Metabolic regulation during early frog development: glycogenic flux in Xenopus oocytes, eggs, and embryos

32P-labeled glucose 6-phosphate and phosphoenolpyruvate were injected into oocytes, fertilized eggs, and early embryos of Xenopus laevis, and the 32P label was followed into glycolytic enzymes and acid-soluble metabolites. The kinetics of labeling of phosphoglucomutase and phosphoglyceromutase and the formation of specific metabolites were used to measure carbon flux through glycolytic intermediates in these cells. In full-grown stage VI oocytes, fertilized eggs, and cells of cleaving embryos, carbon metabolism is in the glycogenic direction. Glycolytic intermediates injected into these cells were metabolized into UDP-glucose and then presumably into glycogen. Carbon flow between phosphoenolpyruvate and glucose 6-phosphate does not utilize fructose 1,6-bisphosphatase; rather, it may depend largely on enzymes of the pentose phosphate pathway. Maturation and fertilization of the oocyte did not result in a change in the qualitative pattern of metabolites formed. Pyruvate kinase, although abundant in oocytes and embryos, is essentially inactive in these cells. Pyruvate kinase also appears to be inactive in small previtellogenic stage II oocytes; however, in these cells injected glycolytic intermediates were not metabolized to UDP-glucose.     

In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model

Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 μM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 μM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 μM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos even though further studies are necessary to confirm this possibility.     

Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro

Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.     

Metabolic regulation during early frog development: flow of glycolytic carbon into phospholipids in Xenopus oocytes and fertilized eggs

32P-labeled glucose 6-phosphate, [32P]phosphoenolpyruvate, and [gamma-32P]ATP were injected into oocytes and fertilized eggs of Xenopus laevis, and the incorporation of the 32P label was followed into phospholipids. Several classes of phospholipids incorporated 32P label from the injected glycolytic intermediates, including lysophosphatidic acid, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol phosphates, inferring de novo synthesis of these lipids from dihydroxyacetone phosphate or glycerol 3-phosphate. Injection of [gamma-32P]ATP into oocytes and fertilized eggs led to labeling of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate, indicating an active phosphatidylinositol cycle in resting oocytes and fertilized eggs. Maturation and fertilization of the oocyte led to a qualitative change in phosphatidylinositol metabolism, increased labeling of phosphatidylinositol phosphate compared to phosphatidylinositol bisphosphate (either from glycerol 3-phosphate or from ATP). This change occurs late in the maturation process, and the new pattern of phosphatidylinositol metabolism is maintained during the rapid cleavage stages of early embryogenesis.     

Translational repression by the oocyte-specific protein P100 in Xenopus

The translational regulation of maternal mRNAs is one of the most important steps in the control of temporal-spatial gene expression during oocyte maturation and early embryogenesis in various species. Recently, it has become clear that protein components of mRNPs play essential roles in the translational regulation of maternal mRNAs. In the present study, we investigated the function of P100 in Xenopus oocytes. P100 exhibits sequence conservation with budding yeast Pat1 and is likely the orthologue of human Pat1a (also called PatL2). P100 is maternally expressed in immature oocytes, but disappears during oocyte maturation. In oocytes, P100 is an RNA binding component of ribosome-free mRNPs, associating with other mRNP components such as Xp54, xRAP55 and CPEB. Translational repression by overexpression of P100 occurred when reporter mRNAs were injected into oocytes. Intriguingly, we found that when P100 was overexpressed in the oocytes, the kinetics of oocyte maturation was considerably retarded. In addition, overexpression of P100 in oocytes significantly affected the accumulation of c-Mos and cyclin B1 during oocyte maturation. These results suggest that P100 plays a role in regulating the translation of specific maternal mRNAs required for the progression of Xenopus oocyte maturation.     

Maternal diabetes adversely affects AMP-activated protein kinase activity and cellular metabolism in murine oocytes

Maternal diabetes is associated with an increased risk of miscarriages and congenital anomalies. Preovulatory oocytes in murine models also experience maturational delay and greater granulosa cell apoptosis. The objective of this study was to examine whether maternal diabetes influences preovulatory oocyte metabolism and impacts meiotic maturation. Streptozotocin-induced diabetic B6SJLF1 mice were superovulated, and oocytes were collected at 0, 2, and 6 h after human chorionic gonadotropin (hCG) injection. Individual oocyte concentrations of ATP, 5'-AMP, glycogen, and fructose-1,6-phosphate (FBP) and enzyme activities of glucose-6-phosphate dehydrogenase (G6PDH), adenylate kinase, hydroxyacyl-CoA dehydrogenase (Hadh2), and glutamic pyruvate transaminase (Gpt2) were measured. Protein levels of phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were also measured. ATP levels were significantly lower in oocytes from diabetic mice, and the percent change in the AMP-to-ATP ratio was significantly higher in these oocytes. In contrast, activities of Hadh2 and Gpt2, two enzymes activated by AMPK, were significantly less in these oocytes. Additionally, glycogen and FBP levels, both endogenous inhibitors of AMPK, were elevated. Phosphorylated ACC, a downstream target of AMPK, and phosphorylated AMPK were both decreased in diabetic oocytes, thus confirming decreased AMPK activity. Finally, addition of the activator AICAR to the in vitro maturation assay restored AMPK activity and corrected the maturation defect experienced by the oocytes from diabetic mice. In conclusion, maternal diabetes adversely alters cellular metabolism leading to abnormal AMPK activity in murine oocytes. Increasing AMPK activity in these oocytes during the preovulatory phase reverses the metabolic changes and corrects delays in meiotic maturation.     

Effects of Upregulation of Hsp27 Expression on Oocyte Development and Maturation Derived from Polycystic Ovary Syndrome

Heat shock protein 27 (Hsp27) is a heat shock protein family member which can inhibit apoptosis. Our previous studies reported down-regulated Hsp27 in ovarian tissue derived from women with polycystic ovary syndrome (PCOS) however, the exact effect of Hsp27 on oocyte maturation and developmental competence in PCOS is unclear. The effect of Hsp27 over-expression was studied in vitro using oocytes derived from PCOS patients. An artificial GFP-plasmid was injected into human oocyte to increase Hsp27 protein level. Oocyte maturation was evaluated by morphological observation. Mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and embryonic developmental competence was evaluated. Critical apoptotic factors and cytokines were measured at both the mRNA and protein level. Our results revealed that Overexpression of HSP27 lowered the maturation rate of oocytes derived from PCOS patients. Meanwhile, fertilization rate and high quality embryo rate were similar between the Hsp27 overexpressing group and controls; however, the blastocyst formation rate in this group was significantly higher than control. Expression analysis revealed that the oocyte-secreted factors, BMP15 and GDF9, and the apoptotic-related regulators, Caspase 3, 8 and 9, were all significantly decreased in Hsp27 overexpressing oocytes. In conclusion, upregulation of Hsp27 inhibits oocyte maturation from PCOS patients, but improves embryonic developmental potential.     

Developmental competence of bovine oocytes is not related to apoptosis incidence in oocytes, cumulus cells and blastocysts

The number of follicles undergoing atresia in an ovary is very high, and isolation of cumulus-oocyte complexes (COCs) from such atretic follicles may impair subsequent embryo development in vitro. Our aim was to study if stringent selection by morphological assessment of COCs can improve embryo development, and to evaluate whether oocyte diameter is related with apoptotic ratio in oocytes and blastocysts. COCs from slaughtered cattle were recovered by follicle aspiration and classified depending on oocyte diameter: (A) <110 microm; (B) 110-120 microm; (C) >120 microm. COCs were matured, fertilized and cultured in vitro. Early and late stages of apoptosis were detected by Annexin-V and TUNEL staining, respectively, in denuded oocytes, COCs and blastocysts. Immature oocytes from Group A showed higher apoptotic ratio assessed by TUNEL assay, and the COCs corresponding to this group also showed a higher proportion of apoptotic cumulus cells. After maturation, no differences were present in the incidence of apoptosis among oocytes from different groups, but COCs corresponding to the largest diameter showed less apoptotic cumulus cells. In addition, the percentage of apoptotic oocytes decreased during in vitro maturation in all groups. Apoptotic cell ratio (ACR) in blastocysts was not related to oocyte diameter. In conclusion, oocyte selection and oocyte morphological evaluation prior to maturation was not sufficient to select non-atretic oocytes. When oocyte diameter was used as an additional selection the embryonic developmental potential increased together with oocyte diameter, but this improvement was not related to a lower incidence of apoptosis in the largest oocytes.     

Leptin improves in-vitro maturation of goat oocytes through MAPK and JAK2/STAT3 pathways and affects gene expression of cumulus cells

We investigated whether the recombinant leptin (1, 10, 100 ng/mL) influences the meiotic maturation of goat oocytes, whether the MAPK and JAK2/STAT3 pathways mediate the effects of leptin during in-vitro maturation, and whether leptin differently affects the abundance of mRNAs relevant to leptin signal transduction and apoptosis in oocytes and cumulus cells. The addition of leptin to the maturation medium positively affected the number of oocytes that completed nuclear maturation. Nuclear oocyte maturation stimulated by leptin was significantly impaired when we added the specific inhibitors of MAPK (U0126) and JAK2/STAT3 (AG490) to the maturation medium. The addition of leptin (10 ng/mL) during maturation did not affect the expression of AMPKα1, PPARα, Caspase 3, and BCL2 genes in oocytes or cumulus cells. The PPARγ and BAX mRNA abundances were significantly reduced in cumulus cells in the leptin group compared to the control group. Our results demonstrate that supplementation of the in-vitro maturation medium with leptin significantly improves nuclear maturation and reveal the important role of the MAPK and JAK2/STAT3 signaling pathways in establishing the leptin-mediated nuclear maturation of goat oocytes. Moreover, leptin treatment affects PPARγ and BAX gene expression in cumulus cells.     

Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.     

Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro

Current methods for detecting complete oocyte maturation and developmental competence are inadequate. The objectives of this study were to (1) examine the relationship between cat oocyte energy metabolism and development in vitro after fertilization and (2) determine if cumulus cell metabolism could be used to predict development of individual oocytes after fertilization in vitro. The hanging drop method was used to assess metabolism of three different types of cat oocytes: immature (IMO), in vitro matured (IVM), and in vivo matured (IVOM). Stage of oocyte nuclear maturation or developmental competence was assessed after metabolic analysis. Glycolysis and oxidation of glucose, glutamine, palmitate, and lactate increased with the resumption of oocyte meiotic maturation (P<0.05). Pyruvate was the preferred substrate, but uptake was not linked to maturation. IVM oocytes had impaired glucose and palmitate metabolism compared to IVOM oocytes (P<0.05). Oocyte glycolytic activity and oocyte glucose oxidation correlated well with embryo development after insemination in vitro (P<0.05). Furthermore, oocytes that had similar glucose metabolism and that were grouped together for culture on this basis had higher (P<0.05) overall rates of development than oocytes grouped randomly. There was no correlation (P>0.05) between cumulus cell metabolism and individual oocyte development after in vitro fertilization. The data reveal that energy metabolism is linked to oocyte maturation in the cat and that glucose metabolic activity can indicate those oocytes most likely to fertilize and develop in vitro. Measuring cumulus cell metabolism does not accurately predict individual oocyte development after insemination in vitro.     

SET improved oocyte maturation by serine/threonine protein phosphatase 2A and inhibited oocyte apoptosis in mouse oocytes

SET is a multifunctional protein involved in a variety of molecular processes such as cell apoptosis and cell-cycle regulation. In ovaries SET is predominantly expressed in theca cells and oocytes. In polycystic ovary syndrome (PCOS) patients the expression of SET was increased than healthy people. The current study was designed to determine whether SET plays a role in oocyte maturation and apoptosis, which may provide clues for the underlying pathological mechanism of follicular development in PCOS patients. Oocytes at germinal vesicle (GV) stage were collected from 6-week-old female ICR mice ovaries. The expression of SET was manipulated by AdCMV-SET and AdH1-SiRNA/SET adenoviruses. SET overexpression improved oocyte maturation whereas SET knockdown inhibited oocyte maturation. Moreover, SET negatively regulated serine/threonine protein phosphatase 2A (PP2A) activity in oocytes. Treatment with PP2A inhibitor okadaic acid (OA) promoted oocyte maturation. Furthermore, PP2A knockdown confirmed the role of PP2A in oocyte maturation, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition on oocyte maturation. The central role of PP2A in SET-mediated regulation of oocyte maturation was confirmed by the finding that SET increased the expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) and PP2A inhibited their expressions. Besides, SET inhibited oocyte apoptosis through decreasing the expression of caspase 3 and caspases 8, while PP2A had no effect on oocyte apoptosis. SET promoted oocyte maturation by inhibiting PP2A activity and inhibited oocyte apoptosis in mouse in-vitro cultured oocytes, which may provide a pathologic pathway leading to impaired oocyte developmental competence in PCOS.     

The mycotoxin beauvericin induces oocyte mitochondrial dysfunction and affects embryo development in the juvenile sheep

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus‐oocyte‐complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short‐term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long‐term carry‐over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late‐stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3–0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.