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

Abstract

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.

Role of sex‐biased miRNAs in teleosts – a review

“…Zebrafish miR-34a knockouts generated using CRISPR/Cas9 system showed enhanced sperm motility in comparison with wild-type fish without exhibiting any change in testicular morphology (Guo et al 2017). Guo et al (2019) explored the synchronicity between the gonadotropin signalling system and miRNA expression for the maintenance of gonadal functions in zebrafish. They identified miR430a-Sox9a and miR218a-Sox9b regulatory circuits in testis and ovary, respectively, that act to promote spermatogenesis and maintain the primordial follicles in the male and female gonads.…”

“…Guo et al . (2019) explored the synchronicity between the gonadotropin signalling system and miRNA expression for the maintenance of gonadal functions in zebrafish. They identified miR430a‐Sox9a and miR218a‐Sox9b regulatory circuits in testis and ovary, respectively, that act to promote spermatogenesis and maintain the primordial follicles in the male and female gonads.…”

A Preview of Selected Articles

“…A recent Stem Cells article from researchers led by Jizhou Yan (Shanghai Ocean University, Shanghai, China) sought to explore the mechanisms controlling zebrafish gonadal stem cell renewal with a focus on the miRNA‐mediated control of the sox9a and sox9b co‐orthologs . The team hoped that the identification of novel regulatory mechanisms could contribute to an increased understanding of how gonadal stem cells renew to maintain hormonal stasis and gametogenesis/fertility throughout a natural lifespan and identify therapeutic targets to treat fertility problems.…”

“…Importantly, recent research has identified miRNAs as vitally important paracrine‐acting factors; they are secreted by cells alongside other RNAs, DNA, and proteins within exosomes/extracellular vesicles and are crucial to the success of various stem cell therapies developed for a range of diseases and disorders. In our first Featured Article this month in Stem Cells , Guo et al establish the importance of two G protein‐coupled receptor (GPCR)‐regulatory circuits in zebrafish—miR430a‐Sox9a in the testis and miR218a‐Sox9b in the ovary—that may offer mechanistic insight into gonadal differentiation and rejuvenation and identify targets for the treatment of fertility problems . In a Related Article from Stem Cells Translational Medicine , Mead and Tomarev report that exosomes derived from mesenchymal stem cells (MSCs) protect retinal ganglion cells from death and preserve function in a rodent model of optic nerve crush through an miRNA‐dependent mechanism .…”

Heterotrimeric G proteins regulate planarian regeneration and behavior

“…Importantly, GPCR signaling regulates regeneration and wound responses throughout the animal kingdom including nematodes, fruit flies, and mammals (Ziegler et al 2009; Doze and Perez 2013; Kiseleva et al 2014; Zugasti et al 2014; Choi et al 2015; Guo et al 2019; O’Connor et al 2021). For example, the Protease Activated Receptor 1 (PAR1) GPCR promotes wound healing in murine skin by stimulating production of keratinocytes (Kiseleva et al 2014).…”