RESUMO
Improvement of in vitro culture methods of Spermatogonial Stem Cells (SSCs) is known to be an effective procedure for further study of the process of spermatogenesis and can offer effective therapeutic modality for male infertility. Tissue decellularization by providing natural 3D and extracellular matrix (ECM) conditions for cell growth can be an alternative procedure to enhance in vitro culture conditions. In the present study, the testicular tissues were taken from brain death donors. After enzymatic digestion, the tissue cells were isolated and cultured for four weeks. Then the identity of the SSCs was confirmed using anti-GFRα1 and anti-PLZF antibodies via immunocytochemistry (ICC). The differentiation capacity of SSCs were evaluated by culture of them on a layer of decellularized testicular matrix (DTM) prepared from sheep testis, as well as under two-dimensional (2D) culture with differentiation medium. After four and six weeks of the initiation of differentiation culture, the pre-meiotic, meiotic and post- meiotic genes at the mRNA and protein levels was examined via qPCR and ICC methods, respectively. The results showed that pre-meiotic, meiotic and post-meiotic genes expressions were significantly higher in the cells cultured in DTM substrate (Pâ¯≤â¯0.01).The present study indicated that, the natural structure of ECM prepare the suitable conditions for further study of the spermatogenesis process in the in vitro and contributes to the maintenance and treatment of male infertility.
Assuntos
Técnicas de Cultura de Células , Matriz Extracelular/química , Espermatogônias/citologia , Células-Tronco/citologia , Testículo/citologia , Animais , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Separação Celular/métodos , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Imuno-Histoquímica , Infertilidade Masculina/terapia , Masculino , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Protaminas/genética , Protaminas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ovinos , Espermatogênese/genética , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Testículo/metabolismoRESUMO
BACKGROUND: In the males, Spermatogonial Stem Cells (SSCs) contribute to the production of sex cells and fertility. In vitro SSCs culture can operate as an effective strategy for studies on spermatogenesis and male infertility treatment. Cell culture in a three-dimensional (3D) substrate, relative to a two-dimensional substrate (2D), creates better conditions for cell interaction and is closer to in vivo conditions. In the present study, in order to create a 3D matrix substrate, decellularized testicular matrix (DTM) was used to engender optimal conditions for SSCs culture and differentiation. MATERIALS AND METHODS: After, testicular cells enzymatic extraction from testes of brain-dead donors, the SSCs were proliferated in a specific culture medium for four weeks, and after confirming the identity of the colonies derived from the growth of these cells, they were cultured on a layer of DTM as well as in 2D condition with a differentiated culture medium. In the Sixth week since the initiation of the differentiation culture, the expression of pre meiotic (OCT4 & PLZF ), meiotic (SCP3 & BOULE) and post meiotic (CREM & Protamine-2) genes were measured in both groups. RESULTS: The results indicated that the expression of pre meiotic, meiotic and post meiotic genes was significantly higher in the cells cultured on DTM (P ≤ 0.001). CONCLUSION: SSCs culture in DTM with the creation of ECM and similar conditions with in vivo can be regarded as a way of demonstrating spermatogenesis in vitro, which can be adopted as a treatment modality for male infertility.
