A novel amino acid combination supports the in vitro maturation and fertilisation of bovine oocytes

In vitro maturation (IVM) of oocytes assists cattle producers to maximise the number of offspring from genetically superior animals. This technology has significant commercial benefits to the cattle industry. Through collection and maturation of multiple oocytes, producers increase the number of embryos produced from those animals with the desired production traits. Suboptimal culture conditions limit the proportion of metaphase II (MII) oocytes following IVM, affecting the viability of IVM as an assisted reproductive technology (ART). Improving IVM culture conditions will increase the proportion of MII oocytes that can be fertilised for the purpose of embryo production. Maturation medium is supplemented with hormones including 17 β-estradiol, follicle stimulating hormone (FSH) and luteinising hormone (LH). FSH and LH activate the mitogen-Activated Protein Kinase 3/1 (MAPK3/1) and protein kinase c (PKC) signalling pathways, which are involved in the resumption of meiosis and germinal vesicle breakdown (GVBD). Maturation medium is also supplemented with amino acids, which are energy sources and can act as pH and osmoregulation during IVM. Additionally, a protein source, either fetal bovine serum (FBS) or bovine serum albumin (BSA) is added to maturation medium. Whilst the use of these supplements in IVM has been extensively studied, their optimal combination remains controversial. The addition of FSH to the commercial medium, Tissue Culture Medium 199 (TCM199), achieved nuclear maturation, but there was no increase in the proportion of MII oocytes with the addition of 17β-estradiol or human chorionic gonadotropin (hCG). To determine the effects of amino acids, bovine cumulus oocyte complexes (COC) were matured in a simple inorganic salt solution (M1) supplemented with multiple amino acids combinations. The data shows that the addition of Glutamine, proline, and isoleucine to M1, results in %MII oocytes, comparable to TCM199. However, when cystine was added to glutamine, proline, and isoleucine, the %MII was significantly reduced. It was hypothesised that cystine activates the SLC7A11 (xCT) antiporter, which exports intracellular glutamate resulting in reduced glutathione (GSH), leading to increased reactive oxygen species (ROS). Live-cell imaging and mass-spectrometry data show ROS production is higher in oocytes cultured with cystine, and that their glutamate content was significantly lower than control oocytes. However, there was no difference in the GSH content of oocytes matured in the presence of cystine. Furthermore, the xCT antiporter was only present in the nuclear envelope at the germinal vesical stage of development. It was hypothesised that cystine affected the maturation promoting effects of glutamine, proline, and isoleucine competitive uptake through membrane transporters. Using radiolabelled glutamine and proline, it was observed that cystine blocked proline uptake at the 24 h timepoint of IVM. This suggests that the maturation promoting effects of proline are inhibited by cystine. Similarly, isoleucine inhibited proline uptake, but it is likely that isoleucine compensates for proline activity. Data presented here show that supplementation of a simplified inorganic salt solution with glutamine, proline, isoleucine and FSH supports reliable and reproducible nuclear maturation of bovine oocytes. It also demonstrates that under certain conditions, amino acids such as systine, cause a reduction in the proportion of MII oocytes following IVM. The data acts as a foundation to further investigate the role of amino acids in oocyte maturation with the aim of improving culture conditions and the overall viability of IVM as an assisted reproductive technology