Optical imaging of cleavage stage bovine embryos using hyperspectral and confocal approaches reveals metabolic differences between on-time and fast-developing embryos.

The assessment of embryo quality aims to enhance subsequent pregnancy and live birth outcomes. Metabolic analysis of embryos has immense potential in this regard. As a step towards this goal, here we assess the metabolism of bovine embryos using label-free optical imaging. We compared embryos defined as either on-time or fast-developing, as fast dividing embryos are more likely to develop to the blastocyst stage. Specifically, bovine embryos at 48 (Day 2) and 96 (Day 4) hours post fertilization were fixed and separated based on morphological assessment: on-time (Day 2: 2 cell; Day 4: 5-7 cell) or fast-developing (Day 2: 3-7 cell; Day 4: 8-16 cell). Embryos with different developmental rates on Day 2 and Day 4 were correlated with metabolic activity and DNA damage. Confocal microscopy was used to assess metabolic activity by quantification of cellular autofluorescence specific for the endogenous fluorophores NAD(P)H and FAD with a subsequent calculation of the optical redox ratio. Separately, hyperspectral microscopy was employed to assess a broader range of endogenous fluorophores. DNA damage was determined using γH2AX immunohistochemistry. Hyperspectral imaging showed significantly lower abundance of endogenous fluorophores in fast-developing compared to on-time embryos on Day 2, indicating a lower metabolic activity. On Day 4 of development there was no difference in the abundance of FAD between on-time and fast-developing embryos. There was, however, significantly higher levels of NAD(P)H in fast-developing embryos leading to a significantly lower optical redox ratio when compared to on-time embryos. Collectively, these results demonstrate that fast-developing embryos present a 'quiet' metabolic pattern on Day 2 and Day 4 of development, compared to on-time embryos. There was no difference in the level of DNA damage between on-time and fast-developing embryos on either day of development. To our knowledge, this is the first collective use of confocal and hyperspectral imaging in cleavage-stage bovine embryos in the absence of fluorescent tags.

A rapid, sensitive and inexpensive method for detection of grapevine red blotch virus without tissue extraction using loop-mediated isothermal amplification.

Grapevine red blotch virus (GRBV) is an emerging virus of significant viticultural importance throughout North America. Here, we report the development of a simple protocol for point-of-use detection of GRBV. Extraction of nucleic acids is not required; instead, the whole intact plant can simply be pricked with a sterile pipette tip, which is then incubated in sterile distilled water to provide the sample template in a loop-mediated isothermal amplification (LAMP) reaction. This method is 10,000 times more sensitive than conventional PCR, costs under a dollar per sample, and can be completed from sampling to readout in just over half an hour.

Fibroblast growth factor 17 and bone morphogenetic protein 15 enhance cumulus expansion and improve quality of in vitro-produced embryos in cattle.

Bone morphogenetic protein 15 (BMP15) and members of the fibroblast growth factor (FGF) family are expressed by the oocyte and are involved in the control of cumulus cell function. We tested the hypothesis that FGF17, alone or combined with BMP15 in the maturation medium, enhances cumulus expansion, meiosis progression, embryonic development, and expression of mRNA encoding key genes regulating expansion (prostaglandin-endoperoxide synthase 2 [PTGS2], hyaluronan synthase 2 [HAS2], tumor necrosis factor-stimulated gene 6 [TNFAIP6], and pentraxin 3 [PTX3]) and markers of oocyte developmental competence (phosphofructokinase [PFKP], gremlin [GREM1], versican [VCAN], and the genomic progesterone receptor [nPR]) in cumulus cells. Fibroblast growth factor 17 and BMP15 increased the percentage of fully expanded cumulus-oocyte complexes (COCs), but there was no additive effect when both were combined. Neither FGF17 nor BMP15 altered the percentage of oocytes reaching meiosis II at the end of COC culture or cleavage and blastocyst rates after IVF. However, embryo quality, as assessed by the number of cells in the inner cell mass, was improved by the combination of FGF17 with BMP15. Fibroblast growth factor 17 alone did not alter gene expression in cumulus cells at the end of IVM, whereas BMP15 increased PTGS2 and PTX3 mRNA levels. The combination of FGF17 and BMP15 increased nPR mRNA abundance in cumulus cells but did not change the expression of other markers of developmental competence. This study provides novel evidence that FGF17 enhances cumulus expansion in bovine COCs submitted to IVM and that the supplementation of the IVM medium with FGF17 and BMP15 may improve embryo quality.

Pentose phosphate pathway activity: effect on in vitro maturation and oxidative status of bovine oocytes.

The relationship between pentose phosphate pathway (PPP) activity in cumulus-oocyte complexes (COCs) and oxidative and mitochondrial activity in bovine oocytes was evaluated with the aim of analysing the impact of two inhibitors (NADPH and 6-aminonicotinamide (6-AN)) and a stimulator (NADP) of the key enzymes of the PPP on the maturation rate, oxidative and mitochondrial activity and the mitochondrial distribution in oocytes. The proportion of COCs with measurable PPP activity (assessed using brilliant cresyl blue staining), glucose uptake, lactate production and meiotic maturation rate diminished when 6-AN (0.1, 1, 5 and 10mM for 22h) was added to the maturation medium (P<0.05). The addition of NADPH did not modify glucose uptake or lactate production, but reduced PPP activity in COCs and meiotic maturation rates (P<0.05). The presence of NADP (0.0125, 0.125, 1.25 and 12.5mM for 22h of culture) in the maturation medium had no effect on PPP activity in COCs, glucose uptake, lactate production and meiotic maturation rate. However, in the absence of gonadotropin supplementation, NADP stimulated both glucose uptake and lactate production at 12.5mM (the highest concentration tested; P<0.05). NADP did not modify cleavage rate, but decreased blastocyst production (P<0.05). During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22h maturation, which was also related to progressive mitochondrial migration. Inhibiting the PPP with 6-AN or NADPH led to reduced oxidative and mitochondrial activity compared with the respective control groups and inhibition of mitochondrial migration (P<0.05). Stimulation of the PPP with NADP increased oxidative and mitochondrial activity at 9h maturation (P<0.05) and delayed mitochondrial migration. The present study shows the significance of altering PPP activity during bovine oocyte IVM, revealing that there is a link between the activity of the PPP and the oxidative status of the oocyte.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes.

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.

Glycolytic pathway activity: effect on IVM and oxidative metabolism of bovine oocytes.

The aim of the present study was to determine the effect of altering glycolytic pathway activity during bovine IVM on the meiotic maturation rate, oxidative activity, mitochondrial activity and the mitochondrial distribution within oocytes. Glycolytic activity was manipulated using two inhibitors (ATP, NaF) and a stimulator (AMP) of key enzymes of the pathway. Inhibition of glucose uptake, lactate production and meiotic maturation rates was observed when media were supplemented with ATP or NaF. The addition of AMP to the maturation medium had no effect on glucose uptake, lactate production or meiotic maturation. In the absence of gonadotrophin supplementation, AMP stimulated both glucose uptake and lactate production. However, AMP also decreased cytoplasmic maturation, as determined by early cleavage. During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22h maturation. Inhibiting glycolysis with ATP or NaF led to a reduced oxidative and mitochondrial pattern compared with the respective control groups. Stimulation of the pathway with AMP increased oxidative and mitochondrial activity. A progressive mitochondrial migration to the central area was observed during maturation; oocytes treated with ATP, NaF or AMP showed limited migration. The present study reveals the effects of altering glycolytic pathway activity in cumulus-oocyte complexes, revealing the link between glycolysis of the cumulus-oocyte complex and the oxidative and mitochondrial activity of the oocyte.

Influence of hyaluronic acid synthesis and cumulus mucification on bovine oocyte in vitro maturation, fertilisation and embryo development.

During cumulus-oocyte complex (COC) maturation, cumulus expansion involves the deposition of mucoelastic compounds, especially hyaluronic acid, synthesised from glucose via the hexosamine biosynthesis pathway. The aim of the present study was to determine the effects of uridine monophosphate (UMP) and 6-diazo-5-oxo-L-norleucine (DON), inhibitors of hyaluronic acid synthesis, during bovine oocyte in vitro maturation (IVM) on cumulus expansion, glucose uptake, protein synthesis, cumulus cell number, meiotic maturation, cleavage rate and subsequent embryo development. A further aim of the study was to examine the effect of hyaluronic acid on sperm capacitation and acrosome reaction in relation to the capacity of COCs to be fertilised in vitro. A low correlation between glucose uptake and degree of cumulus expansion was observed. Total and partial inhibition of cumulus expansion was observed with DON and UMP, respectively, and was accompanied by a decrease in glucose uptake with DON. Total protein content and cumulus cell number per COC increased during IVM, but was unaffected by the presence of DON or UMP, as was oocyte meiotic maturation. Rates of cleavage and blastocyst development decreased in oocytes matured with DON and UMP, although this inhibition was reversed when the in vitro fertilisation (IVF) medium contained heparin. Hyaluronic acid induced capacitation and the acrosome reaction, and in IVF medium prevented the inhibition of cleavage and blastocyst development by DON in a similar fashion to heparin. Hyaluronic acid synthesis during cumulus mucification contributes to the penetration and fertilisation of bovine oocytes, most likely by facilitating the processes of capacitation and acrosome reaction. Mucification during IVM is independent of cumulus cell proliferation, COC protein content, oocyte meiotic maturation and subsequent developmental competence once fertilised.

Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content.

Cysteamine and beta-mercaptoethanol supplementation of in vitro maturation (IVM) medium has been found to increase intracellular glutathione (GSH) content in oocytes and to improve embryo development and quality in several species. The objective of this experiment was to study the effect of cysteamine and beta-mercaptoethanol added during IVM of sheep oocytes on GSH synthesis and embryo development. Furthermore, we examined if cysteamine addition (hence GSH production) had an effect on the reduction of the intracellular peroxide content. We matured oocytes obtained from ovaries collected at a slaughterhouse in vitro in the presence of 0, 50, 100, and 200 microM cysteamine (Experiment 1) or with 0, 50, 100, and 200 microM beta-mercaptoethanol (Experiment 2). Following fertilization and embryo development, there was a increasing level of morula and blastocyst development in the presence of cysteamine, reaching significance in the presence of 200 microM (P < 0.05). However, beta-mercaptoethanol did not influence on the rate of embryo development. GSH levels were measured in oocytes matured in the presence or absence of 200 microM cysteamine (Experiment 3) or 50 microM beta-mercaptoethanol (Experiment 4), with or without buthionine sulfoximide (BSO), an inhibitor of GSH synthesis. Results demonstrated that for both cysteamine and beta-mercaptoethanol, intracellular GSH levels increased against control values (P < 0.01), which was abolished in the presence of BSO. Finally, we reduced intracellular peroxide levels, as measured by the relative fluorescence of the intracellular peroxide probe, carboxy-H2DCFDA, in the presence of either 200 microM cysteamine or 50 microM beta-mercaptoethanol (Experiment 5). These results demonstrate that cysteamine, but not beta-mercaptoethanol, when present during IVM, stimulates sheep embryo development; both cysteamine and beta-mercaptoethanol stimulate GSH synthesis; the increase in intracellular GSH is associated with a decrease in peroxide levels within oocytes.