Functional characterisation of the cumulus oocyte matrix during maturation of oocytes.

Abstract

Female gametes, or oocytes grow and mature in a niche environment maintained by the somatic cells of the ovarian follicle. At ovulation ovarian follicle cells respond to the luteinising hormone (LH) surge coordinating the final maturation, meiotic resumption and release of oocytes. Simultaneously, production of a unique “mucified” extracellular matrix surrounding the oocyte through synthesis of Hyaluronan (HA) and HA cross-linking proteins produces an “expanded” and stabilised cumulus oocyte matrix with a specific composition, structure and function. In vitro maturation (IVM) of oocytes is a procedure by which cumulus oocyte complexes (COCs) are stimulated to produce cumulus matrix and undergo oocyte maturation ex vivo. In vitro maturation is a useful procedure for studying oocyte competence as well as offering health benefits for patients undergoing assisted reproduction. Oocytes derived from IVM have much lower developmental competence than in vivo matured oocytes, likely as a result of altered environmental conditions and gene expression leading to suboptimal maturation and/or inappropriate metabolic control in oocytes. Cumulus matrix expansion is widely used as an indicator of good oocyte developmental potential, however, the mechanism(s) that endow oocyte quality and how these may be influenced by the cumulus matrix are poorly understood. To better understand the process by which cumulus matrix is linked to the final stages of oocyte maturation, I undertook investigation of mouse COC matrix composition and function after in vivo maturation in comparison to IVM. The gene responsible for Hyaluronan synthesis, Has2, was not impaired under IVM conditions. In contrast, two key extracellular matrix proteins; Versican and Adamts1, which are normally selectively incorporated into periovulatory COCs in vivo, were greater than 10-fold reduced in IVM whether stimulated with Egf and/or FSH. This work is the first to show that commonly used IVM conditions result in altered gene expression in cumulus cells. Furthermore, the absence of Adamts1 and Versican suggest that COC matrix may be functionally insufficient. Although associated with good developmental potential, the function of the COC matrix in oocyte maturation is unknown. I assessed the properties of COC matrix that control metabolite supply to oocytes by examining transport of fluorescently labelled glucose and cholesterol across mouse COCs. Profound differences in the control of metabolite supply to oocytes in IVM were observed. In vivo matured complexes were capable of excluding glucose from the entire COC and cholesterol was excluded from oocytes. Conversely IVM COCs were more permissive to rapid equilibration of glucose and cholesterol concentrations across the complex and in oocytes. In fact both metabolites accumulated rapidly in IVM oocytes resulting in inverse gradient patterns of glucose and cholesterol abundance with highest concentrations accumulating in the oocyte after IVM vs highest concentrations surrounding the COC after in vivo maturation conditions. As oocytes are highly sensitive to high glucose my results indicate that metabolic balance in IVM may be disrupted due to impaired molecular filtration properties of the mucified COC matrix that controls supply of hydrophilic and lipophylic substrates. Importantly these novel findings can explain the glucose sensitivity of IVM oocytes and identifies a mechanism by which IVM may lead to poorer oocyte developmental competence. To translate these findings into the improvement of IVM I generated recombinant expression plasmid constructs for several Adamts1 and Versican functional domains. The efficacy of Versican as an IVM supplement that activates cumulus cell signal transduction was proved in principle, by showing enhanced COC matrix expansion when added to mouse IVM cultures. Similar mechanisms are likely to be functional in human COCs since I demonstrated VERSICAN and ADAMTS1 expression in human in vivo matured cumulus and granulosa cells. This work has advanced our understanding of oocyte maturation and will lead to improvements in IVM and healthier outcomes from reproductive therapies.