340 EFFECT OF GLUCOSE ON THE MORPHOLOGY OF GERMINAL VESICLE AND MEIOTIC PROGRESSION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 285
Author(s):  
H. Funahashi ◽  
T. Koike
Keyword(s):  
Critical Role ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Defined Medium ◽  
Pentose Phosphate ◽  
Chemically Defined Medium ◽  
Dibutyryl Camp ◽  
Germinal Vesicle Breakdown ◽  
Maturation Medium ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose metabolism through the pentose phosphate pathway (PPP) seems to play a critical role in meiotic resumption in mouse oocytes (Downs et al. 1998 Biol. Reprod. 58, 1084–1094). However, the role is not clear in porcine oocytes. In the present study, we examined whether glucose affects morphological change of germinal vesicles and the resumption of meiosis in porcine oocytes in a chemically defined medium. In the first experiment, porcine cumulus–oocyte complexes (COCs) were collected from 3–6-mm follicles of slaughterhouse ovaries and cultured in a chemically defined medium, mNCSU37-PVA with/without 5.55 mM glucose in the presence of eCG, hCG, and dibutyryl cAMP for 20–22 h and then in the absence of eCG, hCG, and dibutyryl cAMP for 24 h. In the second experiment, 5.55 mM glucose in the maturation medium was replaced with the same concentration of Na pyruvate. In the third experiment, the PPP inhibitor 6-aminonicotinamide (6-AN) was added to the maturation medium at various concentrations (0, 10, 50, and 100 �M). To determine the activity of glucose-6-phosphate dehydrogenase (G6PD), OCCs were fixed, blocked, and treated with anti-G6PD polyclonal antibody and the secondary antibody labeling a fluorescent material. Results from 3–5 replicates were analyzed by ANOVA and Duncan's multiple range test. When OCCs were cultured in glucose-free chemically defined maturation media, regardless of the presence of hormones and dibutyryl cAMP, germinal vesicle breakdown (GVBD) of oocytes was inhibited (10.0–21.3%), as compared with OCCs cultured in the presence of glucose and hormones (91.4–92.0%). In a majority of oocytes in which GVBD was inhibited, the arrest occurred at the GV-I stage. When OCCs were cultured in maturation media in which glucose was replaced with Na pyruvate, GVBD was not inhibited any more than in control samples that were cultured in the presence of glucose (97.4% vs. 97.1%). However, the incidence of oocytes developing to the metaphase II stage was significantly lower in this condition than in controls (4.8% vs. 49.9%, respectively). A majority of the oocytes were at the metaphase I stage (86.0% vs. 45.5% in controls). The presence of 6-AN in maturation media significantly inhibited GVBD of oocytes (77.3, 29.0, 7.4, and 8.4% at 0, 10, 50, and 100 µM, respectively) and arrested the oocytes at the GV-I stage. Immunocytochemistry with anti-G6PD demonstrated the activity of G6PD in cumulus cells of OCCs. In conclusion, these results demonstrate that glucose plays a critical role in the release of porcine oocytes arrested at the GV-I stage, probably through PPP of cumulus cells. The current results also suggest the possibility of gluconeogenesis in porcine OCCs when glucose in maturation media was replaced with Na pyruvate.

scholarly journals 78 SURVIVAL OF PORCINE OOCYTES AT GERMINAL VESICLE STAGE AFTER VITRIFICATION WITH OPEN PULLED STRAW METHOD

2005 ◽  
Vol 17 (2) ◽  
pp. 189 ◽  
Author(s):  
A. Bali Papp ◽  
T. Somfai ◽  
E. Varga ◽  
M. Marosán
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Penicillin G ◽  
National Committee ◽  
Ministry Of Education ◽  
Germinal Vesicle Breakdown ◽  
Maturation Medium ◽  
Maturation Rate ◽  
Nuclear Stage

The present study was performed to assess the survival of immature denuded or cumulus-covered porcine oocytes (COCs). Immature porcine oocytes were collected from 2–6 mm follicles of slaughterhouse ovaries and subjected to open pulled straw (OPS) vitrification, according to the method of Vajta et al. (1998 Mol Reprod. Dev. 51, 53–58). After vitrification, oocytes were matured in vitro for 48 h at 39°C, 5% CO2 in air. The maturation medium was TCM199 supplemented with 10% pig follicular fluid, 1.25 mM L-glutamine, 0.9 mM Na pyruvate, 150 μM cysteamine, 0.1 mg/mL streptomycin sulfate, 100 IU/mL PG penicillin g potassium, 10 IU/mL PMSG, and 25 IU/mL hCG. After IVM, to assess nuclear stage, all oocytes were fixed with acetic acid–alcohol (1:3) for at least three days and then stained with 0.1% orcein and examined under a phase-contrast microscope at 100× magnification. All data were analyzed by χ2 test (P < 0.05). Immediately after collection, all oocytes were at the germinal vesicle (GV) stage with an intact GV membrane. After vitrification, significantly fewer oocytes had normal morphology (intact plasma membrane) in the denuded and COC groups (4.7% and 8.5%, respectively) than did the denuded and COC control groups (95% and 92%, respectively). By the end of IVM, significantly fewer oocytes were surrounded by expanded cumulus after vitrification of COCs than were the COC controls (28.1% and 63.5%, respectively). After IVM, more of the COC control oocytes underwent germinal vesicle breakdown than did the denuded controls (95% and 78.2%, respectively); the rate of MII oocytes was higher for the COC controls than for the denuded controls (80% and 54.5%, respectively). After vitrification, the number of oocytes that underwent GVBD was significantly less for both the denuded and the COC groups (2.0% and 7.0%, respectively); the percentage of oocytes that reached MII was also lower (0.64% and 2.78%, respectively). Most of the vitrified oocytes had a damaged GV with disrupted membrane and cluster-like or scattered chromatin in both the denuded and the COC groups (96.4% and 90.7%, respectively). These data suggest that vitrification of cumulus-enclosed immature porcine oocytes is preferable compared to vitrification of denuded ones. Loss of cumulus cells compromises competence of oocytes to resume meiosis, which might result in a lower maturation rate after IVM. This research was supported by the grants of the Hungarian Scientific Research Fund (T 031758), the Hungarian National Committee of the Technical Development at the Ministry of Education (00796/2003), and the Ministry of Education (OM-KMUFA; BIO-00086/2002).

343 DISTRIBUTION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND IN VITRO MATURATION OF PORCINE OOCYTE - CUMULUS COMPLEXES FROM SMALL AND MEDIUM FOLLICLES

2007 ◽  
Vol 19 (1) ◽  
pp. 287
Author(s):  
Y. Ishida ◽  
H. Funahashi
Keyword(s):  
In Vitro Maturation ◽  
Critical Role ◽  
Morphological Characteristics ◽  
Cumulus Cells ◽  
Pentose Phosphate ◽  
G6pd Activity ◽  
Cresyl Blue ◽  
Key Enzyme ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose metabolism through the pentose phosphate pathway (PPP) plays a critical role in meiotic maturation and fertilization. However, the relationship between the distribution of a PPP key enzyme, glucose-6-phosphate dehydrogenase (G6PD), in cumulus–oocyte complexes (COCs) and the in vitro maturation (IVM) of the oocytes is not clear. In the present study, we examined the distribution of G6PD, the morphological characteristics in OCCs derived from small (d2 mm in diameter) and medium (3 to 6 mm in diameter) follicles, and the rate of oocyte maturation. Porcine COCs were collected from small or medium follicles of slaughterhouse ovaries. The COCs were cultured in a maturation medium, BSA-free NCSU37 supplemented with 10% porcine follicular fluid, eCG, and hCG, for 20 h and then in the absence of hormones for 24 h. To determine the distribution of G6PD, the COCs were treated with 13 �M brilliant cresyl blue (BCB) in TL-HEPES-PVA for 90 min. Results from 3–6 replicates were analyzed by ANOVA and Duncan&apos;s multiple range test. The mean diameters for COCs collected from small follicles (136.7 &micro;m for the outer zona and 103.1 &micro;m for ooplasm) were significantly less than for those derived from medium follicles (164.1 &micro;m and 122.0 &micro;m, respectively). G6PD activity was detected in the cumulus cells for most of the COCs derived from medium follicles, but it was not significantly different from that of COCs derived from small follicles. In the second group of COCs, very little G6PD activity was found in both the cumulus cells and the oocytes (34.7 &plusmn; 11.5&percnt; and 18.0 &plusmn; 6.7&percnt; in COCS derived from small and medium follicles, respectively). After stimulation by eCG and hCG, the percentages of COCS in which G6PD activity was detected in the cumulus cells, but not in the oocytes, were 56.2 &plusmn; 23.8&percnt; and 72.9 &plusmn; 6.1&percnt; for small and medium follicles, respectively. The percentage of oocytes at the metaphase II stage (53&percnt; and 63.9&percnt; in oocytes from small and medium follicles, respectively) was higher for the COCs that showed higher G6PD activity in their cumulus cells. In conclusion, although no statistical differences were detected in the distribution of G6PD between COCs from small and medium follicles, due to a large variation, a higher percentage of mature oocytes seems to be the result of COCs where the G6PD activity is detected in the cumulus cells, but not in the oocyte, during IVM.

Mice homozygous for an insertional mutation in the Zp3 gene lack a zona pellucida and are infertile

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2903-2910 ◽  
Author(s):  
T. Rankin ◽  
M. Familari ◽  
E. Lee ◽  
A. Ginsberg ◽  
N. Dwyer ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Genetic Defect ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Preimplantation Embryos ◽  
Germinal Vesicle Breakdown ◽  
Female Mice ◽  
Insertional Mutation ◽  
Homozygous Mutant

Mammalian oocytes synthesize and secrete a zona pellucida that surrounds the growing oocytes, ovulated eggs and preimplantation embryos. The extracellular zona matrix is composed of three glycoproteins (ZP1, ZP2, ZP3) that are involved in folliculogenesis, species-specific fertilization, and passage of the early embryo down the oviduct. We have established a mouse line in which Zp3 has been inactivated by homologous recombination with an insertional mutation. Neither Zp3 transcripts nor ZP3 protein was detected in female mice homozygous for the mutation (Zp3−/−), whereas both ZP1 and ZP2 were present in mutant oocytes. Homozygous mutant Zp3−/− mice had follicles with germinal-vesicle-intact oocytes but that lacked a zona pellucida matrix and had a disorganized corona radiata. Although mutant oocytes underwent germinal vesicle breakdown (GVBD) prior to ovulation, the cumulus-oocyte complex was markedly disrupted and the oocytes were often separate from the cumulus cells. After hormone-induced ovulation, cumulus masses were present in the oviducts of homozygous mutant mice, but zona-free eggs were observed in only half of the females and, in these, less than 10% of the normal number [correction of mumber] of eggs were detected. No zona-free 2-cell embryos were recovered from homozygous mutant Zp3−/− female mice after mating with males proven to be fertile, and none became visibly pregnant or produced offspring. These results demonstrate that a genetic defect in a zona pellucida gene causes infertility and, given the conserved nature of the zona pellucida, a similar phenotype is expected in other mammals.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

324 FUNCTIONAL ANALYSIS USING SHORT-INTERFERING (si)RNA OF TRAM-6, A NOVEL TRANSCRIPT ASSOCIATED WITH OOCYTES MATURATION IN CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 269
Author(s):  
C. E. Farin ◽  
J. R. Sommer ◽  
K. F. Rodriguez ◽  
R. M. Petters ◽  
J. E. Alexander
Keyword(s):  
Functional Role ◽  
Germinal Vesicle ◽  
Vascular Endothelial ◽  
Germinal Vesicle Breakdown ◽  
Cumulus Expansion ◽  
Early Maturation ◽  
Novel Transcript ◽  
Maturation Medium ◽  
Si Rna ◽  
Endothelial Growth Factor

Maturation of cumulus–oocyte complexes (COCs) with gonadotropins requires transcription of new mRNAs. When COCs are cultured with FSH and the transcriptional inhibitor DRB, cumulus expansion and germinal vesicle breakdown (GVBD) are arrested. Differential mRNA display was used to identify a novel transcript associated with maturation of COCs (TRAM-6) that is expressed during early maturation but not when maturation is inhibited by DRB. The objective of this study was to use siRNAs targeted against TRAM-6 mRNA to assess its functional role in oocyte maturation. Exp. 1: Pools of 60–10 bovine COCs (n = 5/trt) were randomly assigned to culture in maturation medium consisting of 0.5 mL TCM-199 with 2.5 μg FSH, 0.5 μg estradiol, and 10% estrous cow serum with or without DRB (120 μM) or with increasing doses of siTRAM-6 (25, 50, or 100 nM). Exp. 2: COC pools (n = 4/trt) were cultured in maturation medium with one of the following treatments: control, 120 μM DRB, 100 nM non-specific siRNA (siNS), or 100 nM siTRAM-6. After 4 h of culture, COC pools were used to assess levels of mRNA for TRAM-6, VEGF (vascular endothelial growth factor), and GAPD (glyceraldehyde-3-phosphate dehydrogenase) by semiquantitative RT-PCR. Exp. 3: COCs were cultured in maturation medium for either 8 h (n = 9 pools/trt) or 20 h (n = 7 pools/trt) with one of the following treatments: control, 120 µM DRB, 100 nM siNS, or 100 nM siTRAM-6. Cumulus expansion was graded every 4 h. At the end of culture, a subset of COCs from each treatment was used to assess oocyte meiotic stage. Remaining COCs were used to assess TRAM-6, VEGF, and GAPD mRNA. Data were analyzed using ANOVA and Duncan's test. At 4 h of culture, relative expression of TRAM-6:GAPD mRNA (least squares means ± SEM) was decreased in the DRB and the 100 nM siTRAM-6 treatments but was unaffected by 25 nM TRAM-6, 50 nM siTRAM-6, or 100 nM siNS (Exp. 1: 100 ± 13%a, 17 ± 13%b, 101 ± 13%a, 60 ± 13%ab and 48 ± 13%b for control, DRB, 25 nM siTRAM-6, 50 nM siTRAM-6, and 100 nM siTRAM-6, respectively; abcP < 0.05; Exp. 2: 100 ± 10%a, 14 ± 10%b, 106 ± 10%a and 68 ± 10%c for control, DRB, 100 nM siNS, and 100 nM siTRAM-6, respectively; abcP < 0.05). There was no effect of treatment on expression of VEGF and GAPD mRNA, both of which are present in COCs at the start of culture and are unrelated to TRAM-6 mRNA. At 8 h, GVBD was inhibited by treatment with DRB and siTRAM-6 (68 ± 8%a, 3 ± 8%b, 72 ± 8%a, and 30 ± 8%c for control, DRB, siNSs and siTRAM-6, respectively; abcP < 0.05). At 20 h, the incidence of metaphase II (MII) oocytes was decreased in the DRB and siTRAM-6 groups (96 ± 8%a, 9 ± 8%b, 94 ± 8%a, and 56 ± 8%c for control, DRB, siNS, and siTRAM-6, respectively; abcP < 0.05). Cumulus expansion was inhibited (P < 0.05) by DRB but was not affected by any other treatment. In summary, TRAM-6 siRNA decreased the expression of TRAM-6 mRNA in bovine COCs at 4 h of culture as well as the proportions of oocytes in GVBD at 8 h and in MII at 20 h of culture, but did not affect cumulus expansion. In conclusion, these data are consistent with the identification of a novel mRNA transcript, TRAM-6, that has a functional role in regulating meiotic maturation in bovine COCs. This work was supported by USDA Grant #2002–35205–12810.

Time course of the meiotic arrest in sheep cumulus–oocyte complexes treated with roscovitine

Zygote ◽  
2015 ◽  
Vol 24 (2) ◽  
pp. 310-318 ◽  
Author(s):  
Letícia Ferrari Crocomo ◽  
Wolff Camargo Marques Filho ◽  
Camila Louise Ackermann ◽  
Daniela Martins Paschoal ◽  
Midyan Daroz Guastali ◽  
...  
Keyword(s):  
Exposure Time ◽  
Cell Expansion ◽  
Time Course ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Maturation Medium

SummaryTemporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus–oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

The effect of PD98059 on MAPK regulation in cumulus-enclosed and cumulus-free mouse oocytes

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Jaroslav Kalous ◽  
Michal Kubelka ◽  
Jan Motlík
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Experimental Conditions ◽  
Germinal Vesicle Breakdown ◽  
Mapk Activation ◽  
Mapk Phosphorylation ◽  
Kinase Activation ◽  
Mouse Oocytes ◽  
Significant Difference ◽  
Epidermal Growth

The effect of the p42/44 mitogen-activated kinase (MAPK) inhibitor, PD98059, on MAPK activation and meiosis resumption in mouse oocytes was studied. When germinal vesicle (GV)-stage denuded oocytes (DOs) were cultured continuously in 50 μM PD98059, germinal vesicle breakdown (GVBD) was postponed for 2-3 h. MAPK phosphorylation and activation was delayed as well. However, PD98059 did not impair histone H1 kinase activation. After 14 h of culture there was no significant difference in the rate of DOs reaching metaphase II (MII) arrest in either control or experimental conditions. The effect of PD98059 on MAPK inhibition was further tested in epidermal growth factor (EGF)-treated oocyte–cumulus complexes (OCCs). Exposure of GV-stage OCCs for 5 min to EGF (10 ng/ml) induced a considerable increase in MAPK phosphorylation. After OCCs were further cultured in 50 μM PD98059 a rapid dephosphorylation of MAPK was induced. Already after 1 min of treatment the non-phosphorylated form of MAPK dominated, indicating the high effectivity of PD98059. This result indicates that short EGF/PD98059 treatment of OCCs induced MAPK phosphorylation/dephosphorylation in cumulus cells only. As only a transient delay in MAPK phosphorylation and activation was observed in PD98059-treated DOs we conclude that there is also another PD98059-nonsensitive pathway(s) leading to MAPK activation in mouse oocytes. The data obtained suggest that meiosis resumption in mouse oocytes is somehow influenced by the MEK/MAPK activation pathway.

scholarly journals Cyclic Adenosine 3′,5′-Monophosphate-Dependent Activation of Mitogen-Activated Protein Kinase in Cumulus Cells Is Essential for Germinal Vesicle Breakdown of Porcine Cumulus-Enclosed Oocytes

Endocrinology ◽  
2005 ◽  
Vol 146 (10) ◽  
pp. 4437-4444 ◽  
Author(s):  
Cheng-Guang Liang ◽  
Li-Jun Huo ◽  
Zhi-Sheng Zhong ◽  
Da-Yuan Chen ◽  
Heide Schatten ◽  
...  
Keyword(s):  
Mapk Pathway ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Mitogen Activated Protein Kinase ◽  
Pde4 Inhibitor ◽  
Germinal Vesicle Breakdown ◽  
Germinal Vesicles ◽  
Mitogen Activated Protein ◽  
Culture Models ◽  
Cell Type Specific

MAPK plays an important role during meiotic maturation in mammalian oocytes, whereas the necessity of MAPK during meiotic resumption in porcine oocytes is still controversial. Here, by applying the method of ultracentrifugation to move the opaque lipid droplets to the edge of the oocyte, therefore allowing clear visualization of porcine germinal vesicles, oocytes just before germinal vesicle breakdown (GVBD) and those that had just undergone GVBD were selected for the assay of MAPK activation. Our results showed that phosphorylation of MAPK in oocytes occurred after GVBD in all three different culture models: spontaneous maturation model, inhibition-induction maturation model, and normal maturation model. Moreover, we found that activation of MAPK in cumulus cells but not in oocytes was essential for GVBD in cumulus-enclosed oocytes. Then the cross-talk between cAMP and MAPK in cumulus cells was investigated by using cell-type-specific phosphodiesterase (PDE) isoenzyme inhibitors. Our results showed that PDE3 subtype existed in oocytes, whereas PDE4 subtype existed in cumulus cells. PDE3 inhibitor prevented meiotic resumption of oocytes, whereas PDE4 inhibitor enhanced the ability of FSH or forskolin to activate MAPK in cumulus cells. We propose that increased cAMP resulting from inhibition of PDE3 in oocytes blocks GVBD, whereas increased cAMP resulting from inhibition of PDE4 activates MAPK pathway in cumulus cells, which is essential for GVBD induction.

scholarly journals Activity of key enzymes involved in glucose and triglyceride catabolism during bovine oocyte maturation in vitro

Reproduction ◽  
2002 ◽  
pp. 675-681 ◽  
Author(s):  
P Cetica ◽  
L Pintos ◽  
G Dalvit ◽  
M Beconi
Keyword(s):  
Enzymatic Activity ◽  
Pentose Phosphate Pathway ◽  
In Vitro Maturation ◽  
Specific Activity ◽  
Lipolytic Activity ◽  
Cumulus Cells ◽  
Pentose Phosphate ◽  
Key Enzymes ◽  
Glucose 6 Phosphate Dehydrogenase

Little is known about the metabolic profile of cumulus-oocyte complexes (COCs) during maturation. The aim of this study was to determine the differential participation of enzymatic activity in cumulus cells and the oocyte during in vitro maturation of bovine oocytes, by measuring the activity of key enzymes involved in the regulation of glycolysis (phosphofructokinase), the pentose phosphate pathway (glucose-6-phosphate dehydrogenase) and lipolysis (lipase). COCs were matured in medium 199 plus 10% (v/v) steer serum for 22-24 h at 39 degrees C in 5% CO(2):95% humidified air. Phosphofructokinase, glucose-6-phosphate dehydrogenase and lipase activities were measured in immature and in vitro matured COCs, denuded oocytes and cumulus cells, respectively. Phosphofructokinase and glucose-6-phosphate dehydrogenase activities (enzymatic units) remained constant during in vitro maturation of COCs, but there was a significant decrease in lipase activity (units) (P < 0.05), as activity in cumulus cells decreased significantly (P < 0.05). For the three enzymes studied, enzyme activity (units) remained unchanged in the oocyte during in vitro maturation. Specific activity increased in the oocyte (P < 0.05) and decreased in cumulus cells as a result of maturation (P < 0.05). In cumulus cells, phosphofructokinase was the most abundant of the three enzymes followed by glucose-6-phosphate dehydrogenase and then lipase (P < 0.05), whereas in the denuded oocyte this order was reversed (P < 0.05). Thus, the metabolism of cumulus cells is adapted to control the flow of metabolites toward the oocyte, which maintains its enzymatic activity even when dissociated from cumulus cells during maturation. The high activity of phosphofructokinase in cumulus cells indicates that glucose is metabolized mainly via the glycolytic pathway in these cells. The greater relative activity of glucose-6-phosphate dehydrogenase recorded in the oocyte indicates that glucose uptake could be directed mainly toward the pentose phosphate pathway. The marked lipolytic activity concentrated in the oocyte indicates an active participation in lipid catabolism during maturation.

344 PENTOSE PHOSPHATE PATHWAY ACTIVITY CONTROLS NUCLEAR MATURATION OF PORCINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 279 ◽  
Author(s):  
L. Tubman ◽  
A. Peter ◽  
R. Krisher
Keyword(s):  
Pentose Phosphate Pathway ◽  
Control Level ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Germinal Vesicle ◽  
Pentose Phosphate ◽  
Meiotic Stage ◽  
Germinal Vesicle Breakdown ◽  
Pathway Activity ◽  
Nuclear Maturation

Diphenyleneiodonium (DPI), an inhibitor of the pentose phosphate pathway (PPP), arrests nuclear maturation of porcine oocytes. This inhibition is reversed using products or cofactors of PPP such as nicotinamide adenine dinucleotide phosphate (NADP), phosphoribose diphosphate (PRPP), and ribose-5-phosphate (R5P). The objective of this study was to determine the relationship between DPI-mediated meiotic inhibition, reversal of this inhibition, and metabolism of in vitro-matured (IVM) porcine oocytes. Oocytes were aspirated, searched, and selected in the presence of DPI, with the exception of control oocytes. Oocytes were then matured in one of five treatments for 40 h in 7% CO2 in air at 39°C in defined Purdue Porcine Medium for maturation (PPMmat). Treatments included control, 50 nM DPI (DPI), DPI + 5 mM NADP (NADP), DPI + 12.5 mM PRPP (PRPP), and DPI + 10 mM R5P (R5P). Following IVM, oocytes were denuded by vortexing. Glycolysis and PPP activities were measured in 4 μL hanging drops containing labeled glucose (0.0125 mM 5-3H glucose and 0.482 mM 1-14C glucose, respectively) for 3 h in 6% CO2. Oocytes were then individually fixed in a 3:2:1 solution of ethanol:acetic acid:chloroform and stained with aceto-orcein for determination of meiotic stage (germinal vesicle = 1 through metaphase II = 7). Data were analyzed using one-way ANOVA. The use of DPI inhibited PPP and nuclear maturation; additionally glycolysis was decreased by DPI compared to control. Addition of NADP and PRPP increased both metabolic pathways and nuclear maturation compared to DPI. R5P restored glycolysis and nuclear maturation to control levels, and PPP to above the control level. There were no significant differences among meiotic stages relative to glycolytic activity. PPP activity was significantly different (values with different superscripts; P < 0.05) among oocytes of different meiotic stages (germinal vesicle = 0.24 ± 0.03ad, germinal vesicle breakdown = 0.40 ± 0.05bcde, condensed chromatin = 0.44 ± 0.05bcd, metaphase I = 0.45 ± 0.12abcd, anaphase = 0.76 ± 0.50abcde, telophase = 0.92 ± 0.17be, metaphase II = 0.74 ± 0.08be). Percentages of oocytes reaching MII were 43.48 (control), 2.08 (DPI), 28.30 (NADP), 18.18 (PRPP), and 46.94 (R5P). These results demonstrate that the PPP is a critical control mechanism for nuclear maturation of porcine oocytes, as inhibition of this metabolic pathway resulted in arrest of nuclear maturation. Addition of PPP cofactors or end products to the arresting medium led to reversal of inhibition as demonstrated by restoration of PPP activity resulting in nuclear maturation. Table 1. Meiotic stage, glycolysis, and pentose phosphate pathway activity after in vitro maturation of porcine oocytes

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