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

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.

Keywords: Cumulus-oocyte-complex; Gap junction; Oocyte maturation; α-Amanitin.

Fig. 1.

In vitro maturation of DOs and COCs with α-amanitin treatment for 4 h of initial culture period when GVBD. 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).

Fig. 2.

In vitro maturation of DOs and COCs with α-amanitin pretreatment for 6 h GV stage prior to start In vitro maturation. 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).

Fig. 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).

Fig. 4.

Microphotographs of α-amanitin treated COCs. COCs cultured in M16 supplemented with IBMX for 6 h (A). COCs cultured in M16 supplemented with IBMX and α-amanitin for 6 h (B). COCs cultured in M16 supplemented with IBMX for 6 h followed by culture in plain M16 for 4 h (C). COCs cultured in M16 supplemented with IBMX and α-amanitin for 6 h followed by culture in M16 supplemented with α-amanitin for 4 h (D). Arrows indicate the scattered CCs. Bars = 100 μm.

Fig. 5.

Expression of cumulus expansion marker genes, Ptx3, Has2, and Tnfaip6, in the control and α-amanitin-treated CCs. Gapdh was used as a loading control. (Left panel) Control CCs were collected after COCs were cultured in M16 supplemented with IBMX for 6 h followed by culture in plain M16 for 4 h. (Right panel) CCs were collected after culture COCs in M16 supplemented with IBMX and α-amanitin for 6 h followed by culture in M16 supplemented with α-amanitin for 4 h.

Fig. 6.

Expression of various gap junctional protein genes in oocytes (left panel) and CCs (right panel) that obtained from the COCs after culture in the control and the α-amanitin-treated experimental group. Oocyte-specific genes, Gdf9 and H1foo, were used as markers for pure oocyte collection, while granulosa/cumulus-specific genes, FSHr and LHr, were used as markers for pure CC collection. Gapdh was used as internal control.

Fig. 7.

Expression of various enzymes related to the cellular metabolism in oocytes (left panel) and CCs (right panel) that obtained from the COCs after culture in the control and the α-amanitin-treated experimental group.