RESUMO
SDS-PAGE combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 2-dimensional electrophoresis (2DE) combined with matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (MALDI TOF/TOF) were applied to characterize the turkey seminal plasma proteome. LC-MS/MS led to the identification of 175 proteins, which were classified according to their function and to corresponding biochemical pathways. Using 2DE and MALDI TOF/TOF, 34 different turkey seminal plasma proteins could be identified, of which 20 were found in more than one spot, indicating different proteoforms of these proteins. For validation, antibodies against turkey albumin and ovoinhibitor as well as sperm acrosin were used in 2DE Western blots experiments. The bioinformatic analysis of the results indicates that turkey seminal plasma proteins may be involved in regulation of lipid metabolism [liver X receptor/retinoid X receptor (LXR/RXR) activation and farnesoid X receptor/retinoid X receptor (FXR/RXR) activation pathways)], endocytic entry of proteins and lipids at the plasma membrane (clathrin-mediated endocytosis pathway), and defense against pathogens (acute phase response signaling pathway) and energy production (glycolysis and gluconeogenesis). Moreover, a comparative meta-analysis of seminal plasma proteomes from other species indicated the presence of proteins specific for avian reproduction, but distinct differences between turkey and chicken seminal plasma proteomes were detected. The results of our study provide basic knowledge of the protein composition of turkey seminal plasma highlighting important physiological pathways which may play crucial roles in the sperm environment after ejaculation. This knowledge can be the basis to further develop procedures improving the reproduction of farmed turkeys.
Assuntos
Proteoma/metabolismo , Sêmen/metabolismo , Proteínas de Plasma Seminal/genética , Perus/genética , Animais , Masculino , Proteômica , Proteínas de Plasma Seminal/metabolismo , Perus/metabolismoRESUMO
Despite recent advances in bull epididymal fluid proteome research, significant numbers of proteins secreted to epididymal lumen remain unidentified. The objective of this study was to expand the number of identified cauda epididymal fluid proteins in bulls and to contextualize them in a broader view of their mutual interactions and involvement in biological processes and pathways, to fully elucidate the ways in which epididymal fluid proteins are involved in storage and maturation of spermatozoa in epididymis. We collected postmortem cauda epididymal fluid from 6 mature Holstein Friesian bulls. We performed the identification of proteins using 2-dimensional electrophoresis coupled with MALDI mass spectrometry. Analysis of functionality and pathway involvement of identified proteins was performed using Ingenuity Pathway Analysis software. We identified a total of 189 epididymal fluid proteins, out of which 100 were newly identified in bull epididymal fluid. We have combined our data with 2 previously performed bull epididymal fluid proteome identifications, yielding 280 proteins total, and analyzed it. The main canonical pathways involving epididymal proteins were glycolysis, gluconeogenesis, protein ubiquitination pathway, nuclear factor-erythroid 2-related factor 2-mediated oxidative stress response, and farnesoid X receptor/retinoid X receptor activation. The main biological functions potentially performed by epididymal fluid proteins included carbohydrate metabolism, cellular growth and proliferation, cell death and survival, and small molecule biochemistry. Overall, our results have pointed out multiple novel pathways in bull epididymal fluid that might take part in various aspects of maturation and protection processes of epididymal spermatozoa.
Assuntos
Proteínas de Plasma Seminal/isolamento & purificação , Proteínas de Plasma Seminal/fisiologia , Animais , Líquidos Corporais , Bovinos , Epididimo , Masculino , Proteoma/metabolismo , Glândulas Seminais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , EspermatozoidesRESUMO
The existing knowledge on the bull seminal vesicle proteome, a major seminal plasma constituent, and its relationship with seminal plasma is limited. This knowledge is prerequisite for a better understanding of seminal plasma variability, which is linked to semen quality. The objective of this study was to characterize the proteomes of seminal vesicle fluid and seminal plasma and to compare them to better understand the origin of seminal plasma proteins. We collected ejaculates and seminal vesicle fluid postmortem from 6 mature Holstein Friesian bulls. We performed the analysis and identification of proteins using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. We identified 105 proteins in bull seminal vesicle fluid and 88 proteins in seminal plasma. For both seminal vesicles and seminal plasma proteins described in our study, top biological functions were cellular movement, cell death and survival, and cellular growth and proliferation. Additionally, seminal vesicle fluid proteins were involved in protein degradation and synthesis. Seminal plasma proteins were also involved in cellular assembly and organization and cell-to-cell signaling and interactions. Proteins of both fluids were involved in the following canonical pathways: glycolysis, gluconeogenesis, liver X receptor/farnesoid X receptor, and farnesoid X receptor/retinoid X receptor activation. Additionally, seminal vesicle fluid proteins appeared to be involved in oxidative stress response mediated by nuclear factor E2-related factor 2. Our results described the bull seminal vesicle fluid proteome for the first time and allowed for significant expansion of the current knowledge on the bull seminal plasma proteome. Moreover, analysis indicated that both bull seminal vesicle fluid and seminal plasma proteomes contained interconnected protein groups related to protective functions, glycolysis, and the morphology and physiology of the spermatozoa. These proteins and their interactions could be targeted in future research.