Peer Review #2 of "Aromatisation of steroids in the bivalve Mytilus trossulus (v0.1)"

Abstract

In this study, we demonstrated the presence of the enzymatic complex able to perform aromatization (estrogen synthesis) in both, the microsomal and mitochondrial fractions of gills and gonads from Mytilus trossulus. Based on in vitro experiments, we highlighted the importance of temperature as the limiting factor of aromatisation efficiency (AE) in mussels. After testing range of temperatures (4°C-23°C), the highest AE was found during incubation at 8°C and pH 7.6 (41.66 pmol/h/mg protein in gills and 58.37 pmol/h/mg protein in gonads). The results were confirmed during field studies where the most efficient aromatisation occurred in bivalves collected in spring while the least effective in those collected in winter. During in vitro studies, AE turned out to be more intensive in female gonads than in male gonads. The process was also more intensive in mitochondrial fraction than in microsomal one (62.97 pmol/h/mg protein in male gills and 73.94 pmol/h/mg protein in female gonads). Enzymatic complex (aromatase-like enzyme) catalysing aromatisation in mussels was found to be insensitive to inhibitory effect of selective inhibitors of mammalian aromatase such as letrozole and anastrazole, suggesting its different structure from vertebrate aromatase. Further in vivo studies using 13 C-labelled steroids at 8°C temperature window confirmed that bivalves are able to uptake testosterone and androstenedione from the ambient environment and metabolise them to estrone and 17β-estradiol thus confirming endogenous estrogen' synthesis.