Increased Beta-Oxidation, ATP Levels, and Improved Oocyte Developmental Competence in Response to L-Carnitine.

The metabolic rate of mammalian oocytes and embryos are closely associated with their developmental potential. Lipids are a rich source of energy; however their utilization by the oocyte and early embryo is poorly understood. The generation of ATP from lipids occurs in mitochondria via β-oxidation. We have found that β-oxidation increases in the cumulus oocyte complex (COC) during oocyte maturation and is essential for subsequent embryo development. We also found that carnitine palmitoyl transferase (Cpt1), the rate limiting enzyme in β-oxidation, is hormonally regulated in the COC. L-carnitine is a circulating metabolite required for Cpt1 activity which we hypothesized would be a key regulator of lipid metabolism in the COC and promote oocyte developmental competence. To better understand the importance of lipid metabolism during oocyte maturation and determine whether L-carnitine can be used to manipulate this process we 1) determined the effect of L-carnitine on β-oxidation and ATP levels in the COC, 2) assessed the effect of L-carnitine supplementation on oocyte developmental competence and 3) assessed whether L-carnitine could promote utilization of intracellular lipid stores and enable zygote survival in the absence of exogenous metabolic substrates. To determine the effect of L-carnitine supplementation on β-oxidation in COCs from d21 CBA/C57Bl6 F1 female mice, metabolism of 3H-palmitate by COCs in vitro was measured by the production of 3H2O in the media. The inclusion of L-carnitine (1mM) in the culture media resulted in a significant increase in β-oxidation in immature COCs and in COCs undergoing in vitro maturation. To determine whether increased β-oxidation in response to L-carnitine was associated with increased ATP production, ATP levels were measured in COCs following a 90 min culture period by bioluminescence assay. In a dose dependant manner, L-carnitine supplementation (0-10mM) led to increased ATP levels in the COC. As L-carnitine supplementation was found to increase β-oxidation and ATP levels in the COC we determined what effect this had on oocyte developmental competence. Following in vitro maturation, with or without L-carnitine (1mM), COCs were washed and fertilized in vitro and on-time embryo development assessed. L-carnitine supplementation resulted in a significant increase in the number of hatching blastocysts on day 5 compared to control. To determine whether L-carnitine improves oocyte developmental competence by promoting the utilization of intracellular lipid stores, presumptive zygotes were isolated from CBA/C57Bl6 F1 female mice and cultured in the absence of glucose, lactate, pyruvate and fatty acids for 24h. L-carnitine supplementation, in a dose dependant manner, led to increased numbers of 2-cell embryos, with a significant 2.1-fold increase in the cleavage rate of zygotes cultured in 5mM L-carnitine compared to 0mM. Thus fatty acids are a vital energy source for oocyte and embryo development and the physiological metabolite L-carnitine is an important co-factor for the utilization of these substrates. We have shown that inclusion of L-carnitine during oocyte maturation in vitro is able to increase lipid metabolism, raise ATP levels and improve embryo development. The inclusion of L-carnitine in culture media formulations is therefore expected to better mimic the normal in vivo environment and lead to improvements in oocyte and embryo quality in human clinics and in agriculturally important species.(platform)