Rojas V et al. (2019), Rhinella marina oocytes: a suitable alternative...

XB-ART-55616

Sci Rep 2019 Jan 10;91:18. doi: 10.1038/s41598-018-37069-6.

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Rhinella marina oocytes: a suitable alternative expression system for functional characterization of aquaglyceroporins.

Rojas V , Ortiz YY , Rodríguez S , Araque V , Rodríguez-Acosta A , Figarella K , Uzcátegui NL .

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Amphibian oocytes have been extensively used for heterologous expression of membrane proteins for studying their biochemical and biophysical properties. So far, Xenopus laevis is the main amphibian used as oocytes source to express aquaglyceroporins in order to assess water and solutes permeability. However, this well-established amphibian model represents a threat to the biodiversity in many countries, especially in those from tropical regions. For that reason, the import of Xenopus laevis is subjected to strict control, which essentially has restricted its use in these regions. Therefore, a wider variety of expression systems for aquaglyceroporins is needed. Rhinella marina is extensively distributed in the Americas and its native range spreads from South America to Texas, US. Here we report the use of Rhinella marina oocytes as an alternative expression system for aquaglyceroporins and demonstrated its suitability to determine the permeability to water and non-ionic solutes. Rhinella marina oocytes were able to functionally express channels from human and the protozoan pathogen Trypanosoma brucei, two very distant organisms on the evolutionary scale. Permeability values obtained from Rhinella marina oocytes expressing members of aquaporin family were similar and comparable to those values reported in the literature for the same channels expressed in Xenopus laevis oocytes.

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	Figure 1. Evaluation of the linearity of the standard oocytes hypo-osmotic swelling assay in R. marina oocytes. Oocytes of R. marina injected with RNA encoding for hAQP1, TbAQP1, 2 or 3 were subjected the standard oocytes hypo-osmotic swelling assay and the relative volume change (d(V/V0)/dt) was monitored. Control cells were injected with water.
	Figure 2. Water permeability values in R. marina oocytes expressing hAQP1, TbAQP1, 2, or 3. R. marina oocytes were injected with water (control cells) or 5, 10, and 25 ng of cRNA encoding for hAQP1, TbAQP1, 2, or 3 and subjected to the standard oocytes hypo-osmotic swelling assay every day. Water permeability values were obtained according to Pf equation. Results are shown as mean values ± standard error (SE). Kruskal-Wallis statistical test was used and the statistical significance is represented as >0.05 (), 0.01 (), and 0.001 (). Pf values of oocytes expressing hAQP or TbAQPs compared to control oocytes were in all cases statistically different. For simplicity, the figure only shows the statistical significance of the cRNA amounts compared among them starting from the second day. For instance, (a) day 2 (green square, , inverted blue triangle, *, and orange diamond) indicates that 10 ng is statically different from 5 ng () and from 25 ng (***), but there are no differences between 5 ng and 25 ng.
	Figure 3. Evaluation of the linearity of the standard oocytes iso-osmotic swelling assay in R. marina oocytes. Oocytes of R. marina expressing TbAQP2 or 3 were subjected to the standard oocytes iso-osmotic swelling assay and the relative volume change (d(V/V0)/dt) was monitored. Control cells were injected with water.
	Figure 4. Glycerol permeability values in R. marina oocytes expressing TbAQP2 or 3. R. marina oocytes were injected with water (control cells), 10 ng of TbAQP2-cRNA, or 3-cRNA (alone or co-injected with 5 ng of hAQP1-cRNA). At day 2, 3, and 4 p.i., oocytes were subjected to the standard iso-osmotic swelling assay. Glycerol permeability values were obtained according to Ps equation. Results are shown as mean values ± standard error (SE). The t-student statistical test was used and the significance was represented with asterisks as follows, p < 0.05 (), 0.01() and 0.001(**).

References [+] :

Abousaab, Up-Regulation of the Excitatory Amino Acid Transporters EAAT1 and EAAT2 by Mammalian Target of Rapamycin. 2016, Pubmed, Xenbase