Fertility and endocrine function rely on a tightly regulated synchronicity within the hypothalamic-pituitary-gonadal axis, for which the sex gonad serves as the primary source of sex steroid hormones and germ cells. To maintain hormonal stasis and fertility throughout the lifespan, inducing gonadal stem cell renewal is an attractive strategy. The follicle-stimulating hormone/cAMP/MAPK/Sox9 signaling axis and its regulated specific miRNAs are thought to regulate vertebrate gonadal development and sex differentiation, yet the regulatory networks are largely unknown. By genome-wide transcriptome mining and gonadal microinjections, we identify two G protein-coupled receptor (GPCR)regulatory circuits: miR430a-Sox9a in the testis and miR218a-Sox9b in the ovary. Coinjection of a Sox9a-miR430a mixture promotes spermatogenesis, whereas Sox9b-miR218a mixture increases primordial ovarian follicles. Coimmunoprecipitation and mass spectrometry indicate that the two mixtures differentially modulate Sox9a/Sox9b multiple covalent modifications. We further reveal that miR430a and Sox9a synergistically activate testicular protein kinase C (PKC)/Akt signaling, whereas the miR218a and Sox9b mixture constrains ovary PKC/Akt signaling. pMIR-GFP reporter assay demonstrate that miR430a and miR218a target the 3 0 untranslated region (UTR) of four GPCR targets (lgr4, grk5l, grk4, and grp157). Knockdown of these GPCR genes or two Sox9 genes alters miR430a and miR218a regulation in the above gonad-specific PKC and Akt signaling pathways. These results establish two specific miRNA-GPCR-Sox9 networks and provide mechanistic insight into gonadal differentiation and rejuvenation. STEM CELLS 2019;37:1189-1199 SIGNIFICANCE STATEMENTAccumulating evidence suggests that microRNAs and small molecule cocktails are able to manipulate cell fate and plasticity in stem cells and differentiated cells. However, phenotypic conversion of sex gonad requires intricate coordination of multiple somatic and germline lineages. Zebrafish genome-wide transcriptome mining was performed, and it was found that gonadal injection of miR430a-Sox9a mixture and miR18-Sox9b mixture could increase the testis spermatogonia reserve and ovarian follicle reserve, respectively. This study also predicts that the miRNA-target pair data reported will help identify more potential therapeutic targets and promote the development of new therapeutic interventions for aging-related ovarian failure and testicular regression in humans.
14Fertility and endocrine function rely on a tightly regulated synchronicity within the hypothalamic-pituitary 15 gonadal (HPG) axis. FSH/cAMP/MAPK/ Sox9 axis signaling and its regulated specific miRNAs are thought 16 to regulate vertebrate gonadal development and sex differentiation, and yet the regulatory networks are largely 17 unknown. Here we construct small RNA and mRNA libraries from sexually matured ovary and testis of 18 zebrafish to identify specific miRNA-target pairs. Integration of Targetscan prediction and in vivo induced 19 gene expression highlight four specific miRNAs that conditionally target three G protein-coupled receptor 20 (GPCR) -Sox9 signaling genes, and implicate two regulatory circuits of miR430a-Sox9a in the testis and 21 miR218a-Sox9b in the ovary. Co-injected Sox9a-miR430a mixture increases the proportion of spermatogonia 22 but degenerates primary oocyte, while Sox9b-miR218a mixture induces renewal of ovarian follicles. 23Co-immunoprecipitation and mass-spectrometry analyses further reveal that miR430a and Sox9a 24 synergistically activate testicular PKC/Rock1 signals while miR218a and Sox9b constrict ovary 25 PKC/PI3K/Rock1 signaling. These results clarify specific miRNAs-GPCR regulatory networks of 26 Sox9a/Sox9b switch, and also provide mechanistic insight into gonadal rejuvenation and plasticity. 27 28
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