Specific miRNA‐G Protein‐Coupled Receptor Networks Regulate Sox9a/Sox9b Activities to Promote Gonadal Rejuvenation in Zebrafish

• Published in: Stem cells (Dayton, Ohio) Ohio), 2019-09, Vol.37 (9), p.1189-1199
• Main Authors: Guo, Huiping, Du, Xinlu, Zhang, Ying, Wu, Jiacheng, Wang, Chenghui, Li, Mingyou, Hua, Xianxin, Zhang, Xin A., Yan, Jizhou
• Format: Article
• Language: English
• Subjects: Aging; AKT protein; Cell differentiation; Coinjection; Differentiation; Fertility; Follicles; G protein-coupled receptors; Gene expression; Genes; Genomes; Germ cells; Germline; Gonads; Hormones; Hypothalamic-pituitary-gonadal axis; Hypothalamus; Kinases; Life span; MAP kinase; Mass spectrometry; Mass spectroscopy; Menopause; miRNA; Networks; Pituitary; Protein kinase C; Proteins; Self‐renewal; Sex; Sex differentiation; Signal transduction; Signaling; Sox9 protein; Spermatogenesis; Stem cells; Steroid hormones; Steroids; Tissue regeneration; Vertebrates; Zebrafish
• ISSN: 1066-5099; 1549-4918
• DOI: 10.1002/stem.3040

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′ 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 Coinjection of miR430a and Sox9a mRNA mixture into the testis induces testis somatic and germ cells, and promotes spermatogenesis. Thereby, miR430a and Sox9a reciprocally regulate G protein‐coupled receptor (lgr4, grk5l, grk4) networks, activate p‐PKC/p‐Akt and related signaling pathways, and reversely regulate Sox9a/b post‐translation modifications such as phosphorylation, ubiquitination, acetylation, and oxidation. In the ovary, coinjection of miR218a and Sox9b mixture induces ovarian somatic and germ cells, and increases primordial follicle reserve. Sox9b regulates G protein‐coupled receptor (gpr157, grk5l, grk4) networks, and extensively activates PKC/Akt and related signaling pathways, and reversely modulates Sox9a/b post‐translational modifications, whereas miR218a restrains su