Characterization of Microrna Expression Profiles and Role of Nodal-Related Genes in Zebrafish Ovarian Follicles

Zebrafish is a valuable model to study the biology of reproduction as the processes that regulate follicle development and oocyte maturation are conserved among vertebrates. In zebrafish, early vitellogenic (stage IIIa) ovarian follicles are maturationally incompetent while mid-late vitellogenic (stage IIIb) follicles are able to undergo oocyte maturation in response to maturation-inducing hormone signals. Signaling molecules derived from the ovary, such as microRNAs (miRNAs) and growth factors, are important in controlling ovarian function. To determine whether miRNAs may play a role in maturation competency acquisition, we characterized miRNA expression profiles in follicular cells isolated from stage IIIa and IIIb follicles. Bioinformatics analysis uncovered 214 known, 31 conserved novel and 44 novel miRNAs, of which 24 miRNAs were significantly regulated between stage IIIa and IIIb follicular cells. In addition, gene enrichment and pathway analyses of the predicted targets of the significantly regulated miRNAs supported the involvement of several key signaling pathways in regulating ovarian function. We then investigated the role of Nodal, a member of the transforming growth factor-β family, in regulating zebrafish ovarian function. We used real-time PCR to detect the zebrafish Nodal orthologs, nodal-related (ndr1) and ndr2 and found that they were expressed in ovarian follicles at all stages of development. We also detected the mRNAs for Nodal signaling components in follicular cells of vitellogenic follicles. Recombinant human Nodal activated Smad3, CREB, and ERK, and inhibited cell proliferation in ovarian follicular primary cell cultures. The mRNA levels of cyp17a1, hsd3b2 and paqr8 were increased in response to Nodal treatment. Subsequently, we used CRISPR/Cas9 technology to generate ndr1 and ndr2 null mutants, which caused severe defects in early development. To overcome this lethality in vivo, we developed a fluorescently-labeled, Doxycycline-inducible CRISPR-ON system that expresses single or multiplexed sgRNAs to knockout ndr1, ndr2, and ndr3. Activation of the system induced gene editing in the designated genomic loci. Our findings suggest that miRNAs and Nodal play a role in zebrafish follicles. The CRISPR-ON system will facilitate further investigating the roles of miRNAs and Nodal in adult zebrafish in vivo