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Abstract
ING3 (inhibitor of growth family, member 3) is a subunit of the nucleosome acetyltransferase of histone 4 (NuA4) complex, which activates gene expression. ING3, which contains a plant homeodomain (PHD) motif that can bind to trimethylated lysine 4 on histone H3 (H3K4me3), is ubiquitously expressed in mammalian tissues and governs transcriptional regulation, cell cycle control, and apoptosis via p53-mediated transcription or the Fas/caspase-8 pathway. Thus, ING3 plays a number of important roles in various somatic cells. However, the role(s) of ING3 in germ cells remains unknown. Here, we show that loss of ING3 function led to the failure of asymmetric cell division and cortical reorganization in the mouse oocyte. Immunostaining showed that in fully grown germinal vesicle (GV) oocytes, ING3 localized predominantly in the GV. After germinal vesicle breakdown (GVBD), ING3 homogeneously localized in the cytoplasm. In oocytes where Ing3 was targeted by siRNA microinjection, we observed symmetric cell division during mouse oocyte maturation. In those oocytes, oocyte polarization was not established due to the failure to form an actin cap or a cortical granule-free domain (CGFD), the lack of which inhibited spindle migration. These features were among the main causes of abnormal symmetric cell division. Interestingly, an analysis of the mRNA expression levels of genes related to asymmetric cell division revealed that only mTOR was downregulated, and, furthermore, that genes downstream of mTOR (e.g., Cdc42, Rac1, and RhoA) were also downregulated in siIng3-injected oocytes. Therefore, ING3 may regulate asymmetric cell division through the mTOR pathway during mouse oocyte maturation.
Highlights
Oocyte maturation in mammals is characterized by a unique asymmetric cell division
After washing three times in Phosphate Buffered Saline (PBS)/PVP, oocytes were treated with 0.5% Triton X-100 in PBS for 40 min at room temperature (RT), blocked in blocking solution for 1 h at RT, and incubated overnight at 4uC with one of the following reagents diluted into blocking solution: mouse antiING3 antibody (1:100 dilution, sc-101245, Santa Cruz Biotechnology Inc., Dallas, TX); 2 mg/ml FITC-conjugated lectin (L7381, Sigma-Aldrich); rabbit anti-acetylated H4K12 (AcH4K12) antibody (1:300 dilution 06761, Millipore Corp., Billerica, MA); or rabbit anti-mTOR antibody (1:2000 dilution, ab2732, Abcam Ltd., Cambridge, UK)
Together with the knowledge that ING3 is an important subunit of the nucleosome acetyltransferase of histone 4 (NuA4) histone acetyltransferase complex, these observations suggested that ING3 functions as a chromatin remodeling factor in fully grown germinal vesicle (GV) oocytes
Summary
After germinal vesicle breakdown (GVBD), the centrally positioned spindle migrates to the oocyte cortex; thereafter, asymmetric cell division occurs. The oocyte is transformed into a highly polarized, large metaphase II (MII)-arrested oocyte with an extruded small polar body [1]. Failure of this asymmetric cell division to occur is usually observed in low quality oocytes or those that have experienced postovulatory aging, a cause of mammalian infertility [2,3]. Asymmetric cell division depends upon the position of the spindle that is formed after GVBD. Normal asymmetric cell division is induced after the spindle migrates to the oocyte cortex during meiotic maturation. Details of the molecular mechanisms underlying oocyte polarization are poorly understood
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