The effect of elevated non-esterified fatty acid concentrations on bovine spermatozoa and on oocyte in vitro fertilisation.

Elevated non-esterified fatty acid (NEFA) concentrations, present in follicular and oviductal fluid, have been postulated as a causative link between metabolic disorders and subfertility. High NEFA conditions can directly disrupt oocyte maturation and developmental capacity after fertilisation. However, their influence on sperm function and the fertilisation process is not known. This study investigated the fertilisation process under high NEFA conditions. To differentiate between effects on both spermatozoa and oocytes or on spermatozoa only, different experiments were conducted. In the first experiment both gametes were simultaneously incubated during IVF under different conditions: (1) NEFA-free, solvent-free control conditions, (2) solvent control, (3) physiological concentrations of oleic (OA), palmitic (PA) and stearic (SA) acids or (4) pathophysiological concentrations of OA, PA and SA. In the second experiment spermatozoa were incubated (4h) under the same treatment conditions prior to routine IVF. Gamete co-incubation resulted in reduced fertilisation and cleavage rates and increased prevalence of polyspermy. In the second experiment embryo developmental capacity and quality were not affected, although sperm motility and plasma membrane integrity were decreased. In conclusion, lipolytic conditions affected the fertilisation process mainly through an effect on the oocyte. Spermatozoa were still able to fertilise even though these conditions reduced sperm function.

Exposure of bovine oocytes and embryos to elevated non-esterified fatty acid concentrations: integration of epigenetic and transcriptomic signatures in resultant blastocysts.

BACKGROUND: Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts. RESULTS: Bovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological ("BASAL") concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological ("HIGH COMBI") concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. CONCLUSIONS: Overall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.