During cumulus-oocyte complex (COC) maturation, cumulus expansion involves the deposition of mucoelastic compounds, especially hyaluronic acid, synthesised from glucose via the hexosamine biosynthesis pathway. The aim of the present study was to determine the effects of uridine monophosphate (UMP) and 6-diazo-5-oxo-L-norleucine (DON), inhibitors of hyaluronic acid synthesis, during bovine oocyte in vitro maturation (IVM) on cumulus expansion, glucose uptake, protein synthesis, cumulus cell number, meiotic maturation, cleavage rate and subsequent embryo development. A further aim of the study was to examine the effect of hyaluronic acid on sperm capacitation and acrosome reaction in relation to the capacity of COCs to be fertilised in vitro. A low correlation between glucose uptake and degree of cumulus expansion was observed. Total and partial inhibition of cumulus expansion was observed with DON and UMP, respectively, and was accompanied by a decrease in glucose uptake with DON. Total protein content and cumulus cell number per COC increased during IVM, but was unaffected by the presence of DON or UMP, as was oocyte meiotic maturation. Rates of cleavage and blastocyst development decreased in oocytes matured with DON and UMP, although this inhibition was reversed when the in vitro fertilisation (IVF) medium contained heparin. Hyaluronic acid induced capacitation and the acrosome reaction, and in IVF medium prevented the inhibition of cleavage and blastocyst development by DON in a similar fashion to heparin. Hyaluronic acid synthesis during cumulus mucification contributes to the penetration and fertilisation of bovine oocytes, most likely by facilitating the processes of capacitation and acrosome reaction. Mucification during IVM is independent of cumulus cell proliferation, COC protein content, oocyte meiotic maturation and subsequent developmental competence once fertilised.

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Glycolysis and the pentose phosphate pathway (PPP) were modulated in porcine cumulus-oocyte complexes during IVM by the addition of inhibitors and stimulators of key enzymes of the pathways to analyze their influence on the oxidative status, active mitochondria, and maturation of the oocyte. The influence of pharmacologic and physiological inhibitors of glycolysis (Sodium fluoride and ATP) and PPP (6-Aminonicotinamide and nicotinamide adenine dinucleotide phosphate) was validated by assessing glucose and lactate turnover and brilliant cresyl blue staining in oocytes. Inhibitors of glycolysis and PPP activity significantly perturbed nuclear maturation, oxidative metabolism (Redox Sensor Red CC-1), and active mitochondria (Mitotracker Green FM) within oocytes (P < 0.05). In comparison, physiological stimulators of glycolysis (adenosine monophosphate) and PPP (nicotinamide adenine dinucleotide phosphate) did not affect any of evaluated parameter. In the absence of modulators, fluctuations in the oocyte oxidative activity and active mitochondria were observed during porcine IVM. The inhibition of glycolysis and PPP modified the pattern of oxidation and mitochondrial fluctuation, resulting in impaired meiotic progression. We demonstrated the relationship between carbohydrate metabolism in COC and oocyte redox status necessary for porcine oocyte IVM.

26The relationship between the pentose phosphate pathway (PPP) activity in COCs and 27 oxidative and mitochondrial activity in bovine oocytes was evaluated, with the aim of 28 analyzing the impact of two inhibitors (NADPH, 6-AN) and a stimulator (NADP) of the 29 key enzymes of PPP on the maturation rate, the oxidative and mitochondrial activity, and 30 the mitochondrial distribution in oocytes. The percentage of COCs with PPP activity, 31(assessed using BCB staining), glucose uptake, lactate production, and meiotic maturation 32 rate diminished when 6-AN was added to the maturation media (p<0.05). The addition of 33 NADPH did not modify glucose uptake and lactate production, while the COC PPP activity 34 and the meiotic maturation rates decreased (p<0.05). The presence of NADP in the 35 maturation medium had no effect on COC PPP activity rate, glucose uptake, lactate 36 production and meiotic maturation rate. However, in the absence of gonadotrophin 37 supplementation NADP stimulated both glucose uptake and lactate production at the

 Use the work for non-commercial purposes within their institution subject to the usual copyright licencing agency arrangements  Use the work for further research and presentations at meetings and conferences  Use the illustrations (line art, photographs, figures, plates) and research data in their own future works  Share print or digital copies of their work with colleagues for personal use or study  Include the work in part or in full in a thesis provided it is not published for commercial gain  Place his/her pre-publication version of the work on a pre-print server  Place his/her pre-publication version of the work on a personal website or institutional repository on condition that there is a link to the definitive version on the CSIRO PUBLISHING web site. th February 2014Glycolytic pathway activity: effect on in vitro maturation and oxidative metabolism ofRunning head: Glycolysis activity in COCs. The aim of this study was to determine the influence of altering glycolytic pathway activity 28 during bovine IVM on the meiotic maturation rate, oxidative activity, mitochondrial 29 activity, and the mitochondrial distribution within oocytes. Glycolytic activity was 30 manipulated using two inhibitors (ATP, NaF) and a stimulator (AMP) of key enzymes of 31 the pathway. Inhibition of glucose uptake, lactate production and meiotic maturation rates 32 was observed when media was supplemented with ATP or NaF. The addition of AMP in 33 the maturation medium had no effect on glucose uptake, lactate production and meiotic

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.

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Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the specie to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. To do this purpose we compared in vitro development of synchronic (S) or asynchronic (AS) and asynchronic tetraploid (AST) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA-fragmentation. Additionally, the developmental rates of AST aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. AS aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by AST aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by AST aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of 2cell fused embryos to generate tetraploid blastomeres and demonstrate that AST aggregation generates good quality embryos.