RESUMO
Aeromonas salmonicida is recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. However, the mechanism of immunity to these bacteria in common carp is still not well understood, especially the immune regulation in the gonad to bacterial infection. The aims of our study were to analyze changes in the seminal plasma proteome following A. salmonicida infection in carp males. The observed pathological changes in the tissue (liver, spleen, kidney and testis) morphology and upregulation of immune-related genes (tnfa2, il6a) confirmed the successful infection challenge. Using mass spectrometry-based label-free quantitative proteomics, we identified 1402 seminal plasma proteins, and 44 proteins (20 up- and 24 downregulated) were found to be differentially abundant between infected and control males. Most differentially abundant proteins were involved in the immune response mechanisms, such as acute phase response, complement activation and coagulation, inflammation, lipid metabolism, cell-cell and cell-matrix adhesion, creatine-phosphate biosynthesis and germ cell-Sertoli cell junction signaling. Bacterial infection also caused profound changes in expression of selected genes in the testis and hematopoietic organs, which contributed to changes in seminal proteins. The altered seminal proteins and bacterial proteins in seminal plasma may serve as valuable markers of infection in the testis.
Assuntos
Infecções Bacterianas , Carpas , Doenças dos Peixes , Animais , Infecções Bacterianas/veterinária , Carpas/genética , Genitália Masculina , Imunidade , Masculino , Proteômica , Sêmen/metabolismoRESUMO
In carp aquaculture, hormonal manipulation with an analog of GnRH (Ovopel) and carp pituitary extract (CPE), which act at different levels of the hypothalamic-pituitary-gonadal axis, is a routine practice to enhance sperm production. Our recent studies revealed that hormonal stimulation of male carp was associated with changes in the seminal plasma proteome, including blood origin proteins. Here, we explored whether Ovopel and CPE could affect the blood proteome of male carp. Both preparations induced increases in semen volume, total number of sperm, and testosterone level. However, hormonal stimulation did not affect the plasma cortisol and glucose levels. A comparative proteomic analysis of carp blood plasma between the control (PBS) and the hormonally treated males revealed significant changes (>1.2 <-1.2-fold change, P < 0.05) in the abundance of 30 spots (14 up- and 16 downregulated) and 44 spots (28 up- and 16 downregulated) upon CPE and Ovopel treatment, respectively. The most significantly affected pathways were acute phase response signaling, the coagulation system, LXR/RXR and FXR/RXR activation; however, there were different sets of proteins in Ovopel- and CPE-treated males. The majority of differentially abundant proteins were involved in the regulation of the immune defense response, the response to stress, and complement activation. Moreover hormonal stimulation with CPE markedly increased the bactericidal activity of blood and both preparations caused profound changes in gene expression in hematopoietic organs. This work is important in understanding the biological processes behind the protein-based response to hormonal stimulation of sperm production in fish.
Assuntos
Carpas , Proteoma , Animais , Proteínas Sanguíneas , Carpas/microbiologia , Carpas/fisiologia , Proteínas de Choque Térmico , Masculino , Plasma , Proteômica , Eletroforese em Gel Diferencial BidimensionalRESUMO
Two functionally distinct isoforms of warm-temperature acclimation related 65-kDa protein (Wap65-1 and Wap65-2) with a role in the immune response are present in fish. To our knowledge, contrary to Wap65-1, Wap65-2 has neither been isolated nor functionally characterized in carp especially in reproductive system. The aim of this study was to characterize Wap65-2 and ascertain its functions in immune response and temperature acclimation within reproductive system. Wap65-2 corresponded to one of the most abundant proteins in carp seminal plasma, with a high immunologic similarity to their counterparts in seminal plasma of other fish species and a wide tissue distribution, with predominant expression in the liver. The immunohistochemical localization of Wap65-2 to spermatogonia, Leydig cells, and the epithelium of blood vessels within the testis suggests its role in iron metabolism during spermatogenesis and maintenance of blood-testis barrier integrity. Wap65-2 secretion by the epithelial cells of the spermatic duct and its presence around spermatozoa suggests its involvement in the protection of spermatozoa against damage caused by heme released from erythrocytes following hemorrhage and inflammation. Our results revealed an isoform-specific response of Wap65 to temperature acclimation and Aeromonas salmonicida infection which alters blood-testis barrier integrity. Wap65-2 seems to be related to the immune response against bacteria, while Wap65-1 seems to be involved in temperature acclimation. This study expands the understanding of the mechanism of carp testicular immunity against bacterial challenge and temperature changes, in which Wap65-2 seems to be involved and highlights their potential usefulness as biomarkers of inflammation and temperature acclimation.
Assuntos
Aclimatação/fisiologia , Carpas/fisiologia , Proteínas de Peixes/química , Sêmen/química , Testículo/imunologia , Aeromonas salmonicida , Animais , Clonagem Molecular , Doenças dos Peixes/metabolismo , Doenças dos Peixes/microbiologia , Proteínas de Peixes/metabolismo , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Masculino , Isoformas de Proteínas , TemperaturaRESUMO
The environmental temperature affects plasma biochemical indicators, antioxidant status and hematological and immunological parameters in fish. So far, only single blood proteins have been identified in response to temperature changes. The aim of this study was to compare the proteome of carp blood plasma from males acclimated to warm (30⯰C) and cold (10⯰C) temperatures by two-dimensional differential gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. A total of 47 spots were found to be differentially regulated by temperature (>1.2-fold change, pâ¯<â¯0.05): 25 protein spots were more abundant in warm-acclimated males and 22 were enriched in cold-acclimated males. The majority of differentially regulated proteins were associated with acute phase response signalling involved in: i) activation of the complement system (complement C3-H1), ii) neutralization of proteolytic enzymes (inter-alpha inhibitor H3, fetuin, serpinA1, antithrombin, alpha2-macroglobulin), iii) scavenging of free hemoglobin and radicals (haptoglobin, Wap65â¯kDa), iv) clot-formation (fibrinogen beta and alpha chain, T-kininogen) and v) the host's immune response modulation (ApoA1 and ApoA2). However, quite different sets of these proteins or proteoforms were involved in response to cold and warm temperatures. In addition, cold acclimation seems to be related to the proteins involved in lipid metabolism (apolipoproteins A and 14â¯kDa) and stress response (corticosteroid binding globulin). We discovered a strongly regulated protein Cap31 upon cold acclimation, which can serve as a potential blood biomarker of cold response in carp. These studies significantly extend our knowledge concerning mechanisms underlying thermal adaptation in poikilotherms.
Assuntos
Aclimatação/imunologia , Proteínas Sanguíneas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Metabolismo dos Lipídeos/imunologia , Estresse Fisiológico/imunologia , Reação de Fase Aguda/imunologia , Animais , Proteínas Sanguíneas/imunologia , Proteínas Sanguíneas/metabolismo , Carpas/sangue , Carpas/fisiologia , Temperatura Baixa , Eletroforese em Gel Bidimensional/veterinária , Temperatura Alta , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , ProteômicaRESUMO
Spermatozoa of externally fertilizing freshwater fish possess several different modes of motility activation. Spermatozoa of common carp (Cyprinus carpio L.) are activated by hypoosmolality, whereas spermatozoa of sterlet (Acipenser ruthenus) require Ca2+ and low concentration of K+ for motility activation. Intracellular signaling differs between these two species as well, particularly in terms of utilization of secondary messengers (cAMP and Ca2+), and kinase activities. The current study was performed in order to determine the importance of protein phosphorylation and protein kinases for activation of sperm motility in carp and sterlet. Treatment with kinase inhibitors indicates that protein kinases A and C (PKA and PKC) participate in spermatozoa motility of both species. Immunodetection of phospho-(Ser/Thr) PKA substrates shows that phosphorylated proteins are localized differently in spermatozoa of carp and sterlet. Strong phosphorylation of PKC substrate was observed in flagella of sterlet spermatozoa, whereas in carp sperm, PKC substrates were lightly phosphorylated in the midpiece and flagella. Motility activation induced either phosphorylation or dephosphorylation on serine, threonine and tyrosine residues of numerous proteins in carp and sterlet spermatozoa. Proteomic methods were used to identify proteins whose phosphorylation state changes upon the initiation of sperm motility. Numerous mitochondrial and glycolytic enzymes were identified in spermatozoa of both species, as well as axonemal proteins, heat shock proteins, septins and calcium-binding proteins. Our results contribute to an understanding of the roles of signaling molecules, protein kinases and protein phosphorylation in motility activation and regulation of two valuable fish species, C. carpio and A. ruthenus.
Assuntos
Carpas , Proteínas de Peixes/metabolismo , Peixes , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Animais , Carpas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peixes/metabolismo , Masculino , Fosforilação , Proteína Quinase C/metabolismo , Proteômica , Transdução de SinaisRESUMO
The presence of the low-molecular-mass serine protease inhibitor Kazal-type (Spink) is a characteristic feature of vertebrate semen. Its main function is control of the serine protease in the acrosome, acrosin. Here we showed for the first time that Spink is present in the seminal plasma of carp, which have anacrosomal spermatozoa. Using a three-step isolation procedure that consisted in gel filtration and RP-HPLC and re-RP-HPLC, we isolated this inhibitor and identified it as serine protease inhibitor Kazal-type 2 (Spink2), a reproductive-derived member of the Spink family. The cDNA sequence of this inhibitor obtained from carp testis encoded 77 amino acids, including a 17 amino acids signal peptide; this sequence was distinct from fish Kazal-type inhibitors. The mRNA expression analysis showed that Spink2 is expressed predominantly in carp testis and spermatic duct. Immunohistochemical analysis demonstrated its localization in testis in Sertoli, Leydig and germ cells at all developmental stages (with the exception of spermatozoa) and in the epithelium of the spermatic duct. Aside from strong inhibition of trypsin, this inhibitor acts strongly against subtilisin and possesses bacteriostatic activities against Lactobacillus subtilis, Escherichia coli and Aeromonas hydrophila. The localization of Spink2 in carp reproductive tract suggests an important function in spermatogenesis and in maintenance of the microenvironment in which sperm maturation occurs and sperm are stored. Our results suggest that Spink2 from carp seminal plasma may play a role in antibacterial semen defense, protecting semen against unwanted proteolysis within the reproductive tract.
Assuntos
Carpas/genética , Proteínas de Peixes/genética , Genitália Masculina/metabolismo , Inibidores de Serina Proteinase/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/metabolismo , Sequência de Bases , Carpas/classificação , Carpas/imunologia , Carpas/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli/fisiologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Lactobacillus/fisiologia , Masculino , Filogenia , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sêmen/metabolismo , Alinhamento de Sequência/veterinária , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismoRESUMO
The effect of reactive oxygen species production on the motility parameters, DNA integrity, acid phosphatase activity, and protein tyrosine phosphorylation in spermatozoa of the common carp (Cyprinus carpio) was investigated. Spermatozoa were exposed to different concentrations of xanthine and xanthine oxidase (X-XO) either in the presence or absence of antioxidants for 15 and 60 min. A dose- and time-dependent reduction in spermatozoa motility and velocity was observed. Comet assays showed a dramatic increase in DNA fragmentation after 15 min. Changes in tyrosine phosphorylation of spermatozoa proteins were observed by Western blotting with anti-phosphotyrosine antibodies, and proteins of interest were identified by mass spectrometry. After a 60 min exposure to X-XO, O-linked N-acetylglucosamine transferase, isoform 4 was phosphorylated and septin-8-A was dephosphorylated. Acid phosphatase activity also decreased in a dose-dependent manner after a 60 min exposure to oxidative stress. The results demonstrate that oxidative stress impaired functional variables (sperm motility, velocity, DNA integrity) of carp spermatozoa, and altered intracellular signalling pathways through changes in tyrosine phosphorylation and acid phosphatase activity.
Assuntos
Fosfatase Ácida/metabolismo , Carpas/metabolismo , Fragmentação do DNA , Proteínas de Peixes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Animais , Masculino , FosforilaçãoRESUMO
Antimicrobial proteins such as apolipoproteins A (ApoA-I and ApoA-II) play an important role in the primary defence barrier in vertebrates including fish. The aims of the present study were to isolate and characterise rainbow trout seminal plasma ApoA-I and ApoA-II, to examine the mRNA expression of each apolipoprotein in testis and spermatic ducts, and to test the antibacterial properties of the apolipoproteins. Using a three-step isolation procedure consisting of ion-exchange chromatography, gel filtration and preparative SDS-PAGE, apolipoproteins were purified and identified as ApoA-I and ApoA-II. Both apolipoproteins were represented by several proteoforms. The expression of ApoA-I and ApoA-II mRNA in the reproductive tract and their antibacterial properties against Escherichia coli suggest that seminal apolipoproteins play an important role in innate immunity in the rainbow trout reproductive tract. The functions of seminal ApoA can be related to protection of sperm and reproductive tissue from microbial attack and to the maintenance of sperm membrane integrity.
Assuntos
Antibacterianos/metabolismo , Apolipoproteína A-II/genética , Apolipoproteína A-I/genética , Proteínas de Peixes/genética , Imunidade Inata , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Animais , Apolipoproteína A-I/metabolismo , Apolipoproteína A-II/metabolismo , Escherichia coli/fisiologia , Proteínas de Peixes/metabolismo , Expressão Gênica , Masculino , Oncorhynchus mykiss/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sêmen/química , Testículo/metabolismo , Ducto Deferente/metabolismoRESUMO
Apolipoproteins A are multifunctional proteins that, in addition to contributing to lipid metabolism and transport, are associated with the innate immune system in fish. Using a three step isolation procedure consisting of affinity chromatography on Blue-Sepharose, delipidation and reverse phase HPLC we isolated apolipoproteins from carp seminal plasma and identified them as ApoA-I and Apo-14 kDa. Moreover, we provided the full-length cDNA sequence of ApoA-I encoding 257 amino acids including a 18 amino acid signal peptide and a 4 amino acid propeptide. Apolipoproteins corresponded to the most abundant proteins in carp seminal plasma. Both ApoA-I and Apo-14 kDa were represented by several proteoforms that differ both in molecular mass and isoelectric point. The proteoforms of ApoA-I characteristic for seminal plasma were distinguished from those of blood. Carp seminal plasma ApoA-I and Apo-14 kDa showed a high immunologic similarity to their counterparts in carp blood and seminal plasma of other Cyprinid species. The mRNA expression analysis and immunohistochemical study suggest synthesis and secretion of ApoA-I and Apo-14 kDa in the fish reproductive tract and suggest a role in spermatogenesis and the stabilization of sperm membrane. Moreover, ApoA-I displayed bactericidal activity against Escherichia coli and bacteriostatic activity against Aeromonas hydrophila which suggests that ApoA-I is associated with innate immune system of the fish reproductive tract.
Assuntos
Apolipoproteínas A/genética , Apolipoproteínas A/imunologia , Infecções Bacterianas/veterinária , Carpas , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Sêmen/química , Aeromonas hydrophila , Análise de Variância , Animais , Apolipoproteínas A/análise , Infecções Bacterianas/imunologia , Western Blotting , Cromatografia Líquida de Alta Pressão , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/genética , DNA Complementar/genética , Escherichia coli , Imuno-Histoquímica , Fígado/metabolismo , Masculino , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Testículo/metabolismoRESUMO
Parvalbumins (Pv) are calcium-binding proteins present mainly in the muscle and nervous system where they act as a Ca(2+) buffer. Our previous work demonstrated the presence of Pv-I in carp semen and indicated the presence of a second Pv (Pv-II). The purpose of the present work was to identify, purify and determine the full-length cDNA sequence of Pv-II from carp testis. Pv-II from seminal plasma was purified by ion-exchange chromatography (IEC) and preparative electrophoresis, while the Pv-II from spermatozoa was purified by IEC, gel filtration and preparative electrophoresis. The purified Pv-II was submitted to an analysis of molecular mass, isoelectric point (pI), amino-acid sequence and oligomerisation ability. The amino-acid sequence was used to construct primers and obtain the full-length cDNA sequence of seminal-specific Pv-II from carp testis. Analysis of the cDNA sequence indicated that carp-testis Pv-II was distinct from carp-muscle parvalbumins. Pv-II was distinct from Pv-I regarding sequence, molecular mass and pI. Both parvalbumins had the ability to form oligomers or to bind to other proteins. Carp seminal plasma had a protective effect against parvalbumin oligomerisation. Pv-II underwent post-translational modification such as n-acetylation and cysteinylation. The present study is the first to report the full-length cDNA sequence of parvalbumin from carp testis.
Assuntos
Carpas/genética , DNA Complementar/genética , Parvalbuminas/genética , Parvalbuminas/isolamento & purificação , Sêmen/química , Análise de Sequência de DNA , Espermatozoides/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Carpas/metabolismo , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Ponto Isoelétrico , Masculino , Dados de Sequência Molecular , Peso Molecular , Parvalbuminas/química , Isoformas de Proteínas , Processamento de Proteína Pós-Traducional , Análise de Sequência de ProteínaRESUMO
Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods-liquid chromatographyâmass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability.
Assuntos
Criopreservação , Crioprotetores , Peixes , Proteoma , Preservação do Sêmen , Motilidade dos Espermatozoides , Espermatozoides , Animais , Masculino , Criopreservação/métodos , Crioprotetores/farmacologia , Espermatozoides/metabolismo , Espermatozoides/efeitos dos fármacos , Proteoma/metabolismo , Peixes/metabolismo , Peixes/fisiologia , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Proteômica/métodos , Metanol/farmacologiaRESUMO
The AID/APOBECs are a group of zinc-dependent cytidine deaminases that catalyse the deamination of bases in nucleic acids, resulting in a cytidine to uridine transition. Secreted novel AID/APOBEC-like deaminases (SNADs), characterized by the presence of a signal peptide are unique among all of intracellular classical AID/APOBECs, which are the central part of antibody diversity and antiviral defense. To date, there is no available knowledge on SNADs including protein characterization, biochemical characteristics and catalytic activity. We used various in silico approaches to define the phylogeny of SNADs, their common structural features, and their potential structural variations in fish species. Our analysis provides strong evidence of the universal presence of SNAD1 proteins/transcripts in fish, in which expression commences after hatching and is highest in anatomical organs linked to the immune system. Moreover, we searched published fish data and identified previously, "uncharacterized proteins" and transcripts as SNAD1 sequences. Our review into immunological research suggests SNAD1 role in immune response to infection or immunization, and interactions with the intestinal microbiota. We also noted SNAD1 association with temperature acclimation, environmental pollution and sex-based expression differences, with females showing higher level. To validate in silico predictions we performed expression studies of several SNAD1 gene variants in carp, which revealed distinct patterns of responses under different conditions. Dual sensitivity to environmental and pathogenic stress highlights its importance in the fish and potentially enhancing thermotolerance and immune defense. Revealing the biological roles of SNADs represents an exciting new area of research related to the role of DNA and/or RNA editing in fish biology.
Assuntos
Citidina Desaminase , Ácidos Nucleicos , Animais , Desaminase APOBEC-1/genética , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA , CitidinaRESUMO
Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca2+-binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology.
Assuntos
Preservação do Sêmen , Sêmen , Masculino , Animais , Sêmen/fisiologia , Análise do Sêmen/veterinária , Acrosina/análise , Tubulina (Proteína) , Proteômica , Motilidade dos Espermatozoides/fisiologia , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Espermatozoides/fisiologia , Perus/fisiologiaRESUMO
Two-dimensional difference gel electrophoresis (2D-DIGE) appears to be especially useful in quantitative approaches, allowing the co-separation of proteins of control samples and proteins of treated/disease samples on the same gel, eliminating gel-to-gel variability. The principle of 2D-DIGE is to label proteins prior to isoelectric focusing and use three spectrally resolvable fluorescent dyes, allowing the independent labeling of control and experimental samples. This procedure makes it possible to reduce the number of gels in an experiment, allowing the accurate and reproducible quantification of multiple samples. 2D-DIGE has been found to be an excellent methodical tool in several areas of fish research, including environmental pollution and toxicology, the mechanisms of development and disorders, reproduction, nutrition, evolution, and ecology.
Assuntos
Corantes Fluorescentes , Proteômica , Animais , Proteômica/métodos , Coloração e Rotulagem , Eletroforese em Gel Diferencial Bidimensional/métodos , Focalização Isoelétrica , Corantes Fluorescentes/análise , Proteínas , Peixes , Eletroforese em Gel Bidimensional/métodosRESUMO
Lung cancer is responsible for the most cancer-related mortality worldwide and the mechanism of its development is poorly understood. Proteomics has become a powerful tool offering vital knowledge related to cancer development. Using a two-dimensional difference gel electrophoresis (2D-DIGE) approach, we sought to compare tissue samples from non-small-cell lung cancer (NSCLC) patients taken from the tumor center and tumor margin. Two subtypes of NSCLC, adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were compared. Data are available via ProteomeXchange with identifier PXD032736 and PXD032962 for ADC and SCC, respectively. For ADC proteins, 26 significant canonical pathways were identified, including Rho signaling pathways, a semaphorin neuronal repulsive signaling pathway, and epithelial adherens junction signaling. For SCC proteins, nine significant canonical pathways were identified, including hypoxia-inducible factor-1α signaling, thyroid hormone biosynthesis, and phagosome maturation. Proteins differentiating the tumor center and tumor margin were linked to cancer invasion and progression, including cell migration, adhesion and invasion, cytoskeletal structure, protein folding, anaerobic metabolism, tumor angiogenesis, EMC transition, epithelial adherens junctions, and inflammatory responses. In conclusion, we identified several proteins that are important for the better characterization of tumor development and molecular specificity of both lung cancer subtypes. We also identified proteins that may be important as biomarkers and/or targets for anticancer therapy.
Assuntos
Adenocarcinoma , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Eletroforese em Gel Bidimensional , Humanos , Neoplasias Pulmonares/patologia , Margens de Excisão , Eletroforese em Gel Diferencial BidimensionalRESUMO
The lipocalin protein family is a large and diverse group of small extracellular proteins characterised by their ability to bind hydrophobic molecules. In the present study, we describe the isolation procedure for rainbow trout seminal plasma protein, characterised by a moderate migration rate during polyacrylamide gel electrophoresis, providing information regarding its basic features and immunohistochemical localisation. This protein was identified as a lipocalin-type protein (LTP). The molecular mass of LTP was found to be 18,848 Da and it was found to lack any carbohydrate components. Only a few Salmoniformes contain LTP in their seminal plasma. The abundance of LTP in the Sertoli and Leydig cells of the testes of the rainbow trout, as well as in secretory cells of the efferent duct, suggests that this protein is specific for rainbow trout milt, where it acts as a lipophilic carrier protein. Moreover, the specific localisation of LTP in the flagella of the spermatozoa suggests a role for LTP in sperm motility. Further experiments are necessary to identify the endogenous ligands for LTP in rainbow trout seminal plasma and to characterise the binding properties of this protein.
Assuntos
Lipocalinas/isolamento & purificação , Oncorhynchus mykiss/metabolismo , Sêmen/química , Testículo/química , Sequência de Aminoácidos , Animais , Imuno-Histoquímica , Células Intersticiais do Testículo/química , Lipocalinas/análise , Lipocalinas/química , Masculino , Dados de Sequência Molecular , Células de Sertoli/química , Cauda do Espermatozoide/químicaRESUMO
Calcium ions play an important role in the activation of fish sperm movement. The mechanism of their binding in semen is still unknown. The goal of this study was the development of a method for identifying calcium-binding proteins in fish seminal plasma. Two methods of calcium-binding proteins detection were tested with the use of Quin2 and Stains-all dyes. The first method was useful for the identification of calcium-binding proteins of fish seminal plasma. It consisted of proteins separation using SDS-PAGE, transfer on PVDF membrane, incubation with CaCl2, staining with Quin2 and illumination with UV light to reveal calcium-binding protein bands. Using Quin2 allowed the detection of calcium-binding proteins with low and high molecular weight. Electrophoretic species-specific profiles of calcium-binding proteins were identified in the seminal plasma of carp, whitefish, roach, brook trout, brown trout and rainbow trout. Staining of calcium-binding proteins with Quin2 is a quick and safe method, allowing the identification of calcium-binding proteins in fish semen.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Peixes/fisiologia , Sêmen/química , Sêmen/metabolismo , Aminoquinolinas/química , Animais , Corantes Fluorescentes/química , Masculino , Especificidade da Espécie , Coloração e RotulagemRESUMO
The age of the bull is widely accepted to influence the production of sperm, affecting the amount and quality of produced semen, which in turn impacts the results of cryopreservation. However, the exact influence of the maturation process on cryopreserved sperm, as well as the underlying molecular mechanisms of this process, are not fully understood. The goal of this study was to evaluate changes in the proteome of thawed semen (spermatozoa and supernatant) collected from young and adult bulls (n = 6) using the 2D-DIGE approach. The quality of semen was assessed using a CASA system and flow cytometry. We found no significant age-related variation in semen quality, with the exception of the average path velocity of sperm movement, which was higher in adult bulls. Proteomic analysis indicated 15 spermatozoa proteins and 10 supernatant proteins with significant age-related changes. Our results suggest that semen from adult bulls is better equipped with proteins related to energy production, protection of spermatozoa against oxidative stress and fertilizing ability. Proteins increased in abundance in young bull spermatozoa were connected to the cytoskeleton and its development, which strongly suggests that developmental processes are still in progress. In conclusion, our results provide novel insight into the mechanism of the development of the male reproductive system of cattle.
RESUMO
Different strategies are used to meet optimal reproductive performance or manage reproductive health. Although exogenous human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone (GnRH) agonists (A) are commonly used to trigger ovulation in estrous cycle synchronization, little is known about their effect on the ovarian follicle. Here, we explored whether hCG- and GnRH-A-induced native luteinizing hormone (LH) can affect the endocrine and molecular milieus of ovarian preovulatory follicles in pigs at different stages of sexual development. We collected ovaries 30 h after hCG/GnRH-A administration from altrenogest and pregnant mare serum gonadotropin (eCG)-primed prepubertal and sexually mature gilts. Several endocrine and molecular alternations were indicated, including broad hormonal trigger-induced changes in follicular fluid steroid hormones and prostaglandin levels. However, sexual maturity affected only estradiol levels. Trigger- and/or maturity-dependent changes in the abundance of hormone receptors (FSHR and LHCGR) and proteins associated with lipid metabolism and steroidogenesis (e.g., STAR, HSD3B1, and CYP11A1), prostaglandin synthesis (PTGS2 and PTGFS), extracellular matrix remodeling (MMP1 and TIMP1), protein folding (HSPs), molecular transport (TF), and cell function and survival (e.g., VIM) were observed. These data revealed different endocrine properties of exogenous and endogenous gonadotropins, with a potent progestational/androgenic role of hCG and estrogenic/pro-developmental function of LH.
Assuntos
Gonadotropina Coriônica/farmacologia , Ciclo Estral/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Folículo Ovariano/metabolismo , Ovulação/efeitos dos fármacos , Maturidade Sexual/efeitos dos fármacos , Animais , Feminino , Humanos , SuínosRESUMO
Transferrin (Tf) in fish is recognized as a component of non-specific humoral defense mechanisms against bacteria. It is a major protein of common carp seminal plasma but its structure and localization in carp testis is unknown. In this study we developed a simple and efficient three-step purification procedure consisting of affinity chromatography (Con A-Sepharose), hydrophobic interaction chromatography (Phenyl Sepharose) and gel filtration (Superdex 200). The molecular mass of Tf has been determined to be 73.6 kDa and isoelectric point 5.1. The peculiar characteristics of carp transferrin were the lack of carbohydrate component and binding of iron ions by only one functional iron-binding site. Western blot analysis revealed a strong similarity of carp seminal plasma Tf to carp blood Tf and Tf from seminal plasma of other cyprinids but a lower similarity to salmonid and percid fishes. Tf was localized to the blood vessels of the carp testis which strongly suggest that most Tf of carp seminal plasma originates from blood. In conclusion, seminal plasma Tf has a unique structure and is similar or identical to blood Tf.