Development of a marine fish model for studying in vivo molecular responses in ecotoxicology.

A protocol for fixation and processing of whole adult marine medaka (Oryzias melastigma) was developed in parallel with in situ hybridization (ISH) and immunohistochemistry (IHC) for molecular analysis of in vivo gene and protein responses in fish. Over 200 serial sagittal sections (5microm) can be produced from a single adult medaka to facilitate simultaneous localization and quantification of gene-specific mRNAs and proteins in different tissues and subcellular compartments of a single fish. Stereological analysis (as measured by volume density, V(v)) was used to quantify ISH and IHC signals on tissue sections. Using the telomerase reverse transcriptase (omTERT) gene, omTERT and proliferating cell nuclear antigen (PCNA) proteins as examples, we demonstrated that it is possible to localize, quantify and correlate their tissue expression profiles in a whole fish system. Using chronic hypoxia (1.8+/-0.2 mgO(2)L(-1) for 3 months) as an environmental stressor, we were able to identify significant alterations in levels of omTERT mRNA, omTERT protein, PCNA (cell proliferation marker) and TUNEL (apoptosis) in livers of hypoxic O. melastigma (p<0.05). Overall, the results suggest that O. melastigma can serve as a model marine fish for assessing multiple in vivo molecular responses to stresses in the marine environment.

Impairment of sea urchin sperm quality by UV-B radiation: predicting fertilization success from sperm motility.

Sperm quality of the sea urchin, Anthocidaris crassispina, after exposure to environmentally realistic UV-B irradiances, was assessed by changes in sperm motility (measured by the computer-assisted sperm analysis (CASA) system), and related to subsequent fertilization success. Percentage motile sperm of A. crassispina declined significantly after exposure to a UV-B dose of 16.2 kJ m(-2), while sperm motion velocity as measured by curvilinear velocity (VCL), straight line velocity (VSL), and average path velocity (VAP) showed significant reduction after exposure to a UV-B dose of 5.4 kJ m(-2). A parallel study showed that fertilization success was significantly reduced after sperm were exposed to UV-B doses > or = 5.4 kJ m(-2). Notably, the four sperm motility parameters were strongly correlated with fertilization success (P < 0.001), followed the increasing order: VSL (r = 0.8) < % motile sperm (r = 0.804) < VCL (r = 0.912) < VAP (r = 0.928). Fertilization success is best predicted by VAP using the exponential model: y = 8.678 + 90.202/[1 + exp(82.83 - x)/10.27)] (r(2) = 0.95). Thus, impairment of sperm motility of sea urchin, as measured by the CASA method, can be used to predict reproductive success and ecological effects.