The Role of Glucose Metabolism on Porcine Oocyte Cytoplasmic Maturation and Its Possible Mechanisms

<div><p>In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 μM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 μM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity.</p></div

Typical confocal images showing nuclear DNA and proliferating cells in 7 d porcine blastocysts.

<p>(A) Immunofluorescent staining of BrdU in blastocysts from control, DHEA-treated, and IA-treated groups (×400). Scale bar indicates 100 μm. (B) Percentages of proliferating cells in different groups. Values are the mean ± standard deviation the mean. The numbers of embryos examined in each experimental group are shown in the bars. Differences between bars superscripted with different letters (a, b, or c) were statistically significant (p < 0.05).</p

Effect of adding various concentrations of glucose or pyruvate on IVM of porcine oocytes.

<p>(A) COCs were cultivated in NCSU37 with 10% PFF and different concentrations of glucose or pyruvate. (B) DOs were cultivated in NCSU37 with 10% PFF and supplemented with glucose and/or pyruvate.(C) COCs were cultivated in NCSU37 with glucose and/or pyruvate. Control: no exogenous glucose/pyruvate; G: glucose-only supplementation; P: pyruvate-only supplementation; G + P: supplementation with both glucose and pyruvate. Differences between bars superscripted with different letters (a, b, c, or d within same graph)were statistically significant (p < 0.05). Values are the mean ± standard deviation from three independent experiments.</p

The effects of DHEA and IA on GSH and ROS levels in MII oocytes.

<p>(A) MII oocytes maturated in control IVM medium or medium supplemented with DHEA or IA were dyed with CMF₂HC (Cell Tracker Blue) and H₂DCFDA to detect GSH and ROS levels, respectively. Scale bar indicates 100 μm. (B) Effect of DHEA or IA supplementation during IVM on intracellular GSH levels in mature oocytes. (C) Effect of DHEA or IA supplementation during IVM on intracellular ROS levels in mature oocytes. Differences between bars superscripted with different letters (a or b within same graph) were statistically significant (p <0.05). The experiment was repeated three times.</p

Typical confocal images showing nuclear DNA and apoptotic cells in 7 d porcine blastocysts.

<p>(A) Immunofluorescent staining of apoptotic cells in blastocysts from control, DHEA-treated, and IA-treated groups (×400). Scale bar indicates 100 μm. (B) The total number of cells per blastocyst in different groups. (C) Apoptotic cell rate per blastocyst in different groups. The number of observed oocytes in each experimental group is displayed inside the bars. (D) <i>Bcl-2</i>, <i>Bax</i>, and <i>Casp3</i> mRNA levels in 7 d porcine blastocysts from controls and groups treated with DHEA or IA during IVM. Values are the mean ± standard deviation of the mean. Differences between bars superscripted with different letters (a, b, or c within same graph) were statistically significant (p < 0.05).</p

Intra-oocyte ATP and NADPH levels and MPF activity in MII oocytes.

<p>(A) Intra-oocyte ATP levels, (B) Intra-oocyte NADPH levels, (C) Expression levels of the maternal transcripts <i>cdc2</i> and <i>cyclin B1</i>, and (D) MPF activity in MII oocytes, after porcine COC maturation in NCSU37 with 10% PFF, 5.6 mM glucose, and 2 mM pyruvate, in the absence (controls) or presence of 200 μM DHEA or 2 μM IA. Each treatment was repeated three times. In each experiment, samples contained 50 (for ATP, mRNA, and MPF activity levels) or 100 (for NADPH levels) cumulus-free oocytes. Differences between bars superscripted with different letters (a, b, or c within same graph) were statistically significant (p < 0.05).</p

Effect of various concentrations of glucose or pyruvate during IVM on porcine oocyte development to the blastocyst stage.

<p>(A) Blastocyst rates when COCs were cultivated in NCSU37 with 10% PFF, without or with supplementation with different concentrations of glucose and/or pyruvate. (B) Blastocyst rates after DOs were cultivated in NCSU37 with 10% PFF supplemented with glucose, pyruvate, or both. (C) Blastocyst rates when COCs were cultivated in NCSU37 with glucose and/or pyruvate (P). Control: no exogenous glucose/pyruvate; G: glucose-only supplementation; P: pyruvate-only supplementation; G + P: supplementation with both glucose and pyruvate. Differences between bars superscripted with different letters (a, b, or c within same graph) were statistically significant (p < 0.05). Values are the mean ± standard deviation from three independent experiments.</p