Dynamic developmental states of follicles are regarded to be determinants of sexual maturation in fish ovaries. However, it is still a challenge to identify the critical points at which the developmental processes of different types of follicles interact and affect the ovarian development. In this study, four subtypes of the primary follicle (PF) in the ovarian folliculogenesis of zebrafish, i.e., the so-called PF-i, PF-ii, PF-iii, and PF-iv, are first identified by discontinuous NaCl-Percoll gradient centrifugation, as well as their respective morphological features. Then, for the four subtypes of PFs, stage-specific comparative analysis is employed to identify the differentially expressed genes and the differentially methylated regions, which have been validated to be significantly enriched in biological processes encompassing ribosomal biogenesis, meiotic progression, transcriptional regulation, and mitochondrial respiration. Results from transcriptional analysis further demonstrate significant changes in the expression profiles at different developmental stages from the PF-ii to the PF-iii. By molecular biology identification, it is shown that the enhancement of Notch and mTOR pathways can significantly regulate the ovarian development through the pacing effect of primary follicles. Clearly, all these uncovered results could provide a deeper understanding of the initial regulation of ovarian maturation, as well as a new multidisciplinary analytic tool to study follicle candidate regulators in the developmental process of other fish.

Folliculogenesis is essential for fish reproduction, but the role of ERK signaling during early primary follicle development in teleosts remains unclear. Here, we reanalyzed and integrated previously published transcriptomic and DNA methylation datasets from four primary follicle subtypes in zebrafish to identify MAPK-related candidate genes. No new sequencing was performed. Additional qRT-PCR and immunohistochemical analyses examined gene expression and the localization of total ERK and JNK proteins, respectively. Zebrafish were also exposed to the ERK inhibitor SCH772984 from 25 to 60 days post-fertilization to assess changes in ovarian development. The reanalysis identified MAPK-related genes with stage-associated differences in expression and DNA methylation. SCH772984 exposure was associated with reduced ovarian ERK phosphorylation and a lower proportion of advanced follicles. These results support a role for ERK signaling in the progression of early ovarian follicles in zebrafish. These observations derive from a single ERK inhibitor at nominal exposure; complementary assays could strengthen the evidence for target specificity.