ABSTRACT
An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0-5 µM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh), α6 integrin, ß1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3 µM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 µM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs.
Subject(s)
Cell Proliferation/drug effects , Cell Transdifferentiation/drug effects , Embryonic Stem Cells/drug effects , Spermatogonia/cytology , Tretinoin/pharmacology , Adaptor Proteins, Signal Transducing/drug effects , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Argonaute Proteins/drug effects , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Biomarkers/metabolism , Bone Morphogenetic Protein 4/metabolism , Cdh1 Proteins/drug effects , Cdh1 Proteins/metabolism , Cell Differentiation , Cell Survival/drug effects , Cells, Cultured , Coculture Techniques , DEAD-box RNA Helicases/drug effects , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Embryonic Stem Cells/cytology , Immunohistochemistry , Integrin alpha6beta1/drug effects , Integrin alpha6beta1/genetics , Integrin alpha6beta1/metabolism , Male , Mice , Spermatogonia/metabolism , TranscriptomeABSTRACT
Newly formed oligodendrocytes in the CNS derive survival cues from their target axons. These cues are provided in part by laminins expressed on the axon, which are recognized by alpha6beta1 integrin on the oligdendrocyte and amplify platelet-derived growth factor (PDGF) signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway. The alpha6beta1 integrin is localized in oligodendrocyte lipid rafts. We show here using the sphingolipid synthesis inhibitor fumonisin-B1 to deplete rafts that this localization is important for normal survival signaling, because depletion increases oligodendrocyte apoptosis and inhibits PI3K signaling. We have shown previously that PDGF-mediated integrin activation is an important component of oligodendrocyte proliferation signaling, and here we present evidence that a similar mechanism operates in survival signaling. Integrin activation using manganese increases raft localization and rescues the effects of both raft depletion and PDGF removal on survival and PI3K signaling. Together, these results point to an essential role for rafts in oligodendrocyte survival signaling on the basis of the provision of a favorable environment for growth factor-mediated integrin activation.
