Proteômica do epidídimo suíno: uso do modelo imunocastrado na identificação de marcadores moleculares para a qualidade seminal
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Data
2020-09
Autores
Orientador
Filho, Ivan Cunha Bustamante
Banca
Contini, Verônica
Moura, Arlindo De Alencar Araripe Noronha
Rossi, Carlos Augusto Rigon
Título do periódico
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Resumo
O epidídimo é um órgão especializado responsável pela maturação de células espermáticas pós-testiculares, dando a ele motilidade e capacidade de fertilização. A passagem dos espermatozoides pelo epitélio epididimal promove interações entre as secreções ao longo das três principais regiões cabeça, corpo e cauda. Cada região tem um perfil proteico garantindo diferentes funções em cada etapa da maturação e armazenamento epididimário. Entretanto este processo é dependente de diversos fatores contribuem na qualidade seminal e estes podem estar relacionados ao sistema androgênico e condições patológicas de cada individuo. Deficiência androgênica causada por uma desregulação endócrina altera a síntese e secreção de proteínas envolvidas em importantes processos celulares da reprodução. A infertilidade masculina é associada a interrupção do eixo hipotálamo-hipófise-gônadas a secreção de hormônio liberador de gonadotrofinas-GnRH que caracterizam o hipogonadismo secundário. Esta patologia altera o perfil proteíco e permite identificar, utilizando abortagens proteômicas, grande numero de proteínas candidatas a marcadores proteicos de fertilidade. A presente tese tem por objetivo avaliar as alterações proteômicas nos espermatozoides (CESperm) e no fluído da cauda do epidídimo (CEF) de suínos imunizados contra GnRH sendo este um modelo para hipogonadismos secundário. Foram obtidos tecidos reprodutivos de 10 suínos machos reprodutores saudáveis e 10 de animais previamente imunizados contra GnRH Vivax (Pfizer). Espermatozoides foram coletados da cauda do epidídimo por dissecação e lavagem, o fluído foi retirado e centrifugado para remoção de resíduos celulares. Após dosagem de proteína total, pools com as amostras do grupo controle e imunizado foram realizadas utilizando 300ug de proteína cada grupo e realizada proteômica utilizando a técnica de MudPIT, composto de separação dos peptídeos por HPLC Agilent 1100, seguido por identificação dos peptídeos por espectrometria de massa utilizando LTQ Orbitrap XL. Analises de bioinformática foram realizadas para categorizar as proteínas em relação a Gene Ontology, vias metabólicas por KEGG, mapeamento funcional e identificação dos genes que codificam proteínas especificas de cada condição e estratégias de validação dos resultados obtidos na proteômica. Além disso foram analisadas a expressão genica, por qPCR, no testículo e três regiões epididimárias de genes associados a proteínas que foram diferencialmente expressas em espermatozoides e fluído epididimário de ambos os grupos. A imunização com GnRH dobrou o número de proteínas no CEF, com 417 proteínas sendo encontradas exclusivamente em amostras de suínos imunizados com GnRH. O CEF de machos imunizados com GnRH apresentou aumento no número de proteínas relacionadas a processos celulares e metabólicos, com afinidade por compostos orgânicos cíclicos, pequenas moléculas e compostos heterocíclicos, além de alterar o perfil enzimático. O aumento significativo no número de proteínas associadas ao sistema ubiquitina-proteassoma foi identificado no CEF de animais imunizados com GnRH. Em espermatozoides (CESperm) foram identificados 1.322 proteínas, com abundancia de proteínas envolvidas no metabolimo dos espermatozoides (enolase, piruvato desidrogenase), reação do acrossomo e capacitação (oxoprolinase, proteína acrossomal SP- 10, diidrolipoil desidrogenase) e interações espermatozoides-oócitos (proteína de ligação à zona pelúcida, zonadesina, molécula de adesão de espermatozoides 1). Além disso, a abundância de proteínas mitocondriais foi severamente afetada, com alterações significativas nas proteínas dos Complexos I e II, bem como na ATPase da cadeia de fosforilação oxidativa. Esses resultados trazem fortes evidências do impacto do hipogonadismo secundário no ambiente epididimário, responsável pela maturação e armazenamento dos espermatozoides antes da ejaculação. Finalmente, as proteínas expressas de forma diferente no CEF e CESperm são candidatos a biomarcadores seminais para distúrbios testículares e epididimários causados por hipogonadismo secundário.
The epididymis is a specialized organ responsible for the maturation of post- testicular sperm cells, giving it motility and fertilization capacity. The passage of sperm through the epididymal epithelium promotes interactions between secretions along the three main head, body and tail regions. Each region has a protein profile guaranteeing different functions at each stage of maturation and epididymal storage. However, this process is dependent on several factors that contribute to seminal quality and these may be related to the androgenic system and pathological conditions of each individual. Androgen deficiency caused by endocrine disruption alters the synthesis and secretion of proteins involved in important reproductive processes. Male infertility is associated with an interruption of the hypothalamic-pituitary-gonadal axis to the secretion of gonadotropin-releasing hormone- GnRH that characterize secondary hypogonadism. This pathology alters the protein profile and allows the identification, using proteomic abortions, of the large number of candidate proteins for protein fertility markers. This study aims to evaluate how proteomic changes in sperm (CESperm) and epididymis tail fluids (CEF) of swine immunized against GnRH are a model for secondary hypogonadism. Reproductive tissues were collected from 10 healthy male breeding pigs and 10 animals previously immunized against GnRH Vivax (Pfizer). Sperm were collected from the tail of the epididymis by dissection and washing, the fluid was removed and centrifuged to remove cell debris. The dosage of total protein, pools with the control and immunized groups were performed using 300ug of protein each group and proteomics was performed using MudPIT technique, compound of peptide separation by HPLC Agilent 1100, followed by identification of the peptides by mass spectrometry using LTQ Orbitrap XL. Bioinformatics analyzes were carried out to categorize proteins in relation to a Genetic Ontology, metabolic pathways by KEGG, functional mapping and identification of the genes encoding specific proteins for each condition and clinic for validating the results requested in proteomics. In addition, gene expression was analyzed by qPCR in the testis and three epididymal regions of genes associated with proteins that were differentially expressed in sperm and epididymal fluid from both groups. GnRH immunization doubled the number of proteins in the CEF, with 417 proteins being evaluated exclusively in pigs immunized with GnRH. The CEF of males immunized with GnRH shows an increase in the number of proteins related to cellular and metabolic processes, with affinity for organic compounds, small molecules and heterocyclic compounds, in addition to altering the enzymatic profile. The significant increase in the number of proteins associated with the ubiquitin-proteasome system was identified in the CEF of animals immunized with GnRH. In spermatozoa (CESperm), 1,322 proteins were identified, with abundance of proteins involved in the metabolism of sperm (enolase, pyruvate dehydrogenase), acrosome reaction and capacity building (oxoprolinase, acrosomal protein SP-10, dihydrolipoil dehydrogenase) and sperm- oocyte interactions (protein binding to the pellucid zone, zonadesin, sperm adhesion molecule 1). In addition, the abundance of mitochondrial proteins was severely affected, with significant changes in the proteins of Complexes I and II, as well as in the ATPase of the oxidative phosphorylation chain. These results provide strong evidence of the impact of secondary hypogonadism on the epididymal environment, responsible for the maturation and storage of sperm before privacy. Finally, as proteins expressed differently in CEF and CESperm they are candidate seminal biomarkers for testicular and epididymal disorders caused by secondary hypogonadism.
The epididymis is a specialized organ responsible for the maturation of post- testicular sperm cells, giving it motility and fertilization capacity. The passage of sperm through the epididymal epithelium promotes interactions between secretions along the three main head, body and tail regions. Each region has a protein profile guaranteeing different functions at each stage of maturation and epididymal storage. However, this process is dependent on several factors that contribute to seminal quality and these may be related to the androgenic system and pathological conditions of each individual. Androgen deficiency caused by endocrine disruption alters the synthesis and secretion of proteins involved in important reproductive processes. Male infertility is associated with an interruption of the hypothalamic-pituitary-gonadal axis to the secretion of gonadotropin-releasing hormone- GnRH that characterize secondary hypogonadism. This pathology alters the protein profile and allows the identification, using proteomic abortions, of the large number of candidate proteins for protein fertility markers. This study aims to evaluate how proteomic changes in sperm (CESperm) and epididymis tail fluids (CEF) of swine immunized against GnRH are a model for secondary hypogonadism. Reproductive tissues were collected from 10 healthy male breeding pigs and 10 animals previously immunized against GnRH Vivax (Pfizer). Sperm were collected from the tail of the epididymis by dissection and washing, the fluid was removed and centrifuged to remove cell debris. The dosage of total protein, pools with the control and immunized groups were performed using 300ug of protein each group and proteomics was performed using MudPIT technique, compound of peptide separation by HPLC Agilent 1100, followed by identification of the peptides by mass spectrometry using LTQ Orbitrap XL. Bioinformatics analyzes were carried out to categorize proteins in relation to a Genetic Ontology, metabolic pathways by KEGG, functional mapping and identification of the genes encoding specific proteins for each condition and clinic for validating the results requested in proteomics. In addition, gene expression was analyzed by qPCR in the testis and three epididymal regions of genes associated with proteins that were differentially expressed in sperm and epididymal fluid from both groups. GnRH immunization doubled the number of proteins in the CEF, with 417 proteins being evaluated exclusively in pigs immunized with GnRH. The CEF of males immunized with GnRH shows an increase in the number of proteins related to cellular and metabolic processes, with affinity for organic compounds, small molecules and heterocyclic compounds, in addition to altering the enzymatic profile. The significant increase in the number of proteins associated with the ubiquitin-proteasome system was identified in the CEF of animals immunized with GnRH. In spermatozoa (CESperm), 1,322 proteins were identified, with abundance of proteins involved in the metabolism of sperm (enolase, pyruvate dehydrogenase), acrosome reaction and capacity building (oxoprolinase, acrosomal protein SP-10, dihydrolipoil dehydrogenase) and sperm- oocyte interactions (protein binding to the pellucid zone, zonadesin, sperm adhesion molecule 1). In addition, the abundance of mitochondrial proteins was severely affected, with significant changes in the proteins of Complexes I and II, as well as in the ATPase of the oxidative phosphorylation chain. These results provide strong evidence of the impact of secondary hypogonadism on the epididymal environment, responsible for the maturation and storage of sperm before privacy. Finally, as proteins expressed differently in CEF and CESperm they are candidate seminal biomarkers for testicular and epididymal disorders caused by secondary hypogonadism.
Descrição
Palavras-chave
Epidídimo; Fertilidade; Hipogonadismo; Proteômica; Epididymis; Fertility; Hypogonadism; Proteomics
Citação
SOUZA, Ana Paula Binato De. Proteômica do epidídimo suíno: uso do modelo imunocastrado na identificação de marcadores moleculares para a qualidade seminal. 2020. Tese (Doutorado) – Curso de Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, 04 set. 2020. Disponível em: http://hdl.handle.net/10737/3153.