The role of enzymes of pyruvate and citrate metabolism in control of gluconeogenesis in oocytes and embryos of the loach,Misgurnus fossilis L.

Summary Cessation of gluconeogenesis during oocyte maturation inMisgurnus fossilis L. is accompanied by an increase of pyruvate dehydrogenase activity (EC 1.2.4.1). The activity of other enzymes of citrate and pyruvate metabolism (citrate synthetase, EC 4.1.3.7, pyruvate carboxylase, EC 6.4.1.1., malate dehydrogenase, EC 1.1.1.37) remains constant during oocyte maturation and early embryogenesis.In the course of oocyte maturation the levels of acetyl-CoA, pyruvate and citrate remained unchanged, but the level of malate and oxaloacetate underwent drastic increase. The level of phosphoenolpyruvate increased about two-fold. The mitochondrial (NAD⁺)/(NADH) ratio was calculated by measurement of intermediates of the glutamate dehydrogenase reaction and it was found to increase six-fold during oocyte maturation. The lower mitochondrial (NAD⁺)/(NADH) ratio in oocytes compared to that in the embryos is likely to be responsible for the transfer of reducing equivalents from mitochondria to cytoplasm, while in embryos transfer in the opposite direction takes place.     

Expression pattern of glucose metabolism genes in relation to development rate of buffalo (Bubalus bubalis) oocytes and in vitro–produced embryos

The expression pattern of glucose metabolism genes (hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase [G6PDH], lactate dehydrogenase [LDH], and pyruvate dehydrogenase [PDH]) were studied in buffalo in vitro–matured oocytes and in vitro–produced embryos cultured under different glucose concentrations (0 mM, 1.5 mM, 5.6 mM, and 10 mM) during in vitro maturation of oocytes and culture of IVF produced embryos. The expression of the genes varied significantly over the cleavage stages under different glucose concentrations. Developmental rate of embryos was highest under a constant glucose level (5.6 mM) throughout during maturation of oocytes and embryo culture. Expression pattern of glucose metabolism genes under optimum glucose level (5.6 mM) indicated that glycolysis is the major pathway of glucose metabolism during oocyte maturation and early embryonic stages (pre-maternal to zygotic transition [MZT]) and shifts to oxidative phosphorylation during post-MZT stages in buffalo embryos. Higher glucose level (10 mM) caused abrupt changes in gene expression and resulted in shifting toward anaerobic metabolism of glucose during post-MZT stages. This resulted in decreased development rate of embryos during post-MZT stages. High expression of LDH and PDH in the control groups (0 mM glucose) indicated that in absence of glucose, embryos try to use available pyruvate and lactate sources, but succumb to handle the post-MZT energy requirement, resulting to poor development rate. Expression pattern of G6PDH during oocyte maturation as well early embryonic development was found predictive of quality and development competence of oocytes/ embryos.     

Physiological differentiation of the mitochondria during Bufo bufo development.

1) The oxygen consumption increases during Bufo bufo development in accordance with the two steps which border at the "heart beat" stage. 2) Cytochrome c oxidase activity is not proportional to the oxygen consumption: it is notable and constant in the first step, and it only increases in the second. 3) In the mitochondria of preneural embryos, citrate synthase, NADP+ dependent isocitrate dehydrogenase, and succinate dehydrogenase activities are very low in respect to malate dehydrogenase and glutamate oxaloacetate transaminase activities. The Krebs cycle results lowered at the condensing reaction level with acetyl accumulation when pyruvate is available. The same behavior has been observed in the Xenopus laevis oocytes and differentiated tissues. 4) The presence of a phosphagen system which is different from creatine phosphate and arginine phosphate, supporting ATP level, has been demonstrated in B. bufo embryos. 5) Mitochondria of postneural embryos are able to accomplish a complete Krebs cycle by increasing citrate synthase, and succinate dehydrogenase activities. 6) In all B. bufo development, malate dehydrogenase and glutamate oxaloacetate transaminase constitute a multienzymatic system by which the mitochondria accomplish a decarboxylic amino acid shunt required for the transformation of deutoplasm into protoplasm. This shunt is also operative in the X. laevis oocytes. 7) Through pyruvate production, by oxidative decarboxylation of malate, the NAD(P)+ dependent malic enzyme could carry out a fundamental anaplerotic function in the mitochondria which is specialized in the production of biosynthetic blocks belonging to the embryo in which the carbohydrates metabolism rather than the glycolytic activity is designed for pentose phosphate and glycerol phosphate synthesis for protein and cytomembrane production. 8) Consistent metabolic differences have been highlighted between B. bufo embryos and X. laevis embryos.     

Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis

The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.     

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.     

Cloning and characterization of Plx2 and Plx3, two additional Polo-like kinases from Xenopus laevis

Members of the family of Polo-like kinases are implicated in the regulation of cell cycle progression in all eukaryotes. In Xenopus laevis, only one member of this family, Plx1, has previously been described. Here we report the cloning and characterization of X. laevis Plx2 and Plx3, the likely homologs of mammalian Plk2 (Snk) and Plk3 (Fnk/Prk), respectively. RNA expression studies indicate that all three Xenopus Plks are present in both oocytes and unfertilized eggs. Further analysis by in situ hybridization revealed that Plx1 RNA is ubiquitously expressed in early embryos, but shows more restricted expression at later stages. In contrast, Plx2 and Plx3 expression is highly restricted in both early and late-stage embryos. Using Plx-specific antisera, Plx1 and Plx3 polypeptides could readily be detected on immunoblots of oocyte and egg extracts. Both Plx1 and Plx3 protein levels remained virtually constant during oocyte maturation. However, whereas Plx1 is more active in M phase than in I phase (P. Descombes and E. A. Nigg (1998) EMBO J. 17, 1328-1335), Plx3 protein and activity levels remained constant upon release of meiotic metaphase II-arrested egg extracts into interphase. Finally, microinjection of in vitro-transcribed RNAs for Plx1, Plx2, and Plx3 increased the rate of progesterone-induced oocyte maturation, and concomitantly, all three kinases became activated. Conversely, overexpression of the corresponding catalytically inactive kinases delayed maturation. This suggests that, at least in oocytes, all three kinases may be regulated by similar mechanisms, and they may also share common substrates. However, the strikingly restricted pattern of expression of Plx2 and Plx3 observed in embryos strongly suggests that individual Plk family members perform at least partly distinct functions at later stages of development.     

Effect of lactate dehydrogenase activity and isoenzyme localization in bovine oocytes and utilization of oxidative substrates on in vitro maturation

Oocyte nutritional metabolism changes during maturation in order to increase the energy available to support metabolic requirements. The aim of this work was to study pyruvate and lactate utilization as oxidative substrates on IVM and lactate dehydrogenase (LDH) activity and localization of their isoenzymes in bovine oocytes. Immature cumulus-oocyte complexes (COCs) were recovered by aspiration of antral follicles in ovaries obtained from slaughtered cows. The COCs and denuded oocytes were separately cultured in TCM-199 with steer serum (controls) and were supplemented with pyruvate, lactate or lactate plus NAD for 24 h at 39 degrees C in 5% CO2:95% humidified air. No significant differences were found in IVM rates of COCs matured according to the various treatments (P>0.05). The IVM rate in denuded oocytes without supplementation was 47.8%. The presence of pyruvate in the culture medium resulted in an increased number of matured denuded oocytes (59.4%; P<0.05), but the addition of lactate failed to improve the IVM rate of matured denuded oocytes (47.6%, P>0.05). When the medium was supplemented with lactate plus NAD, the IVM rate of denuded oocytes likewise failed to differ from that obtained with the addition of pyruvate (59.9%, P>0.05). The LDH activity in immature and matured COCs and denuded oocytes was (3.1+/-1.6) 10(-3), (3.3+/-1.6) 10(-3) U/COC, (5.2+/-2.0) 10(-5), (5.4+/-3.5) 10(-5) U/oocyte with pyruvate as substrate, and (1.2+/-0.5) 10(-3), (1.0+/-0.5) 10(-3) U/COC, (2.2+/-0.1) 10(-5), (2.5+/-1.4) 10(-5) U/oocyte respectively, with lactate; no significant differences due to maturation status were observed (P>0.05; n = 9 for each LDH activity). Electrophoresis disclosed that the principal band corresponded to the LDH-1 isoenzyme in oocytes, while there was no predominance of any isoenzyme in cumulus cells. Due to the fact that LDH-1 is the main oocyte isoenzyme, the pyruvate used during oocyte maturation could be partly produced from lactate when the NAD supply is adequate. Cumulus cells would be responsible for providing pyruvate and/or lactate as oxidative substrates to be used by the bovine oocyte and this supply would be regulated by the LDH activity in these cells.     

Use of different oxidation substrates in the energy metabolism of mammalian germ cells and embryos

Mammalian oocytes and embryos on the early stages of development are in need of pyruvate as source of energy. The pyruvate or pyruvate and lactate combination are necessary for the processes of the maturation and fertilisation. The 8-cell stage embryos tend to change their energetical metabolism. The mouse embryos at this stage of development begin to utilise the glucose. alpha-Ketoglutarate without pyruvate, lactate or glucose is necessary for the development of cow and pig embryos after 8-cell stage.     

The role of cAMP in oocyte maturation and the role of the germinal vesicle contents in mediating maturation and subsequent developmental events in hydrozoans

Summary Externally applied membrane permeable cAMP derivatives and the injection of cAMP induce oocyte maturation in several species of hydrozoans. This technique for inducing oocyte maturation has been used to study ion permeability changes, maturation promoting factor activity and surface tension changes during maturation. Oocyte membrane potential remains constant during maturation. Cyclic AMP induced maturation proceeds in the absence of external Ca²⁺, K⁻, Mg²⁺ or Na⁺. Cytoplasm from maturing oocytes that induces oocyte maturation when it is injected into untreated oocytes is produced during cAMP induced maturation. Surface tension, as measured by the application of a standardized force that mechanically deforms individual oocytes, declines during the first part of maturation. This is followed by a sharp rise and fall of surface tension at first and second polar body formation that accompanies a slow rise in the resistance of oocytes to deformation during the last part of maturation. The production of maturation promoting factor activity and some of the changes in surface tension during maturation can occur in the absence of germinal vesicle material. Two early developmental events that follow oocyte maturation are the production of sperm chemoattractant and calcium channel function. Neither of these events occurs in eggs that have undergone maturation in the absence of germinal vesicle material. The addition of germinal vesicle contents from oocytes to eggs that have undergone maturation in the absence of germinal vesicle material initiates calcium channel function. This experiment indicates that the germinal vesicle contains factors that are necessary for post-maturation developmental events.     

A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.     

APPEARANCE OF PRONUCLEUS-INDUCING ACTIVITY DURING HORMONALLY INDUCED MEIOTIC MATURATION IN TOAD OOCYTES*

Detergent-pretreated spermatozoa of the toad, Bufo bufo japonicus, transform into pronuclei when injected into progesterone-matured oocytes at 18 hr post-hormone treatment (PHT). These sperm, however, do not show any change when injected into the oocytes at the same age from which the germinal vesicle (GV) has been removed before the progesterone treatment. In an attempt to determine when and how the pronucleus-inducing activity (PIA) develops in hormonally induced maturation process, enucleated oocytes were injected with GV and sperm at various stages after the hormone treatment and electrically stimulated at 18 hr PHT. It was found that sperm pronuclei are induced only in those oocytes receiving GV before 14 hr PHT. The 1 hr pulse-treatment of maturing oocytes with cycloheximide between 8–18 hr PHT and the injection of sperm at 18 hr PHT revealed that PIA does not occur in the oocytes treated with the inhibitor during 10–14 hr PHT. Injection of α-amanitin into maturing oocytes had no effect in this respect. Determination of DNA synthetic activity in vitro of the oocyte extracts from various maturation stages showed that the net increase of the activity occurs before the formation of PIA. The activity of the cycloheximide-treated oocyte extracts utilizing native DNA did not correlate with the sensitivity of oocytes to the inhibitor with respect to PIA in situ. It is concluded that PIA develops, in association with the GV materials, by way of translational events at 10–14 hr PHT, being quiescent during later maturation stages, and commences to function as an activation response of oocytes at 18 hr PHT.     

Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium

Background

Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes.

Results

Analysis of the cell cycle stages revealed that 91.2 ± 0.2% of confluent cells were at the G0/G1 phase and 54.1 ± 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors.

Conclusions

Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells.     

Regulation of redox metabolism in the mouse oocyte and embryo

Energy homeostasis of the oocyte is a crucial determinant of fertility. Following ovulation, the oocyte is exposed to the unique environment of the Fallopian tube, and this is reflected in a highly specialised biochemistry. The minute amounts of tissue available have made the physiological analysis of oocyte intermediary metabolism almost impossible. We have therefore used confocal imaging of mitochondrial and cytosolic redox state under a range of conditions to explore the oxidative metabolism of intermediary substrates. It has been known for some time that the early mouse embryo metabolises external pyruvate and lactate but not glucose to produce ATP. We now show at the level of single oocytes, that supplied glucose has no effect on the redox potential of the oocyte. Pyruvate is a cytosolic oxidant but a mitochondrial reductant, while lactate is a strong cytosolic reductant via the activity of lactate dehydrogenase. Unexpectedly, lactate-derived pyruvate appears to be diverted from mitochondrial oxidation. Our approach also reveals that the level of reduced glutathione (GSH) in the oocyte is maintained by glutathione reductase, which oxidises intracellular NADPH to reduce oxidised glutathione. Surprisingly, NADPH does not seem to be supplied by the pentose phosphate pathway in the unfertilised oocyte but rather by cytosolic NADP-dependent isocitrate dehydrogenase. Remarkably, we also found that the oxidant action of pyruvate impairs development, demonstrating the fundamental importance of redox state on early development.