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Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions.

Karja NW, Kikuchi K, Fahrudin M, Ozawa M, Somfai T, Ohnuma K, Noguchi J, Kaneko H, Nagai T - Reprod. Biol. Endocrinol. (2006)

Bottom Line: Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups.These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos.Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Support Center, Swine and Poultry Feeding Management Laboratory, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan. karja_nwk@yahoo.com

ABSTRACT

Background: Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined.

Methods: In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6.

Results: Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA fragmentation rate than those of Pyr-Lac blastocysts.

Conclusion: These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos. Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

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Fragmented TUNEL-labeled nuclei (arrow) were present in almost all blastocysts, irrespective of the culture medium. The blastocysts shown were derived from the Pyr-Lac group (A) or the Gluc-20 group (B) and developed under 5% oxygen. Scale bars represent 20 μm.
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Figure 5: Fragmented TUNEL-labeled nuclei (arrow) were present in almost all blastocysts, irrespective of the culture medium. The blastocysts shown were derived from the Pyr-Lac group (A) or the Gluc-20 group (B) and developed under 5% oxygen. Scale bars represent 20 μm.

Mentions: When the number of DNA-fragmented nuclei and the total number of cells per blastocyst were evaluated by TUNEL-staining, DNA-fragmented nuclei were detected in almost all blastocysts, irrespective of the culture conditions (see figure 5). Of the embryos cultured under 5% oxygen tension, the total number of cells in the blastocysts of the Pyr-Lac group was significantly higher (P < 0.01) than that in the blastocysts supplemented with glucose at any concentration (see figure 6). In contrast, when cultured under 20% oxygen tension, no significant differences in cell numbers of the blastocysts were found among the energy substrate supplement groups. The cell numbers within each energy supplement group (except for the Gluc-20 group) were significantly higher (P < 0.05) under 5% oxygen than under 20% oxygen treatment. Only 20 mM glucose group showed a significantly higher (P < 0.05) DNA fragmentation rate than that in the blastocysts of the Pyr-Lac group, however, the fragmentation indices did not differ significantly among the glucose groups (see figure 7). Of the embryos cultured under 20% oxygen, no significant differences in fragmentation rates were found among energy substrate supplement groups. There were no significant differences within each energy supplement group in terms of DNA fragmentation rates across oxygen tension treatments.


Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions.

Karja NW, Kikuchi K, Fahrudin M, Ozawa M, Somfai T, Ohnuma K, Noguchi J, Kaneko H, Nagai T - Reprod. Biol. Endocrinol. (2006)

Fragmented TUNEL-labeled nuclei (arrow) were present in almost all blastocysts, irrespective of the culture medium. The blastocysts shown were derived from the Pyr-Lac group (A) or the Gluc-20 group (B) and developed under 5% oxygen. Scale bars represent 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1636643&req=5

Figure 5: Fragmented TUNEL-labeled nuclei (arrow) were present in almost all blastocysts, irrespective of the culture medium. The blastocysts shown were derived from the Pyr-Lac group (A) or the Gluc-20 group (B) and developed under 5% oxygen. Scale bars represent 20 μm.
Mentions: When the number of DNA-fragmented nuclei and the total number of cells per blastocyst were evaluated by TUNEL-staining, DNA-fragmented nuclei were detected in almost all blastocysts, irrespective of the culture conditions (see figure 5). Of the embryos cultured under 5% oxygen tension, the total number of cells in the blastocysts of the Pyr-Lac group was significantly higher (P < 0.01) than that in the blastocysts supplemented with glucose at any concentration (see figure 6). In contrast, when cultured under 20% oxygen tension, no significant differences in cell numbers of the blastocysts were found among the energy substrate supplement groups. The cell numbers within each energy supplement group (except for the Gluc-20 group) were significantly higher (P < 0.05) under 5% oxygen than under 20% oxygen treatment. Only 20 mM glucose group showed a significantly higher (P < 0.05) DNA fragmentation rate than that in the blastocysts of the Pyr-Lac group, however, the fragmentation indices did not differ significantly among the glucose groups (see figure 7). Of the embryos cultured under 20% oxygen, no significant differences in fragmentation rates were found among energy substrate supplement groups. There were no significant differences within each energy supplement group in terms of DNA fragmentation rates across oxygen tension treatments.

Bottom Line: Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups.These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos.Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Support Center, Swine and Poultry Feeding Management Laboratory, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan. karja_nwk@yahoo.com

ABSTRACT

Background: Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined.

Methods: In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6.

Results: Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA fragmentation rate than those of Pyr-Lac blastocysts.

Conclusion: These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos. Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

Show MeSH
Related in: MedlinePlus