RESUMO
In the field of 'single cell analysis', many classical strategies like immunofluorescence and electron microscopy are the primary techniques of choice. However, these methodologies are time consuming and do not permit direct identification of specific molecular classes, such as lipids. In the present study, a novel mass spectrometry-based analytical approach was applied to bovine oocytes and embryos. This new metabolomics-based application uses mass spectrometry imaging (MSI), efficient data processing and multivariate data analysis. Metabolic fingerprinting (MF) was applied to the analysis of unfertilised oocytes, 2-, 4- and 8-cell embryos and blastocysts. A semiquantitative strategy for sphingomyelin [SM (16:0)+Na](+) (m/z 725) and phosphatidylcholine [PC (32:0)+Na](+) (m/z 756) was developed, showing that lipid concentration was useful for selecting the best metabolic biomarkers. This study demonstrates that a combination of MF, MSI features and chemometric analysis can be applied to discriminate cell stages, characterising specific biomarkers and relating them to developmental pathways. This information furthers our understanding of fertilisation and preimplantation events during bovine embryo development.
Assuntos
Blastocisto/metabolismo , Metabolômica/métodos , Oócitos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Biomarcadores/metabolismo , Bovinos , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Feminino , Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos , Análise Multivariada , Fosfatidilcolinas/metabolismo , Gravidez , Esfingomielinas/metabolismo , Fatores de TempoRESUMO
Overabundant populations of elk (Cervus elaphus) are a significant concern in some areas of the western United States because of potential ecologic damage and spread of brucellosis to domestic livestock. Brucella abortus is transmitted among elk through direct contact with aborted fetuses, placentas and associated fluids, or postpartum discharge of infected animals. Because transmission of brucellosis is dependent on pregnancy, contraception of cows could be used for both disease and population management. The objective of this study was to evaluate the contraceptive efficacy of a gonadotropin-releasing hormone vaccine (GonaCon) in female elk. In September 2004, cows were given a single immunization of either 1,000 microg (n = 12) or 2,000 microg (n = 10) of GonaCon and compared with a group of adjuvant-treated controls (n = 15). In November 2004, 2005, and 2006, cows were grouped with bulls for the breeding season. Blood samples were taken in February 2005 and March 2006 and 2007 for pregnancy testing, progesterone assays, and antibody titers. For cows given 1,000 microg GonaCon the percentages that were infertile for 2005, 2006, and 2007 were 86%, 90%, and 100%, respectively, compared with 90%, 100%, and 100% for cows given 2,000 microg GonaCon. Rates of infertility for control cows were 23%, 28%, and 0% (P<0.0001). The results indicated that either dose of GonaCon prevented pregnancy of elk cows for at least 3 yr. We concluded that GonaCon use for population management of elk warrants consideration as part of a strategy to control brucellosis.
Assuntos
Brucella abortus/patogenicidade , Brucelose/veterinária , Anticoncepção/veterinária , Anticoncepcionais Femininos/farmacologia , Cervos/microbiologia , Animais , Animais Domésticos , Animais Selvagens , Brucelose/prevenção & controle , Brucelose/transmissão , Brucelose Bovina/prevenção & controle , Brucelose Bovina/transmissão , Bovinos , Anticoncepção/métodos , Gerenciamento Clínico , Reservatórios de Doenças/microbiologia , Reservatórios de Doenças/veterinária , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Feminino , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Masculino , Controle da População , Gravidez , Taxa de GravidezRESUMO
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 fluorescence on post-equatorial segment (P<0.05). There were 72% and 78% reductions (P<0.05) of acrosome fluorescence and intensification (P<0.05) in equatorial fluorescence in non-intact acrosome sperm as compared with acrosome intact cells incubated with isthmic and ampullary ODF. In summary, interactions of BSP A1/A2 and 30 kDa and osteopontin with the sperm membrane undergo modifications dictated by the oviductal fluid. The BSP are thought to modulate cholesterol and phospholipid movement from the sperm membrane and help sperm binding to the oviductal epithelium. Furthermore, our model suggests that OPN participates in sperm-oocyte interaction, affecting fertilization and early embryonic development.
Assuntos
Bovinos/fisiologia , Osteopontina/metabolismo , Oviductos/fisiologia , Proteínas de Plasma Seminal/metabolismo , Espermatozoides/fisiologia , Animais , Ejaculação , Feminino , Imuno-Histoquímica , Masculino , Ligação Proteica , Espermatozoides/citologiaRESUMO
INTRODUCTION: The unicellular trophoblast epithelium of all ruminants so far investigated contains 15-20% binucleate cells with numerous secretory granules. Electron microscope (EM) studies of the domesticated cow, ewe, goat and deer species have established that these BNC migrate out of the trophoblast epithelium to fuse with the apposed maternal uterine epithelial cells or derivative to form fetomaternal tissue throughout pregnancy. However there is one careful EM study of the trophoblast of a wild ruminant, the White-tail deer, which found the usual number of BNC but no evidence of any migration or fusion. Since there are up to 200 species of wild ruminants, it was important to establish whether there really are two possible scenarios for BNC function. MATERIALS AND METHODS: This paper reports a light microscope (LM) immunocytochemical study of cell dynamics in ruminant placentas using 1-2â¯mµ deresinated sections. RESULTS: The results clearly demonstrate that the White-tail deer and all of the other 15 (see Table 1) randomly selected wild ruminants show the same BNC migration and fusion pattern. DISCUSSION: These results suggest that this remarkable cellular behaviour is fundamental to the ruminant evolutionary success.
Assuntos
Movimento Celular/fisiologia , Placenta/citologia , Trofoblastos/citologia , Útero/citologia , Animais , Animais Selvagens , Feminino , Imuno-Histoquímica , Placenta/metabolismo , Gravidez , Ruminantes , Trofoblastos/metabolismo , Útero/metabolismoRESUMO
The expression of proteins in accessory sex gland fluid (AGF) of proven, high use mature Holstein bulls was evaluated. Thirty-seven bulls with documented fertility based on their non-return rates were studied. AGF was obtained by artificial vagina after bulls were surgically equipped with cannulae in the vasa deferentia. Samples of AGF were evaluated by two-dimensional SDS-PAGE, gels stained with Coomassie blue and polypeptide maps analyzed by PDQuest software. A master gel generated by the software representing the best pattern of spots in the AGF polypeptide maps was used as a reference for protein identification. Proteins were identified by Western blots and capillary liquid chromatography-nanoelectrospray ionization tandem-mass spectrometry (CapLC-MS/MS). The product ion spectra were processed using Protein Lynx Global Server 2.1 prior to database search with both PLGS and MASCOT (Matrix Science) software. The entire NCBI database was considered for mass fingerprint matching. An average of 52+/-5 spots was detected in the AGF 2D gels, which corresponded to proteins potentially involved in capacitation (bovine seminal plasma protein-BSP-A1/A2 and A3, BSP 30 kDa, albumin); sperm membrane protection, prevention of oxidative stress, complement-mediated sperm destruction and anti-microbial activity (albumin, clusterin, acidic seminal fluid protein--aSFP, 5'-nucleotidase--5'-NT, phospholipase A2--PLA2); acrosome reaction and sperm-oocyte interaction (PLA2, osteopontin); interaction with the extracellular matrix (tissue inhibitor of metalloproteinase 2, clusterin) and sperm motility (aSFP, spermadhesin Z13, 5'-NT). The 20 spots distinguished in all gels were matched to proteins associated with these functions. Proteins identified by tandem mass spectrometry as ecto-ADP-ribosyltransferase 5 and nucleobindin, never described before in the accessory sex gland secretions, were also detected. In summary, we identified a diverse range of components in the accessory sex gland fluid of a select group of Holstein bulls with documented fertility. Known characteristics of these proteins suggest that they play important roles in sperm physiology after ejaculation.
Assuntos
Bovinos/metabolismo , Genitália Masculina/química , Proteômica , Espermatozoides/fisiologia , Animais , Western Blotting , Eletroforese em Gel Bidimensional , Feminino , Fertilidade , Genitália Masculina/anatomia & histologia , MasculinoRESUMO
We evaluated the relationships between proteins in cauda epididymis fluid (CEF) and fertility scores of dairy bulls. Fertility was expressed as the percentage point deviation (PD) of bull nonreturn rate from the average fertility of all bulls at an artificial insemination center. The number of services for each bull ranged from 1074 to 52 820, and PD values ranged from +7.7% to -6.6%. CEF from 20 bulls was obtained from vasa deferentia cannulae and was separated from sperm by centrifugation immediately after collection. Samples were evaluated by 2-dimensional (2-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis gels stained with Coomassie blue, and polypeptide maps were analyzed by PDQuest software. Protein quantities, defined as the total integrated optical density of the spots, were compared between groups of high-fertility sires (n = 12; PD >or= 0) and low-fertility sires (n = 8; PD < 0) and were also used as independent variables in regression analysis. Proteins were identified by capillary liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry. An average of 118 spots was detected in 2-D maps of the CEF, but we were unable to distinguish any protein that was expressed only in high-fertility or in low-fertility bulls. However, the amount of alpha-L-fucosidase 2 and cathepsin D was 2.3- and 2.4-fold greater (P < .05) in high-fertility than in low-fertility bulls, respectively. Conversely, the intensities of 3 isoforms (24-27 kd; pl 6.3-5.8) of prostaglandin D-synthase (PGDS) were from 3.2- to 2.2-fold greater in low-fertility sires (P < .05). An empirical regression model established that a significant proportion (R(2) = 0.72; P < .0001) of the variation in fertility scores (PD values) was explained by the intensities of cathepsin D and 1 isoform of PGDS (24 kd; pl 6.3). Thus, multiple proteins present in the CEF are potential biomarkers of fertility in high-use, mature Holstein bulls.
Assuntos
Líquidos Corporais/química , Epididimo/química , Fertilidade/fisiologia , Proteínas/química , Animais , Catepsina D/genética , Bovinos , Eletroforese em Gel de Poliacrilamida , Oxirredutases Intramoleculares/genética , Lipocalinas , Masculino , Proteoma , alfa-L-Fucosidase/genéticaRESUMO
We evaluated the expression of proteins in the accessory sex gland fluid (AGF) and their relationships with fertility indexes of dairy bulls. Fertility was normalized as the percentage point deviation of their nonreturn rates (PD) from the average fertility of all bulls from a given artificial insemination center. Services associated with each sire ranged from 269 to 77 321 and PD values from +7.7% to -18.1%. AGF, from 37 bulls, was obtained with an artificial vagina after cannulation of the vasa deferentia. Proteins from AGF were separated by 2-dimensional SDS-PAGE followed by staining with Coomassie blue and analysis of polypeptide maps using PDQuest software. Bulls were divided in groups based on PD values and the optical density of spots in the AGF gels used as independent variables to predict bull fertility. Proteins were identified by capillary liquid chromatography nanoelectrospray ionization tandem mass spectrometry (CapLC-MS/MS). An average of 52 +/- 5 spots was detected in the AGF gels, but there were no spots unique to groups of either high- (PD > or = 0) or low- (PD < 0) fertility sires. The former were neither less nor more homogeneous than the latter based on correlations of all matched spots between pairs of AGF maps. However, high fertility of dairy bulls was significantly associated with lower expression of 14-kDa spermadhesin Z13 isoforms and higher amounts of 55-kDa osteopontin and 58-kDa phospholipase A2 (PLA2) isoforms. The average intensity of 5 spots identified as BSP 30 kDa in the AGF gels had a quadratic association with fertility indexes (R2 = .18; P = .03). PD values of bulls were related (R2 = .56) to the quantity of spermadhesin, osteopontin, and BSP 30 kDa in the AGF polypeptide maps. Bull fertility was also determined by another equation (R2 = .53) with spermadhesin, BSP 30 kDa, and PLA2 as independent variables. We conclude that interactions among several proteins in accessory sex gland fluid explain a significant proportion of the variation in fertility scores of mature dairy sires.
Assuntos
Genitália Masculina/química , Proteínas/química , Proteoma , Sequência de Aminoácidos , Animais , Bovinos , Eletroforese em Gel Bidimensional , Fertilidade , Genitália Masculina/anatomia & histologia , Masculino , Dados de Sequência Molecular , Osteopontina , Fragmentos de Peptídeos/química , Proteínas/isolamento & purificação , Sialoglicoproteínas/química , Sialoglicoproteínas/isolamento & purificaçãoRESUMO
Presence of a Fas-Fas ligand (FasL) system defines the immune-privileged status of certain tissues such as placenta. This study examined the fluids and tissue(s) of the bovine oviduct, where both spermatozoa and early embryos escape elimination by the female immune system, for the presence and the distribution of Fas and FasL, which might provide an explanation for the immune-privileged site of this organ. In the present study, the immunolocalisation of FasL and Fas, as well as the gene expression of FasL, were determined in the uterotubal junction (UTJ), isthmic (I) and ampullar (A) segments of the oviduct during oestrus and the luteal phase of the oestrous cycle. The degree of apoptosis of oviductal epithelium was examined by the TUNEL method. Oviductal fluid (ODF), collected chronically via indwelling catheters from the I or A segments during both non-luteal and luteal phases of the cycle, was analysed for the presence of FasL. The Fas immunostaining was scattered along the epithelium of all regions of the oviduct and cycle stages investigated, whereas FasL immunolabelling was more conspicuous in oestrous samples. This staining disappeared during the luteal phase, which was particularly evident in the sperm reservoir (UTJ and I). There were fewer TUNEL-positive cells than Fas- or FasL-positive cells in the oviductal epithelium, suggesting that tubal Fas and FasL are not directly involved in epithelial apoptosis. Western blot analyses detected FasL in ODF collected from both I and A, most conspicuously as a 24-27kDa band but also at a 40-45kDa band level. FasL mRNA was expressed in the epithelial cells from the sperm reservoir and A during both non-luteal and luteal phases. However, the level of expression differed significantly between segments during the luteal phase. The results provide novel evidence that the Fas-FasL system is present in the bovine oviduct and could be involved in mediating survival of spermatozoa and early embryos.
Assuntos
Bovinos/imunologia , Glicoproteínas de Membrana/imunologia , Oviductos/imunologia , Receptor fas/imunologia , Animais , Apoptose/imunologia , Western Blotting/veterinária , Proteína Ligante Fas , Feminino , Imuno-Histoquímica/veterinária , Marcação In Situ das Extremidades Cortadas/veterinária , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Receptor fas/genética , Receptor fas/metabolismoRESUMO
We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in the male mouse reproductive organs. Northern blotting revealed that the mPGES-1 mRNA was expressed intensely in the epididymis and weakly in the lung, spleen, skin, kidney, colon, and brain. In the male reproductive tract, the expression of mPGES-1 increased from the testis to the cauda epididymis and was highest in the vas deferens when examined by Northern blotting, RT-PCR, and Western blotting. By immunohistochemistry, mPGES-1 was detected in Leydig cells of the testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In addition, the caput and cauda regions of the epididymis and the vas deferens in this order showed a progressive increase in the expression of COX-1 mRNA and immunoreactivity, whereas COX-2 was dominantly expressed in the vas deferens. COX-1 was localized in epithelial cells of the caput, corpus and cauda epididymis and of the vas deferens, and COX-2 was evident in epithelial cells of the distal cauda epididymis and vas deferens. These results show that mPGES-1 is expressed coordinately with COX-1 and COX-2 and is involved in PGE(2) production in male genital organs.
Assuntos
Genitália Masculina/enzimologia , Microssomos/enzimologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Northern Blotting , Western Blotting , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Epididimo/diagnóstico por imagem , Epididimo/metabolismo , Imunofluorescência , Genitália Masculina/ultraestrutura , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Oxirredutases Intramoleculares/metabolismo , Isoenzimas/metabolismo , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/ultraestrutura , Masculino , Proteínas de Membrana , Metiltransferases , Camundongos , Camundongos Endogâmicos C57BL , Microssomos/ultraestrutura , Prostaglandina-E Sintases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Túbulos Seminíferos/metabolismo , Túbulos Seminíferos/ultraestrutura , Ultrassonografia , Ducto Deferente/diagnóstico por imagem , Ducto Deferente/metabolismoRESUMO
Identification of norepinephrine (NE) within the microenvironment of the bovine oviduct suggests a potential role for catecholamines in the events surrounding fertilization. Previous studies have shown that the catecholamines capacitate and induce the acrosome reaction in spermatozoa from several species. The current project was undertaken to investigate the role of catecholamines in bovine sperm capacitation and the acrosome reaction. Freshly ejaculated bovine spermatozoa were incubated in NE (0-1000 ng/mL) and induced to acrosome-react with lysophosphatidylcholine (LPC). Additionally, spermatozoa capacitated with heparin were incubated with NE (0-1000 ng/mL) to assess its ability to induce the acrosome-reaction in capacitated spermatozoa. Concentrations of NE were chosen on the basis of physiological concentrations previously determined for bovine oviductal fluid. NE at concentrations of 10 and 20 ng/mL capacitated bovine spermatozoa after 2 hours of incubation. Additionally, spermatozoa incubated for 2 hours with heparin were induced to acrosome-react with 10 and 20 ng/mL NE. Interestingly, higher concentrations of NE inhibited both capacitation and the acrosome reaction. Incubating spermatozoa with dopamine or epinephrine did not result in capacitation or the acrosome reaction, suggesting that the action of NE was specific to that catecholamine. The ability of NE to capacitate or induce the acrosome reaction appears to be dependent on the presence of another membrane-destabilizing factor. Although adrenergic receptors have not been identified on spermatozoa from any species, the action of NE on spermatozoa may be a receptor-mediated event. This study suggests a possible function for oviductal catecholamines in sperm preparation prior to fertilization.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Norepinefrina/farmacologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Simpatomiméticos/farmacologia , Animais , Bovinos , Dopamina/farmacologia , Epinefrina/farmacologia , Técnicas In Vitro , Masculino , Espermatozoides/fisiologiaRESUMO
Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Several observations suggest that the plasminogen activator/plasmin system might also play a role in mammalian fertilization. Movement characteristics of bovine sperm incubated with different concentrations of plasmin were investigated using a computer-assisted automated semen analysis system. Sperm were incubated up to 4h in a modified Tyrode's medium (control) and 0.1, 1, 10 and 100 mU/ml of plasmin. The percentage motile sperm was significantly higher at 0 h for sperm incubated in 1, 10, and 100 mU of plasmin. Relative to sperm incubated in control medium, lateral head displacement (ALH), curvilinear velocity, beat cross frequency, path velocity and straight line velocity (VSL) of sperm treated with 100 mU of plasmin for 0 h were increased. After 2h of incubation, sperm treated with 100 mU of plasmin showed an increase in ALH, but a decrease in VSL, straightness and linearity. The effect of plasmin on most motility parameters appears to be direct since all these parameters were affected at 0 h of incubation. Our results support the notion of hyperactivation of bovine spermatozoa following incubation with different concentrations of plasmin. The present work provides additional information to further characterize motility movement of bovine sperm associated with final preparation for fertilization.
Assuntos
Bovinos , Fibrinolisina/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Animais , Fertilização , Fibrinolisina/administração & dosagem , Masculino , Espermatozoides/fisiologiaRESUMO
Feral swine were targeted for control at Avon Park Air Force Range in south-central Florida to avert damage to sensitive wetland habitats on the 40,000-ha base. We conducted a 5-year study to assess impacts from control to this population that had been recreationally hunted for many years. Control was initiated in early 2009. The feral swine population was monitored from 2008 to 2012 using a passive tracking index (PTI) during the dry and wet seasons and using recreational hunter take rates from the dry season. All three indices showed substantial feral swine declines after implementing control, with indices leveling for the final two study years. Military missions and recreational hunting seasons impacted temporal and spatial consistency of control application, thereby limiting further impacts of control efforts on the feral swine population. The PTI was also able to monitor coyotes, another invasive species on the base, and detect Florida black bear and Florida panther, species of particular concern.
Assuntos
Animais Selvagens , Instalações Militares , Suínos , Animais , Conservação dos Recursos Naturais , Coiotes , Ecossistema , Florida , Controle da População/métodos , Recreação , Estações do AnoRESUMO
This manuscript provides an overview of past wildlife contraception efforts and discusses the current state of research. Two fertility control agents, an avian reproductive inhibitor containing the active ingredient nicarbazin and an immunocontraceptive vaccine, have received regulatory approval with the Environmental Protection Agency and are commercially available in the USA. OvoControl G Contraceptive Bait for Canada Geese and Ovo Control for pigeons are delivered as oral baits. An injectable immunocontraceptive vaccine (GonaCon Immunocontraceptive Vaccine) was registered with the Environmental Protection Agency for use in female white-tailed deer in September 2009. An injectable product (GonaCon Immunocontraceptive Vaccine) is registered for use in female white-tailed deer. Both products are labeled for use in urban/suburban areas where these species are overabundant. Several other compounds are currently being tested for use in wildlife in the USA, Europe, Australia and New Zealand that could have promise in the future. The development and use of reproductive inhibitors for resolving human-wildlife conflicts will depend on a number of factors, including meeting the requirements of regulatory agencies for use in the environment and on the biological and economical feasibility of their use. Use will also be dependent on health and safety issues and on public acceptance of the techniques.
Assuntos
Anticoncepção Imunológica/veterinária , Anticoncepção/veterinária , Anticoncepcionais/normas , Nicarbazina/uso terapêutico , Controle de Pragas/métodos , Vacinas Anticoncepcionais/uso terapêutico , Animais , Animais Selvagens , Aves , Anticoncepção Imunológica/métodos , Cervos , Hormônio Liberador de Gonadotropina/agonistas , Humanos , Nicarbazina/farmacologia , América do Norte , Controle de Pragas/legislação & jurisprudência , Controle de Pragas/estatística & dados numéricos , Opinião Pública , Vacinas Anticoncepcionais/farmacologiaRESUMO
The proteome of cauda epididymal fluid (CEF) from Holstein bulls was defined. Fluid was collected from the vas deferens, subjected to 2-D SDS-PAGE and spots identified by CapLC-MS/MS and MALDI-ToF/ToF. Because albumin accounted for 21.1% of all spot intensities in the gels examined by PDQuest, samples were subjected to albumin depletion and then analyzed again as before. Original CEF gels had 114 ± 3 spots, including as the most abundant: albumin, epididymal secretory protein E1, prostaglandin d-synthase and gelsolin. Epididymal fluid also expressed: clusterin, transferrin, N-acetyl-ß-glucosaminidase, cauxin, glutathione peroxidase, acidic seminal fluid protein (aSFP), aldehyde reductase, α-l-fucosidase, α-1-ß-glycoprotein, apolipoprotein A-1, ß actin, calmodulin, cathepsin D, cystatin E/M, enolase, galectin 3-binding protein, leucine amino-peptidase and nucleobindin. Albumin depletion decreased that very spot to 10% of its original intensity and the resulting gels had, on average, 137 ± 4 spots. Spots identified as dipeptidyl-peptidase 7, angiotensin-converting enzyme, arylsulfatase A, aspartylglucosaminidase, serine protease inhibitors, new isoforms of calmodulin, cystatin E/M and a 17-kDa nucleobindin appeared only in depleted maps. This study is the first to report nucleobindin and aSFP as epididymal components. We suggest that CEF proteins act to facilitate membrane remodeling, transport of lipophilic substances, protect sperm and prevent premature acrosome reaction.
Assuntos
Líquidos Corporais/química , Epididimo/química , Sequência de Aminoácidos , Animais , Bovinos , Eletroforese em Gel Bidimensional , Masculino , Proteoma/análise , Proteômica , Sêmen , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transporte Espermático/fisiologiaRESUMO
PROBLEM: An effective, single-injection, multi-year, GnRH contraceptive agent is needed to control reproduction in overabundant white-tailed deer populations. METHOD OF STUDY: Two GnRH conjugates, GonaCon (GnRH-KLH) and GonaCon-B (GnRH-blue protein), were prepared in emulsion form as one-injection and two-injection immunocontraceptive vaccine formulations. In addition, the GnRH-KLH protein conjugate was lyophilized and suspended in AdjuVac adjuvant to produce a fifth vaccine formulation. Each formulation was administered to a group of five captive adult female white-tailed deer. Reproductive performance of treated female deer was monitored for 5 years to determine the comparative efficacy of the various treatments. RESULTS: The longevity of the contraceptive response (2-5 years) was strongly influenced by the design of the conjugate antigen, the adjuvant used, and the delivery form of the vaccine. CONCLUSION: One-injection and two-injection formulations of GonaCon and GonaCon-B produced multi-year contraception in adult female white-tailed deer. GonaCon-B provided a longer lasting contraceptive effect.
Assuntos
Anticoncepção Imunológica , Cervos , Hormônio Liberador de Gonadotropina/imunologia , Vacinas Anticoncepcionais/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos/sangue , Cervos/imunologia , Cervos/fisiologia , Feminino , Hormônio Liberador de Gonadotropina/administração & dosagem , Hormônio Liberador de Gonadotropina/síntese química , Hemocianinas/imunologia , Progesterona/sangue , Vacinas Anticoncepcionais/administração & dosagemRESUMO
We previously reported that accessory sex gland fluid (AGF) from high fertility (HF) bulls influenced the oocyte-penetrating capacity of cauda epididymal sperm from low fertility (LF) bulls, based on in vitro fertilization (IVF) assays. The present study determined if AGF proteins were associated with these effects. Nineteen IVF assays with 12 bulls were grouped as follows. Group I (n = 8): assays where sperm from LF bulls exposed to AGF from HF bulls had greater oocyte penetration than exposed to homologous AGF. Group II (n = 7): sperm from LF bulls to AGF from HF bulls versus homologous AGF showed no significant differences. Group III (n = 4): sperm from LF bulls treated with homologous AGF had greater fertility than sperm treated with AGF from HF bulls. Sire fertility was based on nonreturn rates (NNR) and AGF collected by artificial vagina from bulls with cannulated vasa deferentia. Two-dimensional SDS-PAGE maps of AGF were analyzed by PDQuest and proteins identified by tandem mass spectrometry and Western blots. Differences in spot intensity between AGF of HF and LF bulls were compared across groups of IVF assays (P < 0.05). The expression of BSP A1/A2 and A3, BSP 30 kDa, clusterin, albumin, phospholipase A(2) (PLA(2)), and osteopontin was greater in the AGF of HF bulls in Group I as compared to Groups II and III. Conversely, there was less nucleobindin in the AGF of HF bulls in Group I than in Groups II and III. This is the first report of nucleobindin (58 kDa/pI 5.6) in male reproductive fluids, using both immunoblots and mass spectrometry. Thus, the effect of AGF from HF bulls on epididymal sperm is likely the result of specific proteins expressed in the AGF.
Assuntos
Epididimo/fisiologia , Oócitos/fisiologia , Animais , Bovinos , Eletroforese em Gel Bidimensional , Feminino , Fertilidade , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Genitália Masculina/fisiologia , Masculino , Espectrometria de Massas , Proteínas/isolamento & purificação , Capacitação EspermáticaRESUMO
The objective of the present study was to characterize and quantify changes in exposed saccharide residues of bovine sperm during capacitation in oviductal fluid (ODF) using flow cytometry (FC). Bovine sperm were incubated with 0% or 50% non-luteal ODF for 30 min or 3.5 h. After incubation, sperm were labelled with 11 fluorescein isothiocyanate-labelled lectins and evaluated for lectin binding with FC. Furthermore, inhibiting sugars were used to determine specificity of lectin binding to oligosaccharides on the sperm surface. After 30 min incubation, there was a 91% decrease in fluorescence intensity of labelled sperm incubated in WGA, a 76% decline for Con A, 75% decline for BS-I and a 36% decline for DBA. These differences remained approximately the same over the 3.5-h incubation. Interestingly, although there was no reduction in UEA-I binding at 30 min, a significant reduction (23%) was observed at 3.5 h. Con A fluorescence was mostly inhibited with either alpha-d-glucose or alpha-d-mannose (86% and 90% respectively). BS-I fluorescence was reduced after prior incubation of the control samples with N-acetyl-galactosamine and galactose by 74% and 80% respectively. After prior incubation with N-acetyl-galactosamine DBA fluorescence reduced by 18% in the control samples. With UEA-I no fluorescence reduction was observed after prior incubation with l-fucose. We have demonstrated that capacitation of bovine sperm in ODF is accompanied by a quantitative reduction in individual lectin binding sites. These modifications may be crucial to the subsequent signalling events involved with sperm-zona binding, zona penetration or interaction with the oolema.
Assuntos
Carboidratos/fisiologia , Líquido Extracelular/fisiologia , Tubas Uterinas/fisiologia , Lectinas/fisiologia , Espermatozoides/fisiologia , Animais , Sítios de Ligação/fisiologia , Carboidratos/análise , Bovinos , Membrana Celular/química , Membrana Celular/fisiologia , Tubas Uterinas/citologia , Feminino , Citometria de Fluxo , Masculino , Transdução de Sinais/fisiologia , Capacitação Espermática/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/citologiaRESUMO
The pregnancy-associated glycoproteins (PAGs) are placental proteins that have been cloned from swine, sheep, goats, and cattle, but never from animals within the Cervidae family. The goal of this work was to characterize PAGs in white-tailed deer. Placenta and uterine tissues were collected from pregnant does at days 85 and 90 of pregnancy. RNA from cotyledons was used to amplify deer PAGs by RT-PCR. Ten distinct cDNAs were cloned and sequenced. Some normally conserved amino acids comprising the catalytic site were found to be altered in deer PAGs 4, 5, and 8; another PAG, (PAG-9) was a splice variant that lacked exon 7. In each case, these mutations would likely preclude proteolytic activity for these proteins. A phylogenetic analysis revealed that most of the deer PAGs fell within the ancient PAG grouping. The remainder fell within the more modern (BNC-specific) PAG group. Western blotting was performed with anti-PAG antibodies and this analysis revealed that deer PAGs comprise a heterogeneous group based on different antigenicities and electrophoretic mobilities. Immunohistochemistry and in situ hybridization revealed some unique localization patterns of PAGs in the deer placentome compared to those in other ruminants. Most notably, deer PAGs 4 and 5, which according to the phylogeny, are "ancient PAGs," were expected to be present in all trophoblasts; instead, they were localized to the BNC. Although many of the PAGs identified here are very similar to those in Bovidae, some are clearly distinct in their expression pattern and probably possess functional roles unique to cervid reproduction.
Assuntos
Cervos/metabolismo , Glicoproteínas/metabolismo , Proteínas da Gravidez/metabolismo , Trofoblastos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , Cervos/genética , Feminino , Glicoproteínas/análise , Glicoproteínas/genética , Imuno-Histoquímica , Dados de Sequência Molecular , Filogenia , Gravidez , Proteínas da Gravidez/análise , Proteínas da Gravidez/genéticaRESUMO
Osteopontin (OPN) is a secreted extracellular matrix phosphoprotein identified in various tissues and fluids including those of the male and female reproductive tracts. OPN was previously identified as a 55 kDa high fertility marker in Holstein bull seminal plasma, produced by the ampulla and the vesicular gland. The objectives of this study were to characterize OPN on ejaculated and cauda epididymal sperm using immunofluorescence and western blot analysis, and to assess the role of sperm OPN in fertilization. Solubilized sperm membrane proteins from ejaculated and cauda epididymal sperm were separated by 1D SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to bovine milk OPN. A 35 kDa protein was detected by this antibody in both ejaculated and cauda epididymal sperm membranes. Analyses also recognized OPN at 55 and 25 kDa in cauda epididymal fluid and testicular parenchyma homogenates respectively. Immunofluorescent analysis of ejaculated and cauda epididymal sperm showed OPN localization in a well-defined band in the postacrosomal region of the sperm head and also on the midpiece. Results of in vitro fertilization experiments showed that sperm treated with an antibody to OPN fertilized fewer oocytes than sperm treated with control medium while increasing incidence of polyspermy, suggesting a role of sperm-associated OPN in fertilization and a block to polyspermy. These studies demonstrate that OPN exists at multiple molecular weight forms in the bull reproductive tract and its presence on ejaculated sperm may signal its importance in fertilization by interacting with integrins or other proteins on the oocyte plasma membrane.
Assuntos
Bovinos/fisiologia , Epididimo , Osteopontina/análise , Sêmen/química , Espermatozoides/química , Testículo/química , Animais , Biomarcadores/análise , Western Blotting/métodos , Ejaculação/fisiologia , Eletroforese em Gel de Poliacrilamida/métodos , Fertilidade/fisiologia , Fertilização in vitro , Masculino , Interações Espermatozoide-Óvulo/fisiologiaRESUMO
Native porcine zona pellucida (PZP) has been shown to be highly effective as an immunocontraceptive in white-tailed deer. However, the immunogenicity of PZP extracted from pig ovaries may vary from lot to lot and the extract has the potential of containing either viral or pathogenic material. Determination of the immunocontraceptive epitopes of PZP would allow portions of the molecule to be synthesized or inserted into a recombinant system for production of a consistent and safe vaccine. In this study, epitopes of PZP were selected and tested by in vitro binding, immunogenicity in rabbits, immunogenicity and immunocontraception in deer. Sera from PZP immunocontracepted deer were tested on ELISA plates containing immobilized peptides from ZP1 and ZP3alpha. Peptides with which sera from infertile deer reacted (six peptides from ZP1 and six peptides from ZP3alpha) were selected, synthesized and tested for immunogenicity in rabbits. Deer were then immunized with combinations of peptides from either the ZP1 or ZP3alpha groups. ZP3alpha peptides induced high immune titers against native PZP, but did not induce infertility in the deer. Although ZP1 peptides induced lower titers, deer immunized with two ZP1 peptides exhibited multiple estrus events and infertility, typical of that for deer immunized with native PZP vaccine. Competitive inhibition assays using the ZP1 peptides demonstrated that the peptide comprising pins 10-16 was most effective in blocking binding by the serum antibody of native PZP immunized deer. This peptide was used to immunocontracept deer, resulting in a significant reduction in fawning for 1 year.