Search

Blastocoel expansion during embryo development is known to be reliant on the Na+/K+-ATPase pump, but little is known about the relative contribution of active (Na+/K+-ATPase pump) and facilitated diffusion (aquaporins) water transport during blastocoel re-expansion after vitrification. The aims of this study were to examine potential effects of artificial blastocoel collapse (ABC) on markers of embryo stress and the contribution of active and facilitated diffusion water transport mechanisms to blastocoel re-expansion. Day 5 mouse embryos were vitrified using either a standard protocol, laser pulse ABC, a hyperosmotic sucrose ABC protocol or both laser pulse and sucrose. Using real-time polymerase chain reaction, no differences were found in the gene expression of the endoplasmic reticulum (ER) stress markers activating transcription factor 4 (Atf4) or heat shock protein 90-alpha (Hsp90α) 2h after warming. Similarly, expression of the Na+/K+-ATPase pump gene, ATPase, Na+/K+ transporting, beta 1 polypeptide (Atp1b1) and protein did not differ between groups. Aquaporin 8 (Aqp8) gene expression was significantly lower in the laser+sucrose ABC group than in fresh controls, and aquaporin 3 (Aqp3) expression significantly higher in standard vitrified embryos compared with all other groups. Ouabain, a potent and specific Na+/K+-ATPase pump inhibitor, inhibited blastocoel re-expansion in both standard protocol- and laser ABC-vitrified embryos, reducing both groups to the same rate of re-expansion 3h after warming. These results demonstrate that ABC before vitrification does not alter mRNA or protein expression of Na+/K+-ATPase, or mRNA levels of ER stress genes Atf4 and Hsp90α. Activity of the pump may be increased in ABC embryos, with potential compensation by AQP3 when it is compromised.

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus–oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.

The function and impact of epidermal growth factor (EGF)-like peptide signalling during ovulation and in vivo oocyte maturation (IVV) has been recently characterized, however, little is currently known about the effect of oocyte in vitro maturation (IVM) on this pathway. The aim of this study was to examine expression and functional aspects of three EGF-like peptides (amphiregulin, epiregulin and betacellulin) and their common receptor (EGFR) in cumulus cells during mouse oocyte IVM compared with IVV. Cumulus-oocyte complexes (COCs) were collected from prepubertal mice either 46 h post-eCG (IVM) or 46 h post-eCG plus 0.5-12 h post-hCG (IVV). Time course experiments showed mRNA expression of all three EGF-like peptides and amphiregulin protein in IVM media were significantly lower for the majority of FSH-supplemented IVM compared with IVV. The supplementation of EGF during IVM yielded EGF-like peptide expression levels comparable with IVV and amphiregulin/epiregulin supplemented IVM. However, despite this, EGF activation of the COC EGFR remained significantly lower at 3 and 6 h of IVM than in vivo, and levels were similar to those observed during FSH-supplemented IVM. The addition of exogenous epiregulin during IVM significantly increased blastocyst rates, and epiregulin and amphiregulin improved blastocyst quality, compared with FSH or EGF. In conclusion, findings from this study suggest that the widely used IVM additives, FSH and EGF, are inadequate propagators of the essential EGF-like peptide signalling cascade. In contrast, the use of epiregulin and/or amphiregulin during IVM leads to improved oocyte developmental competence and therefore may be preferable IVM additives than FSH or EGF.

The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP – inhibiting the hydrolysis of intra-oocyte cAMP – and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect – the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.

The preimplantation embryoinvivois exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture mediain vitro. The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P P in vitroculture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain.

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.

Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous fluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from imaging of native fluorescence has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Multispectral intrinsic fluorescence imaging was applied to patient olfactory neurosphere-derived cells, cell model of a human metabolic disease MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like syndrome). By using an endogenous source of contrast, subtle metabolic variations have been detected between living cells in their full morphological context which made it possible to distinguish healthy from diseased cells before and after therapy. Cellular maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant cell subpopulations, in particular a subpopulation with compromised mitochondrial function. The versatility of our method is further illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes, and assessing the condition of preimplantation embryos. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent.

Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous autofluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from autofluorescence imaging has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent. Label-free classifications are validated by the analysis of Classification Determinant (CD) antigen expression. The versatility of our method is illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes and assessing the condition of preimplantation embryos.

Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM), and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

Hyperspectral imaging based on endogenous contrast provides a new non-invasive method to characterise cells and tissues. Cellular content and maps of native fluorophores help monitor biological processes, with proper account of intrinsic cellular heterogeneity.

Reactive Oxygen Species (ROS) cause DNA damage and defective function in sperm and also affects the developmental competence of embryos. It is therefore critical to monitor ROS in sperm, oocytes and developing embryos. In particular, hydrogen peroxide (H2O2) is a ROS important to normal cell function and signalling as well as its role in oxidative stress. Here we report the development of a fluorescent sensor for H2O2 using carboxyperoxyfluor-1 (CPF1) in solution and attached to a glass slide or multi-mode optical fibre. CPF1 increases in fluorescence upon reaction with H2O2 to non-invasively detect H2O2 near developing embryos. These probes are constructed by immobilising CPF1 to the optical fibre tip a polyacrylamide layer. Also reported is a new dual optical fibre sensor for detecting both H2O2 and pH that is functional at biologically concentrations of H2O2 and can sense pH to 0.1 units. This research shows promise for the use of optical fibre sensors for monitoring the health of developing embryos. Furthermore, these sensors are applicable for use beyond embryos such as detecting stress in endothelial cells involved in cardiovascular dysfunction. Refereed/Peer-reviewed

Citation Robertson SA, Chin PY, Glynn DJ, Thompson JG. Peri-Conceptual Cytokines – Setting the Trajectory for Embryo Implantation, Pregnancy and Beyond. Am J Reprod Immunol 2011; 66 (Suppl. 1): 2–10 Problem The peri-conceptual environment influences the early embryo to impart long-term consequences for the fetus and neonate; however, the underlying mechanisms are not well defined. Method of Study We argue that the cytokine network acting in the female reproductive tract during the pre- and peri-implantation period integrates environmental information to program the embryo and fine-tune the maternal immune response and endometrial remodelling to determine implantation success. Results As well as sex steroid hormones and male seminal fluid factors, female tract cytokines are influenced by agents signalling via the Toll-like receptors including the microbiome and a plethora of metabolic, chemical and other stressors. In mouse models, an altered peri-conceptual cytokine environment induced by cytokine deficiency, inflammatory insults or dysregulated seminal fluid signalling is associated with adverse effects on embryo development, pregnancy viability and reproductive outcome. Conclusion The cytokine network provides a pivotal mechanism through which environmental factors influence both embryo development and receptivity of the uterus.

An innovative approach to in vitro maturation (IVM) for application in infertility treatment and fertility preservation is required to bring this patient-friendly treatment into routine practice. Current approaches to IVM never report more than a 10 to 15% implantation rate per embryo transferred, which is two to three times lower and early pregnancy losses are higher than in conventional in vitro fertilization/intracytoplasmic sperm injection. The cornerstone of such an innovative culture technique is the use of pharmacological compounds that allow synchronization of nuclear and cytoplasmic maturation processes within the oocyte. The rationale of a prolonged oocyte maturation period is to promote a longer interaction between the immature oocyte with adequately conditioned cumulus cells. Successful introduction of a new approach to IVM will reduce the requirement of fertility hormones and will be less invasive to the patient's daily life by reducing the need for monitoring of serum hormone levels and intravaginal ultrasound. The new IVM conditions will reduce a whole range of minor and major complications in assisted reproductive technology and finally will also reduce the total cost for treatment. The minimal invasiveness of this procedure will benefit cancer patients who want to store gonadal tissue before undergoing therapy that devastates subsequent germ-cell competence.

Oocyte and embryo metabolism are closely linked with their subsequent developmental capacity. Lipids are a potent source of cellular energy, yet little is known about lipid metabolism during oocyte maturation and early embryo development. Generation of ATP from lipids occurs within mitochondria via beta-oxidation of fatty acids, with the rate-limiting step catalyzed by carnitine palmitoyl transferase I (CPT1B), a process also requiring carnitine. We sought to investigate the regulation and role of beta-oxidation during oocyte maturation and preimplantation development. Expression of Cpt1b mRNA, assessed by real-time RT-PCR in murine cumulus-oocyte complexes (COCs), increased following hormonal induction of oocyte maturation and ovulation in vivo with human chorionic gonadotropin (5 IU) and in embryos reaching the blastocyst stage. Beta-oxidation, measured by the production of (3)H(2)O from [(3)H]palmitic acid, was significantly increased over that in immature COCs following induction of maturation in vitro with epidermal growth factor (3 ng/ml) and follicle-stimulating hormone (50 mIU/ml). The importance of lipid metabolism for oocyte developmental competence and early embryo development was demonstrated by assessing the rate of embryo development following inhibition or upregulation of beta-oxidation with etomoxir (an inhibitor of CPT1B) or L-carnitine, respectively. Inhibition of beta-oxidation during oocyte maturation or zygote cleavage impaired subsequent blastocyst development. In contrast, L-carnitine supplementation during oocyte maturation significantly increased beta-oxidation, improved developmental competence, and in the absence of a carbohydrate energy supply, significantly increased 2-cell cleavage. Thus, carnitine is an important cofactor for developing oocytes, and fatty acids are an important energy source for oocyte and embryo development.

The environment that the cumulus oocyte complex (COC) is exposed to during eitherin vivoorin vitromaturation (IVM) can have profound effects on the success of fertilisation and subsequent embryo development. Glucose is a pivotal metabolite for the COC and is metabolised by glycolysis, the pentose phosphate pathway (PPP), the hexosamine biosynthesis pathway (HBP) and the polyol pathway. Over the course of oocyte maturation, a large proportion of total glucose is metabolised via the glycolytic pathway to provide substrates such as pyruvate for energy production. Glucose is also the substrate for many cellular functions during oocyte maturation, including regulation of nuclear maturation and redox state via the PPP and for the synthesis of substrates of extracellular matrices (cumulus expansion) andO-linked glycosylation (cell signalling) via the HBP. However, the oocyte is susceptible to glucose concentration-dependent perturbations in nuclear and cytoplasmic maturation, leading to poor embryonic development post-fertilisation. For example, glucose concentrations either too high or too low result in precocious resumption of nuclear maturation. This review will discuss the relevant pathways of glucose metabolism by COCs duringin vivomaturation and IVM, including the relative contribution of the somatic and gamete compartments of the COC to glucose metabolism. The consequences of exposing COCs to abnormal glucose concentrations will also be examined, either during IVM or by altered maternal environments, such as during hyperglycaemia induced by diabetes and obesity.

During fertilization, multiple intracellular calcium (Ca2+) oscillations are initiated after sperm binding to the oocyte vitelline membrane. This Ca2+ signalling has been extensively studied in denuded mouse and Xenopus oocytes but minimally studied in larger mammals. Cows in particular are unusual, as the few studies on oocyte activation have observed fewer Ca2+ oscillations during fertilisation compared with mice. Furthermore, cattle intracytoplasmic sperm injection (ICSI) is inefficient, despite parthenogenetic activation occurring readily. We hypothesise that cumulus cells are important for cattle oocyte activation at fertilisation. Here, we assessed the behaviour of Ca2+oscillations in fertilising intact cattle cumulus–oocyte complexes (COC). Abattoir-derived cattle COC were matured and fertilised in vitro using Bovine Vitro Media Suite (IVF Vet Solutions). The COC were stained 3.5 h after insemination with the Ca2+ fluorescent probe Fluo4AM (5 μM, Molecular Probes Inc., Eugene, OR, USA) for 30 min, washed, and imaged every 5 min for 6 h in a Fluoview FV10i incubating time-lapse confocal microscope (Olympus) before being returned to culture. Embryo development was assessed at Day 8 to confirm fertilisation. Fluo4AM fluorescence intensity was assessed using FIJI ImageJ. Mean relative intensity over time was graphed for specific regions of interest and the area under graphs was calculated to quantify differences for comparison using a Mann-Whitney Test (mean ± SEM). Experiment 1 (4 reps of 10 COC) compared confirmed fertilised v. uninseminated; experiment 2 (2 reps of 10 COC) compared inseminated COC ± 10 μM BAPTA-AM (Ca2+ chelator, Sigma-Aldrich, St. Louis, MO, USA). There were distinct coordinated waves of differing Fluo4AM intensity in both the oocyte and the cumulus cells surrounding the confirmed fertilised oocytes. This contrasted to the random uncoordinated flashes of Fluo4AM fluorescence in the cumulus cells of the uninseminated oocytes. The fluorescence pattern in +BAPTA-AM COC matched the random flashes observed in the uninseminated group of experiment 1. The fluorescence in the media surrounding the COC immediately following the Fluo4AM waves spiked and then plateaued at a higher level of fluorescence. This was quantified by assessing the area under the graph for 1 h of the plateau following the fluorescence spike. There were no differences between confirmed fertilised (346.4 ± 41.62) and uninseminated groups (239.8 ± 32.08; P > 0.05), but this was affected by differences in cumulus dispersal due to the presence or absence of sperm. Experiment 2 used BAPTA-AM to block oocyte activation with sperm present in both groups and showed a significant difference between the fluorescence increase in the media of the 2 groups (–BAPTA-AM: 311.2 ± 31.57, +BAPTA-AM: 201.4 ± 26.59; P

In the first trimester the extravillous cytotrophoblast cells occlude the uterine spiral arterioles creating a low oxygen environment early in pregnancy, which is essential for pregnancy success. Paradoxically, shallow trophoblast invasion and defective vascular remodelling of the uterine spiral arteries in the first trimester may result in impaired placental perfusion and chronic placental ischemia and hypoxia later in gestation leading to adverse pregnancy outcomes. The hypoxia inducible factors (HIFs) are key mediators of the response to low oxygen. We aimed to elucidate mechanisms of regulation of HIFs and the role these may play in the control of placental differentiation, growth and function in both normal and pathological pregnancies. The Pubmed database was consulted for identification of the most relevant published articles. Search terms used were oxygen, placenta, trophoblast, pregnancy, HIF and hypoxia. The HIFs are able to function throughout all aspects of normal and abnormal placental differentiation, growth and function; during the first trimester (physiologically low oxygen), during mid-late gestation (where there is adequate supply of blood and oxygen to the placenta) and in pathological pregnancies complicated by placental hypoxia/ischemia. During normal pregnancy HIFs may respond to complex alterations in oxygen, hormones, cytokines and growth factors to regulate placental invasion, differentiation, transport and vascularization. In the ever-changing environment created during pregnancy, the HIFs appear to act as key mediators of placental development and function and thereby are likely to be important contributors to both normal and adverse pregnancy outcomes.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to promote the development and survival of human and mouse preimplantation embryos; however, the mechanism of action of GM-CSF in embryos is not defined. METHODS: Mouse blastocysts were cultured from zygote stage in vitro with and without recombinant mouse GM-CSF (rmGM-CSF), and in vivo developed blastocysts were flushed from Csf2 null mutant and wild-type mice. The effect of GM-CSF on blastocyst expression of stress response and apoptosis genes was evaluated by microarray, qPCR and immunochemistry. RESULTS: Microarray analysis of the gene transcription profile showed suppression of stress response and apoptosis gene pathways in blastocysts exposed to rmGM-CSF in vitro. qPCR analysis confirmed that rmGM-CSF inhibited expression of heat shock protein (HSP) and apoptosis pathway genes Cbl, Hspa5, Hsp90aa1, Hsp90ab1 and Gas5 in in vitro blastocysts. Immunocytochemical analysis of HSP 1 (HSPA1A/1B; HSP70), BAX, BCL2 and TRP53 (p53) in in vitro blastocysts showed that HSPA1A/1B and BCL2 proteins were less abundant when embryos were cultured with rmGM-CSF. BAX and TRP53 were unchanged at the protein level, but Bax mRNA expression was reduced after GM-CSF treatment. In in vivo developed blastocysts, Csf2 null mutation caused elevated expression of Hsph1 but not other stress response genes. CONCLUSIONS: We conclude that GM-CSF inhibits the cellular stress response and apoptosis pathways to facilitate embryo growth and survival, and the protective effects of GM-CSF are particularly evident in in vitro culture media, whereas in vivo other cytokines can partly compensate for absence of GM-CSF.

Ex vivotwo-cell mouse embryos deprived of glucosein vitrocan develop to blastocysts by increasing their pyruvate consumption; however, zygotes when glucose-deprived cannot adapt this metabolic profile and degenerate as morulae. Prior to their death, these glucose-deprived morulae exhibit upregulation of the H+-monocarboxylate co-transporter SLC16A7 and catalase, which partly co-localize in peroxisomes. SLC16A7 has been linked to redox shuttling for peroxisomal β-oxidation. Peroxisomal function is unclear during preimplantation development, but as a peroxisomal transporter in embryos, SLC16A7 may be involved and influenced by peroxisome proliferators such as peroxisome proliferator-activated receptor-α (PPARA). PCR confirmedPparamRNA expression in mouse embryos. Zygotes were cultured with or without glucose and with the PPARA-selective agonist WY14643 and the developing embryos assessed for expression of PPARA and phospho-PPARA in relation to the upregulation of SLC16A7 and catalase driven by glucose deprivation, indicative of peroxisomal proliferation. Reactive oxygen species (ROS) production and relationship to PPARA expression were also analysed. In glucose-deprived zygotes, ROS was elevated within 2 h, as were PPARA expression within 8 h and catalase and SLC16A7 after 12–24 h compared with glucose-supplied embryos. Inhibition of ROS production prevented this induction of PPARA and SLC16A7. Selective PPARA agonism with WY14643 also induced SLC16A7 and catalase expression in the presence of glucose. These data suggest that glucose-deprived cleavage stage embryos, although supplied with sufficient monocarboxylate-derived energy, undergo oxidative stress and exhibit elevated ROS, which in turn upregulates PPARA, catalase and SLC16A7 in a classical peroxisomal proliferation response.

The nutrient requirements and metabolic pathways used by the developing embryo transition from predominantly pyruvate during early cleavage stages to glucose at the blastocyst; however, the complexities involved in the regulation of metabolism at different developmental stages are not clear. The aims of this study were to examine the role of the malate-aspartate shuttle (MAS) in nutrient metabolism pathways in the developing mouse blastocyst and the consequences of impaired metabolism on embryo viability and fetal and placental growth. Eight-cell-stage mouse embryos were cultured in the presence of the MAS inhibitor amino-oxyacetate, with or without pyruvate as an energy substrate in the media. When the MAS was inhibited, the rate of glycolysis and lactate production was significantly elevated and glucose uptake reduced, relative to control cultured embryos in the presence of pyruvate. Despite these changes in embryo metabolism, this did not influence development to the blastocyst stage, but it did reduce the number of inner cell mass and trophectoderm cells. When these embryos were transferred to psuedopregnant females, inhibition of the MAS significantly reduced the proportion of embryos that implanted and developed into fetuses on Day 18 of pregnancy. Finally, fetal growth was reduced while placental weight was maintained, leading to a decreased fetal:placental weight ratio relative to control embryos. These results suggest that impaired metabolism of glucose in the blastocyst via the MAS alters the ability of the embryos to implant and form a pregnancy and leads to reduced fetal weight, likely via altered placental development and function.

Oxidative stress is a well-established cause of male infertility, with reactive oxygen species (ROS) causing infertility principally by impairing sperm motility and DNA integrity. Currently, most clinics do not test their infertile patients for the presence of oxidative stress because the available tests are expensive or difficult to perform. As antioxidant therapy may improve sperm DNA integrity and pregnancy outcomes, it has become apparent that there is an unmet clinical need for an inexpensive and easy-to-perform assay to identify sperm oxidative stress. The aim of this study was to develop a standardized protocol for performing a photometric nitro blue tetrazolium (NBT) assay for the measurement of seminal ROS production via production of coloured formazan, whilst correlating these results with impaired sperm function (motility and DNA integrity). Semen samples from 21 fertile and 36 male aetiology infertile men were assessed for ROS production (NBT assay), sperm DNA integrity (TUNEL), apoptosis (Annexin V) and sperm motility. Infertile men's semen contained on average fourfold higher levels of ROS than fertile men. The production of ROS by sperm was positively correlated with sperm DNA fragmentation and apoptosis, whilst being negatively correlated with sperm motility. Receiver-operating characteristic plot analysis established a cut-off point of 24 microg formazan/10(7) sperm having a sensitivity of 91.7% and a specificity of 81% for determining the fertility status of an individual. This study has been successful in establishing a standardized protocol for performing a photometric seminal NBT assay that has significant clinical utility in identifying men with impaired fertility because of oxidative stress.

The literature suggests an association between sperm DNA damage and assisted reproductive technology (ART) outcomes. However, previous studies involved the transfer of multiple embryos, which has complicated the interpretation of the results. The aim of this study was to determine the relationship between the levels of sperm DNA damage and fertilization rate, embryo development as well as pregnancy outcome, following single embryo transfer. Patients (n = 113) undergoing in vitro fertilization (IVF) (n = 45) and intra-cytoplasmic sperm injection (ICSI) (n = 68) were assessed for their levels of sperm DNA damage in the sample used for insemination. DNA damage was determined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling (TUNEL). The relationship between DNA damage and outcomes were assessed using regression analysis. Overall data showed no association between sperm DNA damage and fertilization rate, or embryo development in vitro. However, when IVF was the insemination method, there was a significant negative correlation between fertilization rates and sperm DNA damage (p < 0.05). When ICSI was the insemination technique, low sperm DNA damage was associated with successful pregnancy (37.8 +/- 5.7% DNA damaged sperm) compared with failed implantation (52.9 +/- 3.9% DNA damaged sperm, p < 0.05). Our results suggest that sperm DNA damage as measured by the TUNEL assay may provide an indicator for patients with poor fertilization rates and/or those unable to achieve pregnancy following ART treatment.

Oocyte quality is a key limiting factor in female fertility, yet we have a poor understanding of what constitutes oocyte quality or the mechanisms governing it. The ovarian follicular microenvironment and maternal signals, mediated primarily through granulosa cells (GCs) and cumulus cells (CCs), are responsible for nurturing oocyte growth, development and the gradual acquisition of oocyte developmental competence. However, oocyte-GC/CC communication is bidirectional with the oocyte secreting potent growth factors that act locally to direct the differentiation and function of CCs. Two important oocyte-secreted factors (OSFs) are growth-differentiation factor 9 and bone morphogenetic protein 15, which activate signaling pathways in CCs to regulate key genes and cellular processes required for CC differentiation and for CCs to maintain their distinctive phenotype. Hence, oocytes appear to tightly control their neighboring somatic cells, directing them to perform functions required for appropriate development of the oocyte. This oocyte-CC regulatory loop and the capacity of oocytes to regulate their own microenvironment by OSFs may constitute important components of oocyte quality. In support of this notion, it has recently been demonstrated that supplementing oocyte in vitro maturation (IVM) media with exogenous OSFs improves oocyte developmental potential, as evidenced by enhanced pre- and post-implantation embryo development. This new perspective on oocyte-CC interactions is improving our knowledge of the processes regulating oocyte quality, which is likely to have a number of applications, including improving the efficiency of clinical IVM and thereby providing new options for the treatment of infertility.

Glucosamine is a component of hyaluronic acid and an alternative substrate to glucose for the extracellular matrix synthesis of COCs. Its addition to an IVM medium reduces the glucose consumption of bovine COCs. Glucosamine is also metabolized to UDP-N-acetyl glucosamine (UDP-GlcNAc) via the hexosamine biosynthesis pathway and is utilized for O-linked glycosylation by the X-linked enzyme, O-linked GlcNAc transferase (OGT). Moreover, the inactivation of the second X chromosome in female embryos is influential in producing the sex ratio bias observed in vitro when embryos are cultured in the presence of glucose above 2.5mM. Accordingly, the aim of this study is to examine whether the presence of glucosamine during maturation or embryo culture causes a sex ratio bias in bovine blastocysts. Glucosamine was added to the medium in three different embryo developmental periods: in vitro maturation, the one-cell to eight-cell stage (before the maternal-zygotic transition, MZT), and the eight-cell to blastocyst stage (after MZT). When glucosamine was added during in vitro maturation, the developmental competence of oocytes was severely compromised. However, the sex ratio of embryos was not influenced. When glucosamine was added to embryo culture medium during development from one-cell to eight-cell stage (before MZT), it affected neither the development nor the sex ratio of bovine embryos. Finally, when glucosamine was added after MZT, the development rate of embryos was severely decreased, and the sex ratio was skewed toward males. Moreover, an inhibitor of OGT, benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (BADGP), negated the effect of glucosamine on the sex ratio when it was added to embryo culture medium from the eight-cell to blastocyst stage (after MZT). These results suggest that, like glucose, the supplementation of glucosamine into the medium skewed the sex ratio to males and that OGT, an X-linked enzyme, was involved in this phenomenon. Moreover, this effect of glucosamine was limited only to when it was present in the embryo culture medium after MZT.

Intoxication by dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads, among other damages, to early embryo loss, fetal malformations, and cardiovascular toxicity. Apart from binding to the arylhydrocarbon receptor (AhR), the mechanism of TCDD-mediated embryo toxicity is still unclear. We investigated possible modes of a TCDD-mediated toxicity, particularly in glucose metabolism, in pluripotent P19 mouse embryonic carcinoma cells. Undifferentiated P19 cells were exposed to 1–100 nm TCDD and characterized for AhR signaling. For studying cell differentiation, P19 cells were exposed to 10 nm TCDD at stage of embryoid body formation, and analyzed on glucose metabolism and cardiac differentiation during the next 3 wk. TCDD treatment activated the AhR-signaling cascade within 1 h, confirmed by AhR translocation, induction of cytochrome P450 1A1 expression, and activation of the xenobiotic response element. Although cell viability and transcription of the cardiac marker protein α-myosin heavy chain were affected, TCDD did not inhibit the differentiation of P19 cells to pulsating cardiomyocytes. TCDD significantly down-regulated the expression levels of the glucose transporter (GLUT) isoforms 1 and 3. After 24-h TCDD treatment, GLUT1 was no longer localized in the plasma membrane of P19 cells. The impaired GLUT expression correlated with a lower glucose uptake in 5-d-old embryoid bodies. The TCDD effects were mediated by AhR, as shown by preculture with the AhR antagonist α-naphthoflavone. Our data demonstrate that an AhR-mediated disturbance in GLUT expression and insufficient glucose uptake may be major mechanisms in TCDD embryo toxicity.

Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction-with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development.

Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.

The two types of cells that make up the cumulus-oocyte complex (i.e. the oocyte and cumulus cells) have very different metabolic demands, with glucose occupying a central role in metabolic activity. Cumulus cells have a significant requirement for and utilise high levels of glucose, yet appear to have little need for oxidative metabolism. In contrast, oocytes have a requirement for oxidative metabolism, although limited glucose metabolism may also be an important aspect of meiotic and developmental competence. Nevertheless, because of the metabolic and communication link between the cumulus and the oocyte, glucose availability and metabolism within the cumulus can have a significant impact on oocyte meiotic and developmental competence. In particular, the role of the hexosamine biosynthesis pathway within cumulus cells appears critical for the supply of substrate from glucose for extracellular matrix production, yet if overstimulated can significantly decrease developmental competence of the oocyte. Current static systems for in vitro maturation are clearly incompatible with meeting substrate demands, especially glucose. In the future, in vitro maturation will include a more dynamic approach, which will adjust nutrient components to meet the changing functional requirements of cumulus-oocyte complexes during the final process of maturation.

Automation of oocyte maturation and embryo production techniques is a new and exciting development in the field of reproductive technologies. There are two areas where increased automation is having an impact: in the area of embryo diagnostics and in the process of embryo production itself. Benefits include decreased staffing and skill requirements for production and assessment of embryos, as well as increasing quality management systems by removing the "human" factor. However, the uptake of new technologies is likely to be slow, as costs and the conservative nature of the Assisted Reproduction Technology industry to adopt new techniques.

STUDY QUESTION Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? SUMMARY ANSWER Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. WHAT IS KNOWN ALREADY Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. STUDY DESIGN, SIZE, DURATION This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. PARTICIPANTS/MATERIALS, SETTING, METHODS Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. MAIN RESULTS AND THE ROLE OF CHANCE Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P < 0.05), regardless of pre-IVM duration. The final blastocyst yields (proportion of blastocysts/immature oocyte) were 26.3% for the control, compared with 39.2, 35.2 and 34.2%, for the 2, 4 and 6 h pre-IVM FSK + IBMX treatments, respectively. In contrast to standard IVM (control), pre-IVM with cAMP modulators maintained open gap junctions between cumulus cells and oocytes for the duration (6 h) of pre-IVM examined, and persisted for a further 8 h in the IVM phase. Cyclic AMP-modulated pre-IVM increased intra-oocyte GSH levels at the completion of both pre-IVM and IVM, in a pre-IVM duration-dependent manner (P < 0.05), which was ablated when GJC was blocked using CBX (P < 0.05). By 4 h of pre-IVM treatment with cAMP modulators, oocyte H2O2 levels were reduced compared the control (P < 0.05), although this beneficial effect was lost when oocytes were co-treated with BSO. Inhibiting glutathione synthesis with BSO during pre-IVM ablated any positive benefits of cAMP-mediated pre-IVM on oocyte developmental competence (P < 0.01). LIMITATIONS, REASONS FOR CAUTION It is unclear if the improvement in oocyte antioxidant defence and developmental competence reported here is due to direct transfer of total and/or reduced glutathione from cumulus cells to the oocyte via gap junctions, or whether a GSH synthesis signal and/or amino acid substrates are supplied to the oocyte via gap junctions. Embryo transfer experiments are required to determine if the cAMP-mediated improvement in blastocyst rates leads to improved live birth rates. WIDER IMPLICATIONS OF THE FINDINGS IVM offers significant benefits to infertile and cancer patients and has the potential to significantly alter ART practice, if IVM efficiency in embryo production could be improved closer to that of conventional IVF (using ovarian hyperstimulation). Pre-IVM with cAMP modulators is a simple and reliable means to improve IVM outcomes. STUDY FUNDING/COMPETING INTERESTS This work was supported by grants and fellowships from the National Health and Medical Research Council of Australia (1007551, 627007, 1008137, 1023210) and by scholarships from the Chinese Scholarship Council (CSC) awarded to H.J.L. and the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad awarded to S.S. The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. We acknowledge partial support from the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

The preimplantation bovine embryo displays sexual dimorphism in glucose sensitivity and interferon-tau (IFNT) secretion that are negated by inhibition of the pentose phosphate pathway, suggesting that the association between glucose metabolism and IFNT likely underpins the selective loss of female embryos. The aim of this study was to determine if altered glucose metabolism, through glucose supplementation and/or uncoupling oxidative phosphorylation with 2,4-dinitrophenol (DNP), affected embryo development. Bovine blastocyst development, sex, and IFNT production were examined in embryos cultured in the presence or absence of glucose (0, 1.5, 4 mM) with or without exposure to DNP (0, 10, 100 μM) between Days 5 and 8 post-fertilization. The absence or presence of high (4 mM) glucose reduced blastocyst development and favored the development of male embryos (P < 0.001). DNP at 10 μM had no effect, whereas 100 μM had a negative impact on blastocyst development. Notably, in the presence or even absence of glucose, supplementation with 10 μM DNP further skewed the sex ratio toward males (P < 0.05). Sexually dimorphic IFNT production was maintained in these conditions, although total production was reduced in the presence of high glucose and DNP, irrespective of embryo sex. These data suggest that the pentose phosphate pathway can modulate embryonic sex ratio and development. Therefore, bovine embryo culture should be undertaken in a low glucose (

Oocytes within antral follicles are thought to have restricted access to O2, as follicle vascularity is not adjacent and both granulosa and cumulus cells are metabolically active. Indeed, measured follicular antrum partial pressure (pO2) is regarded as low, but accurate and direct measurement represents a technical challenge that has yet to be overcome. The oocyte itself is highly dependent on oxidative phosphorylation for survival and competence for further development following fertilization, and it has been suggested that follicular pO2 levels are correlated with this capacity for further development. It is clear that gonadotropins are involved in regulating antrum formation, follicle vascularization, cellular differentiation, and the hypoxia-inducible factors (HIF), which are mainly regulated by dissolved O2 concentration. A newly discovered player in this story is the intracellular production of hemoglobin by both granulosa and cumulus cells, as well as the oocyte. Furthermore, cellular hemoglobin levels are dynamic, responding to the ovulatory luteinizing hormone (LH) surge. We hypothesize that this gas transport and antioxidant molecule is involved in the prevention of hypoxic response signaling by HIFs within the preovulatory antral follicle; and the transition of granulosa cells to luteal tissue by facilitating the stabilization of HIFs, enabling induction of luteinization signaling. Another possible role is by sequestering nitric oxide (NO) during the ovulatory period, which may facilitate the resumption of meiosis in the oocyte. Testing these hypotheses will be challenging but important if the regulation of ovarian function is to be fully understood.

Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM), and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

In the physiological situation, cytokines are pivotal mediators of communication between the maternal tract and the embryo. Compelling evidence shows that cytokines emanating from the oviduct and uterus confer a sophisticated mechanism for 'fine-tuning' of embryo development, influencing a range of cellular events from cell survival and metabolism, through division and differentiation, and potentially exerting long-term impact through epigenetic remodelling. The balance between survival agents, including GM-CSF, CSF1, LIF, HB-EGF and IGFII, against apoptosis-inducing factors such as TNFα, TRAIL and IFNg, influence the course of preimplantation development, causing embryos to develop normally, adapt to varying maternal environments, or in some cases to arrest and undergo demise. Maternal cytokine-mediated pathways help mediate the biological effects of embryo programming, embryo plasticity and adaptation, and maternal tract quality control. Thus maternal cytokines exert influence not only on fertility and pregnancy progression but on the developmental trajectory and health of offspring. Defining a clear understanding of the biology of cytokine networks influencing the embryo is essential to support optimal outcomes in natural and assisted conception.

The production of reactive oxygen species (ROS) is known to affect the developmental competence of embryos. Hydrogen peroxide (H 2 O 2 ) an important reactive oxygen species, is also known to causes DNA damage and defective sperm function. Current techniques require incubating a developing embryo with an organic fluorophore which is potentially hazardous for the embryo. What we need is a localised ROS sensor which does not require fluorophores in solution and hence will allow continuous monitoring of H 2 O 2 production without adversely affect the development of the embryo. Here we report studies on such a fibre-based sensor for the detection of H 2 O 2 that uses a surface-bound aryl boronate fluorophore carboxyperoxyfluor-1(CPF1). Optical fibres present a unique platform due to desirable characteristics as dip sensors in biological solutions. Attempts to functionalise the fibre tips using polyelectrolyte layers and (3-aminopropyl)triethoxysilane (APTES) coatings resulted in a limited signal and poor fluorescent response to H 2 O 2 due to a low tip surface density of the fluorophore. To increase the surface density, CPF1 was integrated into a polymer matrix formed on the fibre tip by a UV-catalysed polymerisation process of acrylamide onto a methacrylate silane layer. The polyacrylamide containing CPF1 gave a much higher surface density than previous surface attachment methods and the sensor was found to effectively detect H 2 O 2 . Using this method, biologically relevant concentrations of H 2 O 2 were detected, enabling remote sensing studies into ROS releases from embryos throughout early development.

Oocytes within antral follicles are thought to have restricted access to O2, as follicle vascularity is not adjacent and both granulosa and cumulus cells are metabolically active. Indeed, measured follicular antrum partial pressure (pO2) is regarded as low, but accurate and direct measurement represents a technical challenge that has yet to be overcome. The oocyte itself is highly dependent on oxidative phosphorylation for survival and competence for further development following fertilization, and it has been suggested that follicular pO2 levels are correlated with this capacity for further development. It is clear that gonadotropins are involved in regulating antrum formation, follicle vascularization, cellular differentiation, and the hypoxia-inducible factors (HIF), which are mainly regulated by dissolved O2 concentration. A newly discovered player in this story is the intracellular production of hemoglobin by both granulosa and cumulus cells, as well as the oocyte. Furthermore, cellular hemoglobin levels are dynamic, responding to the ovulatory luteinizing hormone (LH) surge. We hypothesize that this gas transport and antioxidant molecule is involved in the prevention of hypoxic response signaling by HIFs within the preovulatory antral follicle; and the transition of granulosa cells to luteal tissue by facilitating the stabilization of HIFs, enabling induction of luteinization signaling. Another possible role is by sequestering nitric oxide (NO) during the ovulatory period, which may facilitate the resumption of meiosis in the oocyte. Testing these hypotheses will be challenging but important if the regulation of ovarian function is to be fully understood.

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.

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.

The oxygen concentration used during embryo culture can influence embryo development and quality. Reducing the oxygen concentration in the atmosphere to 2% during post-compaction culture of mouse embryos perturbs embryonic gene expression. This study examined the effect of culturing mouse embryos under different oxygen concentrations on subsequent fetal and placental development. Embryos were cultured from the zygote to morula stage under 7% oxygen, followed by 20, 7 or 2% oxygen to the blastocyst stage. Cultured and in vivo developed blastocysts were transferred into pseudopregnant recipients. Fetal and placental outcomes were analysed at day 18 of pregnancy. Implantation rate was not influenced by embryo culture conditions, but resorption rates were increased in embryos cultured under 2% oxygen, compared with 7% oxygen. Day 18 fetal weights were reduced following culture under 2%, compared with 7 or 20% oxygen, or in vivo development. Placental weight was not influenced by culture conditions. No differences in the proportion of junctional or labyrinthine exchange regions within the placenta or the morphometry of the labyrinthine region were detected. Surface density (surface area/gram labyrinth) of trophoblast available for exchange was reduced in placentas developed from embryos cultured under 2% oxygen, compared with 7% oxygen. Placental gene expression of Slc2a1, Slc2a3, Igf2, Igf2r and H19 was not influenced by oxygen conditions during embryo culture. Thus, exposure to 2% oxygen during post-compaction pre-implantation embryo development has adverse consequences for fetal development in the mouse. Oxygen is a significant component of the embryonic environment and reductions in oxygen availability can influence both embryonic gene expression and subsequent fetal development.

The presence of ammonium in culture medium has a detrimental effect on embryo physiology and biochemistry; however, the stage at which the embryo is most sensitive to this effect is unknown. The aim of this study was to determine the exact stage at which the embryo is most vulnerable to ammonium by exposing the preimplantation embryo to 300 muM ammonium either at the precompaction stage (between the zygote and two-cell or the two-cell to eight-cell) or at the postcompaction stage (between the eight-cell and blastocyst). This study determined that exposure of embryos to ammonium at the precompaction stage from either the zygote to two-cell stage or from the two-cell to the eight-cell stage did not affect the rate of development to the blastocyst stage; however, the resultant blastocysts had decreased cell numbers and inner cell mass cells. Furthermore, these blastocysts had increased levels of cellular apoptosis and perturbed levels of Slc2a3 expression and glucose uptake. Transfer of these blastocysts revealed that, while implantation was not affected, the number of fetuses was reduced by culture with ammonium at the precompaction stage and fetal development was delayed, as observed by reduced crown-rump length and maturity. In contrast, the later stage embryo was more resistant to the negative effects of ammonium, with only Slc2a3 expression and fetal maturity affected. This raises the possibility that the later stage embryo is more able to protect itself from in vitro-derived stress and that the majority of in vitro-induced damage to mouse embryos is inflicted at the early stages of development.

Cumulus-oocyte complexes (COCs) and early embryos rely on a histotrophic nutrition source for energy production and the synthesis of macromolecules. There is accumulating evidence suggesting that the balance of supply and demand for energy and other anabolic substrates during oocyte maturation and very early stages of development programmes subsequent developmental potential, and this may include subsequent fetal growth trajectory. One example is the role of glucose (Glc) during cumulus-oocyte complex maturation. Glucose is an essential nutrient for maturation, especially its role during cumulus expansion. Our laboratory has shown that during in vitro culture, too little glucose during cumulus-oocyte complex maturation affects meiotic competence. We have focussed on glucose (Glc) metabolism through the hexosamine biosynthesis pathway (HBP) during COC maturation in vitro. The HBP in somatic cells is regarded as a "fuel-sensing" pathway and its interaction with cell signalling systems and transcriptional regulation is increasingly apparent. Up-regulation of the HBP during oocyte maturation in vitro has negative consequences for subsequent development. Another example is the role of hypoxia (low O2) during peri-compaction development. My laboratory believes that ruminant embryos during compaction, blastulation and subsequent development in the uterine cavity lack a key hypoxia responsive element. Because of this, hypoxia is important for normal development in ruminants but perturbs further development in rodents. The implication of these examples to the fundamental concept of peri-conception nutritional programming of development are discussed.

The developmental competence of cumulus oocyte complexes (COCs) can be increased during in vitro oocyte maturation with the addition of exogenous oocyte-secreted factors, such as bone morphogenetic protein 15 (BMP15), in combination with hormones. FSH and BMP15, for example, induce different metabolic profiles within COCs-namely, FSH increases glycolysis while BMP15 stimulates FAD and NAD(P)H accumulation within oocytes, without changing the redox ratio. The aim of this study was to investigate if this BMP15-induced NAD(P)H increase was due to de novo NADPH production. Cattle COCs were cultured with FSH and/or recombinant human BMP15, resulting in a significant decrease in glucose-6-phosphate dehydrogenase activity (P 0.05). Inhibition of isocitrate dehydrogenase (IDH) during this process decreased NAD(P)H intensity threefold in BMP15-treated oocytes, suggesting that BMP15 stimulates IDH and NADPH production via the tricarboxylic acid cycle. As NADPH is a reducing agent, reduced glutathione (GSH), H2O2, and mitochondrial activity were also measured to assess the general redox status of the oocyte. FSH alone decreased GSH levels whereas the combination of BMP15 and FSH sustained higher levels. Expression of genes encoding glutathione-reducing enzymes were also lower in oocytes cultured in the presence of FSH alone. BMP15 supplementation further promoted mitochondrial localization patterns that are consistent with enhanced developmental competence. Metabolomics revealed significant consumption of glutamine and production of alanine by COCs matured with both FSH and BMP15 compared to the control (P 0.05). Hence, BMP15 supplementation differentially modulates reductive metabolism and mitochondrial localization within the oocyte. In comparison, FSH-stimulation alone decreases the oocytes' ability to regulate cellular stress, and therefore utilizes other mechanisms to improve developmental competence.

We have developed a protocol using recombinant human follicle-stimulating hormone (rhFSH) to induce ovarian stimulation in the mouse to investigate its impact on preimplantation embryo development. Embryos were collected from adult female C57Bl/6 × CBA F1 mice treated with rhFSH (0, 2.5, 5.0, 10.0, or 20.0 IU) or 5 IU equine chorionic gonadotropin (eCG). Embryos were also recovered from nontreated control mice. Embryos were cultured in vitro for 88 h, and the stage of development was morphologically assessed. The allocation of cells to the inner cell mass or trophectoderm of blastocysts was determined by differential nuclear staining. The expression of insulin-like growth factor 2 (IGF-II), the insulin-like growth factor receptor (IGF-II receptor), and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time RT-PCR. Blastocyst development was reduced in the 10 (72.3 ± 5.1%) and 20 (77.3 ± 5.6%) IU rhFSH groups compared with control embryos (96.7 ± 1.0%). The number of inner cell mass cells was reduced ( P < 0.001) in the 5, 10, and 20 IU rhFSH groups and the eCG group compared with control embryos. We did not find any effect of rhFSH treatment on IGF-II, IGF-II receptor, or VEGF expression in blastocysts compared with the control group. eCG treatment, however, significantly increased the expression of IGF-II in blastocysts. These results indicate that ovarian stimulation with rhFSH impairs the in vitro development of preimplantation mouse embryos, and these results may have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality.

Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFalpha protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1alpha, HIF2alpha) was unaltered by oxygen concentration. HIF2alpha protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1alpha protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2alpha.

Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 mM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P , 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P , 0.05) increased embryo development to the blastocyst stage by 33%‐39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P , 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential. cumulus cells, cyclic adenosine monophosphate, embryo, meiosis, phosphodiestrases

Glucose is an important metabolite and its presence duringin vitrooocyte maturation (IVM) can have profound effects on the oocyte’s developmental capacity. We have demonstrated that glucose uptake increases over a 24 h IVM period, with most accounted for asl-lactate production. However, as maturation proceeds,l-lactate production remains constant, suggesting an alternative role for glucose metabolism. We hypothesised that in the latter stages of oocyte maturation, glucose not accounted for byl-lactate production is utilised for FSH-stimulated extracellular matrix (ECM) synthesis. To examine precursor utilisation for synthesis of ECM, bovine cumulus–oocyte complexes (COCs) were matured in ± FSH and/or glucosamine (an alternative substrate of matrix components). Measurements included COC diameters, glucose consumption andl-lactate production in spent media and [U-14C]glucose incorporation into ECM. FSH significantly stimulated both diameter and glucose consumption during 20–24 h maturation compared with unstimulated complexes, although co-incubation with glucosamine and FSH decreased total glucose consumption 1.7-fold compared with FSH alone (P< 0.05). Furthermore, there was a linear relationship between glucose andl-lactate metabolism in the presence of glucosamine, suggesting that the majority of glucose was being utilised forl-lactate production via glycolysis. In the presence of glucosamine, twofold less [U-14C]glucose was incorporated into matrix compared with COCs cultured without glucosamine. These results support the hypothesis that there is a link between glucose and glucosamine uptake in FSH-stimulated ECM synthesis. Furthermore, glucose has multiple fates within the COC during maturation and levels of utilisation are dependent on the composition of the maturation environment.

The oxygen concentration used in the incubation atmosphere during embryo culture influences embryo development rates and embryo quality. In somatic cells, oxygen levels can influence the expression of a range of genes, including glucose transporters, glycolytic enzymes, and angiogenic growth factors. Many of these oxygen-regulated genes have important roles in embryonic development and metabolism. The aim of this study was to determine whether oxygen regulates gene expression in the preimplantation mouse blastocyst. Mouse embryos were cultured from the 1-cell to morula stage under 7% oxygen, followed by culture under 20, 7, or 2% oxygen to the blastocyst stage. Expression of glucose transporter (GLUT)-1, GLUT-3, and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time reverse transcription PCR. Development from morula to blastocyst was not altered by culture under different oxygen conditions. Expression of GLUT-1, GLUT-3, and vascular endothelial growth (VEGF) was increased by 2- to 4-fold in embryos cultured under 2% oxygen, when compared to embryos cultured under 20 or 7% oxygen, and when compared to embryos developed in vivo (all P < 0.001). These results suggest that the preimplantation mouse embryo has the capacity to detect and respond to low oxygen availability with changes in expression of oxygen-regulated genes.

Intracellular communication between the cumulus cell complex and the oocyte is essential for numerous processes during oocyte maturation. The aim of this study was to determine the interaction between oocyte-secreted factors and the metabolic activity of bovine cumulus cell complexes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were aspirated from ovaries derived from an abattoir and divided into four treatment groups: (i) intact COCs, (ii) oocytectomized complexes (OOX), in which the ooplasm was microsurgically removed, (iii) OOX co-cultured with denuded oocytes (OOX+DO) and (iv) DO. The complexes were cultured individually in IVM media. After 0-4, 10-14 and 20-24 h of culture, the utilization of oxygen, glucose, pyruvate and L-lactate by the complexes was measured. The metabolic activity of the DO was undetectable. There were no significant differences in metabolic measurement among any of the treatment groups, indicating that the metabolism of the cumulus complex is not affected by the presence of the oocyte. When metabolic activity for the complexes was analysed relative to time in culture, there was an approximate twofold increase in the consumption of oxygen, glucose and pyruvate over the 24 h period (P