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RNA Polymerase II Inhibitor, α-Amanitin, Affects Gene Expression for Gap Junctions and Metabolic Capabilities of Cumulus Cells, but Not Oocyte, during in vitro Mouse Oocyte Maturation.

Park MW, Lee HS, Kim EY, Lee KA - (2013)

Bottom Line: Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in α-amanitin-treated CCs.In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in α -amanitin-treated CCs but not in oocytes.Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Life Science, CHA University, CHA Research Institute, Fertility Center, CHA General Hospital, Seoul 135-081, Korea.

ABSTRACT
A specific inhibitor of RNA polymerase II, α-amanitin is broadly used to block transcriptional activities in cells. Previous studies showed that α-amanitin affects in vitro maturation of cumulus-oocyte-complex (COC). In this study, we evaluated the target of α-amanitin, and whether it affects oocytes or cumulus cells (CCs), or both. We treated α-amanitin with different time period during in vitro culture of denuded oocytes (DOs) or COCs in comparison, and observed the changes in morphology and maturation status. Although DOs did not show any change in morphology and maturation rates with α-amanitin treatment, oocytes from COCs were arrested at metaphase I (MI) stage and CCs were more scattered than control groups. To discover causes of meiotic arrest and scattering of CCs, we focused on changes of cumulus expansion, gap junctions, and cellular metabolism which to be the important factors for the successful in vitro maturation of COCs. Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in α-amanitin-treated CCs. However, these changes were not observed in oocytes. In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in α -amanitin-treated CCs but not in oocytes. Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

No MeSH data available.


In vitro maturation of DOs and COCs with α-amanitin pretreatment for 6 h at GV stage followed by 4 more hours during GVBD occurs. Schematic diagram of culture condition in the control group (A) and α-amanitin treatment in the experimental group (B). Oocytes maturation rates of DOs (C) and COCs (D).
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Figure 3: In vitro maturation of DOs and COCs with α-amanitin pretreatment for 6 h at GV stage followed by 4 more hours during GVBD occurs. Schematic diagram of culture condition in the control group (A) and α-amanitin treatment in the experimental group (B). Oocytes maturation rates of DOs (C) and COCs (D).

Mentions: To determine the effects of α-amanitin to the oocyte maturation, α-amanitin was treated in culture medium at different period of time during in vitro culture. First, DOs and COCs were cultured in M16 medium supplemented with α-amanitin for 4 h followed by culture in the plain M16 medium for 12 h (Fig. 1A, B). Second, DOs and COCs were cultured in M16 medium supplemented with α-amanitin and 0.2 mM IBMX for 6 h followed by culture in the plain M16 medium for further 16 h (Fig. 2A, B). Finally, DOs and COCs were cultured in M16 medium supplemented with α-amanitin and 0.2 mM IBMX for 6 h followed by culture in M16 containing α-amanitin for 4 h and moved into the plain M16 medium for further 12 h (Fig. 3A, B). All cultures were accomplished in 5% CO2 at 37°C.


RNA Polymerase II Inhibitor, α-Amanitin, Affects Gene Expression for Gap Junctions and Metabolic Capabilities of Cumulus Cells, but Not Oocyte, during in vitro Mouse Oocyte Maturation.

Park MW, Lee HS, Kim EY, Lee KA - (2013)

In vitro maturation of DOs and COCs with α-amanitin pretreatment for 6 h at GV stage followed by 4 more hours during GVBD occurs. Schematic diagram of culture condition in the control group (A) and α-amanitin treatment in the experimental group (B). Oocytes maturation rates of DOs (C) and COCs (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4282216&req=5

Figure 3: In vitro maturation of DOs and COCs with α-amanitin pretreatment for 6 h at GV stage followed by 4 more hours during GVBD occurs. Schematic diagram of culture condition in the control group (A) and α-amanitin treatment in the experimental group (B). Oocytes maturation rates of DOs (C) and COCs (D).
Mentions: To determine the effects of α-amanitin to the oocyte maturation, α-amanitin was treated in culture medium at different period of time during in vitro culture. First, DOs and COCs were cultured in M16 medium supplemented with α-amanitin for 4 h followed by culture in the plain M16 medium for 12 h (Fig. 1A, B). Second, DOs and COCs were cultured in M16 medium supplemented with α-amanitin and 0.2 mM IBMX for 6 h followed by culture in the plain M16 medium for further 16 h (Fig. 2A, B). Finally, DOs and COCs were cultured in M16 medium supplemented with α-amanitin and 0.2 mM IBMX for 6 h followed by culture in M16 containing α-amanitin for 4 h and moved into the plain M16 medium for further 12 h (Fig. 3A, B). All cultures were accomplished in 5% CO2 at 37°C.

Bottom Line: Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in α-amanitin-treated CCs.In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in α -amanitin-treated CCs but not in oocytes.Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Life Science, CHA University, CHA Research Institute, Fertility Center, CHA General Hospital, Seoul 135-081, Korea.

ABSTRACT
A specific inhibitor of RNA polymerase II, α-amanitin is broadly used to block transcriptional activities in cells. Previous studies showed that α-amanitin affects in vitro maturation of cumulus-oocyte-complex (COC). In this study, we evaluated the target of α-amanitin, and whether it affects oocytes or cumulus cells (CCs), or both. We treated α-amanitin with different time period during in vitro culture of denuded oocytes (DOs) or COCs in comparison, and observed the changes in morphology and maturation status. Although DOs did not show any change in morphology and maturation rates with α-amanitin treatment, oocytes from COCs were arrested at metaphase I (MI) stage and CCs were more scattered than control groups. To discover causes of meiotic arrest and scattering of CCs, we focused on changes of cumulus expansion, gap junctions, and cellular metabolism which to be the important factors for the successful in vitro maturation of COCs. Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in α-amanitin-treated CCs. However, these changes were not observed in oocytes. In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in α -amanitin-treated CCs but not in oocytes. Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

No MeSH data available.