Laura A. Frank

Effect of Epidermal Growth Factor-Like Peptides on the Metabolism of In Vitro- Matured Mouse Oocytes and Cumulus Cells

ABSTRACT Oocyte in vitro maturation (IVM) is an assisted reproductive technology that involves the maturation of cumulus-oocyte complexes (COCs) that are then capable of normal development. We have shown that epidermal growth factor (EGF)-like peptide signaling is perturbed in mouse COCs undergoing IVM when matured with follicle-stimulating hormone (FSH) and/or EGF, but supplementation of IVM with EGF-like peptides amphiregulin or epiregulin improves oocyte developmental competence. Here we aimed to determine whether EGF-like peptides regulate COC metabolism. Immature 129/Sv mouse COCs underwent IVM with FSH, EGF, amphiregulin, epiregulin, betacellulin, or no treatment (control). Epiregulin significantly increased intraoocyte flavin adenine dinucleotide (FAD) and REDOX (reduction and oxidation) ratio compared to FSH and control. Amphiregulin and epiregulin significantly increased the proportion of J aggregates (from JC-1) in oocyte mitochondria compared to control, FSH, or EGF, and this coupled with FAD and REDOX measures indicates greater mitochondrial activity. There were no differences in glucose consumption, lactate production, or glycolysis between COCs matured with FSH, EGF, and EGF-like peptides. COCs matured with EGF or EGF-like peptides exhibited significantly higher mRNA expression of the hexosamine biosynthesis pathway (HBP) rate-limiting enzyme gene Gfpt2, Has2 expression, and global beta-O-linked glycosylation of proteins, compared to control or FSH, suggesting greater HBP activity. Our findings suggest that 1) EGF-like peptides, particularly epiregulin, induce more oocyte mitochondrial activity than EGF or FSH and 2) EGF-like peptides and EGF induce greater HBP activity, enabling more hyaluronic acid synthesis and protein beta-O-linked glycosylation. These metabolic alterations may be a mechanism by which EGF-like peptides increase oocyte developmental competence.

Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of Heat shock protein 90

STUDY QUESTION What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? SUMMARY ANSWER Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. WHAT IS KNOWN ALREADY O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. STUDY DESIGN, SIZE, DURATION This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. MAIN RESULTS AND THE ROLE OF CHANCE Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client-chaperone relationship. LIMITATIONS, REASONS FOR CAUTION In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. WIDER IMPLICATIONS OF THE FINDINGS This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.

Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence

The effects of hyper- and hypo-glycaemic conditions during the in vitro maturation of mouse cumulus-oocyte complexes on developmental competence were examined, with an emphasis on the role of the hexosamine biosynthesis pathway. A low (1 mM) glucose concentration achieved optimal oocyte competence (3-fold higher blastocyst development rate compared with high (30 mM) glucose, P<0.05). In addition, glucose supplementation during only the first hour after release from the follicle was necessary and sufficient to support oocyte maturation and embryo development to the blastocyst stage. Glucosamine (a known hyperglycaemic mimetic and specific activator of the hexosamine pathway) was able to substitute for glucose during this first hour, indicating that flux through the hexosamine pathway is essential for oocyte competence. In the absence of glucose throughout the maturation period, glucosamine was not able to increase developmental competence, and at higher concentrations (2.5 and 5 mM) had a detrimental effect on MII and blastocyst development rates, compared with controls (P<0.05). These experiments underscore the importance of glucose metabolic pathways during in vitro maturation and support the concept that excess flux through the hexosamine pathway has detrimental consequences.

Hemoglobin: a Gas Transport Molecule That Is Hormonally Regulated in the Ovarian Follicle in Mice and Humans

ABSTRACT An increasing number of nonerythroid tissues are found to express hemoglobin mRNA and protein. Hemoglobin is a well-described gas transport molecule, especially for O2, but also for NO, CO2, and CO, and also acts as a reactive oxygen species scavenger. We previously found Hba-a1 and Hbb mRNA and protein at high levels within mouse periovulatory cumulus cells, but not in cumulus following in vitro maturation. This led us to investigate the temporal and spatial regulation in follicular cells during the periovulatory period. Cumulus-oocyte complexes were collected from equine chorionic gonadotropin/human chorionic gonadotropin-treated peripubertal SV129 female mice and collected and analyzed for gene expression and protein localization at a variety of time points over the periovulatory period. A further cohort matured in vitro with different forms of hemoglobin (ferro- and ferrihemoglobin) under different O2 atmospheric conditions (2%, 5%, and 20% O2) were subsequently fertilized in vitro and cultured to the blastocyst stage. Murine mRNA transcripts for hemoglobin were regulated by stimulation of the ovulatory cascade, in both granulosa and cumulus cells, and expression of HBA1 and HBB was highly significant in human granulosa and cumulus, but erythrocyte cell marker genes were not. Several other genes involved in hemoglobin function were similarly luteinizing hormone-regulated, including genes for heme biosynthesis. Immunohistochemistry revealed a changing localization pattern of HBA-A1 protein in murine cumulus cells and oocytes following the ovulatory signal. Significantly, no positive staining for HBA-A1 protein was observed within in vitro-matured oocytes, but, if coincubated with ferro- or ferrihemoglobin, cytoplasmic HBA-A1 was observed, similar to in vivo-derived oocytes. Addition of ferro-, but not ferrihemoglobin, had a small, positive effect on blastocyst yield, but only under either 2% or 20% O2 gas atmosphere. The identification of hemoglobin within granulosa and cumulus cells poses many questions as to its function in these cells. There are several possible roles, the most likely of which is either an O2 or NO sequestering molecule; perhaps both roles are engaged. The strong endocrine regulation during the periovulatory period suggests to us that one potential function of hemoglobin is to provide a short-lived hypoxic environment by binding very tightly any available O2. This, in turn, facilitates the differentiation of the follicle towards corpus luteum formation by enabling the stabilization of a key transcription factor known to initiate such differentiation: hypoxia inducible factor.

The effect of peri-conception hyperglycaemia and the involvement of the hexosamine biosynthesis pathway in mediating oocyte and embryo developmental competence

The environment that the oocyte is exposed to during the peri‐conception period can have a significant impact on oocyte developmental competence (the ability of the oocyte to support fertilisation and subsequent embryo development) and the long‐term health of the resulting offspring. This is particularly true for maternal hyperglycaemia. While maternal hyperglycaemia during early pregnancy through term development has been extensively studied, the effects on the oocyte itself, and the underlying mechanisms, remain largely unknown. There is increasing evidence, however, for the role of the fuel‐sensing hexosamine biosynthesis pathway in mediating the effects of hyperglycaemia in many different cell types. In this review, we will focus on the reproductive consequences of maternal hyperglycaemia during the peri‐conceptual period and the role of the hexosamine pathway in mediating these processes. Mol. Reprod. Dev. 81: ???–???, 2014.

Oxygen-regulated gene expression in murine cumulus cells

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.