A proposed mechanism of ammonia excretion in the seahorse brood pouch revealed by rhesus glycoprotein localization.

In seahorses (family Syngnathidae), the male brood pouch provides a protected environment for developing embryos. During pregnancy, embryos develop within an enclosed brood pouch, requiring a physiological mechanism to eliminate the nitrogenous waste excreted by the embryos. This study investigated the expression and localization of rhesus (Rh) glycoproteins, expected to function as ammonia transporters, in the brood pouch of the pot-bellied seahorse Hippocampus abdominalis. The expression of the RhAG, RhBG, and RhCG2 genes in the pouch was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). In situ hybridization and immunohistochemistry revealed that RhBG and RhCG2 are localized in the inner epithelium of the pouch, while RhAG is present in erythrocytes and the endothelium of the blood vessels. Based on these findings, we propose the following waste removal pathway. Ammonia, presumably released from the embryo, would be transported from the lumen of the pouch through apical RhCG2 and basolateral RhBG of inner epithelial cells into the connective tissue beneath the inner epithelium. In the connective tissue, ammonia passes through RhAG-expressing endothelial cells into the vascular lumen and is likely taken up by RhAG-expressing erythrocytes, which then travel to the gills via the bloodstream. Then, ammonia is released from the RhAG-expressing erythrocytes through RhAG-expressing pillar cells, basolateral RhBG- and apical RhCG2-expressingpavement cells, finally into the surrounding seawater. This study provides a first molecular and cellular basis for a proposed ammonia removal mechanism within the seahorse brood pouch, highlighting physiological adaptations associated with male pregnancy in syngnathid fish.