Investigating the effect of a physiologically relevant IVM system on the function of bovine cumulus cell-oocyte complexes

There is a world-wide trend of increasing sub-fertility in dairy cows whereby the time interval between the planned dates of mating and successful conception is increasing. This interval is coincident with the timing of peak lactation and thus has been hypothesised to be due to increased metabolic stress leading to cows being in negative energy balance. Thus, it is hypothesised that the follicular microenvironment in which cumulus-oocyte complexes (COC) develop and mature is inadequate in some cows during this time. To test this hypothesis, a better understanding of how different microenvironments affect the functionality of the COC within the developing follicle, and the ability to produce a mature, developmentally-competent oocyte is required.

This study used previous records of concentrations of specific amino acids, glucose, cholesterol, and hormones measured in antral and pre-ovulatory follicles of lactating New Zealand (NZ) dairy cows. This information was used to produce bi-phasic media systems to that represented the changing environment as a follicle matured (i.e. Early and Late) as well as a microenvironment of either a pre-ovulatory follicle that would produce a developmentally-competent oocyte (Good) or a subordinate follicle that would produce an oocyte that, if fertilised, would not be capable of developing into a viable embryo (Bad). The main experiments of the study involved culturing bovine COC in either one of the two PR (PR) IVM systems (Good or Bad) or the common ‘gold-standard’ (Control) IVM system. Differences in COC function after incubation in the PR or Control IVM systems were compared over time in regard to key events involved in oocyte maturation, namely: (i) timing of meiotic resumption; (ii) gap junction (GJ) communication; (iii) expression levels of key genes involved in oocyte maturation; and (iv) amino acid metabolism within the COC.

The methodologies employed to achieve these aims were a fluorescent dye transfer assay to measure gap junction communication between the cumulus cells and oocyte, Taq-man qPCR to quantify gene expression levels and HPLC to measure amino acid metabolism (this was contracted to the University of Auckland). In this study, I present evidence that the PR-IVM system in comparison to the Control IVM system that uses a nutrient-rich media, resulted in an lower levels of amino acid utilisation, lower CC-derived gene expression levels of CX43 (a gene encoding a gap junction protein) and TXNIP (a gene encoding a protein that regulates oxidative stress) as well as lower oocyte-derived gene expression levels of FASN (a gene encoding an enzyme involved in the synthesis of the energy source palmitate). These results suggest the COC incubated in a PR-IVM system are less ‘metabolically-active’ than when cultured in the control system. This is interesting as oocytes with a high metabolic rate are associated with a poor embryological outcome. However, this study also demonstrates that despite these apparent metabolic and molecular differences of COC incubated in the divergent IVM systems, GJ communication and timing of meiotic resumption were not influenced.

This study provides evidence that IVM systems delivering ‘nutrient’ excesses may result in changes to COC function that may not beneficial to the overall attainment of developmental competency in vitro. Moreover, the gold-standard IVM system described herein is not representative of the follicular microenvironment of the NZ dairy cow and this study emphasised the importance of using in vitro systems that best mimic the physiological environment to ensure results are representative of in vivo matured tissues. In summary, this research indicates the need for further development of defined PR IVM systems, which may lead to oocyte maturation events and embryological outcomes that are more representative of in vivo systems.