Embryo development after vitrification of immature and in vitro-matured equine oocytes.

Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO2 or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO2- than Air-equilibrated medium (86 ± 3 vs. 76.9 ± 4%, respectively). Maturation for all vitrified-warmed oocyte treatments (37 ± 6.5 to 45.9 ± 5.8%) was not different from control (50 ± 4.1%), except for MAT CR- CO2 (20.3 ± 4.6%). Cleavage for MAT CR- CO2 and Air groups was similar to control (67.7 ± 12.1, 71.4 ± 8.1, and 78 ± 5.3%, respectively). One blastocyst was produced (MAT CR + CO2), representing the first equine blastocyst reported after vitrification of an in vitro-matured oocyte.

Redox and anti-oxidant state within cattle oocytes following in vitro maturation with bone morphogenetic protein 15 and follicle stimulating hormone.

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.

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.

Bovine oocyte activation with bull or human sperm by conventional ICSI and Piezo-ICSI: Its relationship with PLCɀ activity.

Background: The intracytoplasmic sperm injection (ICSI) technique has low efficiency in cattle. This has mainly been attributed to the oocyte activation failure due to oocyte and/or sperm factors. Aim: Our aim was to evaluate the effect of conventional ICSI and Piezo-ICSI with bull or human sperm on bovine oocyte activation and embryo development and to assess its relationship with the phospholipase C zeta (PLCɀ) activity of both species. Methods: In vitro matured bovine oocytes were randomly divided into five groups and were fertilized as follows: conventional ICSI using bovine sperm with chemical activation (control), conventional ICSI using bovine sperm, Piezo-ICSI using bovine sperm, conventional ICSI using human sperm, and Piezo-ICSI using human sperm. PLCɀ activity was determined in bull and human sperm samples. Results: Within the groups using bull sperm, the oocytes fertilized by conventional ICSI had the lowest values of 2 pronuclei (PN) formation and cleavage, Piezo-ICSI increased both percentages and ICSI + chemical activation presented the highest 2 PN, cleavage, and blastocyst rates (p < 0.05). Within the groups using human sperm, the oocytes fertilized by Piezo-ICSI presented higher 2 PN and cleavage rates than those activated by conventional ICSI (p < 0.05). Piezo-ICSI with human sperm increased bovine oocyte activation as much as conventional ICSI + chemical activation with bovine sperm (p < 0.05). Higher values of PLCɀ activity were found in human sperm compared with bovine sperm (p < 0.05). Conclusion: Our results suggest that the higher stability of the bovine sperm in combination with its relatively low content of PLCɀ impairs bovine oocyte activation after ICSI.

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.

Variations in metabolic parameters of in vitro matured porcine oocytes after vitrification-warming.

Background: As the porcine oocyte is the most sensitive to low-temperature damage, it has been difficult to cryopreserve compared to those from other domestic animals. However, at present, vitrification is used as a method for the cryopreservation of both oocytes and embryos in this species. Aim: Our aim was to analyze alterations in metabolic parameters in vitrified-warmed in vitro matured porcine oocytes at different post-warming recuperation times. In addition, metaphase II plate recovery time analysis, in vitro fertilization, and intracytoplasmic sperm injection were carried out to evaluate oocyte recovery capacity. Methods: Oocytes were vitrified-warmed and then incubated for 0, 3, or 21 hours post-warming to assess biochemical parameters. Results: Oocyte viability and morphology were not affected by vitrification-warming. Cytosolic oxidative status, active mitochondria, and reactive oxygen species levels presented changes at the different time points in control and vitrified-warmed oocytes (p < 0.05) as well as differences between both groups (p < 0.05). Nicotinamide adenine dinucleotide phosphate levels remained constant throughout different recuperation times but were significantly lower in vitrified-warmed oocytes (p < 0.05). Metaphase II plate recovery occurred mostly between 3 and 4 hours post-warming, but the percentage of metaphase II was reduced by vitrification-warming. Sperm head decondensation and pronuclear formation capacities were not modified. Conclusion: In conclusion, vitrification-warming generates biochemical alterations in porcine oocytes that would be, in part, responsible for affecting their performance. Therefore, although the technique is a valid alternative for porcine oocyte cryopreservation, the protocols should be adapted to minimize those alterations.

Reactive oxygen species in bovine oocyte maturation in vitro.

The role of reactive oxygen species (ROS) in the in vitro maturation (IVM) of oocytes remains controversial. The aim of the present study was to determine possible fluctuations in ROS production during bovine oocyte IVM in the presence of different modulators of ROS generation. Cumulus-oocyte complexes were cultured in medium 199 (control) in the absence or presence of 0.6 mM cysteine, 1 mM 1-choro-2,4-dinitro benzene (CDNB), 2 microM diphenyliodonium, 0.5 mM N-nitro-L-arginine methyl ester or 10 microM sodium nitroprusside (SNP) at 39 degrees C, in 5% CO2 in humidified air for 22 h. In addition, the respiratory chain effectors potassium cyanide (KCN; 1 mM) and carbonyl cyanide m-chlorophenylhydrazone (0.42 microM) were used. Meiotic maturation was determined by the presence of MII. ROS production was evaluated in denuded oocytes at different time points as the ratio of 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA) to fluorescein diacetate (FDA). ROS levels, expressed as DCHF-DA:FDA, fluctuated throughout the 22 h of maturation depending on the treatment applied. At 12 h incubation in the presence of KCN and SNP, ROS levels were increased, whereas ROS levels after 12 h in the presence of cysteine were reduced (P<0.05). Both CDNB and SNP impaired meiotic progression. The higher metabolic activity demand during bovine oocyte maturation coincides with a concomitant reduction in ROS generation. These results suggest that 12 h would be a critical point for bovine oocyte IVM because it is closely related to the production of ROS at this time.

Immature oocyte quality and maturational competence of porcine cumulus-oocyte complexes subpopulations.

Porcine immature oocyte quality (i.e., that of live oocytes at the germinal vesicle stage) was evaluated according to features of the surrounding cumulus, aiming to establish maturational competence of different subpopulations of such cumulus-oocyte complexes. Six subpopulations were identified: A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (partly naked oocytes), C (naked oocytes), D (with a dark cumulus). The percent incidence of live oocyte in these subpopulations changed significantly as related to cumulus features, however the occurrence of oocytes in the germinal vesicle stage was lower in class D only. Similar metaphase II rates achieved in A1, A2, B1 and B2 classes after in vitro maturation suggest that the nucleus may in fact mature in vitro, in spite of the different accompanying cumulus features which are typical of these classes. In contrast, a higher cytoplasmic maturation rate obtained in class A may indicate a stronger dependence of this variable upon cumulus features than that shown by nuclear maturation. When different types of cumulus expansion after in vitro maturation were considered (i.e., fully expanded cumulus, partly expanded cumulus, and partly naked oocyte), no differences were found in the percent of oocytes reaching metaphase II or cytoplasmic maturation. It is concluded that morphological features of the collected porcine cumulus-oocyte complexes (rather than cumulus behavior during culture) may be useful for selection of potentially competent oocytes for in vitro fertilization and embryo production.

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.

Implications of glycolytic and pentose phosphate pathways on the oxidative status and active mitochondria of the porcine oocyte during IVM.

Glycolysis and the pentose phosphate pathway (PPP) were modulated in porcine cumulus-oocyte complexes during IVM by the addition of inhibitors and stimulators of key enzymes of the pathways to analyze their influence on the oxidative status, active mitochondria, and maturation of the oocyte. The influence of pharmacologic and physiological inhibitors of glycolysis (Sodium fluoride and ATP) and PPP (6-Aminonicotinamide and nicotinamide adenine dinucleotide phosphate) was validated by assessing glucose and lactate turnover and brilliant cresyl blue staining in oocytes. Inhibitors of glycolysis and PPP activity significantly perturbed nuclear maturation, oxidative metabolism (Redox Sensor Red CC-1), and active mitochondria (Mitotracker Green FM) within oocytes (P < 0.05). In comparison, physiological stimulators of glycolysis (adenosine monophosphate) and PPP (nicotinamide adenine dinucleotide phosphate) did not affect any of evaluated parameter. In the absence of modulators, fluctuations in the oocyte oxidative activity and active mitochondria were observed during porcine IVM. The inhibition of glycolysis and PPP modified the pattern of oxidation and mitochondrial fluctuation, resulting in impaired meiotic progression. We demonstrated the relationship between carbohydrate metabolism in COC and oocyte redox status necessary for porcine oocyte IVM.

Epithelial cadherin is present in bovine oviduct epithelial cells and gametes, and is involved in fertilization-related events.

Fertilization is a calcium-dependent process that involves sequential cell-cell adhesion events of spermatozoa with oviduct epithelial cells (OECs) and with cumulus-oocyte complexes (COCs). Epithelial cadherin (E-cadherin) participates in calcium-dependent somatic cell adhesion; the adaptor protein ß-catenin binds to the E-cadherin cytoplasmic domain and links the adhesion protein to the cytoskeleton. The study was conducted to immunodetect E-cadherin and ß-catenin in bovine gametes and oviduct (tissue sections and OEC monolayers), and to assess E-cadherin participation in fertilization-related events. Epithelial cadherin was found in spermatozoa, oocytes, cumulus cells, and OEC. In acrosome-intact noncapacitated spermatozoa, E-cadherin was mainly localized in the apical ridge and acrosomal cap (E1-pattern; 84 ± 9%; mean ± standard deviation of the mean). After sperm treatment with heparin to promote capacitation, the percentage of cells with E1-pattern (56 ± 12%) significantly decreased; concomitantly, the percentage of spermatozoa depicting an E-cadherin staining pattern similar to E1-pattern but showing a signal loss in the acrosomal cap (E2-pattern: 40 ± 11%) increased. After l-α-lysophosphatidylcholine-induced acrosome reaction, E-cadherin signal was mainly localized in the inner acrosomal membrane (E3-pattern: 67 ± 22%). In IVM COC, E-cadherin was immunodetected in the plasma membrane of cumulus cells and oocytes, but was absent in the polar body. The 120 KDa mature protein form was found in protein extracts from spermatozoa, oocytes, cumulus cells, and OEC. ß-Catenin distribution followed E-cadherin's in all cells evaluated. Epithelial cadherin participation in cell-cell interaction was evaluated using specific blocking monoclonal antibody DECMA-1. Sperm incubation with DECMA-1 impaired sperm-OEC binding (the number of sperm bound to OEC: DECMA-1 = 6.7 ± 6.1 vs. control = 29.6 ± 20.1; P < 0.001), fertilization with COC (% fertilized COC: DECMA-1 = 68.8 ± 10.4 vs. control = 90.7 ± 3.1; P < 0.05) or denuded oocytes (% fertilized oocytes: DECMA-1 = 57.0 ± 15.2 vs. control = 89.2 ± 9.8; P < 0.05) and binding to the oolemma (the number of sperm bound to oolemma: DECMA-1 = 2.2 ± 1.1 vs. control = 11.1 ± 4.8; P < 0.05). This study describes, for the first time, the presence of E-cadherin in bovine spermatozoa, COC, and OEC, and shows evidence of its participation in sperm interaction with the oviduct and the oocyte during fertilization.

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: possible role of the hexosamine pathway as a regulator of developmental competence.

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.

Immature oocyte quality and maturational competence of porcine cumulus-oocyte complexes subpopulations

Porcine immature oocyte quality (i.e., that of live oocytes at the germinal vesicle stage) was evaluated according to features of the surrounding cumulus, aiming to establish maturational competence of different subpopulations of such cumulus-oocyte complexes. Six subpopulations were identified: A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (partly naked oocytes), C (naked oocytes), D (with a dark cumulus). The percent incidence of live oocyte in these subpopulations changed significantly as related to cumulus features, however the occurrence of oocytes in the germinal vesicle stage was lower in class D only. Similar metaphase II rates achieved in A1, A2, B1 and B2 classes after in vitro maturation suggest that the nucleus may in fact mature in vitro, in spite of the different accompanying cumulus features which are typical of these classes. In contrast, a higher cytoplasmic maturation rate obtained in class A may indicate a stronger dependence of this variable upon cumulus features than that shown by nuclear maturation. When different types of cumulus expansion after in vitro maturation were considered (i.e., fully expanded cumulus, partly expanded cumulus, and partly naked oocyte), no differences were found in the percent of oocytes reaching metaphase II or cytoplasmic maturation. It is concluded that morphological features of the collected porcine cumulus-oocyte complexes (rather than cumulus behavior during culture) may be useful for selection of potentially competent oocytes for in vitro fertilization and embryo production.

Immature oocyte quality and maturational competence of porcine cumulus-oocyte complexes subpopulations

Porcine immature oocyte quality (i.e., that of live oocytes at the germinal vesicle stage) was evaluated according to features of the surrounding cumulus, aiming to establish maturational competence of different subpopulations of such cumulus-oocyte complexes. Six subpopulations were identified: A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (partly naked oocytes), C (naked oocytes), D (with a dark cumulus). The percent incidence of live oocyte in these subpopulations changed significantly as related to cumulus features, however the occurrence of oocytes in the germinal vesicle stage was lower in class D only. Similar metaphase II rates achieved in A1, A2, B1 and B2 classes after in vitro maturation suggest that the nucleus may in fact mature in vitro, in spite of the different accompanying cumulus features which are typical of these classes. In contrast, a higher cytoplasmic maturation rate obtained in class A may indicate a stronger dependence of this variable upon cumulus features than that shown by nuclear maturation. When different types of cumulus expansion after in vitro maturation were considered (i.e., fully expanded cumulus, partly expanded cumulus, and partly naked oocyte), no differences were found in the percent of oocytes reaching metaphase II or cytoplasmic maturation. It is concluded that morphological features of the collected porcine cumulus-oocyte complexes (rather than cumulus behavior during culture) may be useful for selection of potentially competent oocytes for in vitro fertilization and embryo production.(AU)