Mammalian nucleolar protein DCAF13 is essential for ovarian follicle maintenance and oocyte growth by mediating rRNA processing

摘要

Expression and conditional knockout of DCAF13 in growing mouse oocytes. a Immunohistochemistry (IHC) of DCAF13 on ovarian sections showing DCAF13 expression in follicles at indicated developmental stages. Scale bars, 50 μm. The arrows indicate oocytes in primordial follicles. The hollow arrows indicate the nucleolus of growing oocytes. The asterisk indicates a nucleolus-like body in the fully grown oocyte. b Immunofluorescence (IF) of DCAF13 in growing and fully grown oocytes. DNA was counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bars, 25 μm. c Western blot result showing DCAF13 expression in growing oocytes and fully grown oocytes isolated from WT mice at postnatal day 12 and 23, respectively. Total proteins from 100 oocytes were loaded in each lane. d Co-localization of DCAF13 and the nucleolus marker fibrilarin (FBL) in oocytes and in HeLa cells. e A schematic diagram showing ovarian follicular development and oocyte-specific Dcaf13 knockout at primordial and primary follicle stages by Gdf9-Cre and Zp3-Cre, respectively. DCAF13 expression level was high in growing NSN oocytes and decreased in fully grown SN oocytes. Red and green curves represent the level of total RNA transcription activity and level of DCAF13 expression, respectively. f Diagram showing the strategy of Dcaf13 conditional knockout and genotyping approach. The sequences of genotyping primers (GP1, GP2, and GP3) were provided in the Supplementary Table 1. g IHC results showing DCAF13 protein expression in oocytes of mice with indicated genotypes. Arrows and hollow arrows indicate primordial follicles and oocyte nuclei of growing follicles, respectively. h Western blot results showing DCAF13 levels in 100 fully grown oocytes isolated from control (WT) and Dcaf13fl/fl;Zp3-Cre (KO) mice. ERK1/2 was blotted as a loading control