Untitled - CNR

Marine research at CNRFigure 3: EROD activity levels (pmol min −1 mg prot −1 ± s.d.) in different size classesof M. barbatus.1 µg of total RNA from each liver samplewas used to synthesize cDNA. The fragmentsof genes of interest, vitellogenin andβ-actin (used as housekeeping gene), weresubsequently amplified in Thermal cyclerMX3000 (Statagene). PCR was carried outin a 20 µl reaction mixture and conductedat 95°C for 15 min, 32 cycles of denaturationat 94 °C for 30 s, annealing at 57 °Cfor 30 s, extension at 72 °C for 1 min, andterminated for 10 min at 72 °C. The PCRproducts were visualized by electrophoresisin agarose with ethidium bromide staining.2.5 Histological analysisGonadal samples were dehydrated in agraded series of ethanol, cleared in xylene,embedded in resin, cut in 5 µm-thicksections and stained with haematoxylineosin.Testes were staged for maturity asfollows: immature/resting (only spermatogonia)(stage I); developing (spermatocytesand spermatids, no spermatozoa) (stage II);maturing (all spermatogenic stages, includingspermatozoa, in the seminiferous lobules(stage III); running (spermatozoa inthe main sperm duct) (stage IV); spentrecovering(residual spermatozoa and spermatogonia)(stage V). The gonads werealso examined for histopathological alterations,such as necrosis, degeneration, feminization,and/or delayed or arrested gametedevelopment.2087