GnRH—Gonadotropes Interactions Revealed by Pituitary Single-cell Transcriptomics in Zebrafish

Abstract GnRH governs reproduction by regulating pituitary gonadotropins. Unlike most vertebrates, gnrh−/− zebrafish are fertile. To elucidate the role of the hypophysiotropic-Gnrh3 and other mechanisms regulating pituitary gonadotropes, we profiled the gene expression of all individual pituitary cells of wild-type and gnrh3−/− adult female zebrafish. The single-cell RNA sequencing showed that LH and FSH gonadotropes express the 2 gonadotropin beta subunits with a ratio of 140:1 (lhb:fshb) and 4:1 (fshb:lhb), respectively. Lh gonadotropes predominantly express genes encoding receptors for GnRH (gnrhr2), thyroid hormone, estrogen, and steroidogenic factor 1. No GnRH receptor transcript was enriched in FSH gonadotropes. Instead, cholecystokinin receptor-b and galanin receptor-1b transcripts were enriched in these cells. The loss of the Gnrh3 gene in gnrh3−/− zebrafish resulted in downregulation of fshb in LH gonadotropes and upregulation of pituitary hormones like TSH, GH, prolactin, and proopiomelanocortin-a. Likewise, targeted chemogenetic ablation of Gnrh3 neurons led to a decrease in the number of fshb+, lhb + and fshb+/lhb + cells. Our studies suggest that Gnrh3 directly acts on LH gonadotropes through Gnrhr2, but the outcome of this interaction is still unknown. Gnrh3 also regulates fshb expression in both gonadotropes, most likely via a non-GnRH receptor route. Altogether, while LH secretion and synthesis are likely regulated in a GnRH-independent manner, Gnrh3 seems to play a role in the cellular organization of the pituitary. Moreover, the coexpression of lhb and fshb in both gonadotropes provides a possible explanation as to why gnrh3−/− zebrafish are fertile.