The Roles of Gonadotropin-releasing Hormone 2 (Gnrh2) in Feeding and Reproduction in Zebrafish: A Potential Mediator of These Interlinked Processes

Abstract

Gonadotropin-releasing hormone (GNRH) is the neuropeptide in vertebrates most well-known for controlling reproduction and stimulating the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), from the pituitary. The gonadotropins circulate to the gonads and stimulate steroidogenesis and gametogenesis. There are three forms of GNRH, named for their location and function in the brain. GNRH1 is known to be the main hypophysiotropic isoform regulating gonadotropin release. GNRH2 is found in the midbrain and, despite being the most evolutionarily conserved and ubiquitous, is the least studied form among the three, most likely due to the absence of GNRH2 protein in common murine research models. Zebrafish, another common vertebrate model for genetic manipulation and biological studies, possesses Gnrh2 and is therefore an ideal organism to study this neuropeptide. Through a combination of gene knockout/knockdown, neuroanatomical imaging, and functional assays, the roles of Gnrh2 in zebrafish were comprehensively explored. Gnrh2 knockout (gnrh2-/-) zebrafish demonstrated decreased lhb expression, compromised oocyte quality, increased feeding and growth, and, most strikingly, major inhibition of spawning and oocyte maturation after long-term fasting. Neuroanatomical assays confirmed that Gnrh2 projected more neurons to gonadotropes under fasting conditions, whereas Gnrh3 was inhibited. Interestingly, Gnrh2 was unable to compensate for Gnrh3 loss, as double knockout (gnrh2-/-;gnrh3-/-) zebrafish displayed normal reproduction. Through transcriptomics and qPCR, novel differentially expressed reproductive factors were discovered in knockout zebrafish, with potential reproductive regulatory roles. Additionally, examination of Gnrh2 neuronal projections identified fibers innervating melatonin cells, neurons of the feeding factor, Agouti-related peptide 1 (Agrp1), and neurons of the reproduction regulator, Gonadotropin-inhibitory hormone (Gnih). Gnrh2 was also able to modulate gnih and agrp1 expression. Additionally, a previously unstudied population of Gnrh2 was identified in the olfactory region and multiple experiments implicated these neurons in transducing pheromonal cues, and thus affecting reproductive behavior. Overall, Gnrh2 most likely has roles in stimulating lhb expression, maintaining oocyte quality, transducing pheromonal cues, and reducing feeding behavior through the modulation of agrp1 and potentially melatonin. Under fasting conditions, Gnrh2 exhibits plasticity to become the main hypophysiotropic stimulator of gonadotropin secretion. Gnrh2 therefore appears to be an important upstream factor mediating feeding and reproductive processes in vertebrates.