It is known that the teleost scale regenerates after being removed. We previously reported that the osteogenesis in regenerating scales was very similar to that in calvarial bone, which suggests that regenerating scale can be used as a model for osteogenesis to analyze the inte raction b e t we e n oste oblasts and osteoclasts. In the present study, we developed an assay system using regenerating scales by modifying our assay system with normal scales. The weight of one regenerating scale cannot be measured precisely because the rate of calcification in regenerating scales is lower than that in normal scales. In regenerating scales, thus, the respective marker enzyme activit y, i.e ., alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts, was normalized by the surface area (mm 2) of each goldfish scale. With this modified method, we were able to detect ALP and TRAP activities with no variation in the lines of regenerating scales. In addition, we found that the ALP activity in the regenerating scales significantly increased under 3G acceleration loading by vibration, while the TRAP activity in the loaded regenerating scales significantly decreased. We strongly believe that the regenerating scale is a good material for the analysis of bone me tabolism unde r dif fe re nt gravit y environments.

Osteocytes, osteoblasts (bone-forming cells), and osteoclasts (bone-resorbing cells) are the primary types of cells that regulate bone metabolism in mammals. Sclerostin produced in bone cells activates osteoclasts, inhibiting bone formation; excess production of sclerostin, therefore, leads to the loss of bone mass. Fish scales have been reported to have morphological and functional similarities to mammalian bones, making them a useful experimental system for analyzing vertebrate bone metabolism in vitro. However, whether fish scales contain cells producing sclerostin and/or osteocytes has not been determined. The current study demonstrated, for the first time, that sclerostin-containing cells exist in goldfish scales. Analysis of the distribution and shape of sclerostin-expressing cells provided evidence that osteoblasts produce sclerostin in goldfish scales. Furthermore, our results found that osteocyte-like cells exist in goldfish scales, which also produce sclerostin. Finally, we demonstrated that microgravity in outer space increased the level of sclerostin in the scales of goldfish, a finding suggesting that the induction of sclerostin is the mechanism underlying the activation of osteoclasts under microgravity.

We have previously reported that microgravity promotes the activation of osteoclasts in cultured regenerating scales. This osteoclastic activation was induced by increased levels of receptor activator of the nuclear factor-κB ligand (RANKL). Therefore, we determined that RANKL is an important factor in evaluating osteoclastogenesis in bone tissue. However, the role of RANKL in fish scales is poorly understood. In the present study, we prepared antiserum against goldfish RANKL in rabbits and detected RANKL-producing cells in regenerating goldfish scales. Furthermore, we studied osteoclastic activation by the addition of RANKL to examine exogenous RANKL on osteoclastogenesis in regenerating goldfish scales. As a result, RANKL immunepositive cells were detected in grooves of regenerating scales. In addition, treating the regenerating scales with mammalian RANKL for 3 h significantly increased the expression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which is essential for osteoclast differentiation. After 6 h of incubation with RANKL, the expression of cathepsin K, a functional osteoclastic gene, significantly increased. Furthermore, the molecules for osteoclast multinucleation and differentiation significantly increased following treatment with mammalian RANKL. Therefore, in fish scales as well as mammalian bone, we concluded that RANKL plays an important role in osteoclastogenesis. ©2020 Jpn. Soc. Biol. Sci.

Fish scale is a calcified tissue which is similar to those found in human bones. Recently, we developed a scale culture system in which osteoblasts and osteoclasts remain intact after being prepared. Using this system, we analyzed the bone metabolism under 2-, 4-, and 7-gravity (G) loading by centrifuge and compared with control (1-G). After loading for 5 and 10 min, the scales were incubated for 6 and 24 h. The osteoblastic and osteoclastic activities were then measured. The osteoblastic activity significantly increased under 2-and 4-G loading. This activity increased remarkably under 7-G loading. On the other hand, we found that the osteoclastic activity significantly decreased under 2-G loading. Under 4-G loading, there was no significant difference between G-loaded scales and control scales. The osteoclastic activity tended to increase under 7-G loading. These results were similar to our previous study of acceleration by vibration. Bone consists of osteoblasts, osteoclasts, and bone matrix. In our culture system including bone matrix, osteoblasts and osteoclasts reacted precisely to G loading by centrifuge. Therefore, we strongly believe that our culture system is useful for the analysis of bone metabolism under G loading.