sperm fitness), signals a male's ability to withstand the deleterious effects of sperm ageing. Instead, polyandry may be a more effective strategy for females to minimize the likelihood of being fertilized by aged sperm. PMID:25392314

  17. Maintenance of Sperm Variation in a Highly Promiscuous Wild Bird

    PubMed Central

    Calhim, Sara; Double, Michael C.; Margraf, Nicolas; Birkhead, Tim R.; Cockburn, Andrew

    2011-01-01

    Postcopulatory sexual selection is an important force in the evolution of reproductive traits, including sperm morphology. In birds, sperm morphology is known to be highly heritable and largely condition-independent. Theory predicts, and recent comparative work corroborates, that strong selection in such traits reduces intraspecific phenotypic variation. Here we show that some variation can be maintained despite extreme promiscuity, as a result of opposing, copulation-role-specific selection forces. After controlling for known correlates of siring success in the superb fairy-wren (Malurus cyaneus), we found that (a) lifetime extra-pair paternity success was associated with sperm with a shorter flagellum and relatively large head, and (b) males whose sperm had a longer flagellum and a relatively smaller head achieved higher within-pair paternity. In this species extrapair copulations occur in the same morning, but preceding, pair copulations during a female's fertile period, suggesting that shorter and relatively larger-headed sperm are most successful in securing storage (defense), whereas the opposite phenotype might be better at outcompeting stored sperm (offense). Furthermore, since cuckolding ability is a major contributor to differential male reproductive output, stronger selection on defense sperm competition traits might explain the short sperm of malurids relative to other promiscuous passerines. PMID:22194918

  18. Silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome.

    PubMed

    Schmidt, Monica A; Barbazuk, W Brad; Sandford, Michael; May, Greg; Song, Zhihong; Zhou, Wenxu; Nikolau, Basil J; Herman, Eliot M

    2011-05-01

    The ontogeny of seed structure and the accumulation of seed storage substances is the result of a determinant genetic program. Using RNA interference, the synthesis of soybean (Glycine max) glycinin and conglycinin storage proteins has been suppressed. The storage protein knockdown (SP-) seeds are overtly identical to the wild type, maturing to similar size and weight, and in developmental ontogeny. The SP- seeds rebalance the proteome, maintaining wild-type levels of protein and storage triglycerides. The SP- soybeans were evaluated with systems biology techniques of proteomics, metabolomics, and transcriptomics using both microarray and next-generation sequencing transcript sequencing (RNA-Seq). Proteomic analysis shows that rebalancing of protein content largely results from the selective increase in the accumulation of only a few proteins. The rebalancing of protein composition occurs with small alterations to the seed's transcriptome and metabolome. The selectivity of the rebalancing was further tested by introgressing into the SP- line a green fluorescent protein (GFP) glycinin allele mimic and quantifying the resulting accumulation of GFP. The GFP accumulation was similar to the parental GFP-expressing line, showing that the GFP glycinin gene mimic does not participate in proteome rebalancing. The results show that soybeans make large adjustments to the proteome during seed filling and compensate for the shortage of major proteins with the increased selective accumulation of other proteins that maintains a normal protein content. PMID:21398260

  19. Identification of PDC-109-like protein(s) in buffalo seminal plasma.

    PubMed

    Harshan, Hiron M; Sankar, Surya; Singh, L P; Singh, Manish Kumar; Sudharani, S; Ansari, M R; Singh, S K; Majumdar, A C; Joshi, P

    2009-10-01

    The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma. PMID:19117702

  20. Sperm Motility in Flow

    NASA Astrophysics Data System (ADS)

    Guasto, Jeffrey; Juarez, Gabriel; Stocker, Roman

    2012-11-01

    A wide variety of plants and animals reproduce sexually by releasing motile sperm that seek out a conspecific egg, for example in the reproductive tract for mammals or in the water column for externally fertilizing organisms. Sperm are aided in their quest by chemical cues, but must also contend with hydrodynamic forces, resulting from laminar flows in reproductive tracts or turbulence in aquatic habitats. To understand how velocity gradients affect motility, we subjected swimming sperm to a range of highly-controlled straining flows using a cross-flow microfluidic device. The motion of the cell body and flagellum were captured through high-speed video microscopy. The effects of flow on swimming are twofold. For moderate velocity gradients, flow simply advects and reorients cells, quenching their ability to cross streamlines. For high velocity gradients, fluid stresses hinder the internal bending of the flagellum, directly inhibiting motility. The transition between the two regimes is governed by the Sperm number, which compares the external viscous stresses with the internal elastic stresses. Ultimately, unraveling the role of flow in sperm motility will lead to a better understanding of population dynamics among aquatic organisms and infertility problems in humans.

  1. Insight into the Assembly of Viruses with Vertical Single β-barrel Major Capsid Proteins.

    PubMed

    Gil-Carton, David; Jaakkola, Salla T; Charro, Diego; Peralta, Bibiana; Castaño-Díez, Daniel; Oksanen, Hanna M; Bamford, Dennis H; Abrescia, Nicola G A

    2015-10-01

    Archaeal viruses constitute the least explored niche within the virosphere. Structure-based approaches have revealed close relationships between viruses infecting organisms from different domains of life. Here, using biochemical and cryo-electron microscopy techniques, we solved the structure of euryarchaeal, halophilic, internal membrane-containing Haloarcula hispanica icosahedral virus 2 (HHIV-2). We show that the density of the two major capsid proteins (MCPs) recapitulates vertical single β-barrel proteins and that disulfide bridges stabilize the capsid. Below, ordered density is visible close to the membrane and at the five-fold vertices underneath the host-interacting vertex complex underpinning membrane-protein interactions. The HHIV-2 structure exemplifies the division of conserved architectural elements of a virion, such as the capsid, from those that evolve rapidly due to selective environmental pressure such as host-recognizing structures. We propose that in viruses with two vertical single β-barrel MCPs the vesicle is indispensable, and membrane-protein interactions serve as protein-railings for guiding the assembly. PMID:26320579

  2. Quinone-induced protein handling changes: Implications for major protein handling systems in quinone-mediated toxicity

    SciTech Connect

    Xiong, Rui; Siegel, David; Ross, David

    2014-10-15

    Para-quinones such as 1,4-Benzoquinone (BQ) and menadione (MD) and ortho-quinones including the oxidation products of catecholamines, are derived from xenobiotics as well as endogenous molecules. The effects of quinones on major protein handling systems in cells; the 20/26S proteasome, the ER stress response, autophagy, chaperone proteins and aggresome formation, have not been investigated in a systematic manner. Both BQ and aminochrome (AC) inhibited proteasomal activity and activated the ER stress response and autophagy in rat dopaminergic N27 cells. AC also induced aggresome formation while MD had little effect on any protein handling systems in N27 cells. The effect of NQO1 on quinone induced protein handling changes and toxicity was examined using N27 cells stably transfected with NQO1 to generate an isogenic NQO1-overexpressing line. NQO1 protected against BQ–induced apoptosis but led to a potentiation of AC- and MD-induced apoptosis. Modulation of quinone-induced apoptosis in N27 and NQO1-overexpressing cells correlated only with changes in the ER stress response and not with changes in other protein handling systems. These data suggested that NQO1 modulated the ER stress response to potentiate toxicity of AC and MD, but protected against BQ toxicity. We further demonstrated that NQO1 mediated reduction to unstable hydroquinones and subsequent redox cycling was important for the activation of the ER stress response and toxicity for both AC and MD. In summary, our data demonstrate that quinone-specific changes in protein handling are evident in N27 cells and the induction of the ER stress response is associated with quinone-mediated toxicity. - Highlights: • Unstable hydroquinones contributed to quinone-induced ER stress and toxicity.

  3. Protein structural and surface water rearrangement constitute major events in the earliest aggregation stages of tau

    PubMed Central

    Pavlova, Anna; Cheng, Chi-Yuan; Kinnebrew, Maia; Lew, John; Dahlquist, Frederick W.; Han, Songi

    2016-01-01

    Protein aggregation plays a critical role in the pathogenesis of neurodegenerative diseases, and the mechanism of its progression is poorly understood. Here, we examine the structural and dynamic characteristics of transiently evolving protein aggregates under ambient conditions by directly probing protein surface water diffusivity, local protein segment dynamics, and interprotein packing as a function of aggregation time, along the third repeat domain and C terminus of Δtau187 spanning residues 255–441 of the longest isoform of human tau. These measurements were achieved with a set of highly sensitive magnetic resonance tools that rely on site-specific electron spin labeling of Δtau187. Within minutes of initiated aggregation, the majority of Δtau187 that is initially homogeneously hydrated undergoes structural transformations to form partially structured aggregation intermediates. This is reflected in the dispersion of surface water dynamics that is distinct around the third repeat domain, found to be embedded in an intertau interface, from that of the solvent-exposed C terminus. Over the course of hours and in a rate-limiting process, a majority of these aggregation intermediates proceed to convert into stable β-sheet structured species and maintain their stacking order without exchanging their subunits. The population of β-sheet structured species is >5% within 5 min of aggregation and gradually grows to 50–70% within the early stages of fibril formation, while they mostly anneal block-wisely to form elongated fibrils. Our findings suggest that the formation of dynamic aggregation intermediates constitutes a major event occurring in the earliest stages of tau aggregation that precedes, and likely facilitates, fibril formation and growth. PMID:26712030

  4. Villin: The major microfilament-associated protein of the intestinal microvillus

    PubMed Central

    Bretscher, Anthony; Weber, Klaus

    1979-01-01

    The major protein associated with actin in the microfilament core of intestinal microvilli has been purified. This protein, for which we propose the name villin, has a polypeptide molecular weight of approximately 95,000. Two arguments suggest that villin may be the microvillus crossfilament protein that links the microfilament core laterally down its length to the cytoplasmic side of the plasma membrane. First, electron microscopy shows that crossfilaments stay attached to isolated membrane-free microvillus cores. Calculation of the expected abundance of the crossfilament protein shows that only villin is present in sufficient quantity to account for these structures. Second, decoration of microvillus cores by antibodies to either actin or villin, followed by ferritin-labeled second antibody in a sandwich procedure, results in specific labeling of the cores in both cases. The antivillin decoration, however, gives rise to a greater increase in diameter, in agreement with a model in which villin projects from the F-actin microfilament core. Villin is distinct from α-actinin, a protein suggested to be involved in membrane anchorage of microfilaments in nonmuscle cells. The two proteins differ in molecular weight. Specific antibodies against villin and α-actinin show no immunological crossreactivity. Immunofluorescence microscopy reveals that villin is located in the microvilli of the brush border whereas α-actinin is absent from the microvilli but is found in the terminal web. In addition, villin is not found in microfilament bundles of tissue culture cells, which are rich in α-actinin. Thus, villin and α-actinin appear to be immunologically and functionally different proteins. Images PMID:287075

  5. Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling.

    PubMed

    Kim, Euikyung; Lee, Seunghwan; Mian, Md Firoz; Yun, Sang Uk; Song, Minseok; Yi, Kye-Sook; Ryu, Sung Ho; Suh, Pann-Ghill

    2006-02-01

    Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST-Src-SH2 fusion proteins. We found MVP as a Src-SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner. MVP overexpression downregulates EGF-dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src-mediated signalling cascades. PMID:16441665

  6. A putative acyl-CoA-binding protein is a major phloem sap protein in rice (Oryza sativa L.).

    PubMed

    Suzui, Nobuo; Nakamura, Shin-ichi; Fujiwara, Toru; Hayashi, Hiroaki; Yoneyama, Tadakatsu

    2006-01-01

    The N-terminal amino-acid sequence of a major rice phloem-sap protein, named RPP10, was determined. RPP10 is encoded by a single gene in the rice genome. Its complete amino-acid sequence, predicted from the corresponding rice full-length cDNA, showed high similarity to plant acyl-CoA-binding proteins (ACBPs). Western blot analysis using anti-ACBP antiserum revealed that putative ACBP is abundant in the phloem sap of rice plants, and is also present in sieve-tube exudates of winter squash (Cucurbita maxima), oilseed rape (Brassica napus), and coconut palm (Cocos nucifera). These findings give rise to the idea that ACBP may involve lipid metabolism and regulation in the phloem. PMID:16804052

  7. Construction and analysis of the protein-protein interaction networks for schizophrenia, bipolar disorder, and major depression

    PubMed Central

    2011-01-01

    Background Schizophrenia, bipolar disorder, and major depression are devastating mental diseases, each with distinctive yet overlapping epidemiologic characteristics. Microarray and proteomics data have revealed genes which expressed abnormally in patients. Several single nucleotide polymorphisms (SNPs) and mutations are associated with one or more of the three diseases. Nevertheless, there are few studies on the interactions among the disease-associated genes and proteins. Results This study, for the first time, incorporated microarray and protein-protein interaction (PPI) databases to construct the PPI network of abnormally expressed genes in postmortem brain samples of schizophrenia, bipolar disorder, and major depression patients. The samples were collected from Brodmann area (BA) 10 of the prefrontal cortex. Abnormally expressed disease genes were selected by t-tests comparing the disease and control samples. These genes were involved in housekeeping functions (e.g. translation, transcription, energy conversion, and metabolism), in brain specific functions (e.g. signal transduction, neuron cell differentiation, and cytoskeleton), or in stress responses (e.g. heat shocks and biotic stress). The diseases were interconnected through several “switchboard”-like nodes in the PPI network or shared abnormally expressed genes. A “core” functional module which consisted of a tightly knitted sub-network of clique-5 and -4s was also observed. These cliques were formed by 12 genes highly expressed in both disease and control samples. Conclusions Several previously unidentified disease marker genes and drug targets, such as SBNO2 (schizophrenia), SEC24C (bipolar disorder), and SRRT (major depression), were identified based on statistical and topological analyses of the PPI network. The shared or interconnecting marker genes may explain the shared symptoms of the studied diseases. Furthermore, the “switchboard” genes, such as APP, UBC, and YWHAZ, are proposed as

  8. Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins

    PubMed Central

    Madeira da Silva, Luciana; Owens, Katherine L.; Murta, Silvane M. F.; Beverley, Stephen M.

    2009-01-01

    Surface glycoconjugates play important roles in the infectious cycle of Leishmania major, including the abundant lipophosphoglycan (LPG) implicated in parasite survival in the sand fly vector and the initial stages of establishment in the mammalian host macrophage. We describe a system for inducible expression of LPG, applying a novel protein-based system that allows controlled degradation of a key LPG biosynthetic enzyme, UDP-galactopyranose mutase (UGM). This methodology relies on a mutated FK506-binding protein (FKBP) destabilizing domain (dd) fused to the protein of interest; in the absence of rapamycin analogs, such as Shld1, the dd domain is destabilized, leading to proteasomal degradation, whereas drug treatment confers stabilization. Tests in L. major using dd fusions to a panel of reporters and cellular proteins confirmed its functionality, with a high degree of regulation and low background, and we established the kinetics of protein activation and/or loss. Two inexpensive and widely available ligands, FK506 and rapamycin, functioned similarly to Shld1, without effect on Leishmania growth or differentiation. We generated parasites lacking UGM through deletion of the GLF gene and substitution with a ddGLF fusion construct, either as chromosomal knockins or through episomal complementation; these showed little or no LPG expression in the absence of inducer, whereas in its presence, high levels of LPG were attained rapidly. Complement lysis tests confirmed the correct integrity of the Leishmania LPG coat. These data suggest that the dd approach has great promise in the study of LPG and other pathways relevant to parasite survival and virulence. PMID:19383793

  9. A double role of sperm in scorpions: the mating plug of Euscorpius italicus (Scorpiones: Euscorpiidae) consists of sperm.

    PubMed

    Althaus, Sarah; Jacob, Alain; Graber, Werner; Hofer, Deborah; Nentwig, Wolfgang; Kropf, Christian

    2010-04-01

    Mating plugs occluding the female gonopore after mating are a widespread phenomenon. In scorpions, two main types of mating plugs are found: sclerotized mating plugs being parts of the spermatophore that break off during mating, and gel-like mating plugs being gelatinous fluids that harden in the female genital tract. In this study, the gel-like mating plug of Euscorpius italicus was investigated with respect to its composition, fine structure, and changes over time. Sperm forms the major component of the mating plug, a phenomenon previously unknown in arachnids. Three parts of the mating plug can be distinguished. The part facing the outside of the female (outer part) contains sperm packages containing inactive spermatozoa. In this state, sperm is transferred. In the median part, the sperm packages get uncoiled to single spermatozoa. In the inner part, free sperm is embedded in a large amount of secretions. Fresh mating plugs are soft gelatinous, later they harden from outside toward inside. This process is completed after 3-5 days. Sperm from artificially triggered spermatophores could be activated by immersion in insect Ringer's solution indicating that the fluid condition in the females' genital tract or females' secretions causes sperm activation. Because of the male origin of the mating plug, it has likely evolved under sperm competition or sexual conflict. As females refused to remate irrespective of the presence or absence of a mating plug, females may have changed their mating behavior in the course of evolution from polyandry to monandry. PMID:20101728

  10. Short-term variation in sperm competition causes sperm-mediated epigenetic effects on early offspring performance in the zebrafish

    PubMed Central

    Zajitschek, Susanne; Hotzy, Cosima; Zajitschek, Felix; Immler, Simone

    2014-01-01

    The inheritance of non-genetic factors is increasingly seen to play a major role in ecology and evolution. While the causes and consequences of epigenetic effects transmitted from the mother to the offspring have received ample attention, much less is known about how variation in the condition of the father affects the offspring. Here, we manipulated the intensity of sperm competition experienced by male zebrafish Danio rerio to investigate the potential for sperm-mediated epigenetic effects over a relatively short period of time. We found that the rapid responses of males to varying intensity of sperm competition not only affected sperm traits as shown previously, but also the performance of the resulting offspring. We observed that males exposed to high intensity of sperm competition produced faster swimming and more motile sperm, and sired offspring that hatched over a narrower time frame but exhibited a lower survival rate than males exposed to low intensity of sperm competition. Our results provide striking evidence for short-term paternal effects and the possible fitness consequences of such sperm-mediated non-genetic factors not only for the resulting offspring but also for the female. PMID:24789902

  11. Human sperm chromatin epigenetic potential: genomics, proteomics, and male infertility

    PubMed Central

    Castillo, Judit; Estanyol, Josep Maria; Ballescà, Josep Lluis; Oliva, Rafael

    2015-01-01

    The classical idea about the function of the mammalian sperm chromatin is that it serves to transmit a highly protected and transcriptionally inactive paternal genome, largely condensed by protamines, to the next generation. In addition, recent sperm chromatin genome-wide dissection studies indicate the presence of a differential distribution of the genes and repetitive sequences in the protamine-condensed and histone-condensed sperm chromatin domains, which could be potentially involved in regulatory roles after fertilization. Interestingly, recent proteomic studies have shown that sperm chromatin contains many additional proteins, in addition to the abundant histones and protamines, with specific modifications and chromatin affinity features which are also delivered to the oocyte. Both gene and protein signatures seem to be altered in infertile patients and, as such, are consistent with the potential involvement of the sperm chromatin landscape in early embryo development. This present work reviews the available information on the composition of the human sperm chromatin and its epigenetic potential, with a particular focus on recent results derived from high-throughput genomic and proteomic studies. As a complement, we provide experimental evidence for the detection of phosphorylations and acetylations in human protamine 1 using a mass spectrometry approach. The available data indicate that the sperm chromatin is much more complex than what it was previously thought, raising the possibility that it could also serve to transmit crucial paternal epigenetic information to the embryo. PMID:25926607

  12. Oviducal sperm storage in poultry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hens are capable of fertilizing a daily succession of ovulated ova due to their ability to store sperm in the oviduct for several weeks. However, the precise biological mechanisms describing how sperm are selected and survive in the oviduct, and which sperm actually reach the site of fertilization c...

  13. Modest truncation of the major capsid protein abrogates B19 parvovirus capsid formation.

    PubMed Central

    Kawase, M; Momoeda, M; Young, N S; Kajigaya, S

    1995-01-01

    In vitro studies have suggested an important role for the minor capsid protein (VP1) unique region and the junction between VP1 and the major capsid protein (VP2) in the neutralizing immune response to B19 parvovirus. We investigated the role of the NH2-terminal region of the major structural protein in capsid structure by expressing progressively more truncated versions of the VP2 gene followed by analysis using immunoblotting and electron microscopy of density gradient-purified particles. Deletion of the first 25 amino acids (aa) of VP2 did not affect capsid assembly. Altered VP2 with truncations to aa 26 to 30, including a single amino acid deletion at position 25, failed to self-assemble but did participate with normal VP2 in the capsid structure. The altered region corresponds to the beginning of the beta A antiparallel strand. Truncations beyond aa 30 were incompatible with either self-assembly or coassembly, probably because of deletion of the beta B strand, which helps to form the core structure of the virus. PMID:7666560

  14. 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. PMID:25968310

  15. Autophagy and ubiquitin-proteasome system contribute to sperm mitophagy after mammalian fertilization.

    PubMed

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

    2016-09-01

    Maternal inheritance of mitochondria and mtDNA is a universal principle in human and animal development, guided by selective ubiquitin-dependent degradation of the sperm-borne mitochondria after fertilization. However, it is not clear how the 26S proteasome, the ubiquitin-dependent protease that is only capable of degrading one protein molecule at a time, can dispose of a whole sperm mitochondrial sheath. We hypothesized that the canonical ubiquitin-like autophagy receptors [sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the nontraditional mitophagy pathways involving ubiquitin-proteasome system and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP), may act in concert during mammalian sperm mitophagy. We found that the SQSTM1, but not GABARAP or LC3, associated with sperm mitochondria after fertilization in pig and rhesus monkey zygotes. Three sperm mitochondrial proteins copurified with the recombinant, ubiquitin-associated domain of SQSTM1. The accumulation of GABARAP-containing protein aggregates was observed in the vicinity of sperm mitochondrial sheaths in the zygotes and increased in the embryos treated with proteasomal inhibitor MG132, in which intact sperm mitochondrial sheaths were observed. Pharmacological inhibition of VCP significantly delayed the process of sperm mitophagy and completely prevented it when combined with microinjection of autophagy-targeting antibodies specific to SQSTM1 and/or GABARAP. Sperm mitophagy in higher mammals thus relies on a combined action of SQSTM1-dependent autophagy and VCP-mediated dislocation and presentation of ubiquitinated sperm mitochondrial proteins to the 26S proteasome, explaining how the whole sperm mitochondria are degraded inside the fertilized mammalian oocytes by a protein recycling system involved in degradation of single protein molecules. PMID:27551072

  16. Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species.

    PubMed

    Hoehn, G T; Clark, V L

    1990-12-01

    The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus. PMID:2123827

  17. Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species.

    PubMed Central

    Hoehn, G T; Clark, V L

    1990-01-01

    The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus. Images PMID:2123827

  18. The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG

    PubMed Central

    2012-01-01

    Background Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes. Results Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein. Conclusions In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the

  19. Revision of the biodistribution of uranyl in serum: is fetuin-A the major protein target?

    PubMed

    Basset, Christian; Averseng, Olivier; Ferron, Pierre-Jean; Richaud, Nicolas; Hagège, Agnès; Pible, Olivier; Vidaud, Claude

    2013-05-20

    Uranium is a natural actinide present as uranyl U(VI) species in aqueous environments. Its toxicity is considered to be chemical rather than radiotoxicological. Whatever the route of entry, uranyl reaches the blood, is partly eliminated via the kidneys, and accumulated in the bones. In serum, its speciation mainly involves carbonate and proteins. Direct identification of labile uranyl-protein complexes is extremely difficult because of the complexity of this matrix. Thus, until now the biodistribution of the metal in serum has not been described, and therefore, little is known about the metal transport mechanisms leading to bone accumulation. A rapid screening method based on a surface plasmon resonance (SPR) technique was used to determine the apparent affinities for U(VI) of the major serum proteins. A first biodistribution of uranyl was obtained by ranking the proteins according to the criteria of both their serum concentrations and affinities for this metal. Despite its moderate concentration in serum, fetuin-A (FETUA) was shown to exhibit an apparent affinity within the 30 nM range and to carry more than 80% of the metal. This protein involved in bone mineralization aroused interest in characterizing the U(VI) and FETUA interaction. Using complementary chromatographic and spectroscopic approaches, we demonstrated that the protein can bind 3 U(VI) at different binding sites exhibiting Kd from ∼30 nM to 10 μM. Some structural modifications and functional properties of FETUA upon uranyl complexation were also controlled. To our knowledge, this article presents the first identification of a uranyl carrier involved in bone metabolism along with the characterization of its metal binding sites. PMID:23527557

  20. The genomic sequence of the murine major vault protein and its promoter.

    PubMed

    Mossink, Marieke; van Zon, Arend; Fränzel-Luiten, Erna; Schoester, Martijn; Scheffer, George L; Scheper, Rik J; Sonneveld, Pieter; Wiemer, Erik A C

    2002-07-10

    Vaults are ribonucleoproteins of unknown function, consisting of three different proteins and multiple copies of small untranslated RNA molecules. One of the protein subunits has been identified as TEP1, a protein that is also associated with the telomerase complex. Another protein appears to contain a functional PARP domain and is hence called VPARP. The third protein, major vault protein (MVP), is believed to make up 70% of the total mass of the vault complex and to be responsible for the typical barrel-shaped structure of vaults. We have isolated the murine MVP cDNA and compared the amino acid sequence with MVP from other species. Over 90% of sequence identity was found between mouse, human and rat, and a considerable degree of identity between mouse and MVPs from lower eukaryotes. We also found that the genomic structure of the murine MVP gene closely resembles the organization of the human MVP gene, both consisting of 15 exons of which most have exactly the same size. Finally we have isolated a genomic region upstream (and partially overlapping) the first untranslated exon, that displayed promoter activity in a luciferase reporter assay. Furthermore, we showed that the sequences from the first exon together with the 5'-end of the first intron enhance the promoter activity, implying the presence of essential promoter elements in this region. Alignment of the murine promoter region with the homologous sequences of the human gene revealed an identity of 58%. The apparent presence of conserved promoter elements suggests a similar regulation of human and murine MVP expression. PMID:12234684

  1. Parkinsonism-associated protein DJ-1/Park7 is a major protein deglycase that repairs methylglyoxal- and glyoxal-glycated cysteine, arginine, and lysine residues.

    PubMed

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-16

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

  2. Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine, Arginine, and Lysine Residues

    PubMed Central

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-01

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

  3. Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals

    PubMed Central

    Shea, Jeremy M.; Boskovic, Ana; Derr, Alan G.; Bing, Xin Y.; Belleannee, Clemence; Kucukural, Alper; Serra, Ryan W.; Sun, Fengyun; Song, Lina; Carone, Benjamin R.; Ricci, Emiliano P.; Li, Xin Z.; Fauquier, Lucas; Moore, Melissa J.; Sullivan, Robert; Mello, Craig C.; Garber, Manuel; Rando, Oliver J.

    2016-01-01

    Several recent studies link parental environments to phenotypes in subsequent generations. Here, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA levels in mature sperm, with decreased let-7 levels and increased levels of 5’ fragments of glycine tRNAs. tRNA fragments are scarce in testicular sperm, but are gained as sperm mature in the epididymis. Epididymosomes – vesicles that fuse with sperm during epididymal transit – carry RNA payloads matching those of mature sperm, and deliver RNAs to immature sperm in vitro. Functionally, tRNA-Gly-GCC fragments repress genes associated with the endogenous retroelement MERVL, both in ES cells and embryos. Our results shed light on small RNA biogenesis, and its dietary regulation, during post-testicular sperm maturation, and link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo. PMID:26721685

  4. Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.

    PubMed

    Sharma, Upasna; Conine, Colin C; Shea, Jeremy M; Boskovic, Ana; Derr, Alan G; Bing, Xin Y; Belleannee, Clemence; Kucukural, Alper; Serra, Ryan W; Sun, Fengyun; Song, Lina; Carone, Benjamin R; Ricci, Emiliano P; Li, Xin Z; Fauquier, Lucas; Moore, Melissa J; Sullivan, Robert; Mello, Craig C; Garber, Manuel; Rando, Oliver J

    2016-01-22

    Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5' fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo. PMID:26721685

  5. A factor in human seminal plasma which affects carnitine accumulation in bovine epididymal sperm.

    PubMed

    Carter, A L; Cho, S H; Bishop, E R; Boldt, J

    1988-05-01

    This study was initiated to determine whether factors are present in human sperm-free seminal plasma (HSP) that regulate the uptake and release of carnitine from sperm. Bovine caput epididymal sperm cells accumulated more carnitine than caudal sperm cells. A significant reduction in carnitine uptake by caput sperm was observed in the presence of HSP from normal subjects, but not from three subjects with reduced motility. A factor has been isolated from HSP that inhibits carnitine uptake by caput sperm and has the following properties: it is nondialyzable, stable to freeze-thawing, soluble in 60% ammonium sulfate, and has an approximate molecular weight of 158 kd. These data are consistent with the existence of a relatively high molecular weight protein in HSP responsible for the preservation of carnitine concentrations in sperm. PMID:3360180

  6. Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications*

    PubMed Central

    Halim, Adnan; Carlsson, Michael C.; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y.; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L.; Wandall, Hans H.

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM1 characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines. PMID:25389185

  7. Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE

    PubMed Central

    Wisedchaisri, Goragot; Park, Min-Sun; Iadanza, Matthew G.; Zheng, Hongjin; Gonen, Tamir

    2014-01-01

    The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins. PMID:25088546

  8. Pneumococcal Surface Protein A Plays a Major Role in Streptococcus pneumoniae-Induced Immunosuppression.

    PubMed

    Saumyaa; Pujanauski, Lindsey; Colino, Jesus; Flora, Michael; Torres, Raul M; Tuomanen, Elaine; Snapper, Clifford M

    2016-05-01

    Intact, inactivated Streptococcus pneumoniae [including the unencapsulated S. pneumoniae, serotype 2 strain (R36A)] markedly inhibits the humoral immune response to coimmunized heterologous proteins, a property not observed with several other intact Gram-positive or Gram-negative bacteria. In this study, we determined the nature of this immunosuppressive property. Because phosphorylcholine (PC), a major haptenic component of teichoic acid in the S. pneumoniae cell wall, and lipoteichoic acid in the S. pneumoniae membrane were previously reported to be immunosuppressive when derived from filarial parasites, we determined whether R36A lacking PC (R36A(pc-)) was inhibitory. Indeed, although R36A(pc-) exhibited a markedly reduced level of inhibition of the IgG response to coimmunized chicken OVA (cOVA), no inhibition was observed when using several other distinct PC-expressing bacteria or a soluble, protein-PC conjugate. Further, treatment of R36A with periodate, which selectively destroys PC residues, had no effect on R36A-mediated inhibition. Because R36A(pc-) also lacks choline-binding proteins (CBPs) that require PC for cell wall attachment, and because treatment of R36A with trypsin eliminated its inhibitory activity, we incubated R36A in choline chloride, which selectively strips CBPs from its surface. R36A lacking CBPs lost most of its inhibitory property, whereas the supernatant of choline chloride-treated R36A, containing CBPs, was markedly inhibitory. Coimmunization studies using cOVA and various S. pneumoniae mutants, each genetically deficient in one of the CBPs, demonstrated that only S. pneumoniae lacking the CBP pneumococcal surface protein A lost its ability to inhibit the IgG anti-cOVA response. These results strongly suggest that PspA plays a major role in mediating the immunosuppressive property of S. pneumoniae. PMID:27029587

  9. Atrazine binds to F1F0-ATP synthase and inhibits mitochondrial function in sperm.

    PubMed

    Hase, Yasuyoshi; Tatsuno, Michiko; Nishi, Takeyuki; Kataoka, Kosuke; Kabe, Yasuaki; Yamaguchi, Yuki; Ozawa, Nobuaki; Natori, Michiya; Handa, Hiroshi; Watanabe, Hajime

    2008-02-01

    Atrazine is a widely used triazine herbicide. Although controversy still exists, a number of recent studies have described its adverse effects on various animals including humans. Of particular interest is its effects on reproductive capacity. In this study, we investigated the mechanisms underlying the adverse effects of atrazine, with a focus on its effects on sperm. Here we show evidence that mitochondrial F(1)F(0)-ATP synthase is a molecular target of atrazine. A series of experiments with sperm and isolated mitochondria suggest that atrazine inhibits mitochondrial function through F(1)F(0)-ATP synthase. Moreover, affinity purification using atrazine as a ligand demonstrates that F(1)F(0)-ATP synthase is a major atrazine-binding protein in cells. The inhibitory activity against mitochondria and F(1)F(0)-ATP synthase is not limited to atrazine but is likely to be applicable to other triazine-based compounds. Thus, our findings may have wide relevance to pharmacology and toxicology. PMID:18060860

  10. Major nonhistone proteins of rat liver chromatin: preliminary identification of myosin, actin, tubulin, and tropomyosin.

    PubMed Central

    Douvas, A S; Harrington, C A; Bonner, J

    1975-01-01

    Two major nonhistone polypeptides from rat liver chromatin have been identified as myosin and actin. Preliminary observations indicate that three other chromatin polypeptides of molecular weights 50,000, 34,000, and 32,000 are tubulin and heavy and light tropomyosin, respectively. A sixth component of molecular weight 65,000 which has been purified and electrophoreses as a single band on sodium dodecyl sulfate-polyacrylamide gels may be composed in part of protease-digested myosin. These six polypeptides together account for as much as 38% of the nonhistone protein mass of chromatin in this tissue. Images PMID:1060072

  11. Effect of sperm cryopreservation on the European eel sperm viability and spermatozoa morphology.

    PubMed

    Asturiano, J F; Marco-Jiménez, F; Peñaranda, D S; Garzón, D L; Pérez, L; Vicente, J S; Jover, M

    2007-04-01

    The main objective of the present work was to study the effect of cryopreservation of European eel sperm both on the sperm viability and the spermatozoa head morphology. Spermatozoa morphology was evaluated with computer-assisted morphology analysis after collection in fresh samples, after adding the freezing medium containing dimethyl sulfoxide as cryoprotectant and, finally, after the cryopreservation process and thawing. Cell viability was assessed, in both fresh and thawed samples, by Hoechst 33258 staining. Computer-assisted sperm analysis (CASA) was used to determine the percentage of motile cells and to measure motility parameters in sperm samples. A significant decrease of head perimeter (12.56%) and area (17.90%) was detected from spermatozoa in fresh to thawed samples, indicating that cells do not recover the original size after the cryopreservation process. CASA was used to measure the percentage of motile cells (51.9%) and spermatozoa motility parameters such as curvilinear, straight line and angular path velocities, as well as beating cross frequency. This technique was employed in the fresh sperm samples but proteins present at the freezing medium (L-alpha-phosphatidylcholine) made impossible to use this last technique in thawed samples. When sperm viability was assessed by Hoechst staining, a significant decrease of approximately 15% (73.10 vs 58.26%) of alive spermatozoa was registered from fresh to thawed samples. The percentage of motile cells measured by CASA in fresh samples (51.9%) was lower than the percentage of alive cells determined by Hoechst stainning, suggesting the existence of different batches of spermatozoa in different stages of development, even during the eight to tenth weeks of treatment, when the highest sperm quality was found. PMID:17348973

  12. Polyandry in the medfly - shifts in paternity mediated by sperm stratification and mixing

    PubMed Central

    2014-01-01

    Background In the Mediterranean fruit fly (medfly), Ceratitis capitata, a highly invasive agricultural pest species, polyandry, associated with sperm precedence, is a recurrent behaviour in the wild. The absence of tools for the unambiguous discrimination between competing sperm from different males in the complex female reproductive tract has strongly limited the understanding of mechanisms controlling sperm dynamics and use. Results Here we use transgenic medfly lines expressing green or red fluorescent proteins in the spermatozoa, which can be easily observed and unambiguously differentiated within the female fertilization chamber. In twice-mated females, one day after the second mating, sperm from the first male appeared to be homogenously distributed all over the distal portion of each alveolus within the fertilization chamber, whereas sperm from the second male were clearly concentrated in the central portion of each alveolus. This distinct stratified sperm distribution was not maintained over time, as green and red sperm appeared homogeneously mixed seven days after the second mating. This dynamic sperm storage pattern is mirrored by the paternal contribution in the progeny of twice-mated females. Conclusions Polyandrous medfly females, unlike Drosophila, conserve sperm from two different mates to fertilize their eggs. From an evolutionary point of view, the storage of sperm in a stratified pattern by medfly females may initially favour the fresher ejaculate from the second male. However, as the second male's sperm gradually becomes depleted, the sperm from the first male becomes increasingly available for fertilization. The accumulation of sperm from different males will increase the overall genetic variability of the offspring and will ultimately affect the effective population size. From an applicative point of view, the dynamics of sperm storage and their temporal use by a polyandrous female may have an impact on the Sterile Insect Technique (SIT

  13. ZP2 peptide beads select human sperm in vitro, decoy mouse sperm in vivo, and provide reversible contraception.

    PubMed

    Avella, Matteo A; Baibakov, Boris A; Jimenez-Movilla, Maria; Sadusky, Anna Burkart; Dean, Jurrien

    2016-04-27

    Gamete recognition in the female reproductive tract occurs at the surface of the zona pellucida surrounding ovulated eggs. The acellular zona matrix is composed of three (mouse) or four (human) proteins (ZP1 to ZP4), and the amino terminus of ZP2 is the primary sperm-binding ligand. Mouse and human sperm bind, respectively, to recombinant moZP2(35-149) and huZP2(39-154) peptides attached to agarose beads. Mouse ZP2 peptide beads markedly inhibited fertilization of ovulated mouse eggs inseminated in vitro and incubated overnight. Similarly, human ZP2 peptide beads prevented sperm binding and penetration of transgenic ZP2(Rescue) zonae pellucidae, in which human ZP2 replaced mouse ZP2. When mouse ZP2 peptide beads were transcervically deposited into the uterus, there was no change in mating behavior and copulatory plugs were present, but bound sperm did not progress into the oviduct and female mice were infertile. On average, contraception lasted >10 estrus cycles but was reversible with no detectable pathology in the reproductive tract. Despite the long-term contraceptive effect, initial sperm binding to the peptide beads was reversible in vitro. We exploited this observation to select human sperm that were better able to penetrate the zonae of human ZP2(Rescue) eggs, and the approach holds promise for identifying superior sperm for human assisted reproductive technologies (ART). We conclude that the amino-terminal ZP2 peptide supports sperm binding, which is initially reversible but, with time, becomes irreversible. Short-term, reversible binding may be useful in selecting sperm for ART, and long-term binding decoys sperm and results in effective contraception in mice. PMID:27122613

  14. Exploring the Cytoskeleton During Intracytoplasmic Sperm Injection in Humans

    NASA Astrophysics Data System (ADS)

    Rawe, Vanesa Y.; Chemes, Héctor

    Understanding the cellular events during fertilization in mammals is a major challenge that can contribute to the improvement of future infertility treatments in humans and reproductive performance in farm animals. Of special interest is the role of the oocyte and sperm cytoskeleton during the initial interaction between gametes. The aim of this chapter is to describe methods for studying cytoskeletal features during in vitro fertilization after intracytoplasmic sperm injection (ICSI) in humans. The following protocols will provide a detailed description of how to perform immunodetection and imaging of human eggs, zygotes, and sperm by fluorescence (confocal and epifluorescence) and electron microscopy.

  15. Purification and characterization of protein H, the major porin of Pasteurella multocida.

    PubMed Central

    Chevalier, G; Duclohier, H; Thomas, D; Shechter, E; Wróblewski, H

    1993-01-01

    Protein H (B. Lugtenberg, R. van Boxtel, D. Evenberg, M. de Jong, P. Storm, and J. Frik, Infect. Immun. 52:175-182, 1986) is the major polypeptide of the outer membrane of Pasteurella multocida, a bacterium pathogenic for humans and animals. We have purified this protein to homogeneity by size exclusion chromatography after selective extraction with surfactants and demonstrated its pore-forming ability after reincorporation into planar lipid bilayers. In these experiments, the current through the pores was a linear function of the applied voltage in the range of -50 to +50 mV. Voltages beyond +/- 50 mV tended to partially close the channels, giving rise to apparent negative resistances. These observations suggest that protein H channels are probably not voltage regulated in vivo. With the patch clamp technique, single-channel conductance fluctuations of 0.33 nS were recorded in 1 M KCl. Electrophoretic and circular dichroism analyses showed that protein H forms homotrimers stable in sodium dodecyl sulfate at room temperature, with a high content of beta-sheet secondary structure. Upon boiling, the trimers were fully dissociated into monomers with an increase of alpha helix and irregular structure, at the expense of beta sheets. The apparent molecular mass of fully denatured monomers ranged between 37 and 41.8 kDa, depending on the electrophoretic system used for analysis. The trimeric arrangement of protein H was confirmed by image analysis of negatively stained, two-dimensional crystal arrays. This morphological study revealed, in agreement with electrophoretical data, a trimeric structure with an overall diameter of 7.7 nm. Each monomer appeared to contain a pore with an average diameter of 1 nm. Quantitative comparisons revealed that the amino acid composition (hydropathy index of -0.40) and the N-terminal sequence (determined over 36 residues) of protein H are similar to those of bacterial general porins, notably porin P2 of Haemophilus influenzae. We conclude

  16. Structure and Evolution of Insect Sperm: New Interpretations in the Age of Phylogenomics.

    PubMed

    Dallai, Romano; Gottardo, Marco; Beutel, Rolf Georg

    2016-01-01

    This comprehensive review of the structure of sperm in all orders of insects evaluates phylogenetic implications, with the background of a phylogeny based on transcriptomes. Sperm characters strongly support several major branches of the phylogeny of insects-for instance, Cercophora, Dicondylia, and Psocodea-and also different infraordinal groups. Some closely related taxa, such as Trichoptera and Lepidoptera (Amphiesmenoptera), differ greatly in sperm structure. Sperm characters are very conservative in some groups (Heteroptera, Odonata) but highly variable in others, including Zoraptera, a small and morphologically uniform group with a tremendously accelerated rate of sperm evolution. Unusual patterns such as sperm dimorphism, the formation of bundles, or aflagellate and immotile sperm have evolved independently in several groups. PMID:26982436

  17. Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

    PubMed

    Daum, G; Schmid, B; MacKintosh, C; Cohen, P; Hofer, H W

    1992-07-13

    In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a. PMID:1321672

  18. A major protein precursor of zebra mussel (Dreissena polymorpha) byssus: deduced sequence and significance.

    PubMed

    Anderson, K E; Waite, J H

    1998-04-01

    The zebra mussel is a nonindigenous invader of North American lakes and rivers and one of the few freshwater bivalve molluscs having a byssus--a sclerotized organ used by the mussel for opportunistic attachment to hard surfaces. We have sequenced a foot-specific cDNA whose composite protein sequence was deduced from a series of overlapping but occasionally nonidentical cDNA fragments. The overall deduced sequence matches tryptic peptides from a major byssal precursor protein--Dreissena polymorpha foot protein 1 (Dpfp1). The calculated mass of Dpfp1 is 49 kDa; but this is known to be extensively hydroxylated and O-glycosylated during maturation. Purified native Dpfp1 analyzed using matrix-assisted laser-desorption ionization mass spectrometry with time-of-flight indicates that the protein occurs as at least two size variants with masses of 48.6 and 54.5 kDa. In all probability, the sequence variants reported in this study are related to the larger mass variant. Dpfp1 has a block copolymer-like structure defined by two consensus motifs that are sharply segregated into domains. The N-terminal side of Dpfp1 has 22 tandem repeats of a heptapeptide consensus (P-[V/E]-Y-P-[T/S/delta]-[K/Q]-X); the C-terminal side has 16 repeats of a tridecapeptide motif (K-P-G-P-Y-D-Y-D-G-P-Y-D-K). Both consensus repeats are unique, with some limited homology to other proteins functioning in tension: marine mussel adhesives, plant extensins, titin, and trematode eggshell precursors. PMID:9604314

  19. Annotation of Selaginella moellendorffii Major Intrinsic Proteins and the Evolution of the Protein Family in Terrestrial Plants

    PubMed Central

    Anderberg, Hanna I.; Kjellbom, Per; Johanson, Urban

    2012-01-01

    Major intrinsic proteins (MIPs) also called aquaporins form pores in membranes to facilitate the permeation of water and certain small polar solutes across membranes. MIPs are present in virtually every organism but are uniquely abundant in land plants. To elucidate the evolution and function of MIPs in terrestrial plants, the MIPs encoded in the genome of the spikemoss Selaginella moellendorffii were identified and analyzed. In total 19 MIPs were found in S. moellendorffii belonging to 6 of the 7 MIP subfamilies previously identified in the moss Physcomitrella patens. Only three of the MIPs were classified as members of the conserved water specific plasma membrane intrinsic protein (PIP) subfamily whereas almost half were found to belong to the diverse NOD26-like intrinsic protein (NIP) subfamily permeating various solutes. The small number of PIPs in S. moellendorffii is striking compared to all other land plants and no other species has more NIPs than PIPs. Similar to moss, S. moellendorffii only has one type of tonoplast intrinsic protein (TIP). Based on ESTs from non-angiosperms we conclude that the specialized groups of TIPs present in higher plants are not found in primitive vascular plants but evolved later in a common ancestor of seed plants. We also note that the silicic acid permeable NIP2 group that has been reported from angiosperms appears at the same time. We suggest that the expansion of the number MIP isoforms in higher plants is primarily associated with an increase in the different types of specialized tissues rather than the emergence of vascular tissue per se and that the loss of subfamilies has been possible due to a functional overlap between some subfamilies. PMID:22639644

  20. Annotation of Selaginella moellendorffii Major Intrinsic Proteins and the Evolution of the Protein Family in Terrestrial Plants.

    PubMed

    Anderberg, Hanna I; Kjellbom, Per; Johanson, Urban

    2012-01-01

    Major intrinsic proteins (MIPs) also called aquaporins form pores in membranes to facilitate the permeation of water and certain small polar solutes across membranes. MIPs are present in virtually every organism but are uniquely abundant in land plants. To elucidate the evolution and function of MIPs in terrestrial plants, the MIPs encoded in the genome of the spikemoss Selaginella moellendorffii were identified and analyzed. In total 19 MIPs were found in S. moellendorffii belonging to 6 of the 7 MIP subfamilies previously identified in the moss Physcomitrella patens. Only three of the MIPs were classified as members of the conserved water specific plasma membrane intrinsic protein (PIP) subfamily whereas almost half were found to belong to the diverse NOD26-like intrinsic protein (NIP) subfamily permeating various solutes. The small number of PIPs in S. moellendorffii is striking compared to all other land plants and no other species has more NIPs than PIPs. Similar to moss, S. moellendorffii only has one type of tonoplast intrinsic protein (TIP). Based on ESTs from non-angiosperms we conclude that the specialized groups of TIPs present in higher plants are not found in primitive vascular plants but evolved later in a common ancestor of seed plants. We also note that the silicic acid permeable NIP2 group that has been reported from angiosperms appears at the same time. We suggest that the expansion of the number MIP isoforms in higher plants is primarily associated with an increase in the different types of specialized tissues rather than the emergence of vascular tissue per se and that the loss of subfamilies has been possible due to a functional overlap between some subfamilies. PMID:22639644

  1. Intracytoplasmic sperm injection

    MedlinePlus Videos and Cool Tools

    ... in which fertilization occurs outside of the body. First, egg cells are harvested and transferred to a special media in a laboratory dish. Within a few hours, a single sperm is injected through a fine needle into the center of an egg cell to aid in the process of fertilization. If successful, the ...

  2. Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits

    PubMed Central

    Verheyen, Toon; Görnemann, Janina; Verbinnen, Iris; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-01-01

    Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, β and γ isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1β emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits. PMID:25990731

  3. Sialylation Facilitates the Maturation of Mammalian Sperm and Affects Its Survival in Female Uterus.

    PubMed

    Ma, Xue; Pan, Qian; Feng, Ying; Choudhury, Biswa P; Ma, Qianhong; Gagneux, Pascal; Ma, Fang

    2016-06-01

    Establishment of adequate levels of sialylation is crucial for sperm survival and function after insemination; however, the mechanism for the addition of the sperm sialome has not been identified. Here, we report evidence for several different mechanisms that contribute to the establishment of the mature sperm sialome. Directly quantifying the source of the nucleotide sugar CMP-beta-N-acetylneuraminic acid in epididymal fluid indicates that transsialylation occurs in the upper epididymis. Western blots for the low-molecular-mass sialoglycoprotein (around 20-50 kDa) in C57BL/6 mice epididymal fluid reflect that additional sialome could be obtained by glycosylphosphatidylinositol-anchored sialoglycopeptide incorporation during epididymal transit in the caput of the epididymis. Additionally, we found that in Cmah (CMP-N-acetylneuraminic acid hydroxylase)-/- transgenic mice, epididymal sperm obtained sialylated-CD52 from seminal vesicle fluid (SVF). Finally, we used Gfp (green fluorescent protein)+/+ mouse sperm to test the role of sialylation on sperm for protection from female leukocyte attack. There is very low phagocytosis of the epididymal sperm when compared to that of sperm coincubated with SVF. Treating sperm with Arthrobacter ureafaciens sialidase (AUS) increased phagocytosis even further. Our results highlight the different mechanisms of increasing sialylation, which lead to the formation of the mature sperm sialome, as well as reveal the sialome's function in sperm survival within the female genital tract. PMID:27075617

  4. Effects of Synthetic Serum Supplementation in Sperm Preparation Media on Sperm Capacitation and Function Test Results.

    PubMed

    Shih, Ying-Fu; Tzeng, Shu-Ling; Chen, Wen-Jung; Huang, Chun-Chia; Chen, Hsiu-Hui; Lee, Tsung-Hsien; Lee, Maw-Sheng

    2016-01-01

    Albumin supplementation of culture media induces sperm capacitation in assisted reproduction technique cycles. Synthetic serum supplementation is clinically used to replace albumin for preventing transmission of infectious agents. However, the effects of synthetic serum supplementation on sperm capacitation have rarely been investigated. Spermatozoa from 30 men with normal basic semen analysis results were collected, divided into five aliquots, and cultured in capacitating conditions in four combinations of two synthetic serum supplements, serum substitute supplement (SSS) and serum protein substitute (SPS), and two fertilization media, Quinns Advantage™ Fertilization (QF) and human tubular fluid (HTF) media. Reactive oxygen species (ROS) levels in spermatozoa were measured through chemiluminescence. Furthermore, acrosome reaction and western blotting for tyrosine phosphorylation were used to evaluate sperm capacitation. HTF+SSS had significantly higher ROS levels than QF+SPS did (11,725 ± 1,172 versus 6,278 ± 864 relative light units). In addition, the spermatozoa cultured in QF+SPS had lower motility, acrosome reaction rates, and tyrosine phosphorylation levels compared with those cultured in HTF+SSS. In conclusion, the effects of synthetic serum supplementation on sperm capacitation varied according to the combination of media. These differences may lead to variations in spermatozoon ROS levels, thus affecting sperm function test results. PMID:27413417

  5. CRYPTIC CHOICE OF CONSPECIFIC SPERM CONTROLLED BY THE IMPACT OF OVARIAN FLUID ON SPERM SWIMMING BEHAVIOR

    PubMed Central

    Yeates, Sarah E; Diamond, Sian E; Einum, Sigurd; Emerson, Brent C; Holt, William V; Gage, Matthew J G

    2013-01-01

    Despite evidence that variation in male–female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species’ ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species’ identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein-rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior. PMID:24299405

  6. Effects of Synthetic Serum Supplementation in Sperm Preparation Media on Sperm Capacitation and Function Test Results

    PubMed Central

    Shih, Ying-Fu; Tzeng, Shu-Ling; Huang, Chun-Chia

    2016-01-01

    Albumin supplementation of culture media induces sperm capacitation in assisted reproduction technique cycles. Synthetic serum supplementation is clinically used to replace albumin for preventing transmission of infectious agents. However, the effects of synthetic serum supplementation on sperm capacitation have rarely been investigated. Spermatozoa from 30 men with normal basic semen analysis results were collected, divided into five aliquots, and cultured in capacitating conditions in four combinations of two synthetic serum supplements, serum substitute supplement (SSS) and serum protein substitute (SPS), and two fertilization media, Quinns Advantage™ Fertilization (QF) and human tubular fluid (HTF) media. Reactive oxygen species (ROS) levels in spermatozoa were measured through chemiluminescence. Furthermore, acrosome reaction and western blotting for tyrosine phosphorylation were used to evaluate sperm capacitation. HTF+SSS had significantly higher ROS levels than QF+SPS did (11,725 ± 1,172 versus 6,278 ± 864 relative light units). In addition, the spermatozoa cultured in QF+SPS had lower motility, acrosome reaction rates, and tyrosine phosphorylation levels compared with those cultured in HTF+SSS. In conclusion, the effects of synthetic serum supplementation on sperm capacitation varied according to the combination of media. These differences may lead to variations in spermatozoon ROS levels, thus affecting sperm function test results. PMID:27413417

  7. Antioxidant treatment in the absence of exogenous lipids and proteins protects rhesus macaque sperm from cryopreservation-induced cell membrane damage

    PubMed Central

    McCarthy, Megan J.; Meyers, Stuart A.

    2011-01-01

    Osmotic stress caused oxidative stress in rhesus macaque sperm, which was alleviated by antioxidant supplementation. The objective of the present study was to demonstrate that cryopreservation of rhesus macaque sperm also induces (reactive oxygen species) ROS production, and to determine whether ROS have an important role in cryopreservation-induced membrane. Additionally, we evaluated the antioxidant capacity of TEST buffer (with 20% egg yolk and 13% skim milk) and supplementation with antioxidants, superoxide dismutase (SOD), catalase (CAT), and α-tocopherol. There was a substantial level of ROS production in both the presence (15% increase in superoxide, P < 0.01; 14% increase in hydrogen peroxide, P < 0.01) and absence of egg yolk (EY) and skim milk (SM; 33% increase in superoxide, P < 0.001; 48% increase in hydrogen peroxide, P < 0.001). Superoxide dismutase provided little membrane protection against ROS, but increased post-thaw total and progressive motility by 10% (P < 0.01) and 15% (P < 0.05), respectively. Supplementation with CAT and α-tocopherol in the presence of EY and SM decreased H2O2 by 55% (P < 0.01) and 49% (P < 0.001), whereas supplementation with CAT and α-tocopherol in the absence of EY and SM reduced the level of lipid peroxidation by 61% (P < 0.05) and 28% (P < 0.01). In conclusion, this is apparently the first report that cryopreservation of rhesus macaque sperm induced a significant increase in ROS and that antioxidant supplementation can significantly decrease the extent of ROS-induced membrane damage. PMID:21458048

  8. Development of the human immune response against the major surface protein (gp190) of Plasmodium falciparum.

    PubMed Central

    Müller, H M; Früh, K; von Brunn, A; Esposito, F; Lombardi, S; Crisanti, A; Bujard, H

    1989-01-01

    The 190-kilodalton glycoprotein (gp190) of Plasmodium falciparum, the precursor of the major surface proteins of merozoites, is considered a promising candidate for a blood stage malaria vaccine. DNA sequences specific for the gp190 of the two isolates K1 and MAD20 were subcloned and expressed in Escherichia coli. The panel of fusion proteins obtained represents about 80% of the polymorphic sequences observed so far within various isolates of P. falciparum. Sera from individuals living in a malaria-endemic area of West Africa were tested in immunoblots against the gp190 fusion proteins, and antibody reactivity was mapped to defined regions of the gp190. Depending on the age of the individual and on the presence of parasites in the blood, distinct regions of gp190 were differentially recognized by the respective antibodies. Similarly, the analysis of sera from German patients with acute malaria revealed a distinct pattern. When grouped according to age and to parasitemia, the reactivity of the sera of people living in malaria-endemic areas may indicate a correlation between certain gp190 regions and protective immune response. Images PMID:2680981

  9. Heat Resistant Characteristics of Major Royal Jelly Protein 1 (MRJP1) Oligomer

    PubMed Central

    Moriyama, Takanori; Ito, Aimi; Omote, Sumire; Miura, Yuri; Tsumoto, Hiroki

    2015-01-01

    Soluble royal jelly protein is a candidate factor responsible for mammiferous cell proliferation. Major royal jelly protein 1 (MRJP1), which consists of oligomeric and monomeric forms, is an abundant proliferative protein in royal jelly. We previously reported that MRJP1 oligomer has biochemical heat resistance. Therefore, in the present study, we investigated the effects of several heat treatments (56, 65 and 96°C) on the proliferative activity of MRJP1 oligomer. Heat resistance studies showed that the oligomer molecular forms were slightly maintained until 56℃, but the molecular forms were converted to macromolecular heat-aggregated MRJP1 oligomer at 65℃ and 96℃. But, the growth activity of MRJP1 oligomer treated with 96°C was slightly attenuated when compared to unheated MRJP1 oligomer. On the other hand, the cell proliferation activity was preserved until 96℃ by the cell culture analysis of Jurkat cells. In contrast, those of IEC-6 cells were not preserved even at 56°C. The present observations suggest that the bioactive heat-resistance properties were different by the origin of the cells. The cell proliferation analysis showed that MRJP1 oligomer, but not MRJP2 and MRJP3, significantly increased cell numbers, suggesting that MRJP1 oligomer is the predominant proliferation factor for mammiferous cells. PMID:26020775

  10. Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin.

    PubMed Central

    Gabay, J E; Blake, M; Niles, W D; Horwitz, M A

    1985-01-01

    We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins. Images PMID:2579942

  11. The level of major urinary proteins is socially regulated in wild Mus musculus musculus.

    PubMed

    Janotova, Katerina; Stopka, Pavel

    2011-06-01

    Major urinary proteins (MUPs) are highly polymorphic proteins that have been shown to perform several important functions in the chemical communication of the house mouse, Mus musculus. Production of these proteins in C57Bl/6 females is cyclic, reaching the maximum just before the beginning of estrus. Social environment is an important factor that increases MUP production in both sexes. We examined responsiveness of MUP production to social stimuli in wild mice, Mus musculus musculus. The direction of change of MUP production in males depended on the sex of the stimulus animal. Males up-regulated MUP production when caged with a female, but down-regulated MUP production when caged with a male. Down-regulation was more pronounced in males that were defeated in a male-male encounter. Females responded to a male's presence with a decrease in MUP production. We conclude that social modulation of MUP production is specific and, in coordination with other mechanisms, facilitates adjustment of the animal's odor profile to different social contexts. Our results also suggest that in males, MUPs may play an important role in advertizing the male's quality to females. Furthermore, we highlight the importance of analyzing data corrected with creatinine, which show MUP production on the (post)translational level as well as raw data (non-corrected with creatinine), which represent actual concentrations of MUPs in the urine. PMID:21594616

  12. Variable Major Proteins as Targets for Specific Antibodies against Borrelia miyamotoi.

    PubMed

    Wagemakers, Alex; Koetsveld, Joris; Narasimhan, Sukanya; Wickel, Melvin; Deponte, Kathleen; Bleijlevens, Boris; Jahfari, Seta; Sprong, Hein; Karan, Lyudmila S; Sarksyan, Denis S; van der Poll, Tom; Bockenstedt, Linda K; Bins, Adriaan D; Platonov, Alexander E; Fikrig, Erol; Hovius, Joppe W

    2016-05-15

    Borrelia miyamotoi is a relapsing fever spirochete in Ixodes ticks that has been recently identified as a human pathogen causing hard tick-borne relapsing fever (HTBRF) across the Northern Hemisphere. No validated serologic test exists, and current serologic assays have low sensitivity in early HTBRF. To examine the humoral immune response against B. miyamotoi, we infected C3H/HeN mice with B. miyamotoi strain LB-2001 expressing variable small protein 1 (Vsp1) and demonstrated that spirochetemia was cleared after 3 d, coinciding with anti-Vsp1 IgM production. Clearance was also observed after passive transfer of immune sera to infected SCID mice. Next, we showed that anti-Vsp1 IgG eliminates Vsp1-expressing B. miyamotoi, selecting for spirochetes expressing a variable large protein (VlpC2) resistant to anti-Vsp1. The viability of Asian isolate B. miyamotoi HT31, expressing Vlp15/16 and Vlp18, was also unaffected by anti-Vsp1. Finally, in nine HTBRF patients, we demonstrated IgM reactivity to Vsp1 in two and against Vlp15/16 in four ∼1 wk after these patients tested positive for B. miyamotoi by PCR. Our data show that B. miyamotoi is able to express various variable major proteins (VMPs) to evade humoral immunity and that VMPs are antigenic in humans. We propose that serologic tests based on VMPs are of additional value in diagnosing HTBRF. PMID:27076681

  13. Structures of Two Arabidopsis thaliana Major Latex Proteins Represent Novel Helix-Grip Folds

    PubMed Central

    Lytle, Betsy L.; Song, Jikui; de la Cruz, Norberto B.; Peterson, Francis C.; Johnson, Kenneth A.; Bingman, Craig A.; Phillips, George N.; Volkman, Brian F.

    2010-01-01

    The major latex proteins (MLP) are a protein family first identified in the latex of opium poppy. They are found only in plants and have 24 identified members in Arabidopsis alone as well as in other plants such as peach, strawberry, melon, cucumber, and soybean. While the function of the MLPs is unknown, they have been associated with fruit and flower development and in pathogen defense responses. Based on modest sequence similarity, they have been characterized as members of the Bet v 1 protein superfamily; however, no structures have yet been reported. As part of an ongoing structural genomics effort, we determined the structures of two Arabidopsis thaliana MLPs: the solution structure of MLP28 (gene product of At1g70830.1) and the crystal structure of At1g24000.1. The structures revealed distinct differences when compared to one another and to the typical Bet v 1 fold. Nevertheless, NMR titration experiments demonstrated that the characteristic Bet v 1 hydrophobic binding pocket of At1g24000.1 is able to bind a ligand, suggesting that it plays a role in the function of the MLPs. A structure-based sequence analysis identified conserved hydrophobic residues in the long alpha helix that contribute to the binding cavity and may specify preferred ligands for the MLP family. PMID:19326460

  14. Expression, Purification, Crystallization of Two Major Envelope Proteins from White Spot Syndrome Virus

    SciTech Connect

    Tang,X.; Hew, C.

    2007-01-01

    White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapor-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 {angstrom} resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 {angstrom}. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 {angstrom}, and diffracts to 2.0 {angstrom} resolution.

  15. Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

    SciTech Connect

    Tang, Xuhua; Hew, Choy Leong

    2007-07-01

    The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.

  16. Autocrine regulation of human sperm motility by tachykinins

    PubMed Central

    2010-01-01

    Background We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. Methods Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). Results The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). Conclusion These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins. PMID:20796280

  17. The quick and the dead? Sperm competition and sexual conflict in sea.

    PubMed

    Bode, Michael; Marshall, Dustin J

    2007-11-01

    Our view of sperm competition is largely shaped by game-theoretic models based on external fertilizers. External fertilization is of particular interest as it is the ancestral mode of reproduction and as such, relevant to the evolution and maintenance of anisogamy (i.e., large eggs and tiny, numerous sperm). Current game-theoretic models have been invaluable in generating predictions of male responses to sperm competition in a range of internal fertilizers but these models are less relevant to marine broadcast spawners, the most common and archetypal external fertilizers. Broadcast spawners typically have incomplete fertilization due to sperm limitation and/or polyspermy (too many sperm), but the effects of incomplete (<100% fertilization rates) fertilization on game-theoretic predictions are unclear particular with regards to polyspermy. We show that incorporating the effects of sperm concentration on fertilization success changes the predictions of a classic game-theoretic model, dramatically reversing the relationship between sperm competition and the evolutionarily stable sperm release strategy. Furthermore, our results suggest that male and female broadcast spawners are likely to be in conflict at both ends of the sperm environment continuum rather than only in conditions of excess sperm as previously thought. Across the majority of the parameter space we explored, males release either too little to too much sperm for females to achieve complete fertilization. This conflict could result in a coevolutionary race that may have led to the evolution of internal fertilization in marine organisms. PMID:17908245

  18. Prospective approaches to avoid flock fertility problems: predictive assessment of sperm function traits in poultry.

    PubMed

    Donoghue, A M

    1999-03-01

    This paper discusses why it is important to evaluate males as individuals and how advances made in understanding and measurement of sperm function can be used to improve reproductive efficiency in poultry. Commercial turkey breeding relies on pooling semen from multiple toms. It generally is assumed that sperm in good quality semen from all toms are equally fecund. (Fecund is defined, for males, as an individual whose semen contains a majority of sperm with the potential of producing fertilized eggs, which includes success at all steps in the fertilization process: sperm movement, storage in the hens' sperm storage tubules, binding and penetrating the perivitelline layer, and fertilization.) However, when DNA fingerprinting was used to determine paternity efficiency after pooling ejaculates from seven or more toms, it was found that 18 of 26 males produced very few, or no, offspring. In addition, the traditional measures of poultry semen quality: semen volume, sperm concentration, sperm viability, and subjective motility assessment, were poor predictors of paternity. In recent years, a concentrated effort has been made to develop and evaluate methods that quantify sperm function in poultry. Methods to measure some of these traits are reviewed: sperm motility, sperm storage in the hen, and sperm binding and penetration of the ovum. Data supporting use of these tools for managing flock fertility from the male perspective are explored. PMID:10090272

  19. Human sperm physiology: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) influence sperm metabolism and may be involved in the pathophysiology of varicocele-associated male infertility.

    PubMed

    Guido, Carmela; Perrotta, Ida; Panza, Salvatore; Middea, Emilia; Avena, Paola; Santoro, Marta; Marsico, Stefania; Imbrogno, Pietro; Andò, Sebastiano; Aquila, Saveria

    2011-12-01

    The mechanisms by which varicocele affects fertility remain undetermined. Estrogens play a key role in the human male reproduction and human sperm expresses the estrogen receptors (ERs) and aromatase. In this study, by Western blotting we evidenced the ERs content concomitantly in healthy sperm and in oligoastenoteratozoospermic (OAT) samples without and with varicocele. In varicocele a strong reduction of the ERβ was observed, while the ERα was almost absent. Besides, transmission electron microscopy (TEM) confirmed the reduction of ERs expression in "varicocele" sperm, indicating that varicocele has a detrimental effect on sperm structure at molecular level. To further define the estrogen significance in male gamete and the pathophysiology of varicocele we investigated both the expression of ERα and ERβ in normal and pathologic sperm samples as well as we evaluated estradiol (E2) action on lipid and glucose sperm metabolism. Responses to E2 treatments on cholesterol efflux, protein tyrosine phosphorylations, motility, and acrosin activity in varicocele sperm were reduced or absent. The evaluation of the triglycerides content, lipase and acyl-CoA dehydrogenase activities, suggest that E2 exerts a lipolytic effect on human sperm metabolism. Concerning glucose metabolism, it appears that E2 induces G6PDH activity concomitantly to the insulin secretion. In "varicocele" sperm, the E2 did not induce energy expenditure. OAT sperm had E2-responsiveness but in a lesser extent with respect healthy sperm. This study discovered a novel role for E2/ERs in human sperm physiology, since they modulate sperm metabolism and new detrimental effects related to the pathophysiology of the varicocele condition. PMID:21344398

  20. G protein-linked signaling pathways in bipolar and major depressive disorders.

    PubMed

    Tomita, Hiroaki; Ziegler, Mary E; Kim, Helen B; Evans, Simon J; Choudary, Prabhakara V; Li, Jun Z; Meng, Fan; Dai, Manhong; Myers, Richard M; Neal, Charles R; Speed, Terry P; Barchas, Jack D; Schatzberg, Alan F; Watson, Stanley J; Akil, Huda; Jones, Edward G; Bunney, William E; Vawter, Marquis P

    2013-01-01

    The G-protein linked signaling system (GPLS) comprises a large number of G-proteins, G protein-coupled receptors (GPCRs), GPCR ligands, and downstream effector molecules. G-proteins interact with both GPCRs and downstream effectors such as cyclic adenosine monophosphate (cAMP), phosphatidylinositols, and ion channels. The GPLS is implicated in the pathophysiology and pharmacology of both major depressive disorder (MDD) and bipolar disorder (BPD). This study evaluated whether GPLS is altered at the transcript level. The gene expression in the dorsolateral prefrontal (DLPFC) and anterior cingulate (ACC) were compared from MDD, BPD, and control subjects using Affymetrix Gene Chips and real time quantitative PCR. High quality brain tissue was used in the study to control for confounding effects of agonal events, tissue pH, RNA integrity, gender, and age. GPLS signaling transcripts were altered especially in the ACC of BPD and MDD subjects. Transcript levels of molecules which repress cAMP activity were increased in BPD and decreased in MDD. Two orphan GPCRs, GPRC5B and GPR37, showed significantly decreased expression levels in MDD, and significantly increased expression levels in BPD. Our results suggest opposite changes in BPD and MDD in the GPLS, "activated" cAMP signaling activity in BPD and "blunted" cAMP signaling activity in MDD. GPRC5B and GPR37 both appear to have behavioral effects, and are also candidate genes for neurodegenerative disorders. In the context of the opposite changes observed in BPD and MDD, these GPCRs warrant further study of their brain effects. PMID:24391664

  1. Reassessing Domain Architecture Evolution of Metazoan Proteins: Major Impact of Gene Prediction Errors

    PubMed Central

    Nagy, Alinda; Szláma, György; Szarka, Eszter; Trexler, Mária; Bányai, László; Patthy, László

    2011-01-01

    In view of the fact that appearance of novel protein domain architectures (DA) is closely associated with biological innovations, there is a growing interest in the genome-scale reconstruction of the evolutionary history of the domain architectures of multidomain proteins. In such analyses, however, it is usually ignored that a significant proportion of Metazoan sequences analyzed is mispredicted and that this may seriously affect the validity of the conclusions. To estimate the contribution of errors in gene prediction to differences in DA of predicted proteins, we have used the high quality manually curated UniProtKB/Swiss-Prot database as a reference. For genome-scale analysis of domain architectures of predicted proteins we focused on RefSeq, EnsEMBL and NCBI's GNOMON predicted sequences of Metazoan species with completely sequenced genomes. Comparison of the DA of UniProtKB/Swiss-Prot sequences of worm, fly, zebrafish, frog, chick, mouse, rat and orangutan with those of human Swiss-Prot entries have identified relatively few cases where orthologs had different DA, although the percentage with different DA increased with evolutionary distance. In contrast with this, comparison of the DA of human, orangutan, rat, mouse, chicken, frog, zebrafish, worm and fly RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences with those of the corresponding/orthologous human Swiss-Prot entries identified a significantly higher proportion of domain architecture differences than in the case of the comparison of Swiss-Prot entries. Analysis of RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences with DAs different from those of their Swiss-Prot orthologs confirmed that the higher rate of domain architecture differences is due to errors in gene prediction, the majority of which could be corrected with our FixPred protocol. We have also demonstrated that contamination of databases with incomplete, abnormal or mispredicted sequences introduces a bias in DA

  2. Reassessing domain architecture evolution of metazoan proteins: major impact of gene prediction errors.

    PubMed

    Nagy, Alinda; Szláma, György; Szarka, Eszter; Trexler, Mária; Bányai, László; Patthy, László

    2011-01-01

    In view of the fact that appearance of novel protein domain architectures (DA) is closely associated with biological innovations, there is a growing interest in the genome-scale reconstruction of the evolutionary history of the domain architectures of multidomain proteins. In such analyses, however, it is usually ignored that a significant proportion of Metazoan sequences analyzed is mispredicted and that this may seriously affect the validity of the conclusions. To estimate the contribution of errors in gene prediction to differences in DA of predicted proteins, we have used the high quality manually curated UniProtKB/Swiss-Prot database as a reference. For genome-scale analysis of domain architectures of predicted proteins we focused on RefSeq, EnsEMBL and NCBI's GNOMON predicted sequences of Metazoan species with completely sequenced genomes. Comparison of the DA of UniProtKB/Swiss-Prot sequences of worm, fly, zebrafish, frog, chick, mouse, rat and orangutan with those of human Swiss-Prot entries have identified relatively few cases where orthologs had different DA, although the percentage with different DA increased with evolutionary distance. In contrast with this, comparison of the DA of human, orangutan, rat, mouse, chicken, frog, zebrafish, worm and fly RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences with those of the corresponding/orthologous human Swiss-Prot entries identified a significantly higher proportion of domain architecture differences than in the case of the comparison of Swiss-Prot entries. Analysis of RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences with DAs different from those of their Swiss-Prot orthologs confirmed that the higher rate of domain architecture differences is due to errors in gene prediction, the majority of which could be corrected with our FixPred protocol. We have also demonstrated that contamination of databases with incomplete, abnormal or mispredicted sequences introduces a bias in DA

  3. Differential proteome association study of freeze-thaw damage in ram sperm.

    PubMed

    He, Yuxuan; Wang, Ke; Zhao, Xingxu; Zhang, Yong; Ma, Youji; Hu, Junjie

    2016-02-01

    In this study proteomics analysis was performed to investigate damage caused to ram sperm by the freeze-thaw process. Sperm motility, viability, reactive oxygen species (ROS) and adenosine triphosphate (ATP) content were measured to evaluate sperm quality. Compared with fresh groups, motility, viability and ATP content were all lower in freeze-thawed sperm (P < 0.001), and ROS content was higher (P < 0.001). Moreover, 25 differential protein spots were detected in two-dimensional gels using PDQuest 8.0 software and the corresponding proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS) coupled with searching of the NCBI protein sequence database. Among these proteins, hexokinase1 (HXK1), the enzyme that catalyzes the first step of glycolysis in the sperm glycolytic pathway, is known to be associated with sperm motility. Casein kinase II subunit alpha (CSNK2A2), a serine/threonine-selective protein kinase, is associated with sperm apoptosis. We used immunoblotting and immunofluorescence to analyze the expression and localization of these two proteins. HXK1 and CSNK2A2 expression levels in fresh sperm were significantly higher than that in freeze-thawed sperm (P < 0.001). HXK1 and CSNK2A2 were detected in the main part of the sperm flagellum, and the immunofluorescence signal from these proteins was weakened in the freeze-thawed group. Decreased expression of HXK1 and CSNK2A2 may be associated with decreased sperm motility and viability following freeze-thawing. PMID:26617253

  4. Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality.

    PubMed

    Holt, William V; Van Look, Katrien J W

    2004-05-01

    Stringent selection mechanisms, in both internal and external fertilisation systems, reject all but a significant minority of the spermatozoa released at ejaculation. Sperm competition theory provides circumstantial evidence that the selection process involves mechanisms by which the quality of the fertilising spermatozoon is controlled, thereby ensuring that females and their offspring receive high quality genetic material. In this review we examine some of these selection processes to see whether they could be exploited for the improvement of laboratory tests of sperm quality. Such tests are not only required for clinical and agricultural purposes, but are increasingly needed in fields such as reproductive and environmental toxicology where the species requirement is much broader. Despite many years of research, sperm quality assessment methods continue to provide imprecise data about fertility; here we suggest that this may be a consequence of using tests that focus on the spermatozoa that would normally be unable to fertilise under natural conditions. To achieve fertilisation a spermatozoon must be capable of responding appropriately to external signalling stimuli; those involving protein kinase-regulated flagellar function seem especially influential in governing effects ranging from non-Mendelian inheritance in mammals to sperm chemotaxis in sea urchins. Examination of the elicited responses reveals considerable heterogeneity in all species. Here we propose that this level of heterogeneity is meaningful both in terms of understanding how spermatozoa from some individuals possess fertility advantages over spermatozoa from their rivals in sperm competition, and in that the heterogeneity should be exploitable in the development of more accurate laboratory tests. PMID:15129008

  5. Analysis of a cDNA encoding the major vault protein from the electric ray Discopyge ommata.

    PubMed

    Herrmann, C; Zimmermann, H; Volknandt, W

    1997-03-25

    The major vault protein is the predominant constituent of vaults ubiquitous large cytosolic ribonucleoprotein particles. A cDNA clone encoding the 100-kDa major vault protein (MVP100) was isolated from an electric lobe library of Discopyge ommata. The complete nucleotide sequence was determined. Northern blot analysis revealed a 2.8-kb transcript with a high expression in neural tissue. Southern blot analysis indicates that the electric ray MVP100 is a single copy-gene with at least two introns. The primary structure of major vault proteins characterized in slime mold, ray, rat and human is evolutionary highly conserved. PMID:9099863

  6. Changes in the distribution and molecular mass of boar sperm acrosome-associated 1 proteins during the acrosome reaction; their validity as indicators for occurrence of the true acrosome reaction.

    PubMed

    Ogura, Yukari; Takagishi, Yuki; Harayama, Hiroshi

    2016-09-01

    The aims of this study were to investigate changes in the distribution and molecular mass of boar sperm acrosome-associated 1 (SPACA1) proteins during the acrosome reaction and to discuss validity of SPACA1 proteins as indicators for occurrence of the true acrosome reaction. Boar ejaculated spermatozoa were used for induction of the extracellular Ca(2+)-dependent acrosome reaction (true acrosome reaction) or acrosomal damages (false acrosome reaction) and then subjected to double staining with the anti-SPACA1 protein antibody and FITC-PNA and Western blotting. Extracellular Ca(2+)-dependently acrosome-reacted spermatozoa were characterized by appearance of SPACA1 proteins in the postacrosomal region (; these spermatozoa were classified into SP-3&AR pattern of double staining). However, SPACA1 proteins were not observed in the postacrosomal region of frozen-thawed spermatozoa with severely damaged acrosomes (; these spermatozoa were classified into SP-2&AR pattern). Moreover, the spermatozoa in which acrosomes were severely damaged by incubation with cyclodextrins and without CaCl2 were classified into either SP-2&AR or SP-3&AR pattern. Although SPACA1 proteins were detected mainly as 36-42kDa proteins in the spermatozoa with intact acrosomes, small types of SPACA1 proteins (15-28kDa) increased in extracellular Ca(2+)-dependently acrosome-reacted spermatozoa as well as frozen-thawed spermatozoa with damaged acrosomes. These results show the increase of boar spermatozoa classified into SP-3&AR pattern after incubation in the medium with CaCl2 and without cyclodextrins indicates occurrence of the true acrosome reaction. Moreover, we suggest the increase of small types of SPACA1 proteins is a valid indicator for occurrence of the acrosomal disintegration arising from the true and false acrosome reactions. PMID:27449406

  7. Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

    PubMed Central

    Kuhtz, Juliane; Schneider, Eberhard; El Hajj, Nady; Zimmermann, Lena; Fust, Olga; Linek, Bartosz; Seufert, Rudolf; Hahn, Thomas; Schorsch, Martin; Haaf, Thomas

    2014-01-01

    The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1–14% GTL2 epimutations. PMID:25625849

  8. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump

    PubMed Central

    Hinchliffe, Philip; Greene, Nicholas P.; Paterson, Neil G.; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-01-01

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. PMID:24996185

  9. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump.

    PubMed

    Hinchliffe, Philip; Greene, Nicholas P; Paterson, Neil G; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-08-25

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. PMID:24996185

  10. Sexual attractiveness in male rats is associated with greater concentration of major urinary proteins.

    PubMed

    Kumar, Vineet; Vasudevan, Anand; Soh, Linda Jing Ting; Le Min, Choo; Vyas, Ajai; Zewail-Foote, Maha; Guarraci, Fay A

    2014-12-01

    Female rats show a distinct attraction for males. This attraction remains consistent without the necessity for the physical presence of the male. However, the identity of the olfactory cues contributing to attraction in rats remains unknown. Rat urine contains copious amounts of major urinary proteins (MUPs). Here, we investigated the hypothesis that MUPs mediate sexual attractiveness in rats. We first demonstrated that a member of a male dyad receiving greater copulatory opportunities in competitive mate choice tests excrete greater amounts of MUPs. Furthermore, the amount of male MUPs positively correlated with both copulatory opportunities received and female exploration of the urine. Using females and a two-choice olfactory attraction test, we demonstrated that urinary fractions containing MUPs were sufficient to induce attraction and that male MUPs activated neurons in the posterodorsal medial amygdala in female rats. Taken together, these results suggest that olfactory cues associated with MUPs act as an attractant to female rats in estrus. PMID:25359898

  11. Presence of common antigens, including major surface protein epitopes, between the cattle (intraerythrocytic) and tick stages of Anaplasma marginale.

    PubMed Central

    Palmer, G H; Kocan, K M; Barron, S J; Hair, J A; Barbet, A F; Davis, W C; McGuire, T C

    1985-01-01

    Epitopes of major surface proteins of the intraerythrocytic cattle stage of Anaplasma marginale were demonstrated in the midgut stage of the organism within the infective tick host Dermacentor andersoni. These proteins were common to all A. marginale isolates tested and at all stages of parasitemia. Sera from cattle immunized with the tick midgut stage of A. marginale immunoprecipitated multiple-erythrocyte-stage proteins, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major proteins recognized (primarily greater than 14 and less than 200 kilodaltons [kDa]) included two major-erythrocyte-stage surface proteins of 36 and 105 kDa molecular size. To confirm the presence of common tick and erythrocyte A. marginale antigens with the immunized cattle sera, we purified the 36-kDa erythrocyte-stage protein by monoclonal immunoaffinity chromatography and developed an enzyme-linked immunosorbent assay based on the purified protein. All sera from cattle immunized with tick-stage A. marginale and cattle infected with various isolates of A. marginale developed antibodies to the 36-kDa protein. The potential immunoprophylactic, diagnostic, and epidemiologic value of the major epitopes common to both the invertebrate and mammalian stages of A. marginale, especially the 36-kDa protein, is discussed. Images PMID:2415457

  12. HSP90 expression correlation with the freezing resistance of bull sperm.

    PubMed

    Wang, Peng; Wang, Yan-Feng; Wang, Hong; Wang, Chun-Wei; Zan, Lin-Sen; Hu, Jian-Hong; Li, Qing-Wang; Jia, Yong-Hong; Ma, Guo-Ji

    2014-05-01

    To date, there has been little improvement in cryopreservation of bull sperm due to lack of understanding of the freezing mechanisms. Therefore, this study set out to investigate expression levels of fertility-associated proteins in bull sperm, and in particular the relationship between the 90 kDa heat-shock protein (HSP90) and the sperm characteristics after freezing-thawing. Semen was collected from eight Holstein bulls by artificial vagina. Characteristics of these fresh semen, including sperm motility, morphology, viability and concentration, were evaluated. Sperm quality was also assessed after freezing-thawing. Eight ejaculates were divided into two groups based on freezing resistance and sperm motility. Sperm proteins were extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and western blotting were performed. SDS-PAGE results showed that there was substantial diversity in 90 kDa proteins in the frozen-thawed sperm and HSP90 was confirmed as one of the 90 kDa proteins by western blot. This study indicated that HSP90 expression correlated positively with sperm quality. The amount of expressed 90 kDa proteins in the high freezing resistance (HFR) group was significantly higher than that in the low freezing resistance (LFR) group (P < 0.05). Thus, higher expression of HSP90 could probably lead to the higher motility and freezing resistance of sperm found after freezing-thawing. Therefore, we concluded that level of HSP90 expression could be used to predict reliably and simply the freezing resistance of bull sperm. PMID:23506739

  13. Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems

    PubMed Central

    Siwaszek, Aleksandra; Ukleja, Marta; Dziembowski, Andrzej

    2014-01-01

    The process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5’-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3’>5’direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsense-mediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main model-species: yeast, Drosophila, plants and mammals. PMID:25483043

  14. The Treponema denticola Major Sheath Protein Is Predominantly Periplasmic and Has Only Limited Surface Exposure

    PubMed Central

    Caimano, Melissa J.; Bourell, Kenneth W.; Bannister, Teresa D.; Cox, David L.; Radolf, Justin D.

    1999-01-01

    The recent discovery that the Treponema pallidum genome encodes 12 orthologs of the Treponema denticola major sheath protein (Msp) prompted us to reexamine the cellular location and topology of the T. denticola polypeptide. Experiments initially were conducted to ascertain whether Msp forms an array on or within the T. denticola outer membrane. Transmission electron microscopy (EM) of negatively stained and ultrathin-sectioned organisms failed to identify a typical surface layer, whereas freeze-fracture EM revealed that the T. denticola outer membrane contains heterogeneous transmembrane proteins but no array. In contrast, a lattice-like structure was observed in vesicles released from mildly sonicated treponemes; combined EM and biochemical analyses demonstrated that this structure was the peptidoglycan sacculus. Immunoelectron microscopy (IEM) subsequently was performed to localize Msp in T. denticola. Examination of negatively stained whole mounts identified substantial amounts of Msp in sonicated organisms. IEM of ultrathin-sectioned, intact treponemes also demonstrated that the preponderance of antigen was unassociated with the outer membrane. Lastly, immunofluorescence analysis of treponemes embedded in agarose gel microdroplets revealed that only minor portions of Msp are surface exposed. Taken as a whole, our findings challenge the widely held belief that Msp forms an array within the T. denticola outer membrane and demonstrate, instead, that it is predominantly periplasmic with only limited surface exposure. These findings also have implications for our evolving understanding of the contribution(s) of Msp/Tpr orthologs to treponemal physiology and disease pathogenesis. PMID:10417176

  15. Solution structure of a two-repeat fragment of major vault protein.

    PubMed

    Kozlov, Guennadi; Vavelyuk, Olga; Minailiuc, Ovidiu; Banville, Denis; Gehring, Kalle; Ekiel, Irena

    2006-02-17

    Major vault protein (MVP) is the main constituent of vaults, large ribonucleoprotein particles implicated in resistance to cancer therapy and correlated with poor survival prognosis. Here, we report the structure of the main repeat element in human MVP. The approximately 55 amino acid residue MVP domain has a unique, novel fold that consists of a three-stranded antiparallel beta-sheet. The solution NMR structure of a two-domain fragment reveals the interdomain contacts and relative orientations of the two MVP domains. We use these results to model the assembly of 672 MVP domains from 96 MVP molecules into the ribs of the 13MDa vault structure. The unique features include a thin, skin-like structure with polar residues on both the cytoplasmic and internal surface, and a pole-to-pole arrangement of MVP molecules. These studies provide a starting point for understanding the self-assembly of MVP into vaults and their interactions with other proteins. Chemical shift perturbation studies identified the binding site of vault poly(ADP-ribose) polymerase, another component of vault particles, indicating that MVP domains form a new class of interaction-mediating modules. PMID:16373071

  16. Assembly of vault-like particles in insect cells expressing only the major vault protein.

    PubMed

    Stephen, A G; Raval-Fernandes, S; Huynh, T; Torres, M; Kickhoefer, V A; Rome, L H

    2001-06-29

    Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although the precise function of vaults is unknown, their wide distribution and highly conserved morphology in eukaryotes suggests that their function is essential and that their structure must be important for their function. The 100-kDa major vault protein (MVP) constitutes approximately 75% of the particle mass and is predicted to form the central barrel portion of the vault. To gain insight into the mechanisms for vault assembly, we have expressed rat MVP in the Sf9 insect cell line using a baculovirus vector. Our results show that the expression of the rat MVP alone can direct the formation of particles that have biochemical characteristics similar to endogenous rat vaults and display the distinct vault-like morphology when negatively stained and examined by electron microscopy. These particles are the first example of a single protein polymerizing into a non-spherically, non-cylindrically symmetrical structure. Understanding vault assembly will enable us to design agents that disrupt vault formation and hence aid in elucidating vault function in vivo. PMID:11349122

  17. In Silico Resurrection of the Major Vault Protein Suggests It Is Ancestral in Modern Eukaryotes

    PubMed Central

    Daly, Toni K.; Sutherland-Smith, Andrew J.; Penny, David

    2013-01-01

    Vaults are very large oligomeric ribonucleoproteins conserved among a variety of species. The rat vault 3D structure shows an ovoid oligomeric particle, consisting of 78 major vault protein monomers, each of approximately 861 amino acids. Vaults are probably the largest ribonucleoprotein structures in eukaryote cells, being approximately 70 nm in length with a diameter of 40 nm—the size of three ribosomes and with a lumen capacity of 50 million Å3. We use both protein sequences and inferred ancestral sequences for in silico virtual resurrection of tertiary and quaternary structures to search for vaults in a wide variety of eukaryotes. We find that the vault’s phylogenetic distribution is widespread in eukaryotes, but is apparently absent in some notable model organisms. Our conclusion from the distribution of vaults is that they were present in the last eukaryote common ancestor but they have apparently been lost from a number of groups including fungi, insects, and probably plants. Our approach of inferring ancestral 3D and quaternary structures is expected to be useful generally. PMID:23887922

  18. In silico resurrection of the major vault protein suggests it is ancestral in modern eukaryotes.

    PubMed

    Daly, Toni K; Sutherland-Smith, Andrew J; Penny, David

    2013-01-01

    Vaults are very large oligomeric ribonucleoproteins conserved among a variety of species. The rat vault 3D structure shows an ovoid oligomeric particle, consisting of 78 major vault protein monomers, each of approximately 861 amino acids. Vaults are probably the largest ribonucleoprotein structures in eukaryote cells, being approximately 70 nm in length with a diameter of 40 nm--the size of three ribosomes and with a lumen capacity of 50 million Å(3). We use both protein sequences and inferred ancestral sequences for in silico virtual resurrection of tertiary and quaternary structures to search for vaults in a wide variety of eukaryotes. We find that the vault's phylogenetic distribution is widespread in eukaryotes, but is apparently absent in some notable model organisms. Our conclusion from the distribution of vaults is that they were present in the last eukaryote common ancestor but they have apparently been lost from a number of groups including fungi, insects, and probably plants. Our approach of inferring ancestral 3D and quaternary structures is expected to be useful generally. PMID:23887922

  19. Mutations in fd phage major coat protein modulate affinity of the displayed peptide

    PubMed Central

    Kuzmicheva, G.A.; Jayanna, P.K.; Eroshkin, A.M.; Grishina, M.A.; Pereyaslavskaya, E.S.; Potemkin, V.A.; Petrenko, V.A.

    2009-01-01

    Multibillion-clone libraries of phages displaying guest peptides fused to the major coat protein pVIII (landscape libraries) are a rich source of probes for proteinaceous and non-proteinaceous targets. As opposed to the pIII-type fusion phages, which display peptides as independent structural domains, the guest peptides in the pVIII-fusion phages can be structurally and functionally influenced by contiguous subunits. To decipher the impact of the locale of a guest peptide on its affinity characteristics, we constructed a library of phages carrying β-galactosidase-binding peptide ADTFAKSMQ at the N-terminus of the pVIII protein surrounded by random amino acids. It was found that mutagenesis of amino acids 12–19 (domain C) has polar effects on target binding affinity of the displayed peptide. The phages with highest affinity are characterized by: (i) a net electrostatic charge around −1 of domain C of the mutated phages at pH 7.0; (ii) a lower radius of cylinder coaxial to α-helix formed by domain C; (iii) a lower higher occupied molecular orbital (HOMO) of domain C leading to a decreased formation of hydrogen bonds and (iv) positively charged surface and torsion energy of domain C, which may require a conformational transition of N-terminal peptide ADTFAKSMQ for its binding with β-galactosidase. Influence of the guest peptide on the diversity of mutations in the neighboring landscape area was also observed. PMID:19633313

  20. Turbulence of swarming sperm.

    PubMed

    Creppy, Adama; Praud, Olivier; Druart, Xavier; Kohnke, Philippa L; Plouraboué, Franck

    2015-09-01

    Collective motion of self-sustained swarming flows has recently provided examples of small-scale turbulence arising where viscous effects are dominant. We report the first observation of universal enstrophy cascade in concentrated swarming sperm consistent with a body of evidence built from various independent measurements. We found a well-defined k^{-3} power-law decay of a velocity field power spectrum and relative dispersion of small beads consistent with theoretical predictions in 2D turbulence. Concentrated living sperm displays long-range, correlated whirlpool structures of a size that provides an integral scale of turbulence. We propose a consistent explanation for this quasi-2D turbulence based on self-structured laminated flow forced by steric interactions and alignment, a state of active matter that we call "swarming liquid crystal." We develop scaling arguments consistent with this interpretation. PMID:26465513

  1. Turbulence of swarming sperm

    NASA Astrophysics Data System (ADS)

    Creppy, Adama; Praud, Olivier; Druart, Xavier; Kohnke, Philippa L.; Plouraboué, Franck

    2015-09-01

    Collective motion of self-sustained swarming flows has recently provided examples of small-scale turbulence arising where viscous effects are dominant. We report the first observation of universal enstrophy cascade in concentrated swarming sperm consistent with a body of evidence built from various independent measurements. We found a well-defined k-3 power-law decay of a velocity field power spectrum and relative dispersion of small beads consistent with theoretical predictions in 2D turbulence. Concentrated living sperm displays long-range, correlated whirlpool structures of a size that provides an integral scale of turbulence. We propose a consistent explanation for this quasi-2D turbulence based on self-structured laminated flow forced by steric interactions and alignment, a state of active matter that we call "swarming liquid crystal." We develop scaling arguments consistent with this interpretation.

  2. Recombinant Major Antigenic Protein 2 of Ehrlichia canis: a Potential Diagnostic Tool

    PubMed Central

    Alleman, A. Rick; McSherry, Leo J.; Barbet, Anthony F.; Breitschwerdt, Edward B.; Sorenson, Heather L.; Bowie, Michael V.; Bélanger, Myriam

    2001-01-01

    The major antigenic protein 2 (MAP2) of Ehrlichia canis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody and immune serum from an experimentally infected dog. The recombinant MAP2 (rMAP2) was tested in an ELISA format using 141 serum samples from E. canis immunofluorescent antibody (IFA)-positive and IFA-negative dogs. Fifty-five of the serum samples were from dogs experimentally or naturally infected with E. canis and were previously demonstrated to contain antibodies reactive with E. canis by indirect immunofluorescence assays. The remaining 86 samples, 33 of which were from dogs infected with microorganisms other than E. canis, were seronegative. All of the samples from experimentally infected animals and 36 of the 37 samples from naturally infected animals were found to contain antibodies against rMAP2 of E. canis in the ELISA. Only 3 of 53 IFA-negative samples tested positive on the rMAP2 ELISA. There was 100% agreement among IFA-positive samples from experimentally infected animals, 97.3% agreement among IFA-positive samples from naturally infected animals, and 94.3% agreement among IFA-negative samples, resulting in a 97.2% overall agreement between the two assays. These data suggest that rMAP2 of E. canis could be used as a recombinant test antigen for the serodiagnosis of canine monocytic ehrlichiosis. PMID:11427559

  3. Expression of the major outer membrane protein of Chlamydia trachomatis in Escherichia coli.

    PubMed Central

    Manning, D S; Stewart, S J

    1993-01-01

    The major outer membrane protein (MOMP) of Chlamydia trachomatis was expressed in Escherichia coli. To assess whether it assembled into a conformationally correct structure at the cell surface, we characterized the recombinant MOMP (rMOMP) by Western immunoblot analysis, indirect immunofluorescence, and immunoprecipitation with monoclonal antibodies (MAbs) that recognize contiguous and conformational MOMP epitopes. Western blot analysis showed that most of the rMOMP comigrated with authentic monomer MOMP, indicating that its signal peptide was recognized and cleaved by E. coli. The rMOMP could not be detected on the cell surface of viable or formalin-killed E. coli organisms by indirect immunofluorescence staining with a MAb specific for a MOMP contiguous epitope. In contrast, the same MAb readily stained rMOMP-expressing E. coli cells that had been permeabilized by methanol fixation. A MAb that recognizes a conformational MOMP epitope and reacted strongly with formalin- or methanol-fixed elementary bodies failed to stain formalin- or methanol-fixed E. coli expressing rMOMP. Moreover, this MAb did not immunoprecipitate rMOMP from expressing E. coli cells even though it precipitated the authentic protein from lysates of C. trachomatis elementary bodies. Therefore we concluded that rMOMP was not localized to the E. coli cell surface and was not recognizable by a conformation-dependent antibody. These results indicate that rMOMP expressed by E. coli is unlikely to serve as an accurate model of MOMP structure and function. They also question the utility of rMOMP as a source of immunogen for eliciting neutralizing antibodies against conformational antigenic sites of the protein. Images PMID:8406797

  4. The sperm mitochondria-specific translocator has a key role in maternal mitochondrial inheritance.

    PubMed

    Hayashida, Kenji; Omagari, Katsuhisa; Masuda, Jun-ichi; Hazama, Hiroaki; Kadokawa, Yoshiko; Ohba, Kazuo; Kohno, Shigeru

    2005-06-01

    The mechanism of maternal mitochondrial inheritance in animals involves the selective elimination of sperm mitochondria by the elimination factor of the egg and the sperm mitochondria-specific factor. In vitro fertilization using sperm from isogenic mice incorporating heterospecific mitochondrial DNA (mtDNA) showed that the number of PCR positives of sperm mtDNA in two-cell embryos was significantly increased following sperm incubation with anti-tetratricopeptide repeat-containing protein involved in spermatogenesis (tpis) protein, anti-translocator of mitochondrial outer membrane (Tom) 22 and anti-Tom40 antibodies. The treatment of fertilized eggs with EGTA and other endonuclease inhibitors increased the sperm mtDNA levels. We conclude that the elimination factor, which is probably an endonuclease, is selectively received by the tpis protein of the sperm mitochondrial outer membrane within the egg. It is then transported into the sperm mitochondria by Tom22 and Tom40, where it destroys the sperm mtDNA, establishing the maternal inheritance of mtDNA. PMID:15979907

  5. Is the major capsid protein of iridoviruses a suitable target for the study of viral evolution?

    PubMed

    Tidona, C A; Schnitzler, P; Kehm, R; Darai, G

    1998-01-01

    Iridoviruses are large cytoplasmic DNA viruses that are specific for different insect or vertebrate hosts. The major structural component of the non-enveloped icosahedral virus particles is the major capsid protein (MCP) which appears to be highly conserved among members of the family Iridoviridae, Phycodnaviridae, and African swine fever virus. The amino acid sequences of the known MCPs were used in comparative analyses to elucidate the phylogenic relationships between different cytoplasmic DNA viruses including three insect iridoviruses (Tipula iridescent virus, Simulium iridescent virus, Chilo iridescent virus), seven vertebrate iridoviruses isolated either from fish (lymphocystis disease virus, rainbow trout virus, European catfish virus, doctor fish virus), amphibians (frog virus 3), or reptiles (turtle virus 3, turtle virus 5), one member of the family Phycodnaviridae (Paramecium bursaria Chlorella virus type 1), and African swine fever virus. These analyses revealed that the amino acid sequence of the MCP is a suitable target for the study of viral evolution since it contains highly conserved domains, but is sufficiently diverse to distinguish closely related iridovirus isolates. Furthermore the results suggest that a substantial revision of the taxonomy of iridoviruses based on molecular phylogeny is required. PMID:9562891

  6. Molecular Evolution of the Yersinia Major Outer Membrane Protein C (OmpC).

    PubMed

    Stenkova, Anna M; Bystritskaya, Evgeniya P; Guzev, Konstantin V; Rakin, Alexander V; Isaeva, Marina P

    2016-01-01

    The genus Yersinia includes species with a wide range of eukaryotic hosts (from fish, insects, and plants to mammals and humans). One of the major outer membrane proteins, the porin OmpC, is preferentially expressed in the host gut, where osmotic pressure, temperature, and the concentrations of nutrients and toxic products are relatively high. We consider here the molecular evolution and phylogeny of Yersinia ompC. The maximum likelihood gene tree reflects the macroevolution processes occurring within the genus Yersinia. Positive selection and horizontal gene transfer are the key factors of ompC diversification, and intraspecies recombination was revealed in two Yersinia species. The impact of recombination on ompC evolution was different from that of another major porin gene, ompF, possibly due to the emergence of additional functions and conservation of the basic transport function. The predicted antigenic determinants of OmpC were located in rapidly evolving regions, which may indicate the evolutionary mechanisms of Yersinia adaptation to the host immune system. PMID:27578962

  7. Molecular Evolution of the Yersinia Major Outer Membrane Protein C (OmpC)

    PubMed Central

    Stenkova, Anna M.; Bystritskaya, Evgeniya P.; Guzev, Konstantin V.; Rakin, Alexander V.; Isaeva, Marina P.

    2016-01-01

    The genus Yersinia includes species with a wide range of eukaryotic hosts (from fish, insects, and plants to mammals and humans). One of the major outer membrane proteins, the porin OmpC, is preferentially expressed in the host gut, where osmotic pressure, temperature, and the concentrations of nutrients and toxic products are relatively high. We consider here the molecular evolution and phylogeny of Yersinia ompC. The maximum likelihood gene tree reflects the macroevolution processes occurring within the genus Yersinia. Positive selection and horizontal gene transfer are the key factors of ompC diversification, and intraspecies recombination was revealed in two Yersinia species. The impact of recombination on ompC evolution was different from that of another major porin gene, ompF, possibly due to the emergence of additional functions and conservation of the basic transport function. The predicted antigenic determinants of OmpC were located in rapidly evolving regions, which may indicate the evolutionary mechanisms of Yersinia adaptation to the host immune system. PMID:27578962

  8. Abundant class III acidic chitinase homologue in tamarind (Tamarindus indica) seed serves as the major storage protein.

    PubMed

    Rao, Devavratha H; Gowda, Lalitha R

    2008-03-26

    The phyla Leguminosae contains protease inhibitors, lectins, chitinases, and glycohydrolases as major defense proteins in their seeds. Electrophoretic analysis of the seed proteins of tamarind ( Tamarindus indica L.), an agri-waste material, indicated the unusual presence of two major proteins comparable to overexpression of recombinant proteins. These proteins were identified by amino-terminal analysis to be (1) Kunitz-type trypsin inhibitor and (2) class III endochitinase (34000 Da). These two proteins were purified to apparent homogeneity by a single-step chitin bead affinity chromatography and characterized. The Kunitz inhibitor was specific toward inhibiting trypsin with a stoichiometry of 1:1. The 33000 +/- 1000 Da protein, accounting for >50% of the total seed protein, is an acidic glycoprotein exhibiting a very low endotype hydrolytic activity toward chitin derivatives. SDS-PAGE followed by densitometry of tamarind seed germination indicates the disappearance of the chitinase with the concomitant appearance of a cysteine endopeptidase. On the basis of its abundance, accumulation without any pathogenesis-related stimulus, temporal regulation, amino acid composition, and very low enzyme activity, this 34000 Da protein designated "tamarinin" physiologically serves as the major storage protein. PMID:18298067

  9. Pre-clinical immunogenicity of human papillomavirus alpha-7 and alpha-9 major capsid proteins

    PubMed Central

    Bissett, Sara L.; Mattiuzzo, Giada; Draper, Eve; Godi, Anna; Wilkinson, Dianna E.; Minor, Philip; Page, Mark; Beddows, Simon

    2014-01-01

    Human papillomavirus (HPV) vaccines confer protection against the oncogenic genotypes HPV16 and HPV18 through the generation of type-specific neutralizing antibodies raised against the constituent virus-like particles (VLP) based upon the major capsid proteins (L1) of these genotypes. The vaccines also confer a degree of cross-protection against some genetically related types from the Alpha-9 (HPV16-like: HPV31, HPV33, HPV35, HPV52, HPV58) and Alpha-7 (HPV18-like: HPV39, HPV45, HPV59, HPV68) species groups. The mechanism of cross-protection is unclear but may involve antibodies capable of recognizing shared inter-genotype epitopes. The relationship(s) between the genetic and antigenic diversity of the L1 protein, particularly for non-vaccine genotypes, is poorly understood. We carried out a comprehensive evaluation of the immunogenicity of L1 VLP derived from genotypes within the Alpha-7 and Alpha-9 species groups in New Zealand White rabbits and used L1L2 pseudoviruses as the target antigens in neutralization assays. The majority antibody response against L1 VLP was type-specific, as expected, but several instances of robust cross-neutralization were nevertheless observed including between HPV33 and HPV58 within the Alpha-9 species and between HPV39, HPV59 and HPV68 in the Alpha-7 species. Immunization with an experimental tetravalent preparation comprising VLP based upon HPV16, HPV18, HPV39 and HPV58 was capable of generating neutralizing antibodies against all the Alpha-7 and Alpha-9 genotypes. Competition of HPV31 and HPV33 cross-neutralizing antibodies in the tetravalent sera confirmed that these antibodies originated from HPV16 and HPV58 VLP, respectively, and suggested that they represent minority specificities within the antibody repertoire generated by the immunizing antigen. These data improve our understanding of the antigenic diversity of the L1 protein per se and may inform the rational design of a next generation vaccine formulation based upon

  10. Cytometry of mammalian sperm

    SciTech Connect

    Gledhill, B.L.

    1983-10-11

    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. The accessibility of male cells makes them well suited for analytical cytology. We might automate the process of determining sperm morphology but should not do so solely for increased speed. Rather, richer tangible benefits will derive from cytometric evaluation through increased sensitivity, reduced subjectivity, standardization between investigators and laboratories, enhanced archival systems, and the benefits of easily exchanged standardized data. Inroads on the standardization of assays for motility and functional integrity are being made. Flow cytometric analysis of total DNA content of individual sperm is an insensitive means to detect exposure to reproductive toxins because of the small size and low frequency of the DNA content errors. Flow cytometry can be applied to determine the proportions of X- and Y-sperm in semen samples.

  11. Sperm function test

    PubMed Central

    Talwar, Pankaj; Hayatnagarkar, Suryakant

    2015-01-01

    With absolute normal semen analysis parameters it may not be necessary to shift to specialized tests early but in cases with borderline parameters or with history of fertilization failure in past it becomes necessary to do a battery of tests to evaluate different parameters of spermatozoa. Various sperm function tests are proposed and endorsed by different researchers in addition to the routine evaluation of fertility. These tests detect function of a certain part of spermatozoon and give insight on the events in fertilization of the oocyte. The sperms need to get nutrition from the seminal plasma in the form of fructose and citrate (this can be assessed by fructose qualitative and quantitative estimation, citrate estimation). They should be protected from the bad effects of pus cells and reactive oxygen species (ROS) (leukocyte detection test, ROS estimation). Their number should be in sufficient in terms of (count), structure normal to be able to fertilize eggs (semen morphology). Sperms should have intact and functioning membrane to survive harsh environment of vagina and uterine fluids (vitality and hypo-osmotic swelling test), should have good mitochondrial function to be able to provide energy (mitochondrial activity index test). They should also have satisfactory acrosome function to be able to burrow a hole in zona pellucida (acrosome intactness test, zona penetration test). Finally, they should have properly packed DNA in the nucleus to be able to transfer the male genes (nuclear chromatic decondensation test) to the oocyte during fertilization. PMID:26157295

  12. Sperm function test.

    PubMed

    Talwar, Pankaj; Hayatnagarkar, Suryakant

    2015-01-01

    With absolute normal semen analysis parameters it may not be necessary to shift to specialized tests early but in cases with borderline parameters or with history of fertilization failure in past it becomes necessary to do a battery of tests to evaluate different parameters of spermatozoa. Various sperm function tests are proposed and endorsed by different researchers in addition to the routine evaluation of fertility. These tests detect function of a certain part of spermatozoon and give insight on the events in fertilization of the oocyte. The sperms need to get nutrition from the seminal plasma in the form of fructose and citrate (this can be assessed by fructose qualitative and quantitative estimation, citrate estimation). They should be protected from the bad effects of pus cells and reactive oxygen species (ROS) (leukocyte detection test, ROS estimation). Their number should be in sufficient in terms of (count), structure normal to be able to fertilize eggs (semen morphology). Sperms should have intact and functioning membrane to survive harsh environment of vagina and uterine fluids (vitality and hypo-osmotic swelling test), should have good mitochondrial function to be able to provide energy (mitochondrial activity index test). They should also have satisfactory acrosome function to be able to burrow a hole in zona pellucida (acrosome intactness test, zona penetration test). Finally, they should have properly packed DNA in the nucleus to be able to transfer the male genes (nuclear chromatic decondensation test) to the oocyte during fertilization. PMID:26157295

  13. Ranavirus phylogeny and differentiation based on major capsid protein, DNA polymerase and neurofilament triplet H1-like protein genes.

    PubMed

    Holopainen, R; Ohlemeyer, S; Schütze, H; Bergmann, S M; Tapiovaara, H

    2009-06-10

    In this study, we developed new methods for differentiation of ranaviruses based on polymerase chain reaction and restriction enzyme analysis of DNA polymerase and neurofilament triplet H1-like (NF-H1) protein gene. Using these methods, we were able to differentiate the 6 known ranaviruses--Bohle iridovirus (BIV), European catfish virus (ECV), epizootic haematopoietic necrosis virus (EHNV), European sheatfish virus (ESV), frog virus 3 (FV3) and Singapore grouper iridovirus (SGIV)--with 3 less characterised virus isolates: short-finned eel ranavirus (SERV), Rana esculenta virus Italy 282/I02 (REV 282/I02) and pike-perch iridovirus (PPIV). Doctor fish virus (DFV) and guppy virus 6 (GV6) were distinguished as a group from the other viruses. In addition, all 11 isolates were analysed and compared based on nucleotide sequences from 3 different genomic regions: major capsid protein (MCP), DNA polymerase and NF-H1. The partial DNA polymerase gene was sequenced from all analysed viruses. The complete sequence of the MCP and a fragment of the NF-H1 gene were obtained from BIV, ECV, EHNV, ESV, FV3, PPIV, REV 282/I02 and SERV. With the exception of GV6, DFV and SGIV, the sequence analyses showed only a few variations within the analysed viruses. The sequence data suggest that PPIV, REV 282/I02 and SERV are new members of the genus Ranavirus. The methods developed in this study provide tools to differentiate between closely related ranaviruses of different host and geographical origin. PMID:19694168

  14. Factors influencing boar sperm cryosurvival.

    PubMed

    Roca, J; Hernández, M; Carvajal, G; Vázquez, J M; Martínez, E A

    2006-10-01

    Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that

  15. Classification of ostrich sperm characteristics.

    PubMed

    Smith, A M J; Bonato, M; Dzama, K; Malecki, I A; Cloete, S W P

    2016-05-01

    The success of assisted reproduction techniques is dependent on a sound foundation of understanding sperm characteristics to evaluate so as to improve semen processing. This study offers a descriptive basis for ostrich semen quality in terms of sperm function characteristics (SFC) that include motility, measured by computer assisted sperm analysis CASA (SCA(®)), viability (SYBR14/PI) and membrane integrity (hypo-osmotic swelling test). Relationships among these SFC's were explored and described by correlations and regressions. Certain fixed effects including the dilution of semen, season, year and male associated with semen collection were interpreted for future applications. The seasonal effect on sperm samples collected throughout the year suggested that it is prudent to restrict collections to spring and summer when SFC's and sperm concentration are maximized, compared to winter when these aspects of sperm quality are suppressed. Dilution of ejaculates helped to maintain important SFC's associated with fertilization success. The SFC's and sperm concentration varied among males, with specific males, having greater values for the percentage of motile (MOT) and progressively motile (PMOT) sperm, as well as sperm velocity (VCL, VSL, VAP) and linearity (LIN) variables. Males may thus be screened on these variables for inclusion in an artificial insemination (AI) programme to optimize fertility success rates. PMID:27039985

  16. Sperm storage in caecilian amphibians

    PubMed Central

    2012-01-01

    Background Female sperm storage has evolved independently multiple times among vertebrates to control reproduction in response to the environment. In internally fertilising amphibians, female salamanders store sperm in cloacal spermathecae, whereas among anurans sperm storage in oviducts is known only in tailed frogs. Facilitated through extensive field sampling following historical observations we tested for sperm storing structures in the female urogenital tract of fossorial, tropical caecilian amphibians. Findings In the oviparous Ichthyophis cf. kohtaoensis, aggregated sperm were present in a distinct region of the posterior oviduct but not in the cloaca in six out of seven vitellogenic females prior to oviposition. Spermatozoa were found most abundantly between the mucosal folds. In relation to the reproductive status decreased amounts of sperm were present in gravid females compared to pre-ovulatory females. Sperm were absent in females past oviposition. Conclusions Our findings indicate short-term oviductal sperm storage in the oviparous Ichthyophis cf. kohtaoensis. We assume that in female caecilians exhibiting high levels of parental investment sperm storage has evolved in order to optimally coordinate reproductive events and to increase fitness. PMID:22672478

  17. Characterization of integral membrane proteins of Leishmania major by Triton X-114 fractionation and analysis of vaccination effects in mice.

    PubMed Central

    Murray, P J; Spithill, T W; Handman, E

    1989-01-01

    The total integral membrane proteins of promastigotes of Leishmania major were extracted by using the Triton X-114 phase separation technique and were characterized by immunoprecipitation, Western blotting (immunoblotting), and lectin chromatography. Of the 40 or more proteins which partitioned into the detergent phase, only about 10 proteins could be surface radioiodinated on live promastigotes, suggesting their surface orientation. The abundance of the gp58-63 antigen varied markedly between two strains of L. major. Sera from patients with visceral leishmaniasis caused by Leishmania donovani chagasi recognized the gp58-63 complex and an additional Mr-42,000 polypeptide shared between L. major and L. donovani chagasi. A subpopulation of six surface proteins, including the abundant gp58-63 antigen and a group of proteins of Mr 81,000 to 105,000, were glycoproteins recognized by antiserum to wheat germ agglutinin- or concanavalin A-binding proteins. The membrane proteins of the LRC-L119 isolate of L. major could successfully vaccinate genetically susceptible mice, thus opening the way for a molecularly defined subunit vaccine composed of glycolipid and membrane protein antigens. Images PMID:2731987

  18. The immunization-induced antibody response to the Anaplasma marginale major surface protein 2 and its association with protective immunity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many vector-borne pathogens evade clearance via rapid variation in immunogenic surface expressed proteins. In the case of A. marginale, the generation of major surface protein 2 (Msp2) variants allows for immune escape and long-term pathogen persistence. In the experiments reported here, we pose t...

  19. Identification of quantitative trait loci (QTL) controlling protein, oil, and five major fatty acids’ contents in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improved seed composition in soybean (Glycine max L. Merr.) for protein and oil quality is one of the major goals of soybean breeders. A group of genes that act as quantitative traits with their effects can alter protein, oil, palmitic, stearic, oleic, linoleic, and linolenic acids percentage in soy...

  20. Sea urchin sperm antigens mediating the acrosome reaction

    SciTech Connect

    Trimmer, J.S.

    1987-01-01

    The study of sea urchin sperm antigens mediating the acrosome reactions (AR) has been undertaken. Monoclonal antibodies (mAbs) have been isolated reacting with a number of sperm surface antigens. These mAbs have been used in functional assays to attempt to infer the roles of these proteins in the induction of the AR. These mAbs have also been used to isolate protein for biochemical characterization and reconstitution studies. mAbs reacting with a 210 kD protein of the sea urchin sperm plasma membrane have been used to identify this protein as playing a role in the regulation of ion fluxes during the induction of the AR. mAbs reacting with certain extracellular regions inhibit the induction of: the AR, the long duration {sup 45}Ca{sup 2+} uptake into the mitochondrion, and H{sup +} efflux. Addition of these same mAbs, however, induces an increase in sperm (Ca{sup 2+}){sub i} to levels much higher than those induced by FSG, as monitored by the fluorescent Ca{sup 2+} indicators fura 2 and indo 1. This (Ca{sup 2+}){sub i} increase occurs without an increase in pH{sub i}, and thus allows for the first time the analysis of the effects of increasing sperm (Ca{sup 2+}){sub i} ion the absence of increased pH{sub i}.

  1. Regulation and Function of Phosphorylation on VP8, the Major Tegument Protein of Bovine Herpesvirus 1

    PubMed Central

    Zhang, Kuan; Afroz, Sharmin; Brownlie, Robert; Snider, Marlene

    2015-01-01

    ABSTRACT The major tegument protein of bovine herpesvirus 1 (BoHV-1), VP8, is essential for virus replication in cattle. VP8 is phosphorylated in vitro by casein kinase 2 (CK2) and BoHV-1 unique short protein 3 (US3). In this study, VP8 was found to be phosphorylated in both transfected and infected cells but was detected as a nonphosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral life cycle. The regulation and function of VP8 phosphorylation by US3 and CK2 were further analyzed. An in vitro kinase assay, site-directed mutagenesis, and liquid chromatography-mass spectrometry were used to identify the active sites for US3 and CK2. The two kinases phosphorylate VP8 at different sites, resulting in distinct phosphopeptide patterns. S16 is a primary phosphoreceptor for US3, and it subsequently triggers phosphorylation at S32. CK2 has multiple active sites, among which T107 appears to be the preferred residue. Additionally, CK2 consensus motifs in the N terminus of VP8 are essential for phosphorylation. Based on these results, a nonphosphorylated VP8 mutant was constructed and used for further studies. In transfected cells phosphorylation was not required for nuclear localization of VP8. Phosphorylated VP8 appeared to recruit promyelocytic leukemia (PML) protein and to remodel the distribution of PML in the nucleus; however, PML protein did not show an association with nonphosphorylated VP8. This suggests that VP8 plays a role in resisting PML-related host antiviral defenses by redistributing PML protein and that this function depends on the phosphorylation of VP8. IMPORTANCE The progression of VP8 phosphorylation over time and its function in BoHV-1 replication have not been characterized. This study demonstrates that activation of S16 initiates further phosphorylation at S32 by US3. Additionally, VP8 is phosphorylated by CK2 at several residues, with T107 having the highest level

  2. S-allyl cysteine ameliorates the quality of sperm and provides protection from age-related sperm dysfunction and oxidative stress in rats.

    PubMed

    Takemura, Shigekazu; Ichikawa, Hiroshi; Naito, Yuji; Takagi, Tomohisa; Yoshikawa, Toshikazu; Minamiyama, Yukiko

    2014-11-01

    Reactive oxygen species play a central role in the pathophysiology of the age-related decrease in male fertility. It has been reported that the total protein of DJ-1 was decreased in a proteomic analysis of seminal plasma from asthenozoospermia patients and a DJ-1 protein acts as a sensor of cellular redox homeostasis. Therefore, we evaluated the age-related changes in the ratio of the oxidized/reduced forms of the DJ-1 protein in the epididymis. In addition, the protective effects of S-allyl cysteine (SAC), a potent antioxidant, were evaluated against sperm dysfunction. Male rats aged 15-75 weeks were used to assess age-associated sperm function and oxidative stress. Sperm count increased until 25 weeks, but then decreased at 50 and 75 weeks. The rate of sperm movement at 75 weeks was decreased to approximately 60% of the rate observed at 25 weeks. Expression of DJ-1 decreased, but oxidized-DJ-1 increased with age. In addition, 4-hydroxy-2-nonenal modified proteins in the epididymis increased until 50 weeks of age. The total number and DNA synthetic potential of the sperm increased until 25 weeks, and then decreased. In rats 75 weeks of age, SAC (0.45% diet) attenuated the decrease in the number, motility, and DNA synthesis of sperm and inhibited the oxidized proteins. These results suggest that SAC ameliorates the quality of sperm subjected to age-associated oxidative stress. PMID:25411519

  3. Involvement of opsins in mammalian sperm thermotaxis

    PubMed Central

    Pérez-Cerezales, Serafín; Boryshpolets, Sergii; Afanzar, Oshri; Brandis, Alexander; Nevo, Reinat; Kiss, Vladimir; Eisenbach, Michael

    2015-01-01

    A unique characteristic of mammalian sperm thermotaxis is extreme temperature sensitivity, manifested by the capacity of spermatozoa to respond to temperature changes of <0.0006 °C as they swim their body-length distance. The identity of the sensing system that confers this exceptional sensitivity on spermatozoa is not known. Here we show that the temperature-sensing system of mammalian spermatozoa involves opsins, known to be G-protein-coupled receptors that act as photosensors in vision. We demonstrate by molecular, immunological, and functional approaches that opsins are present in human and mouse spermatozoa at specific sites, which depend on the species and the opsin type, and that they are involved in sperm thermotaxis via two signalling pathways—the phospholipase C and the cyclic-nucleotide pathways. Our results suggest that, depending on the context and the tissue, mammalian opsins act not only as photosensors but also as thermosensors. PMID:26537127

  4. Proteomics and the genetics of sperm chromatin condensation

    PubMed Central

    Oliva, Rafael; Castillo, Judit

    2011-01-01

    Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies. PMID:21042303

  5. Sperm quality and cryopreservation of Brazilian freshwater fish species: a review.

    PubMed

    Viveiros, A T M; Godinho, H P

    2009-03-01

    The Brazilian freshwater fish diversity is the richest in the world. Only 0.7% of all Brazilian species have had any aspect of their sperm biology addressed up to this date. The majority of the fish species described in this review migrate during the spawning season (a phenomenon known as piracema). Urbanization, pollution, hydroelectric dams and deforestation are some of the causes of stock depletion or even local extinction of some of these species. The knowledge concerning sperm quality and minimum sperm:egg ratio is important to maximize the use of males without reducing hatching rates. Furthermore, sperm cryopreservation and gene banking can guarantee the conservation of genetic diversity and development of adequate breeding programs of native fish species. In this review, we present and evaluate the existing information on Brazilian fish species that have been subject to sperm quality and cryopreservation studies. The following parameters were evaluated: volume of extractable sperm, sperm motility, sperm concentration, freezing media, freezing methods, and post-thaw sperm quality. Although the existing protocols yield relatively high post-thaw motility and fertilization rates, the use of cryopreserved sperm in routine hatchery production is still limited in Brazil. PMID:19189240

  6. Fertility Assessment in Sorraia Stallions by Sperm-Fish and Fkbp6 Genotyping.

    PubMed

    Kjöllerström, H J; do Mar Oom, M; Chowdhary, B P; Raudsepp, T

    2016-06-01

    The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm-FISH and molecular analysis of FKBP6 - a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 64,XY karyotype. The majority of sperm (89%) had normal haploid sex chromosome content, although 11% of sperm carried various sex chromosome aneuploidies. No correlation was found between the percentage of sperm sex chromosome abnormalities and inbreeding, sperm morphology or stallion age. Direct sequencing of FKBP6 exon 4 for SNPs g.11040315G>A and g.11040379C>A revealed that none of the stallions had the susceptibility genotype (A/A-A/A) for IAR. Instead, all animals had a G/G-A/A genotype - a testimony of low genetic variability. The findings ruled out chromosomal abnormalities and genetic predisposition for IAR as contributing factors for subfertility. However, low fertility of the Sorraia stallions could be partly attributed to relatively higher rate of sex chromosome aneuploidies in the sperm. PMID:27020485

  7. Bower-building behaviour is associated with increased sperm longevity in Tanganyikan cichlids.

    PubMed

    Morita, M; Awata, S; Yorifuji, M; Ota, K; Kohda, M; Ochi, H

    2014-12-01

    We investigated the evolutionary relationship between spawning behaviour and sperm motility traits among Tanganyikan mouth-brooding cichlid species that have developed diverse mating behaviours and male sexual traits. Mouth-brooding behaviour is common among these fish, but different species demonstrate a range of spawning behaviours, bower construction, male sexual traits and timing of gamete release. We observed spawning behaviours and compared sperm motility traits of 28 Tanganyikan mouth-brooding cichlids to elucidate the evolutionary correlations between these traits. Sperm longevity was considerably longer in bower-building species that construct crater-shaped spawning sites compared with species that do not build bowers. Male bower builders released sperm in the pit of the bower prior to spawning, and the time from ejaculation to fertilization was longer. Conversely, most mouth-brooding cichlids deposited semen directly into the female buccal cavity, and spawned eggs were immediately picked up to be placed inside the cavity; thus, the time from ejaculation to fertilization was short. These observations suggest that increased sperm longevity is favoured in bower builders. Comparative phylogenetic analyses suggested that bower-building behaviour and greater time from ejaculation to fertilization are associated with the extension of sperm longevity, whereas sperm competition rank does not play a major role. In addition, bower-building behaviour preceded the emergence of increased sperm longevity. These results indicate that the extension of sperm longevity as a result of the emergence of bower builders may have acted as an evolutionary attractor for sperm longevity. PMID:25330280

  8. Sperm Capacitation and Acrosome Reaction in Mammalian Sperm.

    PubMed

    Stival, Cintia; Puga Molina, Lis Del C; Paudel, Bidur; Buffone, Mariano G; Visconti, Pablo E; Krapf, Dario

    2016-01-01

    Physiological changes that endow mammalian sperm with fertilizing capacity are known as sperm capacitation. As part of capacitation, sperm develop an asymmetrical flagellar beating known as hyperactivation and acquire the ability to undergo the acrosome reaction. Together, these processes promote fertilizing competence in sperm. At the molecular level, capacitation involves a series of signal transduction events which include activation of cAMP-dependent phosphorylation pathways, removal of cholesterol, hyperpolarization of the sperm plasma membrane, and changes in ion permeability. In recent years, new technologies have aided in the study of sperm signaling molecules with better resolution, at both spatial and temporal levels, unraveling how different cascades integrate and cooperate to render a fertilizing sperm. Despite this new information, the molecular mechanisms connecting capacitation with acrosomal exocytosis and hyperactivation are not well understood. This review brings together results obtained in mammalian species in the field of sperm capacitation with special focus on those pathways involved in the preparation to undergo the acrosomal reaction. PMID:27194351

  9. Profiles and Majority Voting-Based Ensemble Method for Protein Secondary Structure Prediction

    PubMed Central

    Bouziane, Hafida; Messabih, Belhadri; Chouarfia, Abdallah

    2011-01-01

    Machine learning techniques have been widely applied to solve the problem of predicting protein secondary structure from the amino acid sequence. They have gained substantial success in this research area. Many methods have been used including k-Nearest Neighbors (k-NNs), Hidden Markov Models (HMMs), Artificial Neural Networks (ANNs) and Support Vector Machines (SVMs), which have attracted attention recently. Today, the main goal remains to improve the prediction quality of the secondary structure elements. The prediction accuracy has been continuously improved over the years, especially by using hybrid or ensemble methods and incorporating evolutionary information in the form of profiles extracted from alignments of multiple homologous sequences. In this paper, we investigate how best to combine k-NNs, ANNs and Multi-class SVMs (M-SVMs) to improve secondary structure prediction of globular proteins. An ensemble method which combines the outputs of two feed-forward ANNs, k-NN and three M-SVM classifiers has been applied. Ensemble members are combined using two variants of majority voting rule. An heuristic based filter has also been applied to refine the prediction. To investigate how much improvement the general ensemble method can give rather than the individual classifiers that make up the ensemble, we have experimented with the proposed system on the two widely used benchmark datasets RS126 and CB513 using cross-validation tests by including PSI-BLAST position-specific scoring matrix (PSSM) profiles as inputs. The experimental results reveal that the proposed system yields significant performance gains when compared with the best individual classifier. PMID:22058650

  10. Structures of two Arabidopsis thaliana major latex proteins represent novel helix-grip folds

    SciTech Connect

    Lytle, Betsy L.; Song, Jikui; de la Cruz, Norberto B.; Peterson, Francis C.; Johnson, Kenneth A.; Bingman, Craig A.; Phillips, Jr., George N.; Volkman, Brian F.

    2009-06-02

    Here we report the first structures of two major latex proteins (MLPs) which display unique structural differences from the canonical Bet v 1 fold described earlier. MLP28 (SwissProt/TrEMBL ID Q9SSK9), the product of gene At1g70830.1, and the At1g24000.1 gene product (Swiss- Prot/TrEMBL ID P0C0B0), proteins which share 32% sequence identity, were independently selected as foldspace targets by the Center for Eukaryotic Structural Genomics. The structure of a single domain (residues 17-173) of MLP28 was solved by NMR spectroscopy, while the full-length At1g24000.1 structure was determined by X-ray crystallography. MLP28 displays greater than 30% sequence identity to at least eight MLPs from other species. For example, the MLP28 sequence shares 64% identity to peach Pp-MLP119 and 55% identity to cucumber Csf2.20 In contrast, the At1g24000.1 sequence is highly divergent (see Fig. 1), containing a gap of 33 amino acids when compared with all other known MLPs. Even when the gap is excluded, the sequence identity with MLPs from other species is less than 30%. Unlike some of the MLPs from other species, none of the A. thaliana MLPs have been characterized biochemically. We show by NMR chemical shift mapping that At1g24000.1 binds progesterone, demonstrating that despite its sequence dissimilarity, the hydrophobic binding pocket is conserved and, therefore, may play a role in its biological function and that of the MLP family in general.

  11. Beyond BLASTing: tertiary and quaternary structure analysis helps identify major vault proteins.

    PubMed

    Daly, Toni K; Sutherland-Smith, Andrew J; Penny, David

    2013-01-01

    We examine the advantages of going beyond sequence similarity and use both protein three-dimensional (3D) structure prediction and then quaternary structure (docking) of inferred 3D structures to help evaluate whether comparable sequences can fold into homologous structures with sufficient lateral associations for quaternary structure formation. Our test case is the major vault protein (MVP) that oligomerizes in multiple copies to form barrel-like vault particles and is relatively widespread among eukaryotes. We used the iterative threading assembly refinement server (I-TASSER) to predict whether putative MVP sequences identified by BLASTp and PSI Basic Local Alignment Search Tool are structurally similar to the experimentally determined rodent MVP tertiary structures. Then two identical predicted quaternary structures from I-TASSER are analyzed by RosettaDock to test whether a pair-wise association occurs, and hence whether the oligomeric vault complex is likely to form for a given MVP sequence. Positive controls for the method are the experimentally determined rat (Rattus norvegicus) vault X-ray crystal structure and the purple sea urchin (Strongylocentrotus purpuratus) MVP sequence that forms experimentally observed vaults. These and two kinetoplast MVP structural homologs were predicted with high confidence value, and RosettaDock predicted that these MVP sequences would dock laterally and therefore could form oligomeric vaults. As the negative control, I-TASSER did not predict an MVP-like structure from a randomized rat MVP sequence, even when constrained to the rat MVP crystal structure (PDB:2ZUO), thus further validating the method. The protocol identified six putative homologous MVP sequences in the heterobolosean Naegleria gruberi within the excavate kingdom. Two of these sequences are predicted to be structurally similar to rat MVP, despite being in excess of 300 residues shorter. The method can be used generally to help test predictions of homology via

  12. Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

    PubMed Central

    Mandal, Goutam; Sharma, Mansi; Kruse, Martin; Sander-Juelch, Claudia; Munro, Laura Anne; Wang, Yong; Vilg, Jenny Veide; Tamás, Markus J; Bhattacharjee, Hiranmoy; Wiese, Martin; Mukhopadhyay, Rita

    2012-01-01

    Summary Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defense against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites co-expressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr197 and this phosphorylation requires LmjMPK2 activity. Lys42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. L. mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a MAP kinase. PMID:22779703

  13. Cytochemical evaluation of sperm and lymphocyte DNA content after treatment with 5 N HCl.

    PubMed

    Redi, C A; Garagna, S; Bottiroli, G

    1986-01-01

    In situ as well as extra situm cytochemical methods were used to investigate why the observed Feulgen-DNA value of sperm versus lymphocyte cells is lower than expected. After treatment with 5 N HCl, in situ experiments involving the GCA reaction and the UV cytophotometry showed the loss of DNA in sperm nuclei to be 12% more than that in lymphocyte nuclei. Extra situm study of sperm and lymphocytes treated with 5 N HCl showed the phosphate and DABA contents of sperm to be 35% and 23%, respectively, less than those of lymphocytes. The data suggest that sperm chromatin is much more sensitive than somatic chromatin to HCl depolymerization during the Feulgen reaction, and this can tentatively be attributed to the protein complement of sperm chromatin. PMID:2420758

  14. Sperm Affects Head Sensory Neuron in Temperature Tolerance of Caenorhabditis elegans.

    PubMed

    Sonoda, Satoru; Ohta, Akane; Maruo, Ayana; Ujisawa, Tomoyo; Kuhara, Atsushi

    2016-06-28

    Tolerance to environmental temperature change is essential for the survival and proliferation of animals. The process is controlled by various body tissues, but the orchestration of activity within the tissue network has not been elucidated in detail. Here, we show that sperm affects the activity of temperature-sensing neurons (ASJ) that control cold tolerance in Caenorhabditis elegans. Genetic impairment of sperm caused abnormal cold tolerance, which was unexpectedly restored by impairment of temperature signaling in ASJ neurons. Calcium imaging revealed that ASJ neuronal activity in response to temperature was decreased in sperm mutant gsp-4 with impaired protein phosphatase 1 and rescued by expressing gsp-4 in sperm. Genetic analysis revealed a feedback network in which ASJ neuronal activity regulates the intestine through insulin and a steroid hormone, which then affects sperm and, in turn, controls ASJ neuronal activity. Thus, we propose that feedback between sperm and a sensory neuron mediating temperature tolerance. PMID:27320929

  15. Increased Translocator Protein Distribution Volume, A Marker of Neuroinflammation, in the Brain During Major Depressive Episodes

    PubMed Central

    Setiawan, Elaine; Wilson, Alan A.; Mizrahi, Romina; Rusjan, Pablo M.; Miler, Laura; Rajkowska, Grazyna; Suridjan, Ivonne; Kennedy, James L.; Rekkas, P. Vivien; Houle, Sylvain; Meyer, Jeffrey H.

    2016-01-01

    Importance The neuroinflammatory hypothesis of major depressive disorder (MDD) is supported by several main findings: First, in humans and animals, activation of the immune system causes sickness behaviors that present during a major depressive episode (MDE) such as low mood, anhedonia, anorexia and weight loss. Second, peripheral markers of inflammation are frequently reported in MDD. Third, neuroinflammatory illnesses are associated with high rates of MDE. However, a fundamental limitation of the neuroinflammatory hypothesis is a paucity of evidence for brain inflammation during MDE. To investigate whether microglial activation, an important aspect of neuroinflammation, is present during MDE, [18F]FEPPA positron emission tomography (PET) was applied to measure translocator protein total distribution volume (TSPO VT), an index of TSPO density. Translocator protein density is elevated in activated microglia. Objective To determine whether TSPO VT, is elevated in the prefrontal cortex, anterior cingulate cortex (ACC) and insula in MDE secondary to MDD. Design Case-control study. Setting Tertiary care psychiatric hospital. Participants 20 subjects with MDE secondary to MDD and 20 healthy controls, underwent an [18F]FEPPA PET scan. MDE subjects were medication-free for at least 6 weeks. All participants were otherwise healthy, and non-smoking. Main Outcome Measure TSPO VT was measured in the prefrontal cortex, ACC, and insula. Results In MDE, TSPO VT was significantly elevated in all brain regions examined (multivariate analysis of variance, F15,23=4.46, P=0.001).TSPO VT was increased, on average, by 30% in the prefrontal cortex, ACC and insula. In MDE, greater TSPO VT in the ACC correlated with greater depression severity (ACC: r=0.628, P=0.005). Conclusions and Relevance This finding provides the most compelling evidence to date for brain inflammation, and more specifically microglial activation, in MDE. This is important for improving treatment since it implies

  16. Unraveling the Sperm Bauplan: Relationships Between Sperm Head Morphology and Sperm Function in Rodents.

    PubMed

    Varea-Sánchez, María; Tourmente, Maximiliano; Bastir, Markus; Roldan, Eduardo R S

    2016-07-01

    Rodents have spermatozoa with features not seen in other species. Sperm heads in many rodent species bear one or more apical extensions known as "hooks." The process by which hooks have evolved, together with their adaptive significance, are still controversial issues. In order to improve our understanding of the biological meaning of these sperm head adaptations, we analyzed hook curvature angles, hook length, and overall hook shape in muroid rodents by using geometric morphometrics. We also searched for relationships between hook design and measurements of intermale competition to assess whether postcopulatory sexual selection was an important selective force driving changes in this sperm structure. Finally, we sought possible links between aspects of sperm hook design and sperm velocity as a measure of sperm performance. Results showed that one hook curvature angle is under strong selective pressure. Similarly, hook length appears to be strongly selected by sexual selection, with this selective force also exhibiting a stabilizing role reducing intermale variation in this trait. The adaptive significance of changes in hook structure was supported by the finding that there are strong and significant covariations between hook dimensions and shape and between hook design and sperm swimming velocity. Overall, this study strongly suggests that postcopulatory sexual selection has an important effect on the design of the sperm head that, in turn, is important for enhancing sperm velocity, a function crucial to reaching the vicinity of the female gamete and winning fertilizations under competitive situations. PMID:27281707

  17. Zinc Finger Transcription Factors Displaced SREBP Proteins as the Major Sterol Regulators during Saccharomycotina Evolution

    PubMed Central

    Maguire, Sarah L.; Wang, Can; Holland, Linda M.; Brunel, François; Neuvéglise, Cécile; Nicaud, Jean-Marc; Zavrel, Martin; White, Theodore C.; Wolfe, Kenneth H.; Butler, Geraldine

    2014-01-01

    In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina. PMID:24453983

  18. Major urinary protein (MUP) profiles show dynamic changes rather than individual ‘barcode’ signatures

    PubMed Central

    Thoß, M.; Luzynski, K.C.; Ante, M.; Miller, I.; Penn, D.J.

    2016-01-01

    House mice (Mus musculus) produce a variable number of major urinary proteins (MUPs), and studies suggest that each individual produces a unique MUP profile that provides a distinctive odor signature controlling individual and kin recognition. This ‘barcode hypothesis’ requires that MUP urinary profiles show high individual variability within populations and also high individual consistency over time, but tests of these assumptions are lacking. We analyzed urinary MUP profiles of 66 wild-caught house mice from eight populations using isoelectric focusing. We found that MUP profiles of wild male house mice are not individually unique, and though they were highly variable, closer inspection revealed that the variation strongly depended on MUP band type. The prominent (‘major) bands were surprisingly homogenous (and hence most MUPs are not polymorphic), but we also found inconspicuous (‘minor’) bands that were highly variable and therefore potential candidates for individual fingerprints. We also examined changes in urinary MUP profiles of 58 males over time (from 6 to 24 weeks of age), and found that individual MUP profiles and MUP concentration were surprisingly dynamic, and showed significant changes after puberty and during adulthood. Contrary to what we expected, however, the minor bands were the most variable over time, thus no good candidates for individual fingerprints. Although MUP profiles do not provide individual fingerprints, we found that MUP profiles were more similar among siblings than non-kin despite considerable fluctuation. Our findings show that MUP profiles are not highly stable over time, they do not show strong individual clustering, and thus challenge the barcode hypothesis. Within-individual dynamics of MUP profiles indicate a different function of MUPs in individual recognition than previously assumed and advocate an alternative hypothesis (‘dynamic changes’ hypothesis). PMID:26973837

  19. Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation

    PubMed Central

    Saucedo, Lucía; Buffa, Gabriela N.; Rosso, Marina; Guillardoy, Tomás; Góngora, Adrian; Munuce, María J.

    2015-01-01

    Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility. PMID:25970615

  20. Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation.

    PubMed

    Saucedo, Lucía; Buffa, Gabriela N; Rosso, Marina; Guillardoy, Tomás; Góngora, Adrian; Munuce, María J; Vazquez-Levin, Mónica H; Marín-Briggiler, Clara

    2015-01-01

    Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility. PMID:25970615

  1. Structure of IZUMO1-JUNO reveals sperm-oocyte recognition during mammalian fertilization.

    PubMed

    Ohto, Umeharu; Ishida, Hanako; Krayukhina, Elena; Uchiyama, Susumu; Inoue, Naokazu; Shimizu, Toshiyuki

    2016-06-23

    Fertilization is a fundamental process in sexual reproduction, creating a new individual through the combination of male and female gametes. The IZUMO1 sperm membrane protein and its counterpart oocyte receptor JUNO have been identified as essential factors for sperm-oocyte interaction and fusion. However, the mechanism underlying their specific recognition remains poorly defined. Here, we show the crystal structures of human IZUMO1, JUNO and the IZUMO1-JUNO complex, establishing the structural basis for the IZUMO1-JUNO-mediated sperm-oocyte interaction. IZUMO1 exhibits an elongated rod-shaped structure comprised of a helical bundle IZUMO domain and an immunoglobulin-like domain that are each firmly anchored to an intervening β-hairpin region through conserved disulfide bonds. The central β-hairpin region of IZUMO1 provides the main platform for JUNO binding, while the surface located behind the putative JUNO ligand binding pocket is involved in IZUMO1 binding. Structure-based mutagenesis analysis confirms the biological importance of the IZUMO1-JUNO interaction. This structure provides a major step towards elucidating an essential phase of fertilization and it will contribute to the development of new therapeutic interventions for fertility, such as contraceptive agents. PMID:27309808

  2. Supplementation with Major Royal-Jelly Proteins Increases Lifespan, Feeding, and Fecundity in Drosophila.

    PubMed

    Xin, Xiao-Xuan; Chen, Yong; Chen, Di; Xiao, Fa; Parnell, Laurence D; Zhao, Jing; Liu, Liang; Ordovas, Jose M; Lai, Chao-Qiang; Shen, Li-Rong

    2016-07-27

    The major royal-jelly proteins (MRJPs) are the main constituents responsible for the specific physiological role of royal jelly (RJ) in honeybees. Male and female Drosophila flies were fed diets containing either no MRJPs (A) or casein (B) at 1.25% (w/w) of diet or MRJPs at 1.25% (C), 2.50% (D), or 5.00% (E). Diets B, C, D, and E increased mean lifespan by 4.3%, 9.0%, 12.4%, and 13.9% in males and by 5.8%, 9.7%, 20.0%, and 11.8% in females in comparison to results from diet A, respectively. The diet supplemented with 2.50% MRJPs seems to have the optimal dose to improve both physiological and biochemical measures related to aging in both sexes. Interestingly, lifespan extension by MRJPs in Drosophila was positively associated with feeding and fecundity and up-regulation of copper and zinc-superoxide dismutase (CuZn-SOD) and the Egfr-mediated signaling pathway. This study provides strong evidence that MRJPs are important components of RJ for prolonging lifespan in Drosophila. PMID:27388939

  3. Functional and Immunological Relevance of Anaplasma marginale Major Surface Protein 1a Sequence and Structural Analysis

    PubMed Central

    Cabezas-Cruz, Alejandro; Passos, Lygia M. F.; Lis, Katarzyna; Kenneil, Rachel; Valdés, James J.; Ferrolho, Joana; Tonk, Miray; Pohl, Anna E.; Grubhoffer, Libor; Zweygarth, Erich; Shkap, Varda; Ribeiro, Mucio F. B.; Estrada-Peña, Agustín; Kocan, Katherine M.; de la Fuente, José

    2013-01-01

    Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5′-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5′-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes. PMID:23776456

  4. Penicillamine prevents ram sperm agglutination in media that support capacitation.

    PubMed

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

    2016-02-01

    Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7 ± 2.7% to 2.8 ± 1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation. PMID:26705263

  5. Dephosphorylation of sperm guanylate cyclase during sea urchin fertilization

    SciTech Connect

    Ward, G.E.

    1985-01-01

    When intact Arbacia punctulata spermatozoa are exposed to solubilized egg jelly, the electrophoretic mobility of an abundant sperm flagellar membrane protein changes from an apparent molecular mass of 160 kDa to 150 kDa. A. punctulata spermatozoa can be labeled in vivo with /sup 32/P-labeled cells it was demonstrated that the mobility shift of the 160-kDa protein is due to dephosphorylation. The peptide resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH/sub 2/) is the component of egg jelly which is responsible for inducing the dephosphorylation. The 160/150-kdal sperm membrane protein has been purified to homogeneity by affinity chromatography on concanavalin A-agarose, and identified as sperm guanylate cyclase. The enzymatic activity of the guanylate cyclase is tightly coupled to its phosphorylation state. Resact has been shown to act as a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration-dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for external calcium. This work represents the first demonstration of animal sperm chemotaxis in response to a precisely-defined molecule of egg origin. The results established a new, biologically meaningful function for resact, and may implicate sperm guanylate cyclase and cGMP in flagellar function and the chemotactic response.

  6. Sperm Morphology Assessment in Captive Neotropical Primates.

    PubMed

    Swanson, W F; Valle, R R; Carvalho, F M; Arakaki, P R; Rodas-Martínez, A Z; Muniz, Japc; García-Herreros, M

    2016-08-01

    The main objective of this study was to evaluate sperm morphology in four neotropical primate species to compare the sperm morphological traits and the sperm morphometric parameters as a basis for establishing normative sperm standards for each species. Data from 80 ejaculates collected from four primate species, Callithrix jacchus, Callimico goeldii, Alouatta caraya and Ateles geoffroyi, were analysed for detection of sperm morphological alterations using subjective World Health Organization (WHO-2010) standards and Sperm Deformity Index (SDI) criteria, objective computer-assisted sperm morphometry analysis (CASMA) and subpopulation sperm determination (SSD) methods. There were multiple differences (p < 0.01) observed among primate species in values obtained from WHO-2010, SDI, CASMA and SSD sperm analysis methods. In addition, multiple significant positive and negative correlations were observed between the sperm morphological traits (SDI, Sperm Deformity Index Head Defects, Sperm Deformity Index Midpiece Defects, Sperm Deformity Index Tail Defects, Normal Sperm, Head Defects, Midpiece Defects and Tail Defects) and the sperm morphometric parameters (SSD, Area (A), Perimeter (P), Length (L), Width (W), Ellipticity, Elongation and Rugosity) (p ≤ 0.046). In conclusion, our findings using different evaluation methods indicate that pronounced sperm morphological variation exists among these four neotropical primate species. Because of the strong relationship observed among morphological and morphometric parameters, these results suggest that application of objective analysis methods could substantially improve the reliability of comparative stu