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Diffusion of small molecules into medaka embryos improved by electroporation.

Jung G, Hug M, Halter C, Friesenhengst A, Walzer J, Czerny T - BMC Biotechnol. (2013)

Bottom Line: Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules.The diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion.These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department for Applied Life Sciences, University of Applied Sciences, FH Campus Wien, Helmut-Qualtinger-Gasse 2, A-1030 Vienna, Austria.

ABSTRACT

Background: Diffusion of small molecules into fish embryos is essential for many experimental procedures in developmental biology and toxicology. Since we observed a weak uptake of lithium into medaka eggs we started a detailed analysis of its diffusion properties using small fluorescent molecules.

Results: Contrary to our expectations, not the rigid outer chorion but instead membrane systems surrounding the embryo/yolk turned out to be the limiting factor for diffusion into medaka eggs. The consequence is a bi-phasic uptake of small molecules first reaching the pervitelline space with a diffusion half-time in the range of a few minutes. This is followed by a slow second phase (half-time in the range of several hours) during which accumulation in the embryo/yolk takes place. Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules. Testing electroporation we could establish conditions to overcome the diffusion barrier. Applying this method to lithium chloride we observed anterior truncations in medaka embryos in agreement with its proposed activation of Wnt signalling.

Conclusions: The diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion. These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos.

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Electroporation affects the transfer of fluorescein through inner membranes, but not the chorion. Embryos were incubated with 10 μg/ml fluorescein for 40 minutes (A, blue bars; B-D); subsequent electroporation (A, red bars; E-G) was performed using 35 kHz, 15 V, 5000 ms, 1 pulse. Pictures were taken after 30 minutes washing time. Embryos are shown in lateral (B,E) and dorsal (D,G) view with anterior to the top. Scale bar 250 μM. Abbreviations: ELPO, electroporation; DIFF, diffusion.
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Figure 5: Electroporation affects the transfer of fluorescein through inner membranes, but not the chorion. Embryos were incubated with 10 μg/ml fluorescein for 40 minutes (A, blue bars; B-D); subsequent electroporation (A, red bars; E-G) was performed using 35 kHz, 15 V, 5000 ms, 1 pulse. Pictures were taken after 30 minutes washing time. Embryos are shown in lateral (B,E) and dorsal (D,G) view with anterior to the top. Scale bar 250 μM. Abbreviations: ELPO, electroporation; DIFF, diffusion.

Mentions: In order to verify that electroporation improves the rate limiting diffusion through inner membrane systems, we performed the same experiments with dead and dechorionated embryos (Figure 5). Electroporation did not change the signal intensity of fluorescein in dead embryos, indicating that it has no effect on the uptake through the chorion (Figure 5A,C,F), whereas dechorionated embryos showed a nearly 15-fold increase of dye intensity after electroporation (Figure 5A,D,G). Therefore, similar to whole embryos (Figure 5A,B,E), the dechorionated embryos still contain membranes acting as diffusion barriers, which can be made permeable by electroporation.


Diffusion of small molecules into medaka embryos improved by electroporation.

Jung G, Hug M, Halter C, Friesenhengst A, Walzer J, Czerny T - BMC Biotechnol. (2013)

Electroporation affects the transfer of fluorescein through inner membranes, but not the chorion. Embryos were incubated with 10 μg/ml fluorescein for 40 minutes (A, blue bars; B-D); subsequent electroporation (A, red bars; E-G) was performed using 35 kHz, 15 V, 5000 ms, 1 pulse. Pictures were taken after 30 minutes washing time. Embryos are shown in lateral (B,E) and dorsal (D,G) view with anterior to the top. Scale bar 250 μM. Abbreviations: ELPO, electroporation; DIFF, diffusion.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716799&req=5

Figure 5: Electroporation affects the transfer of fluorescein through inner membranes, but not the chorion. Embryos were incubated with 10 μg/ml fluorescein for 40 minutes (A, blue bars; B-D); subsequent electroporation (A, red bars; E-G) was performed using 35 kHz, 15 V, 5000 ms, 1 pulse. Pictures were taken after 30 minutes washing time. Embryos are shown in lateral (B,E) and dorsal (D,G) view with anterior to the top. Scale bar 250 μM. Abbreviations: ELPO, electroporation; DIFF, diffusion.
Mentions: In order to verify that electroporation improves the rate limiting diffusion through inner membrane systems, we performed the same experiments with dead and dechorionated embryos (Figure 5). Electroporation did not change the signal intensity of fluorescein in dead embryos, indicating that it has no effect on the uptake through the chorion (Figure 5A,C,F), whereas dechorionated embryos showed a nearly 15-fold increase of dye intensity after electroporation (Figure 5A,D,G). Therefore, similar to whole embryos (Figure 5A,B,E), the dechorionated embryos still contain membranes acting as diffusion barriers, which can be made permeable by electroporation.

Bottom Line: Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules.The diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion.These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department for Applied Life Sciences, University of Applied Sciences, FH Campus Wien, Helmut-Qualtinger-Gasse 2, A-1030 Vienna, Austria.

ABSTRACT

Background: Diffusion of small molecules into fish embryos is essential for many experimental procedures in developmental biology and toxicology. Since we observed a weak uptake of lithium into medaka eggs we started a detailed analysis of its diffusion properties using small fluorescent molecules.

Results: Contrary to our expectations, not the rigid outer chorion but instead membrane systems surrounding the embryo/yolk turned out to be the limiting factor for diffusion into medaka eggs. The consequence is a bi-phasic uptake of small molecules first reaching the pervitelline space with a diffusion half-time in the range of a few minutes. This is followed by a slow second phase (half-time in the range of several hours) during which accumulation in the embryo/yolk takes place. Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules. Testing electroporation we could establish conditions to overcome the diffusion barrier. Applying this method to lithium chloride we observed anterior truncations in medaka embryos in agreement with its proposed activation of Wnt signalling.

Conclusions: The diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion. These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos.

Show MeSH
Related in: MedlinePlus