RESUMO
Spermatogonial stem cells (SSCs) self-renew and produce a large number of differentiated germ cells to maintain normal spermatogenesis. However, the growth factors crucial for SSC self-renewal and the mechanism underlying this process remain unclear. In the present study, a serum-free culture media was used to evaluate the effect of several growth factors on the expression of some SSC markers and self-renewal related genes. The putative SSCs were cultured on buffalo Sertoli cell feeder layer in KO-DMEM +10% KOSR. The colony formation was observed between 7 and 10 days. The putative SSC colonies also expressed markers specific for undifferentiated type A spermatogonia and pluripotency markers. After 15 days, relative mRNA expression study revealed that 20 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of PLZF, TAF4B, and THY1. Furthermore, supplementation of a combination of 20 ng/mL GDNF, 10 ng/mL basic fibroblast growth factor (bFGF), 1000 IU/mL leukemia inhibitory factor (LIF), and 1 ng/mL colony stimulating factor 1 (CSF1) upregulated the expression of PLZF, TAF4B, BCL6B, and ID4 genes. These results demonstrated that our defined culture media in combination with GDNF, bFGF, LIF, and CSF1 well supported SSC self-renewal.
Assuntos
Células-Tronco Adultas/citologia , Proliferação de Células , Meios de Cultura Livres de Soro/química , Fator 2 de Crescimento de Fibroblastos/química , Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , Fator Inibidor de Leucemia/química , Fator Estimulador de Colônias de Macrófagos/química , Animais , Búfalos , Células Cultivadas , Masculino , Células de Sertoli/citologia , Espermatogênese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In this study, we investigated the effect of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2, and epidermal growth factor (EGF) on the expression of some self-renewal-related microRNAs (miRs) in putative buffalo spermatogonial stem cells (SSCs). The SSCs were cultured on a buffalo Sertoli cell feeder layer, colony formation was observed between 7 and 10 days. The SSC colonies expressed markers specific for undifferentiated type A spermatogonia and pluripotency markers. After 15 days of initial culture, the colonies were subcultured as treatment (supplemented with 20 ng mL-1 GDNF +10 ng mL-1 FGF2 + 10 ng mL-1 EGF) and control groups. The number and area of SSC colonies were significantly (p < 0.05) higher in the treatment group than in the control group. The relative abundance of miR-20b, miR-21, and miR-106a in SSCs supplemented with growth factors was significantly higher (p < 0.001) than that in the control. The results indicate that supplementation of SSC culture medium with growth factors (GDNF, FGF2, and EGF) may promote the expression of miR-20b, miR-21, and miR-106a, which is essential for self-renewal and maintenance of SSCs.
Assuntos
Proliferação de Células , Fator de Crescimento Epidérmico/química , Fator 2 de Crescimento de Fibroblastos/química , Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , MicroRNAs/genética , Animais , Biomarcadores/metabolismo , Búfalos , Separação Celular/veterinária , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura/veterinária , Ensaio de Unidades Formadoras de Colônias/veterinária , Meios de Cultura/química , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Células de Sertoli/citologia , Espermatogônias/citologia , Células-Tronco/citologia , Regulação para CimaRESUMO
AIM: To investigate the effect of extended photoperiod on growth rate, hormonal levels, and puberty in Murrah heifers. MATERIALS AND METHODS: About 14 Murrah buffalo heifers were divided into normal day photoperiod (NDP; n=7) and extended NDP (ENDP; n=7) groups. The ENDP group was exposed to 4 h of extended photoperiod with artificial light (160 lux) after sunset for 3 months during winter. RESULTS: Group, age and group-by-age interaction effects on plasma glucose concentrations were non-significant (p>0.05). A significant effect of age on non-esterified fatty acids (p<0.05), cholesterol (p<0.01), and triglycerides (p<0.05) concentrations was observed. Group and group-by-age interaction effects on plasma T3, T4, leptin, 17 ß estradiol, prolactin and melatonin concentrations were non-significant (p>0.05) while significant (p<0.05) age effect on T4, leptin and melatonin concentrations was observed. With respect to the circadian pattern of melatonin and prolactin, the group, time and group-by-time interaction effects were non-significant (p>0.05). Average daily gain and dry matter intake of heifers were non-significant between the NDP and ENDP groups but were comparatively higher in ENDP group. By the end of the experiment, 6 out of 7 heifers attained puberty in ENDP group in comparison to 4 out of 7 in NDP group. CONCLUSION: Extending the photoperiod by artificial light for 4 h during winter season resulted in better growth rate and early onset of puberty in Murrah buffalo heifers.