Effects of glucose metabolism during in vitro maturation on cytoplasmic maturation of mouse oocytes

Abstract

Although there are many reports on the effect of glucose metabolism on oocyte nuclear maturation, there are few studies on its effect on ooplasmic maturation. By manipulating glucose metabolism pathways using a maturation medium that could support oocyte nuclear maturation but only a limited blastocyst formation without glucose, this study determined effects of glucose metabolism pathways on ooplasmic maturation. During maturation of cumulus-oocyte-complexes (COCs) with glucose, the presence of PPP inhibitor, DHEA or glycolysis inhibitor, iodoacetate significantly decreased blastocyst rates, intraoocyte glutathione and ATP. While blastocyst rates, GSH/GSSG ratio and NADPH were higher, ROS was lower significantly in COCs matured with iodoacetate than with DHEA. Fructose-6-phosphate overcame the inhibitory effect of DHEA on PPP. During maturation of COCs with pyruvate, electron transport inhibitor, rotenone or monocarboxylate transfer inhibitor, 4-CIN significantly decreased blastocyst rates. Cumulus-denuded oocytes had a limited capacity to use glucose or lactate, but they could use pyruvate to support maturation. In conclusion, whereas glycolysis promoted ooplasmic maturation mainly by supplying energy, PPP facilitated ooplasmic maturation to a greater extent by both reducing oxidative stress and supplying energy through providing fructose-6-phosphate for glycolysis. Pyruvate was transferred by monocarboxylate transporters and utilized through mitochondrial electron transport to sustain ooplasmic maturation.

Figure 1. Concentration of GSX, the ratio of GSH/GSSG and levels of ROS in mouse oocytes after COCs maturation in the simplified α-MEM containing 5.6-mM glucose and 0.5-mM lactate (GL) in the absence or presence (+) of 150-μM DHEA (D) or 1.5-μM iodoacetate (I).

Panel A and B show concentration of GSX and the ratio of GSH/GSSG, respectively. Each treatment was repeated 3 times with each replicate containing about 25 oocytes. Panel C shows levels of ROS. The concentrations of ROS were expressed as average fluorescence intensity value (AFIV), which was calculated from fluorescence intensity values (FIV) of multiple oocytes. The FIV for each oocyte was calculated by subtracting the basal intensity value from the peak value. Each treatment was repeated 3–4 times and each replicate contained about 10 oocytes. (a,b) Values without a common letter above their bars differ significantly (P < 0.05). Photographs D, E and F show ROS levels in GL, GL + D and GL + I oocytes, respectively, when observed under a laser confocal microscope following DCHFDA staining.

Figure 2. Intra-oocyte ATP and NADPH contents after mouse COCs maturation in simplified α-MEM containing 5.6-mM glucose and 0.5-mM lactate (GL) in the presence (+) or absence of 150-μM DHEA (D) or 1.5-μM iodoacetate (I) and/or 10-mM F-6-P (F).

For ATP assays, each treatment was repeated 3 times with each replicate containing 60–80 cumulus-free oocytes, and for NADPH assays, each treatment was repeated 3 times with each replicate containing 100–120 cumulus-free oocytes. (a,b) Values without a common letter above their bars differ significantly (P < 0.05).

Figure 3. Effects of treatment with rotenone, 4-CIN or oxamate during maturation on maturation and blastocyst formation of mouse oocytes.

Panels A and B show percentages of matured (MII) oocytes after COCs were matured with 2-mM pyruvate alone or with different concentrations of rotenone or 4-CIN, respectively. Each treatment was repeated 3 times with each replicate having about 25 oocytes. Panel C shows blastocyst formation after COCs were matured with 2-mM pyruvate (P) or 5.6-mM glucose (G) alone or with (+) 0.025-μM rotenone (R) or 25-μM 4-CIN (C). Each treatment was repeated 4–5 times and each replicate contained around 25 oocytes. Panel D shows percentages of MII oocytes after COCs were matured with 1.5-mM lactate (L) or 2-mM pyruvate (P) alone or in the presence of (+) 30 mM oxamate (O). Each treatment was repeated 3–4 times with each replicate including about 25 oocytes. (a–d) Values without a common letter above their bars differ significantly (P < 0.05).

Figure 4. A proposed model for the glucose catabolism in cumulus cells and the maturing oocyte.

Whereas the oocyte itself can utilize pyruvate, it cannot use glucose or lactate efficiently to support ooplasmic maturation unless in the presence of cumulus cells. However, the oocyte itself does have the capacity to use lactate for a limited nuclear maturation. Within the cumulus cell, glucose is utilized through PPP and glycolysis. Whereas NADPH and F-6-P/Glyceraldehyde-3-P are produced during PPP, pyruvate and ATP are generated during glycolysis. The PPP-derived F-6-P and Glyceraldehyde-3-P enter glycolysis and produce ATP and pyruvate. Both the glycolysis-derived pyruvate and pyruvate in the medium enters the oocyte and is oxidized by the TCA cycle within the ovum mitochondrion (dotted line box) to generate FADH₂, NADH, NADPH, and GTP. Lactate in medium enters the cumulus cells and produces NADH via LDH-catalyzed oxidation to pyruvate. Cysteine produced from the reduction of cystine by cysteamine in the oocyte participates in the synthesis of GSH, a process of energy consumption. Whereas NADPH from both PPP and TCA reduces the GSSG, overcoming the oxidizing effect of ROS, NADH produced by TCA and lactate oxidation generates ATP through electron transfer in the respiratory chain. The present results also suggested that whereas the culture medium- and glycolysis-derived pyruvate entered the TCA, the lactate-derived pyruvate was poorly metabolized by the oocyte mitochondria.