Genetic Evidence for the Gatekeeping Role of Ybx1 in Controlling Follicle Activation and Early Folliculogenesis by Regulation of Cdkn1a in the Zebrafish

Y box-binding protein 1 (YB-1; Ybx1/ybx1) regulates transcription and translation of targeted genes by DNA/RNA-binding. Our previous proteomic study demonstrated a dramatic drop in Ybx1 protein during follicle activation in zebrafish ovary. In this study, we created an ybx1 mutant with CRISPR/Cas9, and showed that the folliculogenesis in the mutant ovary (ybx1-/-) was blocked at pre-vitellogenic (PV)-to-early vitellogenic (EV) transition, leading to small ovaries. The mutant females were therefore sub-fertile with reduced fecundity. RNA-seq and Western blot analyses identified a variety of genes that showed differential expression between mutant ovary (ybx1-/-) and the control (ybx1+/-) including cdkn1a (p21). Disruption of cdkn1a resulted in embryonic lethality. In p21 heterozygotes (cdkn1a+/-), however, follicle activation and maturation in the ovary were both enhanced in contrast to ybx1 mutant (ybx1-/-). Interestingly, partial loss of p21 in heterozygotes (cdkn1a+/-) could rescue the phenotype of ybx1 mutant (ybx1-/-). Folliculogenesis resumed in ybx1-/-;p21+/- females with normal follicle activation, in contrast to the PV-EV blockade in ybx1-/- mutant. Interestingly, the follicle cells from the ybx1-/- mutant follicles displayed a poor proliferative activity in vitro; however, the cells from the ybx1-/-p21+/- follicles resumed normal proliferation compared to that from the wildtype fish. In summary, we demonstrated in this study that Ybx1 played a gatekeeping role in controlling PV-to-EV transition and it might act by suppressing the expression of cdkn1a, a cell cycle inhibitor. Considering our histological PV-to-EV follicle blockade, we further isolated PV follicles from both WT (ybx1+/+) and ybx1-/- zebrafish. After RNA isolation, we then estbalished bulk-RNAseq libraries according to NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. After libraries preparation, we performed sequencing using the Genomics, Bioinformatics and Single Cell Analysis Core at the Univerisity of Macau.