Shotgun Proteomics Analysis Discards Alkali Labile Phosphate as a Reliable Method To Assess Vitellogenin Levels in Mytilus galloprovincialis

Abstract

Vitellogenin, the egg yolk precursor, is a well-known biomarker of endocrine disruption in oviparous vertebrates. In invertebrates, such as bivalves, it has been used in the last 10 years for the same purpose, despite the limited knowledge of invertebrate endocrinology. In bivalves, vitellogenin levels are usually estimated using an indirect technique, alkali labile phosphate (ALP), that assumes that vitellogenin is the most abundant phosphorylated protein in the analyzed tissue. In this study, we applied shotgun proteomics for the identification and quantification of vitellogenin in marine mussel gonads and compared the results with those obtained with the ALP method. The proteomic analysis revealed that vitellogenin is only detected in female gonads with expression levels that are rather variable among female mussels at different stages of gonad development. ALP analysis, on the contrary, detected similar amounts of phosphorylated proteins regardless of sex or gonad development stage. These results show evidence that the ALP method is not providing reliable information about Vtg levels, at least in marine mussel gonads. ALP is not a good proxy to assess Vtg levels in marine mussels, and careful verification of the adequacy of the procedure should be done before ALP is further assumed as a proxy of Vtg in other bivalve mollusks.

Critical review: Grand challenges in assessing the adverse effects of contaminants of emerging concern on aquatic food webs

“…The determination of transcription levels of target genes involved in essential cellular functions and pathways could be of great interest in evaluating the molecular responses to CECs. Proteomics has also become a useful tool to identify and understand processes involved in the bioactivity of CECs because it provides unique evidence about structure, function, posttranslational modifications, interactions, and abundance of proteins (Gouveia et al 2017;S anchez-Mar n et al 2017). Benzotriazole and its associated derivatives-as emphasized by proteomics-exhibit hepatotoxicity in the rare Chinese adult male minnow (Gobiocypris rarus) and the underlying mechanism associated with the damage (Liang et al 2017).…”

The utility of vitellogenin as a biomarker of estrogenic endocrine disrupting chemicals in molluscs

“…In this study, Vtg proteins were identified and quantified in male and female gonads by a shotgun label-free proteomic approach and the results were then compared with those measured by ALP. The results indicated that the proteomic approach detected Vtg only in females and their levels varied at different stages of gonadal development, whereas the ALP method detected similar amounts of phosphorylated protein regardless of sex or gonadal development stage (Sańchez-Marín et al, 2017).…”

“…ALP also provided unreliable information about Vtg levels in marine mussel gonads (Sańchez-Marín et al, 2017). In this study, Vtg proteins were identified and quantified in male and female gonads by a shotgun label-free proteomic approach and the results were then compared with those measured by ALP.…”

From Dark to Light – An Overview of Over 70 Years of Endocrine Disruption Research on Marine Mollusks

“…Finally, reference must be made that the robustness of the bioassay data in most of the papers which claim that mollusks use sex steroids as hormones in the same way as vertebrates, or seem affected by sex steroids, has been questioned (e.g., single experiments only, lack of replication, questionable endpoints, low effect sizes, lack of dose-responsiveness) (8). Highlighting two iconic examples: in contrast to the initial findings/ conclusions felt to be encouraging, 1) TBT was clearly demonstrated to exert its effect not via T but the retinoic X receptor (73-77) and 2) estrogens were shown to have absolutely no effect on vitellogenin production in bivalves (78)(79)(80)(81)(82). In his critical evaluation, Scott indicated the importance of a monotonic dose-response relationship for demonstrating a specific ligand-dependent effect and presented its lack in most of the steroid-effect relationships investigated in mollusks (8).…”