Expanded equine cumulus–oocyte complexes exhibit higher meiotic competence and lower glucose consumption than compact cumulus–oocyte complexes

Abstract

Expanded equine cumulus-oocyte complexes exhibit higher meiotic competence and lower glucose 1 consumption than compact cumulus-oocyte complexes To test this 24 hypothesis eCOCs, cCOCs, and expanded or compact mural granulosa cells (EC and CC respectively) were 25 matured in vitro for 30 hours and the maturation rate, glucose metabolism, and expression of genes involved in 26 glucose transport, glycolysis, apoptosis and meiotic competence were determined. Significant differences were 27 found between eCOCs and cCOCs maturation rates (50% vs. 21.7 %; n = 192 and 46 respectively, p < 0.001), 28 glucose consumption (1.8 ± 0.5 vs. 27.9 ± 5.9 nmol/COC; mean ± SEM), pyruvate production (0.1 ± 0.0 vs. 2.4 ± 29 0.8 nmol/COC; mean ± SEM) and lactate production (4.7 ± 1.3 vs. 64.1 ± 20.6 nmol/COC; mean ± SEM) 30 respectively (p < 0.05). Moreover, similar glucose consumption was observed for EC and CC. Hyaluronan 31 binding protein (TNFAIP6) expression was increased in eCOCs and EC, solute carrier family 2 (facilitated glucose 32 transporter) member 1 (SLC2A1) was increased in eCOCs, while glycolysis-related enzymes and solute carrier 33 family 2 (facilitated glucose transporter) member 3 (SLC2A3) expression did not vary between COCs or mural 34 granulosa cell type. Our data demonstrate that metabolic and genomic differences exist between eCOCs and 35 cCOCs and mural granulosa cells in the horse. 36 37Additional Keywords: in vitro maturation, glycolysis, nuclear magnetic resonance, horse. 38 39

Analysis of the equine “cumulome” reveals major metabolic aberrations after maturation in vitro

“…Bovine cumulus responded to hypoxia during IVM through upregulation of GLUT1 gene expression (Slc2a1), and HIF1α seems to play a role in mediating this response [64]. For equine cumulus cells, the expression of the GLUT1 gene (Slc2a1) was previously documented, with significantly higher abundance in expanded CC than in compact cumulus cells [71]. These studies corroborate the observed GLUT1 underrepresentation after maturation under the hyperoxic (20%) in vitro condition in this study.…”

“…These COCs also exhibited higher maturational competence (50% versus 21.7%). These expanded COCs showed significantly lower glucose consumption along with decreased pyruvate and lactate production [71].…”

Ascorbic acid–cyclodextrin complex alters the expression of genes associated with lipid metabolism in bovine in vitro produced embryos

“…Poly (A) RNA was extracted using the Dynabeads ® mRNA DIRECT™ Micro Kit (Ambion ® ; Thermo Fisher Scientific Inc., Oslo, Norway) following the manufacturer's instructions with minor modifications (González‐Fernández et al, ). After 10 min of incubation in lysis buffer with Dynabeads, poly (A) RNA attached to the Dynabeads was extracted with a magnet and washed twice in washing buffer A and washing buffer B. RNA was eluted with Tris‐HCl.…”

A high glucose concentration during early stages of in vitro equine embryo development alters expression of genes involved in glucose metabolism

“…Transport was performed at 15°C within 8 hours of slaughter. Immature cumulus‐oocyte complexes (COCs) were obtained by follicular scraping as previously described 19 using TCM199 supplemented with HEPES, 1 mg/mL BSA, 10 µg/mL heparin and 2.2 g/L bicarbonate. The oocytes (n = 641) were collected and maintained in holding medium (HM, TCM199 supplemented with HEPES + 20% fetal calf serum (FCS) + 0.5% gentamycin) for a maximum of 12 hours at room temperature in the dark.…”