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.

341 IN VITRO MATURATION AND IN VITRO FERTILIZATION OF ALPACA (VICUGNA PACOS) OOCYTES: EFFECT OF TIME OF INCUBATION ON NUCLEAR MATURATION AND CLEAVAGE

2010 ◽  
Vol 22 (1) ◽  
pp. 327 ◽  
Author(s):  
W. Huanca ◽  
R. Condori ◽  
J. Cainzos ◽  
M. Chileno ◽  
L. Quintela ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hypodermic Needle ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Time ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Maturation Medium

Experiments were carried out to evaluate the effect of incubation time on nuclear maturation (Experiment 1) and determine the cleavage rate of alpaca oocytes after of IVF time (Experiment 2) In Experiment 1, CCOs were collected from slaughterhouse ovaries and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. CCOs were aspirated from follicles >2 mm and pooled in a conical tube to sedimentation previous to evaluation under stereomicroscope and CCOs with a cytoplasm homogeneous and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of maturation medium TCM-199 supplemented with 10% FCS (v : v) plus 0.5 μg mL-1 FSH, 10 μg mL-1 hCG, 0.2 mM sodium pyruvate, 50 μg mL-1 gentamicine, and 1 μg mL-1 Estradiol under mineral oil with 10-12 oocytes/drop. Oocytes were incubated under the following maturation times: 30, 34, and 38 h at 39°C in an atmosphere of 5% CO2 and high humidity. After each maturation time, CCOs were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mgmL-1 of hyaluronidase and fixed in ethanol: acetic acid (3 : 1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min The slides were examined under a phase contrast microscope at × 400 to evaluate status of nuclear maturation and classified as germinal vesicle (GV); metaphase I (M-I), anaphase-telophase; metaphase II (M-II) and degenerated. Experiment 2: The same maturation method as Experiment 1 was used. Testes were collected of mature males from slaughterhouse and transported to the laboratory. Caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation: 700 g on a Percoll discontinuous gradient (22.5 :45.0%) for 25 min. The supernatant was removed by aspiration and pellet (containing viable spermatozoa) was resuspended in TL stock. Spermatozoa and oocytes were co-incubated for 18-20 h at 39°C with 5% CO2 and then cultivated in TCM-199 supplemented with 10% FCS (v: v), 0.2 mM sodium pyruvate, and 50 μg mL-1 gentamicine and evaluated at 48 h. Data were subjected to ANOVA. For Experiment 1, the proportions of oocytes reaching M-II stage was 18.9 ± 15.7, 42.9 ± 16.2, and 65.8 ± 8.1% for the 30, 34, and 38 h of culture, respectively, with difference to maturation time (P < 0.05). For Experiment 2, the cleavage rate was 9.5, 7.7, and 15.4% to 30, 34, and 38 h after of fertilization time 48 h culture. These results indicate that 38 or more h is required for the maturation and fertilization of alpaca oocytes. Grant 064 FINCyT-PIBAP 2008.

Effect of maturation medium on in vitro cleavage of canine oocytes fertilized with fresh and cooled homologous semen

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 43-53 ◽  
Author(s):  
B. de Ávila Rodrigues ◽  
L. Carboneiro dos Santos ◽  
J.L. Rodrigues
Keyword(s):  
Cell Expansion ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Final Concentration ◽  
Comparative Experiment ◽  
Cell Stage ◽  
Maturation Medium ◽  
Developmental Capacity ◽  
Fresh Semen

SUMMARYThis study evaluated the effect of three maturation media on the development of in vitro-matured and in vitro-fertilized dog oocytes. In Experiment 1 (non-comparative experiment) canine cumulus–oocyte complexes (COCs) were matured in vitro in TCM199 supplemented with estrous cow serum (10%) + gonadotropins + steroid (treatment A), TCM199 + estrous cow serum (10%) (treatment B), or TCM199 + polyvinylpyrrolidone (PVP) (4%) (treatment C). All maturation media contained a final concentration of 1 μg/ml of human somatotropin (hST). Oocytes were fertilized with fresh ejaculated sperm and development was assessed by cleavage. The objective of Experiment 2 (comparative experiment) was to compare the rates of cleavage and developmental capacity of COCs matured in vitro in same medium as in Experiment 1, and fertilized either with fresh ejaculated or with cooled extended homologous spermatozoa. In Experiments 1 and 2, oocytes fertilized with fresh semen were in vitro-matured for 48 h, while in Experiment 2 COCs fertilized with cooled semen were matured in vitro for 72 h. The results of Experiments 1 and 2 demonstrated that cleavage was not influenced by the oocyte's maturation environment. The results of Experiment 1 showed that pronucleus formation + cleavage (day 7 after IVF) was similar among treatments A, B and C (p = 0.277). Also, in Experiment 2, pronucleus formation + cleavage (day 7 after IVF) was not different for oocytes fertilized in vitro either with fresh or cooled semen and maturated in media A (p = 0.190), B (p = 0.393) or C (p = 0.687). In both experiments, the numbers of embryos that developed to the 6–8-cell stage were higher for oocytes matured in medium A and fertilized with fresh semen, when compared with numbers of oocytes matured in media B and C. Embryo development to the 6–8-cell stage of oocytes fertilized either with fresh or cooled sperm was observed in treatments A and C in Experiment 2. Cumulus cell expansion was similar among treatments in Experiment 1. In Experiment 2, cumulus cell expansion among treatments A, B and C was similar after 48 h or 72 h of IVM. In both experiments, the greatest expansion category seen was for category 2 (outer cumulus cells slightly expanded). No correlation between cumulus expansion and cleavage were observed. Polyspermy rates in oocytes matured in medium A, and fertilized with fresh sperm were not significantly different from polyspermy rates observed using media B and C, in both experiments. Our findings indicate that treatments A, B and C are similarly effective for the cleavage of dog oocytes. Furthermore, it was demonstrated that canine oocytes matured in vitro could be fertilized by homologous cooled spermatozoa and progress to cleavage.

scholarly journals Prediction of maturational competence of feline oocytes using supravital staining of cumulus cells by propidium iodide

Zygote ◽  
2011 ◽  
Vol 20 (4) ◽  
pp. 333-337 ◽  
Author(s):  
Kenzo Uchikura ◽  
Masashi Nagano ◽  
Mitsugu Hishinuma
Keyword(s):  
Propidium Iodide ◽  
Cumulus Cell ◽  
Membrane Integrity ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Non Invasive ◽  
Maturation Rate ◽  
Cell Layers ◽  
The Relationship

SummaryWe examined the relationship between integrity of cumulus cells and nuclear maturation rate after in vitro culture to determine a non-invasive prediction of the maturational competence of feline oocytes. Feline cumulus–oocyte complexes (COCs) were collected from either small (400–800 μm) or large (≥800 μm) follicles. Immediately after collection, cumulus cells were evaluated morphologically (thickness of cumulus cell layers) and stained with propidium iodide (PI), which penetrates only non-viable cells. Cumulus cells without PI staining were judged as having good membrane integrity. After evaluation, COCs were cultured for 30 h and their nuclear maturation rate was determined. The nuclear maturation rate of oocytes derived from large follicles (89.8%) was higher (p < 0.05) than that from small follicles (60.8%). There was no difference in the maturation rate of oocytes from follicles with the same size regardless of cumulus morphology. In contrast, oocytes that had cumulus cells with good membrane integrity showed a higher maturation rate (93.8%) than oocytes with poor cumulus integrity (76.9%) in large follicles (p < 0.05). We conclude that evaluation of membrane integrity of cumulus cells by propidium iodide staining can be used to predict the maturational competence of oocytes.

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

83 VITRIFICATION OF IMMATURE AND IN VITRO-MATURED HORSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 149 ◽  
Author(s):  
L. Bogliolo ◽  
F. Ariu ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Survival Rates ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hoechst 33342 ◽  
Nuclear Maturation ◽  
And Control ◽  
Cryopreservation Protocol

Few attempts have been carried out to cryopreserve equine oocytes, and an effective cryopreservation protocol is not defined yet. Studies were conducted to compare the viability of immature and in vitro-matured horse oocytes vitrified by the minimal volume cooling (MVC) cryotop vitrification method (Kuwayama et al. 2005 Reprod. BioMed. Online 11, 300–308). Oocytes were recovered from slaughterhouse ovaries and divided, on the basis of the morphology of cumulus cells, into cumulus-expanded (CE) and cumulus-compacted (CC) oocytes. Groups of CC and CE oocytes were vitrified immediately after recovery [germinal vesicle (GV) stage] or matured in vitro (IVM) and cryopreserved at the MII stage as follows: oocytes were incubated 30 min in TCM-199 + 20% FCS + 10% ethylene glycol (EG) + 10% DMSO, followed by 20 min in TCM-199 + 20% FCS + 20% EG + 20% DMSO + 0.25 M sucrose, loaded in cryotops (2 µL), and plunged into liquid nitrogen. Warming was performed at 38.5°C by washing the oocytes in TCM-199 + 20% FCS with decreasing sucrose concentrations (1.25 M, 0.62 M, 0.31 M). After warming oocytes cryopreserved at the GV stage were matured in vitro for 24 h (CE) or 36 h (CC) in TCM-199 + 10% FCS + FSH, LH each at (0.1 UI/mL) + cysteamine, fixed, and stained with glycerol-Hoechst 33342 to assess nuclear maturation. Oocytes vitrified at the MII stage were in vitro cultured for 2 h to evaluate their morphological survival on the basis of the presence of an intact zona pellucida and membrane. Nonvitrified oocytes undergoing the same maturation protocol were used as controls. Results (Table 1) indicated that the survival rate of oocytes vitrified at the GV stage, after IVM, was similar between CE and CC oocytes (43.6% vs 42.6%). Significantly (P < 0.01) higher numbers of vitrified CE MII oocytes (52.9%) survived, compared to CC (34.8%), after 2-h culture. The percentages of viable MII oocytes from CE and CC GV vitrified oocytes were 43.6% and 40.9% respectively and were comparable to those from vitrified MII oocytes (CE, 52.9%; CC, 34.8%) and control oocytes (CE, 56.4%; CC, 53.3%). In conclusion, the results of this study showed that vitrification by the MCV Cryotop method of horse oocytes at either the GV or the MII stage allows a similar number of viable mature oocytes to be recovered. Table 1. Maturation and survival rates of immature and mature equine oocytes vitrified by the MCV Cryotop method

342 EFFECTS OF MELATONIN ON PREIMPLANTATION DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 327 ◽  
Author(s):  
J.-T. Kang ◽  
O.-J. Koo ◽  
D.-K. Kwon ◽  
S.-J. Park ◽  
M. Atikuzzaman ◽  
...  
Keyword(s):  
Treatment Group ◽  
Beneficial Effect ◽  
Real Time ◽  
Cell Expansion ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Pcr Analysis ◽  
Melatonin Treatment ◽  
Parthenogenetic Embryos

In mammalian species, melatonin is a multi-functional pineal gland hormone that regulates several circadian and seasonal rhythms including reproduction. However, the melatonin study was not common as to the oocytes in the pig. Recently, we reported that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine oocyte IVM and we also reported an existence of melatonin receptor on the cumulus cells and granulose cells (Kang JT et al. 2009 J. Pineal Res. 46(1), 22-28). In this study, as adding further experiments rather than our previous study, we investigated effect of exogeneous melatonin (10 ng mL-1) on the porcine oocytes and analyzed possible factors which can be responsible for that results. Oocytes were recovered by aspiration of slaughterhouse ovaries, and then matured in TCM-199 supplemented with EGF, insulin, pyruvate, cystine, and gonadotropin. Expression of apoptosis-related genes mRNA in oocytes cultured with melatonin were evaluated by real-time PCR (Exp 1), cumulus cell expansion on COC was assessed on the microscopes during in vitro maturation (Exp 2), and developmental effects between melatonin treatement group and non-treatment group on the in vitro culture of parthenogenetically activated oocytes was investigated (Exp 3). In results, oocytes matured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl (anti-apoptotic gene) and Bax (proapoptotic gene) by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher compared to the control while the expression of Bax was decreased relative to the control (P < 0.05). Cumulus cell expansion was evaluated under a stereomicroscope at 22 h, 44 h during IVM. Representative photomicrographs of porcine COC at the start of the IVM, after 22 h and 44 h treatment with melatonin, are shown in Figure. After 22 h of melatonin treatment, cumulus cells were visually expanded compared with non-treatment group. We analyzed significantly greater proportion of parthenogenetically activated oocytes developed to blastocyst when the IVM medium was supplemented with melatonin. Melatonin treatment in the IVM has consequently beneficial effect on the blastocyst formation rates on the development of porcine parthenogenetic embryos (15.4%) compared to non-treatment group (10.7%, P < 0.05). However, cleavage frequency was not affectedby the treatment. In conclusion, the present study demonstrated that melatonin had a beneficial effect on the development of parthenogenetically activated porcine embryos, probably through decreased apoptosis rate and increased cumulus cell expansion. This study was supported by Korean MKE, MEST (BK21 program), and Hanhwa L&C

336 MEIOTIC DEVELOPMENT AND CORTICAL GRANULES DISTRIBUTION IN CANINE OOCYTES DURING IN VITRO MATURATION

2010 ◽  
Vol 22 (1) ◽  
pp. 324 ◽  
Author(s):  
M. De los Reyes ◽  
D. Luna ◽  
J. Palomino
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Cumulus Cells ◽  
Homogeneous Distribution ◽  
Cortical Granules ◽  
Dapi Staining ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Low development of IVM canine oocytes could be in part attributed to an impaired cytoplasmic maturation. In mammalian oocytes, migration and the redistribution of cortical granules (CGs) around the periphery of the oocyte contribute to the inhibition of polyspermy and it is an important criterion to evaluate cytoplasmic maturation. The state of nuclear maturation and the distribution of CGs were evaluated in canine oocytes cultured for different periods in order to compare the synchrony of nuclear and cytoplasmic maturation during in vitro maturation. Bitch ovaries at different stages of the estrous cycle were obtained following ovariectomy. COCs with compact cumulus cells showing a homogeneous cytoplasm were selected for experiments. Thirty-six COCs were processed at immature stage, placed in PBS medium until evaluation. A total of 275 COCs were matured in vitro for 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM Hepes, 10% FCS, 0.25 mM pyruvate, 10 IU mL-1 of hCG, 300 IU mL-1 penicillin, and 20 mg mL-1 streptomycin, at 38.5°C and 5% CO2. At each culture period, the oocytes were stained with Lens culinaris agglutinin (LCA), labeled with fluorescein isothiocyanate, and the CGs distributions were examined under a fluorescent microscope. The nuclear status of the denuded oocytes was determined by DAPI staining under a fluorescence microscope. For each treatment, at least four replicates were performed and the data was analyzed by ANOVA using Tukey’s test to determine the differences P < 0.05. Three types of CGs distribution were distinguished during canine oocyte maturation: (1) homogeneous distribution throughout the cytoplasm including the cortex; (2) heterogeneous (clusters) within the cytoplasm and (3) densely distributed beneath the oolemma. Nuclear stages were classified as immature or germinal vesicle (GV) stage; resumption of meiosis or germinal vesicle break down (GVBD); metaphase I to telophase I (MI toTel I); and mature or second metaphase (MII). The distribution patterns of GCs were different (P < 0.05) among oocytes cultured for different periods and the nuclear maturation status also differed between oocytes cultured for different intervals (P < 0.05). Most (>84%) of the immature oocytes at GV showed a uniform distribution of CGs throughout the cytoplasm. At 48 h of culture, CGs distribution was mainly Type 2 (25%) and 3 (61%) and the oocytes were at GVBD (33%) and MI-Tel I (33%) stages. Most nuclei of the type 3 oocytes were in the MI (40%) and MII (11%) stages, corresponding to those oocytes matured for 72 (88%) or 96 h (71%). These results indicate that canine oocytes migrate to the cortex during IVM and this process is not finished before 72 h of culture. In addition, although the re-distribution of the CGs occurred in parallel with nuclear maturation, the oocytes cannot always proceed to the MII stage; however, in such oocytes the CGs are distributed beneath the oolemma. Supported by Grant FONDECYT 1080618.

60 EFFECTS OF AY9944 A-7 ON MEIOTIC RESUMPTION OF PORCINE OOCYTES AND CUMULUS CELL EXPANSION

2016 ◽  
Vol 28 (2) ◽  
pp. 160
Author(s):  
S. Lee ◽  
C. Khoirinaya ◽  
J.-X. Jin ◽  
G. A. Kim ◽  
B.-C. Lee
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Cell Expansion ◽  
Cholesterol Biosynthesis ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Cumulus Expansion

In vitro studies on mammalian oocytes have shown that follicular fluid-meiosis activating sterol (FF-MAS) can overcome the inhibitory effect of hypoxanthine (Hx) on the resumption of meiosis. FF-MAS, an intermediate in the cholesterol biosynthesis pathway, is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. AY9944 A-7, an inhibitor of Δ14-reductase and Δ7-reductase, induces accumulation of FF-MAS by inhibiting its metabolism. The aim of this study was to evaluate the effects of AY9944 A-7 on meiotic resumption of porcine oocytes, cumulus cell expansion, and gene expression related to M-phase-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and oocyte maturation in oocytes and related to cumulus expansion in cumulus cells. In experiment 1, 1136 cumulus-oocyte complexes (COCs) were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in addition to a meiotic inhibitor (Hx, 4 mM) for 44 h. Oocytes treated with 10 and 20 μM AY9944 A-7 in the presence of Hx had significantly higher GVBD and M2 rates than the control group. However, 40 μM AY9944 A-7 significantly decreased GVBD and M2 rates and increased degeneration of oocytes compared with other groups. In experiment 2, 600 COCs were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in the absence of Hx for 44 h. Cumulus expansion of 40 μM AY9944 A-7 treated group was significantly decreased compared with other groups. In experiment 3, we evaluate the effects of AY9944 A-7 on gene expression, and the experiment was replicated four times. Data on gene expression were analysed using Student’s t-test. Oocytes treated with 10 μM AY9944 A-7 increased expression of genes involved in MPF (Cyclin B and Cdc2), MAPK (C-mos), and oocyte maturation (GDF9 and BMP15). Cumulus cells treated with 10 μM AY9944 A-7 decreased cumulus expansion-related genes (Has2, Tnfaip6, Ptgs2, and Ptx-3). In conclusion, our results suggest that although 10 μM AY9944 A-7 decreased cumulus expansion-related genes, there was no difference in cumulus expansion and it induced meiotic resumption of porcine oocytes with increased MPF, MAPK, and oocyte maturation-related genes. Further studies are needed to evaluate the effect of AY9944 A-7 on porcine embryo development. This study was supported by Ministry Of Trade, Industry & Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

scholarly journals Obox4-silencing-activated STAT3 and MPF/MAPK signaling accelerate nuclear membrane breakdown in mouse oocytes

Reproduction ◽  
2016 ◽  
Vol 151 (4) ◽  
pp. 369-378 ◽  
Author(s):  
Hyun-Seo Lee ◽  
Kyeoung-Hwa Kim ◽  
Eun-Young Kim ◽  
Su-Yeon Lee ◽  
Jung-Jae Ko ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Molecular Mechanisms ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Mapk Signaling ◽  
Interferon Α ◽  
Membrane Breakdown ◽  
Nuclear Maturation ◽  
Mouse Oocytes

Mouse oocytes begin to maturein vitroonce liberated from ovarian follicles. Previously, we showed that oocyte-specific homeobox 4 (Obox4) is critical for maintaining the intact nuclear membrane of the germinal vesicle (GV) in oocytes and for completing meiosis at the metaphase I–II (MI–MII) transition. This study further examines the molecular mechanisms of OBOX4 in regulating GV nuclear membrane breakdown. Maturation-promoting factor (MPF) and MAPK are normally inactive in GV stage oocytes but were activated prematurely in arrested GV stage oocytes by 3-isobutyl-1-metyl-xanthine (IBMX)in vitroafterObox4RNA interference (RNAi). Furthermore, signal transducer and activator of transcription 3 (STAT3) was significantly activated byObox4RNAi. We confirmed that thisObox4RNAi-induced premature STAT3 and MPF/MAPK activation at the GV stage provoked subsequent GV breakdown (GVBD) despite the opposing force of high cAMP in the IBMX-supplemented medium to maintain intact GV. When cumulus–oocyte complexes were exposed to interferon α (IFNA), a STAT3 activator, oocytes matured and cumulus cells expanded to resume nuclear maturation in IBMX-supplemented medium, suggesting that STAT3 activation is sufficient for stimulating the continuation of meiosis. Using Stattic, a specific STAT3 inhibitor, we confirmed that GVBD involves STAT3 activation inObox4-silenced oocytes. Based on these findings, we concluded that i)Obox4is an important upstream regulator of MPF/MAPK and STAT3 signaling, and ii)Obox4is a key regulator of the GV arrest mechanism in oocytes.

Bovine in vitro oocyte maturation as a model for manipulation of the γ-glutamyl cycle and intraoocyte glutathione

2008 ◽  
Vol 20 (5) ◽  
pp. 579 ◽  
Author(s):  
E. C. Curnow ◽  
J. Ryan ◽  
D. Saunders ◽  
E. S. Hayes
Keyword(s):  
Oocyte Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Buthionine Sulfoximine ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Maturation Medium

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.

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