Sublethal hypoxia exposure during medaka (Oryzias latipes) embryonic development induced persistent detrimental effects on hepatic growth and functions

Abstract

Aquatic hypoxia, a pressing global environmental concern, is among the most widespread, deleterious anthropogenic influence on freshwater, estuarine and marine environments. Increasing in vitro and in vivo evidence supports the proposition that hypoxia can detrimentally affect the structure and function of the liver in adult vertebrates, from fish to mammals. Surprisingly, the impacts of early life stage exposure to hypoxia on embryonic liver development and function have never been reported in any vertebrates. The liver is central in maintaining organism homeostasis, and liver growth is intimately associated with the overall growth of the individual. If impairments to embryonic liver growth and function are irreversible and unrecoverable upon abatement of hypoxia, it is likely that significant long-term impacts on the vitality and fitness of surviving adults will occur. This postulation, however, has never been validated. Given that hypoxia is a potent teratogen, it is hypothesized that sublethal exposure to hypoxia during early life stages can impair embryonic liver growth and function, the impairments are not recoverable, and will persist into adulthood. In this study, Japanese medaka (Oryzias latipes) was employed to determine the adult consequences of early life stage exposure to aquatic hypoxia. Embryos were exposed to sublethal levels of hypoxia (1.5±0.1 mg O2/L) from Stage 9 (late morula) to Stage 39 (hatched eleutheroembryo, EE). Hypoxia exposure not only resulted in a delay of hatching, but also led to retardation of body growth, as indicated by a significant reduction in body length. The small size of medaka embryos renders isolation of liver and subsequent molecular analyses difficult within the intact organism. A cost effective, high throughput platform (named the 'embryo chip'), which allows synchronical processing and sectioning of multiple chorionated embryos, was developed for single embryo parallel analyses of gene and protein expressions by quantitative in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. This high-throughput embryo chip was used to quantify cell proliferation (marked by immno-staining of proliferative cell nuclei antigen, a.k.a. PCNA) and apoptosis (marked by terminal deoxynucleotidyl transferase dUTP nick end labeling, a.k.a. TUNEL) in multiple normoxia- and hypoxia- exposed embryos simultaneously. A reduced body size and, particularly, a 'stunted' liver in hypoxia-exposed embryos were mainly mediated via suppression of cell proliferation rather than alteration in apoptosis. The impact on the liver was strikingly severe as indicated by PCNA:TUNEL of < 1, suggesting proliferation rate was likely out-balanced by basal apoptotic rate and resulted in a net reduction in liver mass (measured in hepatosomatic index, a.k.a. HSI). The livers of exposed embryos were likely functionally impaired, as indicated by aberrant expression of a set of marker genes directly involved in vital hepatic functions, including (i) body growth regulation: igfbp1a, igfbp1b, igfbp2; (ii) host defense: c9, f2, hamp, plg; and (iii) biotransformation: cyp1a, cyp2j, cyp3a. Changes in expression patterns of these genes (measured by real-time PCR) were consistent with the observed stunted body growth and compromised immunocompetence in hypoxia-exposed embryos. Subsamples of hypoxia-exposed embryos were allowed to recover under normoxic conditions (>7 mg O2 L-1) for three months (reaching sexual maturity). Liver functions were re-evaluated for reversibility of impacts stemming from early life stage hypoxic stress. The hepatic genes that are related to body growth regulation (igfbp1b) and anti-microbial peptide production (hamp) remained differentially expressed in hypoxia-exposed, surviving individuals. The results imply an incomplete recovery of the liver's coordination of body growth and host defenses. This postulation was confirmed by further experimental findings showing the growth of these "pre-hypoxia" adults could not fully recover (as indicated by a reduced Fulton's condition factor) and the surviving adults remained more susceptible to infection by opportunistic bacteria. This study is the first report indicating that sub-lethal aquatic hypoxia during medaka embryonic development is detrimental to liver growth and function, and that the perturbations persisted from the embryo through to adulthood, signaling a long-term threat to the fitness of the surviving individuals. Future risk assessments of aquatic hypoxia on fish/wildlife populations should therefore take into consideration the lifelong and irreversible impairments caused in pre-hypoxia exposed individuals.

Date of Award	15 Jul 2013
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Wai Ting Doris AU (Supervisor)