Multiplex in situ hybridization chain reaction reveals the spatial organization of six pituitary hormone mRNAs in the Japanese eel.

The anterior pituitary gland regulates growth, development, metabolism, reproduction, and stress responses. In this study, we established a two-round multiplex in situ hybridization chain reaction (isHCR) procedure combined with a fluorescence-quenching device to visualize the spatial distribution of six pituitary hormone mRNAs (gh, lhb, fshb, tshb, prl, and pomc) in cultured immature Japanese eels. In pituitary sections, prl and tshb were localized to the rostral pars distalis (RPD), whereas lhb, fshb, and gh were distributed in the proximal pars distalis. Pomc was detected in both the RPD and pars neurointermedia, corresponding to adrenocorticotropic hormone- and α-melanocyte-stimulating hormone-producing cells, respectively. Owing to efficient tissue penetration of probes and hairpin DNAs, the isHCR protocol was also applicable to whole-mount pituitary preparations, enabling comprehensive three-dimensional visualization of all six hormone mRNAs. Spatial analyses revealed an orderly organization of hormone-producing cells and demonstrated co-expression of lhb and fshb within the same cells, indicating the presence of bihormonal gonadotrophs in teleosts, with regional variation in their expression. To assess quantitative capability, prl signal intensity-given its established role as a freshwater- (FW) adaptation hormone-was compared between FW- and seawater-(SW) acclimated eels. Prl signals were significantly reduced in SW-acclimated eels, consistent with RT-qPCR results, indicating that isHCR can provide semi-quantitative gene expression information. Furthermore, this method enabled subcellular localization of transcripts, including nuclear heteronuclear RNA of lhb containing intronic sequences. This multiplex spatial transcriptomic approach provides a powerful tool for pituitary research and is broadly applicable to non-model organisms in comparative endocrinology.