Stage-dependent effects of inhibiting ribonucleic acids and protein synthesis on meiotic maturation of bovine oocytes in vitro

Ribonucleic acid synthesis continues at a low rate within 1 h of germinal vesicle breakdown. To determine if this newly synthesized mRNA is required for the resumption of meiosis in cattle oocytes, cumulus-enclosed oocytes were removed from 2 to 4-mm antral follicles directly into Dulbecco's medium with or without the RNA inhibitor, alpha-amanitin, or the protein synthesis inhibitor, cycloheximide (10 micrograms/ml). They were washed twice and matured for 28 or 48 h in medium 199, with Earle's salts, 2.2 g/L NaHCO3 and L-glutamine supplemented with 20% heated fetal calf serum to which were added gonadotropins (10 micrograms/ml NIH-LH-S18; 10 micrograms/ml NIH-FSH-S8; 20 ng/ml NIH-P-S9), estradiol-17 beta (1.5 micrograms/ml), solcoseryl (30 microliter/ml), and Dibekacin sulfate (100 micrograms/ml) with or without the inhibitors. After 28 h of maturation, 95.8% of control oocytes had undergone germinal vesicle breakdown and formation of a metaphase plate compared with only 1.3% of those continuously exposed to cycloheximide and 38% of those continuously exposed to amanitin. Exposure to cycloheximide or amanitin for only the first 16 h of culture followed by 12 h of inhibitor free culture resulted in 96.6% germinal vesicle breakdown with cycloheximide but only 56.5% germinal vesicle breakdown with amanatin. We conclude newly synthesized mRNA and protein synthesis is required for both full cumulus cell expansion and germinal vesicle breakdown.     

Disparate consequences of heat stress exposure during meiotic maturation: embryo development after chemical activation vs fertilization of bovine oocytes

Consequences of heat stress exposure during the first 12 h of meiotic maturation differed depending on how and when bovine oocytes were activated. If heat-stressed oocytes underwent IVF at ~24 h, blastocyst development was less than for respective controls and similar to that obtained for nonheat-stressed oocytes undergoing IVF at 30 h (i.e. slightly aged). In contrast, if heat-stressed oocytes underwent chemical activation with ionomycin/6-dimethylaminopurine at 24 h, blastocyst development was not only higher than respective controls, but also equivalent to development obtained after activation of nonheat-stressed oocytes at 30 h. Developmental differences in chemically activated vs IVF-derived embryos were not related to fertilization failure or gross alterations in cytoskeletal components. Rather, ionomycin-induced calcium release and MAP kinase activity were less in heat-stressed oocytes. While underlying mechanisms are multifactorial, ability to obtain equivalent or higher development after parthenogenetic activation demonstrates that oocytes experiencing heat stress during the first 12 h of meiotic maturation have the necessary components to develop to the blastocyst stage, but fail to do so after fertilization.     

Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes

The aim of this study was to investigate whether apoptosis occurs in cumulus cells during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes (CEOs). The bovine CEOs obtained from ovaries from an abattoir were cultured for 24 h in IVM medium in the presence or absence of 10% (v/v) fetal bovine serum. The developmental competence of enclosed oocytes, as assessed by the development of the blastocyst after IVF, was significantly higher in the serum-treated group than in the control group. The morphological features of apoptosis that were analysed by orcein staining were hardly detectable in the cumulus cells at the start (0 h) of IVM, but were evident at the end (24 h) of IVM both in the control and serum-treated groups. Genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect apoptotic internucleosomal DNA fragmentation. DNA fragmentation was hardly detectable at the start of IVM, but increased in a time-dependent manner as the IVM culture proceeded. DNA fragmentation was not observed in the oocytes, indicating that fragmentation occurs in cumulus cells. The degree of fragmentation was lower in the serum-treated group compared with the control group. The LM-PCR analysis of DNA extracted from CEOs at 24 h of IVM, in which the DNA had been pretreated with Klenow enzyme or T4 DNA polymerase, revealed that the characteristic forms of the DNA ends generated during cumulus cell apoptosis were mainly 3'-overhangs and blunt ends. In conclusion, the results of the present study demonstrate that cumulus cells in bovine CEOs spontaneously undergo apoptosis during IVM. The degree of apoptosis may be correlated with the developmental competence of the enclosed oocytes.     

Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro

The overall objective was to evaluate the effectiveness of the S-enantiomer of roscovitine (inhibitor of p34cdc2/cyclin B kinase) to maintain bovine cumulus-oocyte complexes at the germinal vesicle (GV) stage for extended times after removal from antral follicles without compromising subsequent maturation, fertilization and embryo development. Oocytes were cultured in 0, 12.5, 25 or 50 micromol/l S-roscovitine for 24 h. Hoechst staining showed that 50 micromol/l S-roscovitine maintained >90% of oocytes at the GV stage and inhibited gonadotropin-induced cumulus expansion. Fewer oocytes underwent nuclear maturation after in vitro maturation (Hoechst staining) when cultured in 50 micromol/l S-roscovitine for 66 versus 21 or 42 h. Zona pellucida (ZP) hardening (pronase resistance), cortical granule types (lens culinaris agglutinin-fluorescein isothiocyanate), nuclear maturation and fertilization with frozen-thawed spermatozoa (Hoechst staining) were assessed after culture of oocytes in 50 micromol/l S-roscovitine for 0, 24 or 48 h. Neither ZP hardening, nor nuclear maturation nor fertilization were altered by roscovitine culture for 48 h. A higher proportion of oocytes had a type III cortical granule pattern (premature translocation to the oolemma) after roscovitine culture for 48 h. However, embryo development was not compromised as cleavage, development to 8-16 cell and blastocyst stages were at least comparable in control and roscovitine-treated oocytes. In conclusion, the studies have shown that S-roscovitine reversibly maintained bovine oocytes at the GV stage for 48 h. However, maintenance of oocytes in static culture for 48 h was not sufficient to improve development above non-treated controls.     

Independent activation of MAP kinase and MPF during the initiation of meiotic maturation in pig oocytes

Mitogen-activated protein (MAP) kinase is universally activated during oocyte maturation in all vertebrates studied to date. Its role in the resumption of meiosis and in the activation of maturation-promoting factor (MPF) remains unclear, especially in domestic species such as the pig. This study aimed to clarify the temporal and causal relationships between MAP kinase and MPF during meiotic maturation, particularly during the resumption of meiosis. Pig oocytes were matured synchronously in culture by treatment with cycloheximide. Kinase activities were analysed using a sensitive in vitro double-kinase assay and the specific MAP kinase pathway inhibitor U0126. MAP kinase and MPF were activated simultaneously at the time of germinal vesicle breakdown (GVBD; 6 h after removal of cycloheximide); they reached significant activity at 7 h (P < 0.05). The activities increased in parallel during GVBD (6-10 h) and peaked when the oocytes entered metaphase I (MI; 10 h). Whereas MAP kinase remained stable at peak activity thereafter, MPF activity significantly declined during the MI-MII transition (16-20 h) but increased to a second peak at MII (22 h). MAP kinase activity in denuded and cumulus-cell enclosed oocytes was completely inhibited by 20 and 80 mmicro mol U0126 l(-1), respectively. Oocytes without detectable MAP kinase activity underwent normal GVBD in terms of nuclear morphology and timing, although later meiotic stages were abnormal. The kinetics of MPF activity during GVBD were unaffected by U0126. This study has demonstrated that MAP kinase is activated simultaneously with MPF at GVBD, but that its activation is not essential for the activation of MPF nor for the resumption of the first meiosis in pig oocytes.     

Chemicals influencing maturation, activation, and degeneration of bovine oocytes in culture

The effects of the dibutyryl cyclic adenosine 3',5'-monophosphate, calmodulin antagonist W7, and ovarian glycosaminoglycans on polar body extrusion, "nucleus" formation, and segmentation of bovine oocytes were examined in vitro. Dibutyryl cyclic adenosine 3',5'-monophosphate (100 microM) accelerated the extrusion of first polar body; however, W7 (20 microM) suppressed the formation of the first polar body. Spontaneous "nucleus" formation resembling pronucleus formation in oocytes was accelerated in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate at concentrations of greater than 20 microM. The spontaneous segmentation, which is a degenerative change, was suppressed in the presence of bovine follicular fluid-glycosaminoglycans at concentrations of 250 micrograms/ml or greater. These results may indicate that maturation, activation, and degeneration of bovine oocytes in culture are modulated by dibutyryl cyclic adenosine 3',5'-monophosphate, W7, and ovarian glycosaminoglycans.     

Disruption of nuclear maturation and rearrangement of cytoskeletal elements in bovine oocytes exposed to heat shock during maturation

Meiotic maturation in mammalian oocytes is a complex process which involves extensive rearrangement of microtubules, actin filaments and chromosomes. Since cytoskeletal elements are sensitive to disruption by heat shock, a series of experiments were performed to determine whether physiologically relevant heat shock disrupts the progression of the oocyte through meiosis, fertilization and zygote formation. Cumulus-oocyte complexes were cultured at 38.5, 40.0 or 41.0 degrees C for the first 12 h of maturation. Incubation during the last 10 h of maturation and 18 h after fertilization was at 38.5 degrees C and in 5% (v/v) CO2 for both treatments. Examination of the cytoskeleton and the chromosome organization in matured oocytes revealed that oocytes matured at 38.5 degrees C were mostly at metaphase II (MII) stage, while the majority of heat-shocked oocytes were blocked at the first metaphase (MI), first anaphase or first telophase stages. A subset of heat-shocked oocytes possessed misshapen MI spindles with disorganized microtubules and unaligned chromosomes. A higher percentage of TUNEL-positive oocytes was noted for oocytes matured at 41.0 degrees C. Addition of 50 nmol/l sphingosine 1-phosphate to maturation medium blocked the effect of heat shock on progression through meiosis and apoptosis and increased the proportion of oocytes matured at 41.0 degrees C that were at MII. Following insemination, a high percentage of heat-shocked oocytes were unfertilized, while the majority of the control zygotes were fertilized and had two visible pronuclei. In conclusion, heat shock disrupts nuclear maturation and induces apoptosis. These alterations are likely to be involved in the mechanism underlying heat-shock-induced disruption of oocyte capacity for fertilization and subsequent development.     

Retinol improves development of bovine oocytes compromised by heat stress during maturation

The objectives of this study were to evaluate: 1) effects of a physiologically relevant elevated temperature on in vitro development of maturing oocytes, 2) effects of retinol on in vitro development of maturing oocytes, and 3) effects of retinol to improve development of oocytes compromised by an elevated temperature. Bovine oocytes were matured for 24 h at 38.5 or 41.0 degrees C (first 12 h) in 0 or 5 microM retinol. After insemination, cleavage and blastocyst development were assessed on d 3 and 8, respectively. Temperature, retinol, and their interaction were included in the statistical model. Culture of oocytes at 41.0 degrees C decreased the proportion of 8- to 16-cell embryos and increased that of 2-cell embryos. In addition, culture at 41.0 degrees C decreased the ability of oocytes to develop to the blastocyst stage. Blastocysts derived from oocytes cultured at 41.0 degrees C had fewer total nuclei. In 3 of the 7 experimental replicates, effects of 41.0 degrees C to reduce blastocyst development were minimal (difference in the development of the control vs. heat stress group was <20%). To provide a more precise test of our hypothesis (retinol administration may improve development of oocytes compromised by heat stress), data were analyzed, including only those replicates (n = 4) in which heat stress reduced development to blastocyst >20%. When this was done, a significant temperature x retinol interaction was noted. The addition of retinol to the maturation medium prevented heat-induced reductions in development of oocytes to blastocyst stage. Results indicate that retinol may protect oocytes from some of the deleterious effects of heat stress.     

Inactivation of glucocorticoids by 11beta-hydroxysteroid dehydrogenase enzymes increases during the meiotic maturation of porcine oocytes

Recent reports have shown that glucocorticoids can modulate oocyte maturation in both teleost fish and mammals. Within potential target cells, the actions of physiological glucocorticoids are modulated by 11beta-hydroxysteroid dehydrogenase (HSD11B) isoenzymes that catalyse the interconversion of cortisol and cortisone. Hence, the objective of this study was to establish whether HSD11B enzymes mediate cortisol-cortisone metabolism in porcine oocytes and, if so, whether the rate of glucocorticoid metabolism changes during oocyte maturation. Enzyme activities were measured in cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) using radiometric conversion assays. While COCs and DOs oxidised cortisol to inert cortisone, there was no detectable regeneration of cortisol from cortisone. The rate of cortisol oxidation was higher in expanded COCs than in compact COCs containing germinal vesicle (GV) stage oocytes (111+/-6 vs 2041+/-115 fmol cortisone/oocyte.24 h; P<0.001). Likewise, HSD11B activities were 17+/-1 fold higher in DOs from expanded COCs than in those from compact COCs (P<0.001). When GV stage oocytes were subject to a 48 h in vitro maturation protocol, the enzyme activities were significantly increased from 146+/-18 to 1857+/-276 fmol cortisone/oocyte.24 h in GV versus MII stage oocytes respectively (P<0.001). Cortisol metabolism was inhibited by established pharmacological inhibitors of HSD11B (glycyrrhetinic acid and carbenoxolone), and by porcine follicular and ovarian cyst fluid. We conclude that an HSD11B enzyme (or enzymes) functions within porcine oocytes to oxidise cortisol, and that this enzymatic inactivation of cortisol increases during oocyte maturation.     

Localization of centromere proteins and their association with chromosomes and microtubules during meiotic maturation in pig oocytes

Centromere proteins (CENPs) are required for the attachment of microtubules to chromosomes. However, their structure and mechanism of action are not well understood, especially in mammalian meiosis. The present study was conducted to examine (i). whether a human nuclear centromere autoantibody can be used to localize the CENPs in pig oocytes and (ii). the dynamics of CENPs and their association with microtubules and chromosomes during meiosis in pigs. Oocytes at various stages were double-labelled for CENPs, chromosomes or microtubules and examined by confocal fluorescence microscopy. Quantification of tubulin and CENPs in the oocytes was determined by immunoblotting. CENPs were detected in all oocytes from germinal vesicle (GV) to metaphase II (MII) stages. The changes in the location were associated with chromosome movement and spindle formation. Tubulin was detected in the oocytes from GV to MII stages and no differences in content were observed. Two major CENPs at 80 kDa (CENP-B) and 50 kDa (CENP-D) were also found in the oocytes by the autoantibody and its content was significantly lower in the oocytes at GV stage compared with oocytes at other stages. These results indicate that the autoantibody used in this study can be used to detect CENPs in the kinetochores, and the proteins are expressed in pig oocytes at all stages during meiosis. As the localization of CENPs is associated with spindle formation and chromosome movement, CENPs may participate in cell cycle changes during meiosis in mammals.     

Possible mechanism for acceleration of meiotic progression of bovine follicular oocytes by growth factors in vitro

The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles. COCs from small follicles were cultured with individual growth factors and sampled for kinase activity. The frequencies of polar body extrusion in EGF only (67%) and EGF + IGF-I (75%) treatment groups were significantly higher than those in the control (no growth factor) group (49%), but not significantly higher than in the IGF-I only group (63%). The H1 kinase activity at 6-8 h of culture in each group increased significantly from the baseline value at 0 h of culture, and the H1 kinase activities in the EGF only, IGF-I only and EGF + IGF-I treatment groups were significantly higher than those in the control group at 8 h of culture. MAP kinase activity was significantly higher than the baseline value and significantly higher than that in the control group at 6 h of culture in the EGF treatment group only. In conclusion, EGF and IGF-I act on COCs from small follicles to accelerate the meiotic cell cycle of the oocytes. This accelerating effect may be related to increased H1 and MAP kinase activities during the early stages of maturation.     

Exposure to a physiologically relevant elevated temperature hastens in vitro maturation in bovine oocytes

The objective of this study was to evaluate nuclear (progression to metaphase II) and cytoplasmic (translocation of cortical granules to the oolemma) maturation in control (38.5° C) and heat-stressed (41.0° C) oocytes. Hoechst staining indicated that a similar proportion of control and heat-stressed oocytes progressed to metaphase II. More heat-stressed oocytes had type III cortical granule distribution suggesting that heat stress accelerated cytoplasmic maturation. The kinetics of nuclear maturation was examined in a second experiment in which a higher proportion of heat-stressed oocytes progressed to metaphase I by 8 h and arrested at metaphase II at 16 and 18 h after placement into maturation medium. However, differences related to maturation temperature were no longer apparent by 21 h. Heat-induced alterations in kinetics of nuclear and cytoplasmic maturation prompted a third experiment to evaluate if earlier insemination of heat-stressed oocytes ameliorates heat-induced reductions in development. A significant temperature × insemination time interaction was noted when evaluating blastocyst development. Blastocyst development was reduced when heat-stressed oocytes were inseminated with sperm 24 h after placement into maturation medium compared with controls. In contrast, blastocyst development was similar to controls when heat-stressed oocytes were inseminated at 19 h. Based on this interaction, earlier insemination in vitro prevented heat-induced reductions in oocyte development. Collectively, these studies suggest a cumulative effect of heat stress to hasten in vitro maturation in bovine oocytes.     

Effects of follicle size and electrolytes and glucose in maturation medium on nuclear maturation and developmental competence of bovine oocytes

The concentrations of electrolytes (Na, K, Cl, Mg and Ca) and glucose in small follicle (SF) follicular fluid (SFF) and large follicle (LF) follicular fluid (LFF) from slaughterhouse-derived ovaries were studied. Oocytes were matured in medium based on synthetic oviductal fluid. The effects of various concentrations of electrolytes (Na, K, Ca and Mg) and glucose in the maturation medium on the progression of nuclear maturation and subsequent development were also studied. K in SFF was significantly greater than that in LFF. The Mg concentration in follicular fluid (FF) is 2.0-2.3 mM, which is greater than the concentration present in medium generally used for culture. The glucose concentration in FF is about 3.5-3.9 mM and rapidly decreases during the preservation of ovaries. LF oocytes resumed nuclear maturation and progressed to the M2 stage significantly faster than those collected from SF oocytes. In addition, more LF oocytes developed to blastocysts than did SF oocytes. Changing the Na/K ratio in the maturation medium from 16 to 24 did not affect either the progression of nuclear maturation or the rate of development. A low concentration of Mg (0.5 mM) combined with a low Ca concentration (0.5 mM) inhibited the rate of development, but did not affect the progression of nuclear maturation. On the other hand, increasing the Mg concentration to 2.0 mM from 0.5 mM hastened the progression of nuclear maturation and improved the rate of blastulation, irrespective of the Ca concentration. The progression of nuclear maturation was faster and the rate of development was greater with 5.56 mM glucose than with 1.5 mM glucose. The difference in time needed to progress to M2 among the experiment was about 2-3 h. Therefore, prolonging the maturation periods from 21 to 24 h did not change the rate of development. Our results show that the concentrations of Mg and glucose in the maturation medium and the follicle size enveloping the oocyte affect the progression of nuclear maturation and subsequent development. The time requirement for oocytes to reach M2 is strongly related to the developmental competence of the oocytes.     

Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation

Mammalian oocytes grow and undergo meiosis within ovarian follicles. Oocytes are arrested at the first meiotic prophase, held in meiotic arrest by the surrounding follicle cells until a surge of LH from the pituitary stimulates the immature oocyte to resume meiosis. Meiotic arrest depends on a high level of cAMP within the oocyte. This cAMP is generated by the oocyte, through the stimulation of the G(s) G-protein by the G-protein-coupled receptor, GPR3. Stimulation of meiotic maturation by LH occurs via its action on the surrounding somatic cells rather than on the oocyte itself. LH induces the expression of epidermal growth factor-like proteins in the mural granulosa cells that act on the cumulus cells to trigger oocyte maturation. The signaling pathway between the cumulus cells and the oocyte, however, remains unknown. This review focuses on recent studies highlighting the importance of the oocyte in producing cAMP to maintain arrest, and discusses possible targets at the level of the oocyte on which LH could act to stimulate meiotic resumption.     

Leydig cell function in mice lacking connexin43

Connexin43 (Cx43) is the most abundantly expressed member of the connexin (gap junction protein) family and the only one so far identified in mouse Leydig cell gap junctions. Mice lacking Cx43 were used to investigate its role in testicular androgen production and regulation. Testes from term fetuses were grafted under the kidney capsules of castrated adult males. After 3 weeks, serum from host mice was analyzed for androgens. In order to test their response to stimulation, the grafted testes were incubated in vitro with varying concentrations of LH and their androgen end products analyzed. Incubation with radiolabeled progesterone was followed by high performance liquid chromatography to quantify the androgen-intermediate metabolites. Radiolabeled testosterone in the presence of NADPH was used to determine the activity of testosterone-metabolizing enzymes 17beta-hydroxysteroid dehydrogenase (17betaHSD), 5alpha-reductase (5alphaR), and 3alpha-hydroxysteroid dehydrogenase (3alpha HSD). Serum androgen levels did not differ between hosts carrying wild-type versus null mutant grafts although Cx43-deficient testes had more 17betaHSD and 5alphaR activity than wild-type controls. Furthermore, the genotype of grafted testes did not influence LH-stimulated androgen production in vitro. These results indicate that the steroidogenic function of Leydig cells is not compromised by the absence of Cx43, perhaps because other gap junction proteins are present. Dye transfer experiments demonstrated that Cx43-deficient Leydig cells retain intercellular coupling, indicating that Cx43 is not the only protein contributing to their gap junctions. Thus, despite their prominence in Leydig cells, Cx43 gap junctions are not essential for androgen production.     

In vitro maturation of bovine cumulus enclosed primary oocytes and their subsequent in vitro fertilization and cleavage

Cumulus enclosed primary oocytes from 2 to 4-mm bovine follicles were matured in vitro in Minimum Essential Medium containing follicle-stimulating hormone (0, .1, 1, 10, 50, or 100 micrograms/ml) or human chorionic gonadotropin (0, .1, 1, or 10 IU/ml) for 48 h at 37 degrees C under paraffin oil. Cumulus mass expansion comparable to that seen in vivo occurred in 18% of the control oocytes, 39% of those cultured in human chorionic gonadotropin, and 56% of those cultured in follicle-stimulating hormone. The optimum follicle-stimulating hormone concentration for cumulus expansion was 1 microgram/ml, and this was then used to mature oocytes individually or in groups of 5 for in vitro fertilization. Ejaculated bovine semen, extended 1:10 with yolk-TES-Tris extender and stored 24 to 48 h at 4 degrees C, was warmed, washed once with Minimum Essential Medium, and 500,000 motile sperm/ml were used to inseminate the matured oocyte-cumulus cell complexes. Criteria for fertilization was cleavage to the two-cell stage 48 h after insemination. Oocytes, inseminated individually, cleaved with a frequency of 5%, whereas 15% of those inseminated in groups of 5 cleaved, perhaps as the result of cumulus factors enhancing capacitation. The cleavage rate for the parthenogenetic control with killed spermatozoa was 0%. Therefore, primary oocytes matured in vitro to secondary oocytes were successfully fertilized in vitro and cleaved to at least the two-cell stage in the Minimum Essential Medium. Individual differences between bulls in ability to fertilize in vitro were noted.     

Involvement of enzymes of amino acid metabolism and tricarboxylic acid cycle in bovine oocyte maturation in vitro

Few studies demonstrate at a biochemical level the metabolic profile of both cumulus cells and the oocyte during maturation. The aim of the present study was to investigate the differential participation of enzymatic activity in cumulus cells and in the oocyte during in vitro maturation (IVM) by studying the activity of enzymes involved in the control of amino acid metabolism, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and the tricarboxylic acid (TCA) cycle, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). No NAD-dependent isocitrate dehydrogenase (NAD-IDH) activity was recorded in cumulus-oocyte complexes (COCs). ALT, AST, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and MDH enzymatic units remained constant in cumulus cells and oocytes during IVM. Specific activities increased in oocytes and decreased in cumulus cells as a result of IVM (P<0.05). Similar activity of both transaminases was detected in cumulus cells, unlike in the oocyte, in which activity of AST was 4.4 times greater than that of ALT (P<0.05). High NADP-IDH and MDH activity was detected in the oocyte. Addition of alanine, aspartate, isocitrate + NADP, oxaloacetate or malate + NAD to maturation media increased the percentage of denuded oocytes reaching maturation (P<0.05), in contrast to COCs in which differences were not observed by addition of these substrates and co-enzymes. The activity of studied enzymes and the use of oxidative substrates denotes a major participation of transaminations and the TCA cycle in the process of gamete maturation. The oocyte thus seems versatile in the use of several oxidative substrates depending on the redox state.     

Germinal vesicle position and meiotic maturation in mouse oocyte

During meiotic maturation, mammalian oocytes undergo an asymmetric division which is crucial for the formation of a functional gamete. In various organisms, accurate positioning of the nucleus before M-phase plays a major role in asymmetric cell divisions. However, the role of the position of the nucleus (or germinal vesicle, GV) during the prophase I arrest has not been investigated in mammalian oocytes. Here, we show that incompetent mouse oocytes possess a peripheral GV, while competent oocytes mainly exhibit a central position of the GV. At that time, the position of the GV correlates with the ability of the oocyte to complete meiotic maturation. Moreover, a lower efficiency in GV centering and a reduced ability to progress through meiosis are observed in oocytes from old mice. Thus, the position of the GV could be used as a simple morphological marker of oocyte quality.     

Fertilization and activation currents in bovine oocytes

One of the first events that occurs at fertilization is a transient modification of the electrical properties of the oocyte plasma membrane. The whole-cell voltage clamp technique was used to demonstrate an outward ion current and a hyperpolarization of the plasma membrane after fertilization in bovine oocytes. These electrical events, together with measurement of internal calcium concentrations, were also recorded after injection with sperm factor and exposure to parthenogenetic activators, such as Ca(2+) ionophore, ethanol and thapsigargin. Experiments were carried out simultaneously in immature and in vitro matured oocytes. Significant differences were recorded in the activation current and hyperpolarization among oocyte activators and between immature and matured oocytes. However, outward ion current and Ca(2+) release showed similar dynamics. The injection of the calcium chelator EGTA completely abolished both ion current and hyperpolarization, indicating that these electrical events are calcium dependent. Addition of specific calcium releasers, such as 1,4,5-inositol trisphosphate (IP(3)) and caffeine, triggered ion activation current and hyperpolarization indicating that IP(3) and ryanodine receptors are active in both immature and matured oocytes. Different ion channel inhibitors were used to characterize the channels underlying outward currents. Only addition of rIberiotoxin caused a complete inhibition of the current, indicating the involvement of high conductance Ca(2+)-activated K(+) channels in generating activation current. In conclusion, these findings provide evidence that bovine oocyte activation is associated with Ca(2+)-dependent electrical events. Oocytes have the potential to react to different activators even when immature; however, oocyte maturation seems to increase sensitivity to physiological activators, such as spermatozoa and sperm factor, and chemicals, such as ethanol.     

Functional maturation during bovine granulopoiesis

Granulocytic precursor cells undergo morphologic changes in the nucleus and the cytoplasm during the process of granulopoiesis, which takes place in the bone marrow. These changes are associated with the development of stage-specific proteins necessary for the highly specialized roles of polymorphonuclear leukocytes in phagocytosis, bacterial killing, and in mediating the inflammatory process. The objective of the current study was to sequence the various events that occur upon functional development of granulocytic bone marrow cells in the bovine species. Cells were obtained from the bone marrow of clinically healthy cows and separated into different stages of maturation using density gradient centrifugation. Three cellular fractions were obtained that were enriched for either early immature, late immature or mature granulocytic cells. Functions and receptor expressions assessed in the three maturation stages were:Fc-IgG2 receptor and CD11b expression, phagocytosis of Escherichia coli, respiratory burst activity, and cellular myeloperoxidase activity. Immature cells expressed already Fc-IgG2 receptor and CD11b on their cytoplasma membrane. Phagocytic ability was acquired in the myelocytic stage, but only the more mature forms were readily capable of phagocytosis. Promyelocytes, myelocytes and metamyelocytes showed no respiratory burst activity. Only band and segmented cells produced reactive oxygen species. Myeloperoxidase was present at all stages of maturity. Thus, each of the maturation stages was characterized by a selective expression of one or more functions and receptors. Therefore, sequential biochemical maturation is postulated during bovine granulopoiesis.