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1.
Andrologia ; 49(1)2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27037571

RESUMO

Sperm genomic integrity has a significant effect on intracytoplasmic sperm injection (ICSI) outcomes, especially post-implantation. Spermatozoa selected based on motility and morphology do not guarantee the genomic integrity of spermatozoa. Nearly fifty percentage of spermatozoa in infertile men with normal morphology present different degrees of DNA fragmentation. However, capacitated or hyperactivated spermatozoa show lower degrees of DNA fragmentation. Therefore, selection of hyperactivated spermatozoa may improve ICSI outcome. Routinely, for ICSI, fast-moving spermatozoa with A or B motility pattern are mainly selected for injection. The result of this study shows that in processed semen samples, hyperactivated spermatozoa are mainly observed in B motility pattern while, in viscous medium like polyvinylpyrrolidone (PVP), hyperactivated spermatozoa are mainly present in spermatozoa with C pattern of motility (nonprogressive). Therefore, we propose spermatozoa with C motility pattern which contains the main population of physiological or hyperactivated spermatozoa should be selected for ICSI.


Assuntos
Injeções de Esperma Intracitoplásmicas/métodos , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/citologia , Espermatozoides/fisiologia , Reação Acrossômica/fisiologia , Forma Celular/fisiologia , Humanos , Masculino
2.
Growth Factors ; 33(3): 181-91, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26154310

RESUMO

Growth factors are increasingly considered as important regulators of spermatogonial stem cells (SSCs). This study investigated the effects of various growth factors (GDNF, IGF1, bFGF, EGF and GFRalpha-1) on purification and colonization of undifferentiated goat SSCs under in vitro and in vivo conditions. Irrespective of the culture condition used, the first signs of developing colonies were observed from day 4 of culture onwards. The number of colonies developed in GDNF + IGF1 + bFGF culture condition was significantly higher than the other groups (p < 0.05). In contrast, the size of colonies developed in GDNF + EGF + LIF culture condition was significantly higher than the other groups (p < 0.05). Immunocytochemical stationing for specific biomarkers of somatic cells (vimentin, alpha-inhibin and α-SMA) and spermatogonial cells (PLZF, THY 1, VASA, alpha-1 integrin, bet-1 integrin and DBA) revealed that both cell types existed in developing colonies, irrespective of the culture condition used. Even though, the relative abundance of VASA, FGFR3, OCT4, PLZF, BCL6B and THY1 transcription factors in GDNF + IGF1 + bFGF treatment group was significantly higher than the other groups (p < 0.05). Additionally, goat SSCs developed in the latter culture condition could colonize within the seminiferous tubules of the germ-cell depleted recipient mice following xenotransplantation. Obtained results demonstrated that combination of GDNF with IGF1 and bFGF promote in vitro culture of goat SSCs while precludes uncontrolled proliferation of somatic cells.


Assuntos
Células-Tronco Adultas/transplante , Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Cabras , Fator de Crescimento Insulin-Like I/farmacologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/fisiologia , Animais , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Camundongos , Fatores de Crescimento Neural/farmacologia , Neuroglia/metabolismo , Túbulos Seminíferos/citologia , Espermatogênese/fisiologia , Testículo/citologia , Fatores de Transcrição/metabolismo , Transplante Heterólogo
3.
Theriogenology ; 77(8): 1519-28, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22289219

RESUMO

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, ß1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.


Assuntos
Bovinos/fisiologia , Técnicas de Cocultura/veterinária , Espermatogônias/citologia , Células-Tronco/citologia , Testículo/citologia , Animais , Biomarcadores/metabolismo , Células Cultivadas , Citometria de Fluxo , Masculino , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Maturidade Sexual
4.
J Assist Reprod Genet ; 29(1): 39-46, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22160429

RESUMO

PURPOSE: To investigate the effect of serum supplementing on short-term culture, fate determination and gene expression of goat spermatogonial stem cells (SSCs). METHODS: Crude testicular cells were plated over Datura-Stramonium Agglutinin (DSA) for 1 h, and non-adhering cells were cultured in the presence of different serum concentrations (1, 5, 10, and 15%) for 7 days in a highly enriched medium initially developed in mice. Colonies developed in each group were used for the assessment of morphology, immunocytochemistry, and gene expression. RESULTS: Brief incubation of testicular cells with DSA resulted in a significant increase in the number of cells that expressed the germ cell marker (VASA). The expression of THY1, a specific marker of undifferentiated spermatogonia, was significantly higher in colonies developed in the presence of 1% rather than 5, 10 and 15% serum. CONCLUSION: Goat SSCs could proliferate and maintain in SSC culture media for 1 week at serum concentrations as low as 1%, while higher concentrations had detrimental effects on SSC culture/expansion.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Cabras/genética , Espermatogônias/citologia , Células-Tronco/citologia , Aglutininas/química , Animais , Diferenciação Celular/genética , Datura stramonium/química , Expressão Gênica , Masculino , Camundongos , Soro/química , Antígenos Thy-1/metabolismo
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