Fig 2: Fam70A interacts directly with oocyte‐predominant Wnt5a. A, SDS‐PAGE and silver staining of Fam70A antibody or control IgG IP complex showed many distinct bands; we cut and sent these distinct bands for IP‐MALDI to identify the potential interacting proteins of Fam70A (see also Figure S2A). B, Among all Wnt members, we found that Wnt5a (red dot‐line square) is the most abundant within mouse oocytes. C, Quantification of (B). D, Immunofluorescence showed that the Fam70A signal on the oocyte membrane highly overlapped with Wnt5a. Fam70A in green, Wnt5a in red, DNA in blue. E, Co‐IP in oocyte lysate showed that endogenous Fam70A and Wnt5a interacted with each other. F, Co‐IP in the lysate of NIH3T3 cells co‐transfected with Fam70A and Wnt5A plasmids showed that exogenous Fam70A and Wnt5a also interacted with each other. G, Yeast 2‐hybrid assays showed that Fam70A directly binds Wnt5a on both Trp/Leu‐deficient and Trp/Leu/His/Ade‐deficient Agar plate. H, Immuno‐EM showed that Fam70A localized close (<20 nm) to Wnt5a on oocyte membrane or microvillus. Fam70A and Wnt5a were labelled with 35 and 15 nm gold particles, respectively. Scale bar in E, 20 µm. Scale bar in F, 400 nm. GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II

Fig 3: Fam70A working model. At cell membrane, Fam70A directly binds Wnt5a, which results in the inhibition of APC and activation of Akt (p‐Akt). APC promotes the phosphorylation (inactive form) of β‐catenin and stabilizes microtubules as well. β‐Catenin (active form) can get into the nucleus to promote gene transcription (but not in fully grown oocytes). Proper microtubule stability and p‐Akt together promote meiosis and quality of mouse oocytes

Fig 4: The “female fertility factor” Fam70A is enriched on the oocyte membrane. A, Immunohistochemistry showed that Fam70A was enriched on the oocyte membrane of growing oocytes. Fam70A was developed in brown; DNA was stained with haematoxylin. Two secondary follicles were shown. B, Western blot showed that Fam70A was more abundant in oocytes (Oos) than in granular cells (GCs). C, Western blot showed that the Fam70A protein level remarkably elevated as follicles were initially recruited (PND 21). D, Western blot showed that the Fam70A protein level kept constant during meiosis. E, Z‐slices of Fam70A immunofluorescent image showed that Fam70A was enriched on the oocyte membrane. F, Immunofluorescence showed that Fam70A remained on the oocyte membrane during meiosis. Scale bar, 20 µm. GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; PND, post‐natal day

Fig 5: Fam70A and Wnt5a regulate adenomatous polyposis coli (APC) level and MT stability. A, Western blot showed that APC protein remarkably increased after Fam70A depletion. B, Quantification of (A). C, Immunofluorescence showed that APC was enriched within spindles and remarkably increased after Fam70A depletion. APC in green, tubulin in red, DNA in blue. D, Western blot showed that Wnt5a was efficiently reduced by siRNA (Table S2). E, Western blot showed that APC remarkably increased after Wnt5a knockdown. F, Quantification of (E). G, RT‐PCR showed that APC was efficiently reduced by siRNA (Table S3). H, Fam70A depletion and Wnt5a knockdown both remarkably increased acetylated tubulin level within spindles, while APC knockdown remarkably reduced acetylated tubulin level. Acetylated tubulin in green, DNA in blue. I, Quantification of (H). Scale bar, 5 µm. *P < .05