Modulation of Glycolysis and the Pentose Phosphate Pathway Influences Porcine Oocyte In Vitro Maturation

Abstract

Contents Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus–oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose‐dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6‐AN) and the physiological (NADPH) inhibitors of PPP induced a dose‐dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.

Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

“…In porcine species, glycolysis appears to be the main fate of glucose during IVM (Krisher et al 2007;Alvarez et al 2012Alvarez et al , 2013. This is consistent with the high activity of PFK detected in porcine COCs remaining to be established the relative participation of this enzyme in oocytes and cumulus cells separately, as mammalian oocytes have low capacity to utilize this substrate (Steeves and Gardner 1999;Purcell and Moley 2009;Sutton-McDowall et al 2010), possibly due to the limited amount of PFK (Cetica et al 2002).…”

“…; Alvarez et al. , ). This is consistent with the high activity of PFK detected in porcine COCs remaining to be established the relative participation of this enzyme in oocytes and cumulus cells separately, as mammalian oocytes have low capacity to utilize this substrate (Steeves and Gardner ; Purcell and Moley ; Sutton‐McDowall et al.…”

“…Live cells were distinguished from dead ones based on their green fluorescence (Figure S1; Alvarez et al. ).…”

“…COCs were washed in PBS before being observed in an epifluorescence microscope (Jenamed 2, Carl Zeiss, Jena, Germany) using 450-490 nm and 520 nm excitation and emission filters, respectively, at 9100 magnification. Live cells were distinguished from dead ones based on their green fluorescence ( Figure S1; Alvarez et al 2013).…”

Nutrient pathways regulating the nuclear maturation of mammalian oocytes

“…Moreover, pharmacological modification of the PPP demonstrated that its activity was associated with meiotic resumption . A prominent role for this pathway in nuclear maturation is supported by additional studies in mice and other species Sakkas 1999, 2005;Herrick at al 2006;Alvarez et al 2013;Gutnisky et al 2014). Kim et al (2012) reported that the PPP is not involved in GVBD in mouse oocytes but, rather, progression beyond MI; however, they tested spontaneous maturation in DOs as opposed to meiotic induction in CEOs, as was the case in our studies.…”