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In vitro maturation (IVM) offers significant benefits for human infertility treatment and animal breeding, but this potential is yet to be fully realised due to reduced oocyte developmental competence in comparison with in vivo matured oocytes. Cumulus cells occupy an essential position in determining oocyte developmental competence. Here we have examined the areas of deficient gene expression, as determined within microarrays primarily from cumulus cells of mouse COCs, but also other species, between in vivo matured and in vitro matured oocytes. By retrospectively analysing the literature, directed by focussing on downregulated genes, we provide an insight as to why the in vitro cumulus cells fail to support full oocyte potential and dissect molecular pathways that have important roles in oocyte competence. We conclude that the roles of epidermal growth factor signalling, the expanded extracellular matrix, cumulus cell metabolism and the immune system are critical deficiencies in cumulus cells of IVM COCs., (© 2017 Society for Reproduction and Fertility.)

Cumulus cell-oocyte communication is an essential feature of mammalian reproduction. Established mechanisms involve the bidirectional transfer of ions and small molecules through gap junctions that fundamentally regulate the process of oocyte maturation. Also, well established is the paracrine signaling from the oocyte to the cumulus, which regulates much of the flow of ions and molecules to the oocyte and orchestrates many of the associated local signaling events around ovulation, which is the key to establishing oocyte competence to sustain early embryo development. Less well-characterized and new potential players include exosomal transfer of noncoding RNAs from cumulus to oocytes and the recent observations of the presence of hemoglobin in oocytes and cumulus cells. The impact of these new communication pathways is either poorly defined or even unknown. Finally, signaling between the two cell types most likely continues after ovulation and even fertilization; however, this too is largely undefined but may play roles in substrate transport, sperm chemotaxis and "trapping", and potential signaling to the rest of the reproductive tract., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In vitro maturation of oocytes is suboptimal to in vivo maturation with altered gene expression and compromised oocyte quality. The large proteoglycan versican is abundant in mouse cumulus-oocyte complexes (COCs) matured in vivo but is absent in cultured COCs. Versican is also positively correlated with human oocyte quality. Versican contains an epidermal growth factor (EGF) motif, and based on EGF-like activities in other systems we hypothesized that versican acts as an EGF-like signaling factor during COC maturation. Here, we purified recombinant versican and compared its function with that of EGF during in vitro maturation (IVM). Versican significantly increased cumulus expansion and induced cumulus-specific genes Ptgs2, Tnfaip6, and Has2, which was blocked by EGF receptor antagonist. Microarray analysis revealed that versican has overlapping function with EGF; however, a subset of genes was uniquely altered following 6 h of IVM with either treatment. Following 6 h of IVM, both Areg and Ereg were significantly increased by both treatments, whereas Egln3, Nr4a1, Nr4a2, Nr4a3, and Adamts5 were significantly higher following versican treatment compared with EGF. In contrast, Sprr1a and Aqp3 were increased after 6 h of EGF but not versican treatment. To determine whether there were temporal differences, COCs were cultured with EGF or versican for 0-12 h. Versican-induced expression occurred later but remained elevated for longer compared with EGF for Ptgs2, Ereg, and Nr4a3. The unique expression profiles of Aqp3 and Nr4a3 during IVM were similarly regulated in vivo. These data indicate that versican has EGF-like effects on COC gene expression, but with distinct temporal characteristics., (© 2015 by the Society for the Study of Reproduction, Inc.)

Oxygen is an important component of the environment of the cumulus-oocyte complex (COC), both in vivo within the ovarian follicle and during in vitro oocyte maturation (IVM). Cumulus cells have a key role in supporting oocyte development, and cumulus cell function and gene expression are known to be altered when the environment of the COC is perturbed. Oxygen-regulated gene expression is mediated through the actions of the transcription factors, the hypoxia-inducible factors (HIFs). In the present study, the effect of oxygen on cumulus cell gene expression was examined following in vitro maturation of the murine COC at 2%, 5% or 20% oxygen. Increased expression of HIF-responsive genes, including glucose transporter-1, lactate dehydrogenase A and BCL2/adenovirus E1B interacting protein 3, was observed in cumulus cells matured at 2% or 5%, compared with 20% oxygen. Stabilisation of HIF1α protein in cumulus cells exposed to low oxygen was confirmed by western blot and HIF-mediated transcriptional activity was demonstrated using a transgenic mouse expressing green fluorescent protein under the control of a promoter containing hypoxia response elements. These results indicate that oxygen concentration influences cumulus cell gene expression and support a role for HIF1α in mediating the cumulus cell response to varying oxygen.

While formation of the expanded cumulus matrix and its importance for oocyte maturation and ovulation are well described, its function in these processes remains unknown. The degree of expansion and expression of cumulus matrix genes are positively correlated with oocyte quality, suggesting that this matrix plays a key role in oocyte maturation. Based on recognized filtration properties of analogous matrices, we investigated whether the cumulus matrix acts as a molecular filter by assessing diffusion of fluorescently labeled dextrans (neutral and negatively charged) and hydrophilic (glucose) and hydrophobic (cholesterol) metabolites in cumulus oocyte complexes (COCs). Expanded in vivo-matured COCs resisted absorption of glucose and cholesterol compared to unexpanded COCs. In vitro-matured (IVM) COCs have a pronounced deficiency in cumulus matrix proteins and have poor oocyte quality. Here we demonstrate that IVM cumulus matrix has deficient filtration properties, with dextran and glucose and cholesterol molecules diffusing more readily into IVM than in vivo-matured COCs. Taking the inverse approach, we found that prostaglandin E2 (PGE2), synthesized by cumulus cells, is retained within the matrix of in vivo-matured COCs but IVM COCs have reduced capacity to retain PGE2, secreting significantly more into the medium. This is the first demonstration of a biophysical property of the cumulus matrix. The ability to regulate metabolite supply from the surrounding environment while sequestering vital signaling factors, such as PGE2, is likely to impact oocyte maturation. Thus, IVM may reduce oocyte quality due to dysregulated control of metabolites and signaling molecules.

Remodeling of ovarian follicle extracellular matrix is essential for ovulation and vascularization of the corpus luteum (CL). Formation of the cumulus matrix around oocytes also plays an important role in ovulation and subsequent fertilization of oocytes. ADAMTS1 is an extracellular metalloprotease induced in ovarian follicles by ovulatory hormones and is required for fertility. In this study, we identified ADAMTS1-mediated structural and morphological changes in remodeling of the follicle and cumulus oocyte complex (COC). In Adamts1(-/-) mice, the ovulation rate was 77% reduced and fertilization of ovulated oocytes was reduced a further 63%, resulting in a reduced number of litters and pups per litter. Morphological assessment of peri-ovulatory ovaries revealed abnormal morphogenesis with a lack of thecal/vascular invagination in the basal region of follicles. Cleavage of the ADAMTS1 substrate, versican, at these invaginating regions was abundant in Adamts1(+/-) but undetectable in Adamts1(-/-) ovaries, indicating that processing of versican by ADAMTS1 is involved in ovulating follicle remodeling. Versican and hyaluronan localization was abnormal during COC matrix expansion, and versican persisted beyond the expected time of fertilization in Adamts1(-/-) but was catabolized and cleared from control COC. The results demonstrate that ADAMTS1 is critical in both ovulation and fertilization processes in vivo. The protease activity of ADAMTS1 mediates neomorphogenesis of the ovulating follicle wall and COC matrix necessary for successful ovulation and fertilization, as well as subsequent catabolism of versican required for degradation of COC matrix after fertilization.

Background: In vitro maturation (IVM) of mammalian oocytes has potential health benefits for patients undergoing assisted reproduction as an alternative to gonadotrophin treatment. This procedure is also useful for studying the process of oocyte and early embryo development. However, oocytes undergoing IVM have much lower competence than in vivo matured oocytes. Efforts to optimize IVM success have focused on replicating in vivo timing, hormonal milieu and cumulus cell responses associated with maturing oocytes. We have previously identified two extracellular matrix proteins, the protease Adamts1 and hyaluronan-binding proteoglycan Versican, produced by mural granulosa cells that selectively incorporate into the periovulatory cumulus-oocyte complex (COC)., Methods: Murine COC were cultured in the presence of epidermal growth factor and/or FSH. mRNA and protein were measured by real time PCR and Western blot and compared to in vivo derived COC., Results: COCs from mice that underwent IVM for 6 or 20 h in the presence of epidermal growth factor, FSH or in combination had a > 10-fold reduction in mRNA (P < 0.05) for Adamts1 and Vcan when compared with in vivo matured COCs. Hyaluronan synthase 2 expression was up-regulated up to 8-fold (P < 0.05) over the unstimulated control, demonstrating successful induction of cumulus gene expression by the IVM conditions. While in vivo matured COCs showed abundant levels of these proteins, COCs that underwent IVM had neither detectable Adamts1, nor intact or Adamts1-cleaved Vcan. Human cumulus and granulosa cells matured in vivo contained abundant mRNA for Adamts1 and Vcan, demonstrating the potential relevance to human IVM., Conclusion: These results indicate that extensively altered COC matrix composition is present during IVM and may contribute to the observed poorer competence of the derived oocytes.