Quantitative proteomics analysis of seminal plasma and its association to sperm functional aspects and to seminal plasma lipid peroxidation levels

funcionais nos espermatozoides e o nivel seminal de peroxidacao lipidica. Metodo: Um estudo transversal foi realizado incluindo 156 pacientes normozoospermicos. Apos a coleta do semen por masturbacao, uma aliquota foi utilizada para a analise seminal e outra para a avaliacao da atividade mitocondrial, da integridade do acrossoma e da integridade do DNA dos espermatozoides. O volume remanescente de semen foi centrifugado e o plasma seminal sobrenadante foi utilizado para a avaliacao do nivel seminal de peroxidacao lipidica e para a analise proteomica. Posteriormente, os pacientes foram divididos em percentis (15%) para formacao dos grupos experimentais de cada estudo: Estudo 1 - alta (grupo controle) e baixa (grupo alterado) atividade mitocondrial dos espermatozoides, Estudo 2 - alta (grupo controle) e baixa (grupo alterado) integridade do acrossoma dos espermatozoides, Estudo 3 - baixa (grupo controle) e alta (grupo alterado) fragmentacao do DNA dos espermatozoides e Estudo 4 - baixo (grupo controle) e alto (grupo alterado) niveis seminais de peroxidacao lipidica. A analise proteomica foi realizada utilizando LCMS/MS. Os grupos foram comparados por meio de analise univariada (teste t de Student) e analise multivariada (PLS-DA e analise discriminante). As proteinas significantes foram posteriormente submetidas a analise de enriquecimento funcional. Resultados: Nos estudos 1, 2, 3 e 4 foram observadas 506, 493, 474 e 629 proteinas, respectivamente. As funcoes enriquecidas no estudo 1 foram detoxificacao de EROs e ligacao a NADP (controle) e atividade de oxidoredutase intramolecular, catabolismo de aminoglicanos, inibicao de endopeptidases, lisossomos e resposta imune de fase aguda (alterado). As principais funcoes enriquecidas no estudo 2 foram resposta imune (controle) e inibicao de fosfolipase, metabolismo do acido araquidonico, exocitose, resposta inflamatoria aguda, resposta ao peroxido de hidrogenio e transporte lisossomal (alterado). As principais funcoes enriquecidas no estudo 3 foram metabolismo de carboidratos, regulacao de lipoproteinas, regulacao negativa da apoptose, metabolismo de hormonios, atividade de metalopeptidases, ligacao ao NAD e lisossomos (controle) e biossintese de prostaglandinas e ligacao a acidos graxos (alterado). As principais funcoes enriquecidas no estudo 4 foram biossintese de acidos graxos insaturados, atividade de oxidantes e antioxidantes e resposta celular ao estresse termico (alterados). Nos estudos 1, 2, 3 e 4 foram sugeridos 8, 6, 8 e 7 biomarcadores seminais de atividade mitocondrial, integridade acrossoma, fragmentacao de DNA e peroxidacao lipidica, respectivamente. Conclusoes: O perfil proteomico do plasma seminal reflete alteracoes funcionais dos espermatozoides e o nivelseminal de peroxidacao lipidica e diversas funcoes pos-genomicas estao relacionadas as alteracoes estudadas. Proteinas relacionadas as alteracoes funcionais dos espermatozoides e ao nivel seminal de peroxidacao lipidica constituem potenciais biomarcadores seminais para cada alteracaoObjective: To verify if the seminal plasma proteomic profile reflects sperm functional alteration and semen lipid peroxidation levels. Method: A cross-sectional study was performed including 156 normozoospermic patients. After semen retrieval by masturbation, an aliquot was utilized for semen analysis and another for evaluation of sperm mitochondrial activity, acrosome integrity and DNA fragmentation. The remaining semen volume was centrifuged and the supernatant seminal plasma was utilized for semen lipid peroxidation levels evaluation, and proteomic analysis. Patients were divided into percentiles (15%) to form the experimental groups: Study 1 – high (control group) and low (altered group) sperm mitochondrial activity; Study 2 – high (control group) and low (altered group) sperm acrosome integrity; Study 3 – low (control group) and high (altered group) sperm DNA fragmentation; Study 4 – low (control group) and high (altered group) semen lipid peroxidation levels. Proteomic analysis was performed by a LC-MS/MS approach. Groups were compared using univariate (Student’s t test) and multivariate (PLS-DA and discrimant analysis) analyses. Differentially expressed proteins were then utilized for functional enrichment analysis. Results: 506, 493, 474, and 629 proteins were observed in studies 1, 2, 3, and 4, respectively. Enriched functions in study 1 were reactive oxygens species detoxification, and NADP binding (control group), and intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (altered group). In study 2, main enriched functions were acute-phase response (control group), and phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammatory response, response to hydrogen peroxide and lysosomal transport (altered group). In study 3, main enriched functions were carbohydrates metabolism, lipoprotein regulation, negative regulation of apoptosis, hormone metabolism, metalopeptidases activity, NAD binding, and lysosomes (control group), and prostaglandin biosynthesis, and fatty acid binding (altered group). In study 4, enriched functions were unsaturated fatty acid biosynthesis, oxidants and antioxidants activity, and cellular response to heat stress (altered group). In total, 8, 6, 8, and 7 seminal biomarkers were proposed for studies 1 (mitochondrial activity), 2 (acrosome integrity), 3 (DNA fragmentation), and 4 (lipid per oxidation), respectively. Conclusions: The seminal plasma proteomic profile reflects sperm functional alterations and semen lipid perodixation levels, and several post-genomic functions are related to the studied alterations. Proteins related to sperm functional alterations, and semen lipid peroxidation levels constitute potential seminal biomarkers for each alteration.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)BV UNIFESP: Teses e dissertaçõe

Proteomic profile of seminal plasma in adolescents and adults with treated and untreated varicocele

Varicocele, the most important treatable cause of male infertility, is present in 15% of adult males, 35% of men with primary infertility, and 80% of men with secondary infertility. On the other hand, 80% of these men will not present infertility. Therefore, there is a need to differentiate a varicocele that is exerting a deleterious effect that is treatable from a "silent" varicocele. Despite the growing evidence of the cellular effects of varicocele, its underlying molecular mechanisms are still eluding. Proteomics has become a promising area to determine the reproductive biology of semen as well as to improve diagnosis of male infertility. This review aims to discuss the state-of-art in seminal plasma proteomics in patients with varicocele to discuss the challenges in undertaking these studies, as well as the future outlook derived from the growing body of evidence on the seminal proteome.Sao Paulo Research Foundation (FAPESP)National Council for Scientific and Technological Development (CNPq)Univ Fed Sao Paulo, Div Urol, Human Reprod Sect, Dept Surg, R Embau 231, BR-04039060 Sao Paulo, BrazilSao Paulo Hosp, Sao Paulo, BrazilUniv Fed Sao Paulo, Div Urol, Human Reprod Sect, Dept Surg, R Embau 231, BR-04039060 Sao Paulo, BrazilSao Paulo Hosp, Sao Paulo, BrazilFAPESP: 2012/15039-7FAPESP: 2014/11493-0FAPESP: 2014/17185-6CNPq: 306616/2013-0Web of Scienc

Effect of the exposure to fine inhalable particulate matter (pm2.5) on sperm functional quality of mice

Objective: To evaluate the effect of exposure to pollution (fine inhalable particulate matter - PM2.5) from the city of São Paulo on sperm functional quality. Design: Male isogenic BALB/c mice were used, distributed in two groups, control (n=6) and polluted air (n=8). For the polluted air group, after weaning (21 days), animals were daily exposed to 600 μg/m3 of PM2,5 for 96 days in an Ambient Particle Concentrator (APC). Control group was simultaneously exposed to filtered air in the APC. On postnatal day 118, animals were sacrificed (isoflurane overdose), body was weighted and the epididymis were collected. Materials and Methods: Sperm obtained from the cauda epididymis were used for the evaluation of motility, mitochondrial activity (DAB staining), acrosome integrity (PNA staining), DNA fragmentation (alkaline comet assay), oxidative stress (DHE staining) and cell viability (PI staining). Groups were compared using an unpaired Student’s t test (p<0.05). Results: Groups did not differ regarding body weight, and sperm motility. Furthermore, air pollution did not alter sperm functional quality (Table 1). Conclusions: Exposure to high concentrations of PM2.5 does not affect sperm motility and functional parameters.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Estudo Das Vias Proteômicas Do Plasma Seminal E Dos Espermatozoides Associadas À Infertilidade Masculina

Objective: The Seminal Plasma And Sperm Protein Profile Is Essential For Adequate Sperm Function, And, Consequently, For Male Fertility. Thus, Proteomic Studies Have Been Undertaken In Order To Understand Underlying Post-Genomic Mechanisms And To Identify Altered Proteins In Male Infertility. Therefore, The Aim Of This Study Was To Evaluate Seminal Plasma And Sperm Proteins And Their Association With Sperm Functional Alterations, And With Primary Or Secondary Male Infertility. Methods: This Study Was Subdivided In Two. In Study 1, Semen Samples From 197 Normozoospermic Men Were Used. After Semen Collection And Liquefaction, An Aliquot Was Utilized For Semen Analysis, Another For Evaluation Of Sperm Functional Integrity, And The Remaining Volume Was Centrifuged For Seminal Plasma Separation. Seminal Plasma Was Then Utilized For The Evaluation Of The Proposed Biomarkers: (I) Of Sperm Mitochondrial Activity Alterations "Gstm3, (Ii) Of Sperm Acrosome Defects " Pltp, And (Iii) Of Sperm Dna Integrity " Crispld1, CrObjetivo: A Composição Proteica Do Plasma Seminal E Dos Espermatozoides É Essencial Para A Sua Função Correta E, Consequentemente, Para A Fertilidade. Assim, Estudos Proteômicos Vêm Sendo Realizados A Fim De Entender Os Mecanismos Pós-Genômicos Subjacentes E De Identificar As Proteínas Alteradas Na Infertilidade Masculina. Portanto, O Objetivo Deste Estudo Foi Avaliar Proteínas De Plasma Seminal E De Espermatozoides E Sua Associação Com Alterações Funcionais Dos Espermatozoides E Com A Infertilidade Masculina Primária Ou Secundária. Métodos: Este Trabalho Foi Subdividido Em Dois Estudos. No Estudo 1, Amostras Seminais De 197 Homens Normozoospérmicos Foram Utilizadas. Após A Coleta E Liquefação Do Sêmen, Uma Alíquota Foi Utilizada Para A Análise Seminal, Outra Para A Avaliação Da Integridade Funcional Dos Espermatozoides E O Volume Remanescente Foi Centrifugado Para A Separação Do Plasma Seminal. Este Foi Então Utilizado Para A Avaliação Dos Níveis Seminais Dos Biomarcadores Sugeridos: (I) De Alterações Na AtiDados abertos - Sucupira - Teses e dissertações (2018

Understanding the seminal plasma proteome and its role in male fertility

Résumé Le plasma séminal est un liquide complexe comprenant les sécrétions des vésicules séminales, de la prostate, des glandes bulbo-urétrales, et des sécrétions provenant de la lumière des tubes séminifères/épididymes/canaux déférents. Bien qu’il a été établi que le plasma séminal n’est pas seulement un milieu servant à transporter, protéger et nourrir les spermatozoïdes après l’éjaculation et jusqu’à la fécondation, mais qu’il constitue aussi un modulateur fonctionnel des fonctions spermatiques, il demeure nécessaire de caractiser de manière appropriée la constitution moléculaire du plasma séminal des hommes féconds, et de comprendre comment celle-ci est altérée dans les différentes causes d’infertilité masculine. Le principal objectif de cet article est de passer en revue les études du protéome du plasma séminal, en allant de celles ayant caractérisé une carte protéomique du plasma séminal fertile aux études ayant comparé le plasma séminal d’hommes féconds et inféconds et à celles ayant comparé le plasma séminal d’hommes féconds ou normozoospermiques à celui d’hommes présentant diverses causes d’infertilité. Pour finir, la présente revue est centrée sur l’association entre d’une part la qualité fonctionnelle du sperme et des spermatozoïdes et d’autre part le protéome du plasma séminal dans le but de démontrer les mécanismes cellulaires et moléculaires de l’infertilité masculine. En raison de la nature non ciblée de la majorité des études présentées dans cette revue, et de la grande diversité des techniques utilisées pour étudier la composition protéomique du plasma séminal, de nombreuses protéines différentiellement exprimées ont été observées. Cependant, d’une façon globale, il semblerait qu’il y ait un protéome séminal associé à la fertilité masculine et que des situations biologiques ou des phénotypes cellulaires particuliers l’éloignerait de son point d’équilibre vers des états associés à une production énergétique altérée. De plus, il semblerait exister une composante inflammatoire du plasma séminal chez les hommes infertiles. En conclusion, il existe de nombreuses études centrées sur la composition protéomique du plasma séminal humain; de futures études de confirmation seront utiles à la compréhension des voies spécifiques de l’infertilité dans ses différentes conditions biologiques