Androgens and androgen receptor directly induce the thickening, folding, and vascularization of the seahorse abdominal dermal layer into a placenta-like structure responsible for male pregnancy via multiple signaling pathways

Abstract

Seahorses exhibit the unique characteristic of male pregnancy, which incubates numerous embryos in a brood pouch that plays an essential role in enhancing offspring survivability. The pot-belly seahorse (Hippocampus abdominalis) possesses the largest body size among seahorses and is a significant species in Chinese aquaculture. In this study, we revealed the cytological and morphological characteristics, as well as regulatory mechanisms, throughout the entire brood pouch development in H. abdominalis. The brood pouch originated from the abdominal dermis, extending towards the ventral midline. As the dermal layers thicken, the inner epithelium folds, the stroma loosens, and vascularization occurs, culminating in the formation of the brood pouch. Furthermore, through transcriptomic analysis of brood pouches at various developmental stages, 8 key genes (tgfb3, fgf2, wnt7a, pgf, mycn, tln2, jund, ccn4) closely related to the development of brood pouch were identified in the MAPK, Rap1, TGF-β, and Wnt signaling pathways. These genes were highly expressed in the pseudoplacenta and dermal layers at the newly formed stage as examined by in situ hybridization (ISH). The angiogenesis, densification of collagen fibers, and proliferation of fibroblasts and endothelial cells in seahorse brood pouch formation may be regulated by these genes and pathways. Additionally, the expression of the androgen receptor gene (ar) was significantly upregulated during the formation of the brood pouch, and ISH confirmed the expression of the ar gene in the dermis and pseudoplacenta of the brood pouch, highlighting its role in the developmental process. Androgen and flutamide (androgen receptor antagonist) treatments significantly accelerated the formation of the brood pouch and completely inhibited its occurrence respectively, concomitant to the upregulated expression of differentially expressed genes involved above signaling pathways. These findings demonstrated that formation of the brood pouch is determined by androgen and the androgen receptor activates the above signaling pathways in the brood pouch through the regulation of fgf2, tgfb3, pgf, and wnt7a. Interestingly, androgen even induced the formation of the brood pouch in females. We firstly elucidated the formation of the seahorse brood pouch, demonstrating that androgens and their receptors directly induce the thickening, folding, and vascularization of the abdominal dermal layer into a placenta-like structure through multiple signaling pathways. These findings provide foundational insights to further exploring the evolution of male pregnancy and adaptive convergence in viviparity across vertebrates.