Fatty acid oxidation and meiotic resumption in mouse oocytes

Abstract

We have examined the potential role of fatty acid oxidation (FAO) in AMP-activated protein kinase (AMPK)-induced meiotic maturation. Etomoxir and malonyl CoA, two inhibitors of carnitine palmitoyl transferase-1 (CPT1), and thus FAO, blocked meiotic induction in dbcAMP-arrested cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) by the AMPK activator, AICAR. C75, an activator of CPT1 and FAO, stimulated meiotic resumption in CEO and DO. This effect was insensitive to the AMPK inhibitor, compound C, indicating an action downstream of AMPK. Palmitic acid or carnitine also promoted meiotic resumption in DO in the presence of AICAR. Since C75 also suppresses the activity of fatty acid synthase (FAS), we tested another FAS inhibitor, cerulenin. Cerulenin stimulated maturation in arrested oocytes, but to a lesser extent, exhibited significantly slower kinetics and was effective in CEO but not DO. Moreover, etomoxir completely blocked C75-induced maturation but was ineffective in cerulenin-treated oocytes, suggesting that the meiosis-inducing action of C75 is through activation of FAO within the oocyte, while that of cerulenin is independent of FAO and acts within the cumulus cells. Finally, we determined that long chain, but not short chain, fatty acyl carnitine derivatives were stimulatory to oocyte maturation. Palmitoyl carnitine stimulated maturation in both CEO and DO, with rapid kinetics in DO; this effect was blocked by mercaptoacetate, a downstream inhibitor of FAO. These results indicate that activation of AMPK stimulates meiotic resumption in mouse oocytes by eliminating a block to FAO.

Figure 1

Flow diagrams of fatty acid metabolism. A, Modulation of fatty acid metabolism by different molecules. AMPK, stimulated by AICAR, phosphorylates and inactivates acetyl CoA carboxylase (ACC), thereby lowering malonyl CoA levels. Malonyl CoA and etomoxir are negative regulators of carnitine palmitoyltransferase-1 (CPT1) and restrict fatty acid entry into mitochondria, thereby blocking fatty acid oxidation. C75 has an opposite, positive effect on CPT1 and promotes fatty acid entry and β–oxidation. C75 also suppresses fatty acid synthase (FAS) activity and thereby reduces fatty acid synthesis, an effect mimicked by cerulenin. MCD, malonyl CoA decarboxylase. B, Regulation of fatty acid entry into mitochondria. Fatty acids are transported into the mitochondria following replacement of the CoA moiety with carnitine through the action of CPT1. This action is blocked by malonyl CoA that is produced by ACC1, which is associated with the outer membrane of the mitochondrion. Oxidation occurs after the carnitine is replaced with CoA by the action of CPT2. Cytoplasmic ACC1 produces the malonyl CoA that serves as precursor for fatty acid synthesis. Figure B is adapted from Tong, 2005.

Figure 2

Inhibition of meiotic induction by etomoxir and malonyl CoA. Denuded oocytes were maintained in meiotic arrest with 3000 μM dbcAMP and were stimulated to resume maturation with 250 μM AICAR. Etomoxir (A) or malonyl CoA (B) was added in increasing concentrations and GVB was assessed 4 h later. The effects of etomoxir were then tested under similar conditions but with meiotic arrest maintained with milrinone (C) or hypoxanthine (D). Groups with no common letter are significantly different.

Figure 3

Effect of etomoxir and malonyl CoA on nonstimulated oocytes. Denuded oocytes were cultured 3 h in control medium or in medium supplemented with 100 μM dbcAMP, in the presence or absence of 250 μM etomoxir or 4 mM malonyl CoA, and then assessed for GVB.

Figure 4

Effect of C75 and cerulenin on meiotic maturation. A, CEO and DO were cultured 17-18 h in medium containing 300 μM dbcAMP plus increasing concentrations of C75. B, CEO or DO were cultured 17-18 h in medium containing 1 mM guanosine (Guan), 2 μM milrinone (Mil) or 4 mM hypoxanthine (Hyp), in the presence or absence of 100 μM C75. In all groups, C75 stimulated a significant increase in GVB. CEO and DO were cultured 17-18 h in medium containing 300 μM dbcAMP (C) or 4 mM hypoxanthine (D) plus increasing concentrations of cerulenin. Cerulenin had no effect on DO but did stimulate maturation in CEO.

Figure 5

Comparative actions of C75 and cerulenin on oocyte maturation. A, Kinetics of meiotic induction. CEO were cultured in 4 mM hypoxanthine, plus or minus 100 μM C75 or 10 μM cerulenin, and GVB was assessed after varying culture times. B,C, Effects of etomoxir on meiotic induction by C75 and cerulenin. CEO were cultured 18 h in 300 μM dbcAMP (B) or 4 mM hypoxanthine (C) and stimulated to undergo maturation with 100 μM C75 or 10 μM cerulenin. Etomoxir was administered at a concentration of 100 μM in dbcAMP-supplemented medium and 250 μM in hypoxanthine-supplemented medium. D, CEO were cultured 17-18 h in medium containing dbcAMP and stimulated to undergo maturation with 250 μM AICAR or 100 μM C75. They were treated with 2.5 μM compound C and 17-18 h later assessed for GVB. Groups with no common letter are significantly different.

Figure 6

Effect of palmitic acid and carnitine on meiotic induction in DO. DO were cultured 4 h in medium containing 300 μM dbcAMP plus or minus 125 μM AICAR and were exposed to increasing concentrations of palmitic acid (A) or carnitine (B). An asterisk denotes a significant difference from controls.

Figure 7

Meiotic induction by carnitine derivatives of fatty acids. A, Dose response effect of C75. CEO and DO were cultured 17-18 h in medium containing 4 mM hypoxanthine plus increasing concentrations of palmitoyl carnitine. B, Time course of meiotic induction. DO were cultured 1-4 h in medium containing hypoxanthine in the presence or absence of 75 μM palmitoyl carnitine. C, effect of etomoxir on meiotic induction. DO were cultured 4 h in 4 mM hypoxanthine plus 75 μM palmitoyl carnitine or 250 μM AICAR, plus or minus 250 μM etomoxir. An asterisk denotes a significant difference from the –etomoxir group. D, Effect of fatty acid chain length on meiotic induction. DO were cultured 4 h in 4 mM hypoxanthine plus fatty acyl carnitines at a concentration of 75 μM. Palmitoyl and stearoyl carnitines were also added at 50 μM. Numbers at the top of the bar denote the mean percentage of dead oocytes for each treatment. An asterisk denotes a significant difference from the control group.

Figure 8

Effect of fatty acid oxidation inhibitors on meiotic induction by palmitoyl carnitine. DO were cultured 0.5 h in 4 mM hypoxanthine plus or minus increasing concentrations of mercaptoacetate (MA, A) or 8-bromo-octanoic acid (B) and then 75 μm palmitoyl carnitine was added to each tube. Cultures were continued for 4 h before oocytes were assessed for GVB. Numbers at the top of the bar denote the mean percentage of dead oocytes for each treatment. Groups with no common letter are significantly different.