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Previous studies have shown fertilization and development of marine species can be significantly inhibited when the pH of sea water is artificially lowered. Little mechanistic understanding of these effects exists to date, but previous work has linked developmental inhibition to reduced cleavage rates in embryos. To explore this further, we tested whether common cell cycle checkpoints were involved using three cellular biomarkers of cell cycle progression: (1) the onset of DNA synthesis, (2) production of a mitotic regulator, cyclin B, and (3) formation of the mitotic spindle. We grew embryos of the purple sea urchin, Strongylocentrotus purpuratus, in seawater artifically buffered to a pH of ∼7.0, 7.5, and 8.0 by CO(2) infusion. Our results suggest the reduced rates of mitotic cleavage are likely unrelated to common cell cycle checkpoints. We found no significant differences in the three biomarkers assessed between pH treatments, indicating the embryos progress through the G(1)/S, G(2)/M and metaphase/anaphase transitions at relatively similar rates. These data suggest low pH environments may not impact developmental programs directly, but may act through secondary mechanisms such as cellular energetics.
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22479526
???displayArticle.pmcLink???PMC3313954 ???displayArticle.link???PLoS One
Figure 2. Impact of low pH on DNA synthesis.The onset of DNA synthesis in embryos developing in different pH seawater was monitored by incorporation of a modified uridine molecule, BrdU, during DNA replication. (A) No BrdU incorporation was detected in embryos developing in the absence of Brdu (BrdU−). Embryos developing in the presence of BrdU alone show strong incorporation into newly synthesized DNA within 30 minutes of fertilization, suggesting DNA synthesis is unaffected. When embryos were allowed to develop in the presence of both BrdU and the DNA synthesis inhibitor, Aphidocholin (BrdU+/Aph+), no BrdU incorporation was observed, suggesting the positive BrdU incorporation was due to synthesis of new DNA and not the activity of DNA repair enzymes. (B) Scoring of embryos (n = 300 per experimental culture) show no statistical difference in the number of BrdU positive embryos across treatments: pH 8.0 (white bars) pH 7.5 (grey bars) pH 7.0 (black bars).
Figure 3. Relative level of cyclin-B protein expression.The temporal changes of the mitotic regulator cyclin-B was determined during the first cellular cleavage of urchin embryos developing in seawater maintained at various pH levels. (A) No difference in the expression of cyclin-B protein relative to GADPH was observed between embryos developing at a pH of 8.0 (white bars), 7.5 (grey bars) or 7.0 (black bars). (B) A representative Western blot showing the specificity of the primary antibodies.
Figure 4. Impact of low pH on mitotic spindle formation.(A) Mitotic spindle formation was observed by staining the microtubules of sea urchin embryos during the first mitotic division. (B) Triplicate blind counts of a minimum 300 embryos from each culture found no significant difference in the number of malformed mitotic spindles observed in embryos reared in seawater held at a pH of 8.0 (white bar) 7.5 (grey bar) or 7.0 (black bar) for 90 min post-fertilization.
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