Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes

Zygote ◽  
2003 ◽  
Vol 11 (3) ◽  
pp. 199-206 ◽  
Author(s):  
Tamás Somfai ◽  
Kazuhiro Kikuchi ◽  
Akira Onishi ◽  
Masaki Iwamoto ◽  
Dai-ichiro Fuchimoto ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Intracellular Level ◽  
Dibutyryl Cyclic Amp ◽  
Oocyte Collection ◽  
Porcine Oocyte ◽  
Blastocyst Rate

We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 μg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.

241 DIBUTYRYL CYCLIC AMP AND EGF-LIKE FACTORS SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM WITHOUT GONADOTROPINS

2009 ◽  
Vol 21 (1) ◽  
pp. 218
Author(s):  
Y. Akaki ◽  
K. Yoshioka ◽  
H. Funahashi
Keyword(s):  
Cyclic Amp ◽  
In Vitro Maturation ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Chemically Defined Medium ◽  
Dibutyryl Cyclic Amp ◽  
Vitro Fertilization ◽  
Porcine Oocyte

Exposure of porcine oocyte–cumulus complexes (OCC) to gonadotropins induces meiotic resumption, but the details of this mechanism are still unknown. The present study was undertaken to examine combinational effects of EGF-like factors and dibutyryl cyclic AMP (dbcAMP) in a chemically defined medium on in vitro maturation (IVM) of porcine oocytes. The OCC were aspirated from 3- to 6-mm-diameter follicles of prepuberal ovaries and used in the current study. The basic culture medium was a chemically defined medium, Porcine Oocyte Medium (POM; Research Institute for the Functional Peptides, Yamagata, Japan). In the first experiment, various concentrations (0, 10, and 1000 ng mL–1) of EGF-like factors (EGF, amphiregulin, and betacellulin) were added to POM during an entire IVM period (44 h). In the second experiment, to determine the additive effect of EGF-like factors, each EGF-like factor with an effective concentration was combined with the others. In the last experiment, to examine the combined effect with dbcAMP, OCC were exposed to EGF (10 ng mL–1), amphiregulin (1000 ng mL–1), and dbcAMP (1 mm) during the first 20 h of IVM and then the culture was continued in the absence of EGF-like factors and dbcAMP. After culture, in all experiments, meiotic resumption and the progress of oocytes were examined after denuding, fixing, and staining. Statistical analyses was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In the first experiment, all treatments without supplementation with 10 ng mL–1 amphiregulin increased the incidence of oocytes maturing to the MII phase, as compared with controls (29.1 to 39.3% v. 11.1%, P < 0.05). In the second experiment, combinations with 2 kinds of EGF-like factor slightly (but not significantly) improved the percentage of oocytes at the MII stage (37.7 to 47.4%). In the last experiment, supplementation with 1 mm dbcAMP during the first 20 h of IVM, regardless of the presence of EGF-like factors, significantly increased the incidence of MII oocytes as compared with controls, whereas the incidence was the highest when 1 mm dbcAMP, 10 ng mL–1 EGF, and 1000 ng mL–1 amphiregulin were supplemented (75.5%). When those oocytes were cultured in a chemically defined medium after in vitro fertilization, the developmental competence of oocytes to the blastocyst stage (25.0%) was not different from oocytes matured in the presence of gonadotropins and dbcAMP during the first 20 h of IVM (17.3%). These observations indicate that supplementation of a chemically defined maturation medium with EGF-like factors and dbcAMP during the first 20 h of IVM can support the meiotic progress and developmental competence of porcine oocytes well. Currently, we are examining the developmental competence of those oocytes after embryo transfer. The results will be presented at the meeting. This study was supported by MAFF AgriBio1605.

Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression

1976 ◽  
Vol 22 (3) ◽  
pp. 531-545
Author(s):  
P.M. Wassarman ◽  
W.J. Josefowicz ◽  
G.E. Letourneau
Keyword(s):  
Cyclic Amp ◽  
Cytochalasin B ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Meiotic Maturation ◽  
Dibutyryl Cyclic Amp ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
First Polar Body

In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

Histone deacetylase inhibitor trichostatin A affects porcine oocyte maturation in vitro

2014 ◽  
Vol 26 (6) ◽  
pp. 806 ◽  
Author(s):  
Yong-Xun Jin ◽  
Ming-Hui Zhao ◽  
Zhong Zheng ◽  
Jung-Suk Kwon ◽  
Seul-Ki Lee ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Germinal Vesicle ◽  
Oocyte Quality ◽  
Histone Deacetylation ◽  
Developmental Competence ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Germinal Vesicle Breakdown ◽  
Porcine Oocyte

Previous studies show that porcine oocyte aging resulting from asynchronised IVM impairs embryo developmental competence. In the present study we investigated whether trichostatin A (TSA; an inhibitor of histone deacetylation) prolongs the maturation time and prevents the aging of oocytes. Porcine oocytes were cultured in medium containing increasing concentrations of TSA (300 nM) for 24, 44 or 64 h. The percentage of oocytes that underwent germinal vesicle breakdown was significantly lower in the TSA-treated group (300 nM) than in the control group. TSA did not affect oocyte quality at MII based on levels of maturation-promoting factor, the phosphorylation status of mitogen-activated protein kinase or histone H3K9 acetylation analysis. We also compared the preimplantation developmental competence and the viability of pathenogenetic embryos treated with 100 nM TSA for 24 h and then continuously cultured for another 24 h in TSA free condition. No significant differences were observed for either parameter between the TSA-treated and control groups. These results indicate that TSA prolongs the IVM of porcine oocytes but that oocyte quality and aging are not affected. These findings provide a feasible option by which to adjust the initiation time of downstream experiments based on porcine matured oocytes.

266 THE SHORT TIME TREATMENT WITH SODIUM BUTYRATE ON GERMINAL VESICLE STAGE OOCYTES IMPROVES OOCYTE QUALITY AND DEVELOPMENTAL COMPETENCE IN PIGS

2011 ◽  
Vol 23 (1) ◽  
pp. 231
Author(s):  
L. M. Liu ◽  
F. Gao ◽  
M. Hua ◽  
J. Y. Guan ◽  
B. Tang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Sodium Butyrate ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Rate ◽  
Group 2 ◽  
Short Time ◽  
Group 1

Oocyte maturation is a complex process during which the epigenetic modifications are dramatically changed, especially the histone acetylation and phosphorylation. Sodium butyrate (NaBu) is a histone deacetylase inhibitor that results in a more open structure of DNA. The aim of the present study was to analyse the role of NaBu in the meiosis of porcine oocytes and the subsequent embryonic developmental competence. Cumulus–oocyte complexes (COC) were collected from ovaries obtained at a local slaughterhouse. The COC were randomly divided into 3 groups and matured in vitro in medium (Hao et al. 2006) supplemented with 1 μM NaBu for 2 h [germinal vesicle (GV) stage, group 1] or for 22 h [GV to GV breakdown (GVBD) stage, group 2] or without treatment (control, group 3). After 44 h of in vitro maturation, the oocytes were denuded by 0.2% hyaluronidase, and oocytes with evenly dark ooplasm and visible first polar bodies were considered matured. The cortical granule distribution of matured oocytes was examined with immunostaining. The relative expression of CyclinB and Cdc2 of 3 group oocytes was determined with real-time PCR. Some matured oocytes from each group were collected and stimulated with electric pulse (2 direct current pulses of 1.2 kV cm–1 for 30 μs). The rate of parthenogenetic blastocyst was recorded, and cell number of each blastocyst was determined under an inverted fluorescence microscope after staining with 10 μg mL–1 of Hoechst 33342. The following results were found. 1) Compared with the control group (n = 70, 67.74 ± 1.64), oocyte maturation rates of group 1 and group 2 decreased significantly along with the extended treatments (n = 70, 59.57 ± 5.29 and 46.99 ± 1.22, respectively; P < 0.05). 2) The long time (22 h) treatment with NaBu inhibited the developmental competence (blastocyst rate) of oocytes (n = 30, 15.33 ± 3.47 v. 27.16 ± 2.10 P < 0.05), and the short time (2 h) treatment with NaBu on GV-stage oocytes inhibited the meiotic process slightly but improved the blastocyst rate (n = 30, 33.93 ± 2.51 v. 27.16 ± 2.10; P < 0.05). 3) The short time (2 h) treatment resulted in the migration of more cortical granules into the plasmasmic membrane and formed a monolayer with the membrane (compared with the control). 4) The exposure to NaBu from GV to GVBD stage induced the expression of the CyclinB and Cdc2 in the matured oocytes (4.68 ± 0.45 and 5.80 ± 0.58, respectively; P < 0.05) compared with the control. 5) Short time (2 h) exposure to NaBu on GV-stage oocytes inhibited the expression of the Cdc2 but increased the expression of the CylinB in the matured oocytes (0.43 ± 0.06 and 1.65 ± 0.26, respectively; P < 0.05) compared with the control. In conclusion, results of this study demonstrate that exposure to NaBu inhibits porcine oocyte meiosis in proportion to treatment length. However, a 2-h treatment with 1 μM NaBu improves oocyte developmental competence to the blastocyst stage. These results are useful for improving the developmental competent of oocytes for IVF and in vitro embryo production. This work was supported by a grant (No. 2009CB941001) from the National Basic Research Program of China.

scholarly journals Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes

Animals ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 163
Author(s):  
Payungsuk Intawicha ◽  
Li-Kuang Tsai ◽  
Shih-Ying Yen ◽  
Neng-Wen Lo ◽  
Jyh-Cherng Ju
Keyword(s):  
Oocyte Maturation ◽  
Mammalian Cells ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
Mitogen Activated Protein ◽  
Porcine Oocyte

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.

A note on the inhibition of in vitro meiotic maturation of mammalian oocytes by dibutyryl cyclic AMP

1981 ◽  
Vol 218 (2) ◽  
pp. 309-311 ◽  
Author(s):  
Georgiana Jagiello ◽  
Mercedes B. Ducayen ◽  
William D. Goonan
Keyword(s):  
Cyclic Amp ◽  
Meiotic Maturation ◽  
Dibutyryl Cyclic Amp

scholarly journals 285 SUPPRESSION OF MPF AND MAPK ACTIVITIES BY DIBUTYRYL CAMP DURING FIRST MEIOTIC MATURATION IMPROVES SUBSEQUENT DEVELOPMENT OF PORCINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 293
Author(s):  
J.-S. Kim ◽  
D.-B.n Koo ◽  
J.-I. Chae ◽  
Y.-K. Choo ◽  
K.-K. Lee ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Subsequent Development ◽  
Treated Group ◽  
Early Embryo ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Dibutyryl Camp ◽  
Vitro Fertilization ◽  
Nuclear Maturation

Maintaining the germinal vesicle (GV) stage in growing oocytes is essential for developmental competence of the eggs. In pig oocytes, MPF and MAPK activities are low during the GV stage, and their activities increase with progression of meiosis I. In general, cAMP that exists at high levels in GV oocytes inhibits germinal vesicle break down (GVBD). After gonadotrophin stimulation, the amount of cAMP in oocyte cytoplasm is decreased gradually for meiotic resumption. This study was conducted to examine the effect of dibutyryl cAMP (dbcAMP) on nuclear maturation, fertilization, and early embryo development of porcine oocytes. Oocytes were cultured in NCSU23 medium with or without dbcAMP for 22 h, and then cultured in fresh maturation medium for an additional 22 h prior to fertilization. The activities of MPF and MAPK were evaluated by Western blot analysis by using specific antibodies such as anti-cdc2 and anti-ERK1/2 in maturing pig oocytes at 22 h and 44 h. In vitro fertilization was performed with fresh ejaculated spermatozoa in the modified TRIS-buffered medium, and fertilized embryos were cultured in NCSU23 medium. When treated with dbcAMP for 22 h, most oocytes (204/224, 91.9%) were arrested in GV stage by blocking meiotic resumption. The activities of constituent proteins (cdc2 and ERK1/2) of MPF and MAPK were also suppressed in dbcAMP-treated oocytes. After completion of IVM, dbcAMP-treated oocytes showed a higher proportion of the metaphase II stage than non-treated ones (156/171, 91.3% vs. 121/167, 72.8%; P < 0.05). Furthermore, incubation of 44 h matured oocytes with dbcAMP for 22 h increased the MPF and MAPK activities. In the dbcAMP-treated group, penetration rate was increased (126/145, 80.0%) and polyspermy rate was reduced (26/126, 22.4%) as compared to the nontreated group (97/140, 69.3% and 46/97, 47.4%, respectively; P < 0.05). Furthermore, blastocyst formation of dbcAMP-treated eggs was also improved compared to the nontreated group (47/126, 37.0% vs. 28/146, 17.2%; P < 0.05). Our results suggest that the synchronization of meiotic resumption by dbcAMP may support meiotic maturation and in vitro development of pig eggs.

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