Effects of EDTA saturated with Ca2+ (Ca-EDTA) on pig, bovine and mouse oocytes at the germinal vesicle stage during maturation culture and the involvement of chelation of Zn2+ in pronuclear formation induction by Ca-EDTA

EDTA saturated with Ca(2+), Fe(3+) or Cu(2+) can induce parthenogenetic activation of pig oocytes at the germinal vesicle stage, whereas EDTA saturated with Zn(2+), which is unable to chelate Zn(2+), does not, indicating that chelation of Zn(2+) with EDTA saturated with Ca(2+) (Ca-EDTA) in maturing pig oocytes plays a pivotal role in the induction of parthenogenetic activation of oocytes. In the present study, the involvement of Zn(2+) chelation in the induction of parthenogenetic activation of pig oocytes at the germinal vesicle stage was confirmed first by examining the effects of concomitant addition of Zn(2+), Cu(2+) or Ni(2+) at various concentrations together with 1 mmol Ca-EDTA l(-1) to the maturation medium. The titration experiments revealed that the pronuclear formation induced by 1 mmol Ca-EDTA l(-1) was completely inhibited by the addition of > 30 micromol Zn(2+) l(-1) to the medium, but not by the addition of Cu(2+) and Ni(2+) at any concentration examined. Second, bovine and mouse oocytes at the germinal vesicle stage were cultured in medium with or without 1 mmol Ca-EDTA l(-1) for 48 h to examine the effects of Ca-EDTA treatment on these oocytes during maturation culture. Most (70-86%) of the bovine oocytes that underwent germinal vesicle breakdown matured to the MII stage via the MI phase, regardless of whether Ca-EDTA was present for the first 24 h of culture. However, 61% of oocytes that had been cultured with Ca-EDTA for 48 h formed a pronucleus without a second polar body, whereas oocytes cultured in the absence of Ca-EDTA were not observed to form a pronucleus at any time during culture. However, even when mouse oocytes at the germinal vesicle stage were cultured for up to 48 h in maturation medium containing Ca-EDTA, pronuclear formation was not observed. Finally, when bovine oocytes that had been cultured with 1 mmol Ca-EDTA l(-1) for 48 h from the germinal vesicle stage were cultured further in medium without Ca-EDTA that was supplemented with 5% fetal calf serum, only 26% of the oocytes developed to the cleaved stage, and none could develop further.

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271 ASSESSMENT OF NUCLEAR STATUS OF ACTIVATED BOVINE OOCYTES MATURED IN DIFFERENT MATURATION CONDITIONS IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab271 ◽

2005 ◽

Vol 17 (2) ◽

pp. 285

Author(s):

J.I. Park ◽

Y. Jang

Keyword(s):

Oocyte Maturation ◽

Polar Body ◽

Calf Serum ◽

Chi Square ◽

Cell Stage ◽

Maturation Medium ◽

Second Polar Body ◽

Humidified Air ◽

Bovine Oocytes

This study was carried out to assess the nuclear status after parthenogenetic activation in in vitro matured oocytes under different conditions. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles of 3–8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL l-cysteine, 20 mg/mL sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 mg/mL epidermal growth factor, with or without 5 mM hypotaurine and taurine. Oocytes were cultured at 38.9°C in 5% CO2 in humidified air. After 24 h of culture, oocytes with polar body were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 μM for 5 min) followed by incubation with 6-DMAP (2 mM), roscovitine (50 μM), or 6-DMAP + roscovitine for 3.5 h. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9°C in 5% CO2, 5% O2 in humidified air for 16 h and stained with Hoechst 33342 or aceto-orcein for assessment of nuclear status. Nuclear status was recorded as follows: 1PB (polar body) + 1PN (pronucleus), 2PB + 1PN and others. Data were analyzed using chi-square test. The maturation rate of bovine oocytes cultured in maturation medium containing hypotaurine/taurine (89.3%, n = 84) was higher (P < 0.05) than those cultured without hypotaurine/taurine (72%, n = 93). In the oocytes matured with hypotaurine/taurine, the rates of diploid activation (1PB + 1PN) were 84% (n = 50) in oocytes treated with 6-DMAP + roscovitine, 78.6% (n = 56) with 6-DMAP, and 52% (n = 50) with roscovitine. In the oocytes matured without hypotaurine/taurine, the rates of diploid activation were 80% (n = 60) in oocytes treated with 6-DMAP + roscovitine, 72% (n = 50) with 6-DMAP, and 54% (n = 50) with roscovitine. The rates of diploid activation were not different in oocytes matured with or without hypotaurine/taurine and among activation treatments. The oocytes treated with roscovitine showed a lower rate (P < 0.05) of diploid activation and higher rate (39.3–40%) of second polar body extrusion (1PN + 2PB) than the other activation groups in both maturation conditions. Cleavage rates to 2-cell stage were 40–45% in all groups. Development rate of blastocysts were 7–10% in all the groups treated with 6-DMAP and 6-DMAP + roscovitine and no blastocysts were obtained from the groups treated with roscovitine alone. Hypotaurine/taurine are known to be stable and potent antioxidants, and have shown the properties of supporting oocyte maturation and further embryonic development (Guerin and Menezo 1995 Zygote 3, 333–43; Mizushima and Fukui 2001 Theriogenology 55, 1432–45). In this study, although the effectiveness of hypotaurine/taurine on promoting oocyte maturation was observed, there were no significant improvements in the rate of diploid activation in oocytes matured with hypotaurine/taurine. These results suggest that the nuclear status of activated oocytes may not have a direct relationship with the enhanced maturation condition. This work was supported by BioGreen 21 Program(#1000520030100000-1), Republic of Korea.

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233 DEHIDROLEUCODINE INDUCES PARTHENOGENETIC ACTIVATION OF BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab233 ◽

2009 ◽

Vol 21 (1) ◽

pp. 214

Author(s):

N. Canel ◽

D. Salamone

Keyword(s):

Polar Body ◽

Metaphase Plate ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Control Group ◽

Germinal Vesicle Breakdown ◽

Parthenogenetic Activation ◽

Polar Bodies ◽

Bovine Oocytes ◽

Full Activation

Dehydroleucodine (DhL) is a sesquiterpene lactone that inhibits germinal vesicle breakdown in Bufo arenarum oocytes. Its action takes place over early stages of the cdc25 activation cascade (Bühler MI et al. 2007 Zygote 15, 183–187). The aim of this study was to evaluate the potential of DhL to induce parthenogenetic activation by observing nuclear dynamics and second polar body (2PB) extrusion of bovine oocytes, in the presence or absence of Cytochalasin B (CB), comparing these treatments with 6-Dimethylaminopurine (DMAP), an activation agent widely used. Cumulus–oocyte complexes were collected from cow ovaries obtained from a slaughterhouse. They were matured in TCM 199, supplemented with 5% FCS, 10 UI mL–1 penicillin, 10 μg mL–1 FSH, 100 μM cysteamine, 0.3 mm sodium pyruvate and 2 mm glutamine, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were selected and treated with 5 μm ionomycin (Io) for 4 min. Afterwards, oocytes were randomly allocated into one of the following treatments: a) incubation with 2 mm DMAP for 3 h (DMAP); b) incubation with 5 μm DhL for 3 h (DhL); and c) incubation with 5 μm DhL and 5 μg mL–1 CB, for 3 h (DhL-CB). A control group was only treated with Io. Activated oocytes were cultured in the maturation medium during 4, 11 or 17 h (Io exposure = 0 h), stained with Hoechst 33342 and analyzed under fluorescence microscope to evaluate nuclear stage and 2PB extrusion. Activation data are presented in Table 1. Oocytes with two extruded polar bodies and a metaphase plate were considered as partially activated (PA) and those exhibiting one pronucleus (PN) or already cleaved, as fully activated (FA). Oocytes that remained arrested at MII were not included in the table. Rates of 2PB emission were 98.3, 4.9, 83.6 and 61.5% for Io, DMAP, DhL and DhL-CB, respectively. These percentages were determined over total number of activated oocytes (PA and FA) within each group, including results from all evaluation times because no differences were found between them. Nuclear evaluation suggests that DhL is as effective as DMAP to induce full activation when combined with CB, and its use does not induce the early PN formation observed with DMAP at 4 h post Io. Most of the oocytes activated with DhL extruded a 2PB; these results were statistically different from those observed for other groups. These results indicate that DhL might be a useful agent to induce parthenogenesis, allowing 2PB extrusion and avoiding early PN formation in bovine oocytes. Table 1.Partial and full activation of bovine oocytes at 4, 11 and 17 h post treatments

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Meiotic maturation of bovine oocytes in vitro: improvement of meiotic competence by dibutyryl cyclic adenosine 3′,5′-monophosphate.

Journal of Animal Science ◽

10.2527/1990.6841182x ◽

1990 ◽

Vol 68 (4) ◽

pp. 1182-1187 ◽

Cited By ~ 25

Author(s):

E. Sato ◽

M. Matsuo ◽

H. Miyamoto

Keyword(s):

Polar Body ◽

Calf Serum ◽

Germinal Vesicle ◽

Razor Blade ◽

Germinal Vesicle Breakdown ◽

First Polar Body ◽

Polar Body Formation ◽

Bovine Oocytes ◽

Meiotic Competence

Abstract The present study was undertaken to determine the precise stage of growth at which the ability to resume meiosis is acquired in bovine oocytes. Oocytes of various sizes were isolated from ovaries by mechanical dissection using an 18-gauge needle followed by a razor blade. This method yielded an average of 26.2 ± 7.4 growing and fully grown oocytes from an ovary. Cumulus-enclosed oocytes were cultured in vitro in tissue culture medium 199 containing 10% fetal calf serum. Oocytes ≤ 90 µm in diameter did not resume meiosis. However, germinal vesicle breakdown was observed in oocytes whose diameters exceeded 91 µm. Polar body formation was observed in oocytes with diameters exceeding 101 µm. About 80% of the oocytes with diameters ≥ 121 µm were able to extrude the polar body. The percentage of large oocytes (101 to 120 µm) with first polar body increased when incubated in medium containing dibutyryl cyclic adenosine 3′,5′-monophosphate; however, oocytes 90 to 101 µm did not extrude the first polar body even when cultured in a medium containing dibutyryl cyclic adenosine 3′,5′-monophosphate. These observations indicate that the capability to resume meiosis is acquired gradually during development of oocytes and that dibutyryl cyclic adenosine 3′,5′-monophosphate can improve the meiotic competence of bovine oocytes in culture.

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Activation of bovine oocytes penetrated after germinal vesicle breakdown

Zygote ◽

10.1017/s0967199400002094 ◽

1994 ◽

Vol 2 (4) ◽

pp. 273-279 ◽

Cited By ~ 3

Author(s):

Lalantha R. Abeydeera ◽

Kiyoshi Okuda ◽

Koji Niwa

Keyword(s):

Culture Medium ◽

Calf Serum ◽

Germinal Vesicle ◽

Nuclear Transformation ◽

Metaphase Chromosomes ◽

Germinal Vesicle Breakdown ◽

Maturation Medium ◽

Sperm Penetration ◽

Sperm Nuclei ◽

Bovine Oocytes

SummaryThe present study was designed to examine the ability of bovine oocytes, after germinal vesicle breakdown (GVBD), to be activated by sperm penetration and the sequence of sperm nuclear transformation. Bovine oovytes cultured for 8 h in maturation medium (tissue culture medium TCM-199 containing 10% fetal calf serum) were inseminated in Brackett and Oilphant's medium supplemented with bovine serum albumin (10 mg/ml), caffeine (5mM) and heparin (10 μg/ml). When oocytes were transferrred to the maturation medium 8 h after insemination and additionally cultured for 5−40 h at 39°C in 5% CO2 in air, 71−76% of oocytes were penetrated and polyspermy (67–75%) was common. The proportions of penetrated oocytes that were activated significantly increased with the lapse of the additional culture time, reaching 88% and 87% by 25 and 40 h after additional culture, respectively. When compared with unpenetrated oocytes, signifcantly higher proportions of penetrated oocytes, reached metaphase II or beyond 15 and 25 h after additional culture. After penetration, sperm nuclei were transformed into metaphase chromosomes and then to telophase chromomes before the formation of male pronuclei. These results provide evidence that bovine oocytes acquire the ability to respond to sperm-mediated activation soon after GVBD.

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Pleiotropic effect of okadaic acid on maturing mouse oocytes

Development ◽

10.1242/dev.112.4.971 ◽

1991 ◽

Vol 112 (4) ◽

pp. 971-980 ◽

Cited By ~ 1

Author(s):

H. Alexandre ◽

A. Van Cauwenberge ◽

Y. Tsukitani ◽

J. Mulnard

Keyword(s):

Protein Kinase ◽

Okadaic Acid ◽

Protein Phosphatases ◽

Chromatin Condensation ◽

Polar Body ◽

Germinal Vesicle ◽

Inhibitory Effect ◽

Negative Control ◽

Germinal Vesicle Breakdown ◽

Mouse Oocytes

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.

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The localization of LAP2β during pronuclear formation in bovine oocytes after fertilization or activation

Zygote ◽

10.1017/s0967199406003613 ◽

2006 ◽

Vol 14 (2) ◽

pp. 157-167 ◽

Cited By ~ 2

Author(s):

Mamiko Isaji ◽

Hisataka Iwata ◽

Hiroshi Harayama ◽

Masashi Miyake

Keyword(s):

Chromatin Condensation ◽

Polar Body ◽

Somatic Cells ◽

Histone H3 ◽

Cumulus Cells ◽

Polar Bodies ◽

Second Polar Body ◽

Pronuclear Formation ◽

Bovine Oocytes

SummaryWe have shown that the assembly of lamin-associated polypeptide (LAP) 2β was detected surrounding the chromatin mass around the time of extrusion of the second polar body (PB) in some fertilized oocytes, but not in most activated oocytes, by using A23187 and cycloheximide (CaA + CH). Here, we immunohistologically analysed the correlation between LAP2β assembly and chromatin condensation in fertilized and activated oocytes during the second meiosis. In bovine cumulus cells, the onset of LAP2β assembly was observed around anaphase chromosomes with strongly phosphorylated histone H3. No LAP2β assembled around the chromosomes in the first and second polar bodies and the alternative oocyte chromatin (oCh) if histone H3 was phosphorylated. Only histone H3 of oCh was completely dephosphorylated during the telophase II/G1 transition (Tel II/G1), and then LAP2β assembled around only the oCh without phosphorylated histone H3. In the oocytes activated by CaA + CH, LAP2β did not assemble around the condensed oCh during the Tel II/G1 transition, although their histone H3 dephosphorylation occurred rather rapidly compared with that of the fertilized oocytes. The patterns of histone H3 dephosphorylation and LAP2β assembly in oocytes activated by CaA alone showed greater similarity to those in fertilized oocytes than to those in oocytes activated by CaA + CH. These results show that LAP2β assembles around only oCh after complete dephosphorylation of histone H3 after fertilization and activation using CaA alone, and that the timing of histone H3 dephosphorylation and LAP2β assembly in these oocytes is different from that of somatic cells. The results also indicate that CH treatment inhibits LAP2β assembly around oCh but not histone H3 dephosphorylation.

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Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Experimental Cell Research ◽

10.1016/0014-4827(86)90603-8 ◽

1986 ◽

Vol 165 (2) ◽

pp. 507-517 ◽

Cited By ~ 72

Author(s):

Elayne A. Bornslaeger ◽

William T. Poueymirou ◽

Peter Mattei ◽

Richard M. Schultz

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Polar Body ◽

Germinal Vesicle ◽

Germinal Vesicle Breakdown ◽

Mouse Oocytes ◽

Kinase C ◽

Protein Kinase C Activators

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Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro

Zygote ◽

10.1017/s0967199403002156 ◽

2003 ◽

Vol 11 (2) ◽

pp. 119-129 ◽

Cited By ~ 5

Author(s):

R.C. Chian ◽

J.T. Chung ◽

K. Niwa ◽

M.A. Sirard ◽

B.R. Downey ◽

...

Keyword(s):

Protein Phosphorylation ◽

Gel Electrophoresis ◽

Germinal Vesicle ◽

Two Dimensional Gel Electrophoresis ◽

Germinal Vesicle Breakdown ◽

One Dimensional ◽

Cell Cycle Transition ◽

Pronuclear Formation ◽

Bovine Oocytes

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.

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Dynamic alterations in H4K12 acetylation during meiotic maturation and after parthenogenetic activation of mouse oocytes

Zygote ◽

10.1017/s0967199420000192 ◽

2020 ◽

Vol 28 (5) ◽

pp. 367-370

Author(s):

Ze Zhang ◽

Baobao Chen ◽

Haoliang Cui ◽

Haixu Gao ◽

Ming Gao ◽

...

Keyword(s):

Germinal Vesicle ◽

Histone Acetyltransferases ◽

Meiotic Maturation ◽

Histone H4 ◽

Germinal Vesicle Breakdown ◽

Parthenogenetic Activation ◽

Expression Levels ◽

Mouse Oocytes

SummaryThe aim of the study was to investigate the continuous changing pattern of H4K12 acetylation, and the expression levels of histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs) in mouse oocytes during meiosis and after parthenogenetic activation (PA). The immunofluorescence results showed hyperacetylation of lysine-12 on histone H4 (H4K12) in the germinal vesicle (GV) oocytes that then decreased during germinal vesicle breakdown (GVBD), and disappeared in metaphase II (MII). However, it reappeared in the early 1-cell embryos derived after 4 h of PA. The expression levels of some selected HATs and HDACs also validated the changing pattern of H4K12 acetylation during meiosis and PA. In conclusion, H4K12 is deacetylated in GVBD and MII, and re-hyperacetylated after PA.

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Activation of p90rsk during meiotic maturation and first mitosis in mouse oocytes and eggs: MAP kinase-independent and -dependent activation

Development ◽

10.1242/dev.122.6.1957 ◽

1996 ◽

Vol 122 (6) ◽

pp. 1957-1964 ◽

Cited By ~ 1

Author(s):

P. Kalab ◽

J.Z. Kubiak ◽

M.H. Verlhac ◽

W.H. Colledge ◽

B. Maro

Keyword(s):

Kinase Activity ◽

Polar Body ◽

Ribosomal Subunit ◽

Germinal Vesicle ◽

Maximum Level ◽

Meiotic Maturation ◽

S6 Kinase ◽

Mouse Oocytes ◽

M Phase ◽

Second Polar Body

Mitogen-activated protein kinases (MAPK) become activated during the meiotic maturation of oocytes from many species; however, their molecular targets remain unknown. This led us to characterize the activation of the ribosomal subunit S6 kinase of Mr 82 X 10(3) - 92 X 10(3) (p90rsk; a major substrate of MAPK in somatic cells) in maturing mouse oocytes and during the first cell cycle of the mouse embryo. We assessed the phosphorylation state of p90rsk by examining the electrophoretic mobility shifts on immunoblots and measured the kinase activity of immunoprecipitated p90rsk on a S6-derived peptide. Germinal vesicle stage (GV) oocytes contained a doublet of Mr 82 × 10(3) and 84 × 10(3) with a low S6 peptide kinase activity (12% of the maximum level found in metaphase II oocytes). A band of Mr 86 × 10(3) was first observed 30 minutes after GV breakdown (GVBD) and became prominent within 2 to 3 hours. MAPK was not phosphorylated 1 hour after GVBD, when the p90rsk-specific S6 kinase activity reached 37 % of the M II level. 2 hours after GVBD, MAPK became phosphorylated and p90rsk kinase activity reached 86% of the maximum level. The p90rsk band of Mr 88 × 10(3), present in mature M II oocytes when S6 peptide kinase activity is maximum, appeared when MAPK phosphorylation was nearly complete (2.5 hours after GVBD). In activated eggs, the dephosphorylation of p90rsk to Mr 86 X 10(3) starts about 1 hour after the onset of pronuclei formation and continues very slowly until the beginning of mitosis, when the doublet of Mr 82 X 10(3) and 84 X 10(3) reappears. A role for a M-phase activated kinase (like p34cdc2) in p90rsk activation was suggested by the reappearance of the Mr 86 X 10(3) band during first mitosis and in 1-cell embryos arrested in M phase by nocodazole. The requirement of MAPK for the full activation of p90rsk during meiosis was demonstrated by the absence of the fully active Mr 88 X 10(3) band in maturing c-mos −/− oocytes, where MAPK is not activated. The inhibition of kinase activity in activated eggs by 6-DMAP after second polar body extrusion provided evidence that both MAPK- and p90rsk-specific phosphatases are activated at approximately the same time prior to pronuclei formation.

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