Constraints-based stoichiometric analysis of hypoxic stress on steroidogenesis in fathead minnows, Pimephales promelas

Abstract

In this study, an in silico genome-scale metabolic model of steroidogenesis was used to investigate the effects of hypoxic stress on steroid hormone productions in fish. Adult female fathead minnows (Pimephales promelas) were exposed to hypoxia for 7 days with fish sub-sampled on days 1, 3 and 7 of exposure. At each time point, selected steroid enzyme gene expressions and steroid hormone productions were quantified in ovaries. Fold changes in steroid enzyme gene expressions were used to qualitatively scale transcript enzyme reaction constraints (akin to the range of an enzyme's catalytic activity) in the in silico model. Subsequently, in silico predicted steroid hormone productions were qualitatively compared with experimental results. Key findings were as follows. (1) In silico gene deletion analysis identified highly conserved 'essential' genes required for steroid hormone productions. These agreed well (75%) with literature-published genes downregulated in vertebrates (fish and mammal) exposed to hypoxia. (2) Quantification of steroid hormones produced ex vivo from ovaries showed a significant reduction for 17β-estradiol and 17α,20β-dihydroxypregnenone production after 24 h (day 1) of exposure. This lowered 17β-estradiol production was concomitant with downregulation of cyp19a1a gene expression in ovaries. In silico predictions showed agreement with experimentation by predicting effects on estrogen (17β-estradiol and estrone) production. (3) Stochastic sampling of in silico reactions indicated that cholesterol uptake and catalysis to pregnenolone along with estrogen methyltransferase and glucuronidation reactions were also impacted by hypoxia. Taken together, this in silico analysis introduces a powerful model for pathway analysis that can lend insights on the effects of various stressor scenarios on metabolic functions.