RESUMEN
The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.
Asunto(s)
Células Madre Germinales Adultas/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Células Madre Germinales Adultas/citología , Animales , Línea Celular , China , Técnicas de Cocultivo , Masculino , Ratones , Espermatogénesis , Porcinos , Porcinos Enanos , Testículo/citología , Factores de Transcripción/metabolismoRESUMEN
Delivery of exogenous glial cell line-derived neurotrophic factor (GDNF) increases locomotor activity in rodent models of aging and Parkinson's disease in conjunction with increased dopamine (DA) tissue content in substantia nigra (SN). Striatal GDNF infusion also increases expression of GDNF's cognate receptor, GFRα1, and tyrosine hydroxylase (TH) ser31 phosphorylation in the SN of aged rats long after elevated GDNF is no longer detectable. In aging, expression of soluble GFRα1 in the SN decreases in association with decreased TH expression, TH ser31 phosphorylation, DA tissue content, and locomotor activity. Thus, we hypothesized that, in aged rats, replenishing soluble GFRα1 in SN could reverse these deficits and increase locomotor activity. We determined that the quantity of soluble GFRα1 in young adult rat SN is ~3.6 ng. To replenish age-related loss, which is ~30 %, we infused 1 ng soluble GFRα1 bilaterally into SN of aged male rats and observed increased locomotor activity compared to vehicle-infused rats up to 4 days following infusion, with maximal effects on day 3. Five days after infusion, however, neither locomotor activity nor nigrostriatal neurochemical measures were significantly different between groups. In a separate cohort of male rats, nigral, but not striatal, DA, TH, and TH ser31 phosphorylation were increased 3 days following unilateral infusion of 1 ng soluble GFRα1into SN. Therefore, in aged male rats, the transient increase in locomotor activity induced by replenishing age-related loss of soluble GFRα1is temporally matched with increased nigral dopaminergic function. Thus, expression of soluble GFRα1 in SN may be a key component in locomotor activity regulation through its influence over TH regulation and DA biosynthesis.