Spatial and quantitative gene expression analysis of SREB receptors in the gonads of green-spotted pufferfish (Dichotomyctere nigroviridis)

Abstract

Super-conserved Receptors Expressed in Brain (SREB) are a highly conserved family of orphan G protein-coupled receptors that consist of three members in most vertebrates: SREB1 (GPR27), SREB2 (GPR85), and SREB3 (GPR173). Each receptor is associated with diverse physiological processes and expressed in both ovaries and testes, but reproductive functions are only beginning to be understood. In addition, some fishes gained a novel fourth gene, SREB3B, which may have unique functions. The purpose of this study was to conduct a spatial and quantitative analysis of SREBs in the gonads of pufferfish (Dichotomyctere nigroviridis), which expresses all four genes. Multiplex RNAscope and absolute qPCR were used to assess gene expression patterns in both ovaries and testes. Expression was detected in early ovaries and dominated by sreb1 (approximately 2500 copies/ng RNA vs. 300 or less for others), with notable expression of all receptors in primary oocytes, granulosa cells, and small numbers of extra-follicular cells. Within primary oocytes, sreb1 and sreb3b exhibited diffuse patterns that may indicate early functions, while sreb2 and sreb3a were granular and may reflect stored mRNA. Early testicular development was dominated by sreb1 and sreb2 (∼5000 copies/ng RNA) in spermatogonia. These patterns were somewhat reduced in late testes (∼1000–2600 copies/ng RNA), but sreb3b exhibited a novel spatial pattern (∼380 copies/ng RNA) within spermatogenic cysts. These results highlight diverse roles for the SREB family, and sreb3b is hypothesized to have unique roles in fish reproduction.