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Identification of integrin heterodimers expressed on the surface of spermatogonial stem cells in hybrid mice
pp.112-113
Microenvironments surrounded with various extracellular matrix (ECM) components can decide specifically the fate of spermatogonial stem cells (SSCs) and integrin heterodimers recognizing directly ECM proteins play an important role in transporting ECM-derived signals into cytoplasm, resulting in inducing a variety of biological functions such as cell attachment, self-renewal and differentiation. However, to date, studies on type of integrin heterodimers expressed functionally on the undifferentiated SSCs derived from mouse with hybrid strain remain unclear. Therefore, we tried to investigate systematically what kind of integrin heterodimers are expressed transcriptionally, translationally and functionally in the SSCs derived from testis of hybrid (B6CBAF1) mouse. For these, magnetic activated cell sorting (MACS) using Thy1 antibody was used for isolating SSCs from testis, and real-time PCR or fluorescence immunoassay was conducted for measuring transcriptional or translational level of integrin α and β subunits in the isolated SSCs. Subsequently, antibody inhibition assay was conducted for confirming functionality of presumed integrin heterodimers. As the results, transcriptional levels of genes encoding total 25 integrin subunits were quantified, 7 integrin α (α4, α6, α7, α9, αV, αL and αE) and 2 integrin β (β1 and β5) subunit genes showed significantly increased transcriptional up-regulation, compared to the other integrin subunit genes. In contrast, integrin α3, α5, α10 and α11, and integrin β2, β3, β4 and β7 were weakly transcribed. When translational levels of the integrin α subunits showing high transcription level (α4, α6, α7, α9, αV, αL and αE) were measured, significantly strong translational up-regulation of integrin α6, α7, α9, αV and αL subunit genes were detected, whereas integrin α4 and αE subunit genes were weakly. In case of integrin β subunit, β1 evaluated more expression than β5. Based on these results, we speculated that the undifferentiated SSCs derived from B6CBAF1 mouse might express integrin α4β 1, α6β1, α7β1, α9β1, αVβ1 or αVβ5 on plasma membrane. Subsequently, the hybrid strain SSCs showed significantly increased adhesion to fibronectin, laminin, tenascine-C and vitronectin and functional blocking of integrin α4β1, α6β1, α9β1, and αVβ1 or αVβ5 in SSCs significantly inhibited attachment to fibronectin, laminin, tenascin-C and vitronectin, respectively. Accordingly, we could identify that the hybrid (B6CBAF1) mouse-derived SSCs had integrin α4β1, α6β1, α9β1, αVβ1 or αVβ5 on plasma membrane. Moreover, this information will greatly contribute to constructing non-cellular niche supporting self-renewal of SSCs in the future.
