Expression of the adaptor protein m-Numb in mouse male germ cells

Abstract

Numb is an adaptor protein that is asymmetrically inherited at mitosis and controls the fate of sibling cells in different species. The role of m-Numb (mammalian Numb) as an important cell fate-determining factor has extensively been described mostly in neural tissues, particularly in progenitor cells, in the mouse. Biochemical and genetic analyses have shown that Numb acts as an inhibitor of the Notch signaling pathway, an evolutionarily conserved pathway involved in the control of cell proliferation, differentiation, and apoptosis. In the present study, we sought to determine m-Numb distribution in germ cells in the postnatal mouse testis. We show that all four m-Numb isoforms are widely expressed during postnatal testis development. By reverse transcriptase-PCR and western blot analyses, we further identify p71 as the predominantly expressed isoform in germ cells. Moreover, we demonstrate through co-immunoprecipitation studies that m-Numb physically associates with Ap2a1, a component of the endocytotic clathrin-coated vesicles. Finally, we employed confocal immunofluorescence microscopy of whole mount seminiferous tubules and isolated germ cells to gain more insight into the subcellular localization of m-Numb. These morphological analyses confirmed m-Numb and Ap2a1 co-localization. However, we did not observe asymmetric localization of m-Numb neither in mitotic spermatogonial stem cells nor in more differentiated spermatogonial cells, suggesting that spermatogonial stem cell fate in the mouse does not rely on asymmetric partitioning of m-Numb.