The distribution and requirements of microtubules and microfilaments in bovine oocytes during in vitro maturation

Zygote ◽  
2000 ◽  
Vol 8 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Nam-Hyung Kim ◽  
Seong Koo Cho ◽  
Seok Hwa Choi ◽  
Eun Young Kim ◽  
Se Pill Park ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Cytochalasin B ◽  
Germinal Vesicle ◽  
Condensed Chromatin ◽  
Meiotic Spindle ◽  
Cell Cortex ◽  
Germinal Vesicle Breakdown ◽  
Metaphase Stage ◽  
Bovine Oocytes

Microtubules and microfilaments are major cytoskeletal components and important modulators for chromosomal movement and cellular division in mammalian oocytes. In this study we observed microtubule and microfilament organisation in bovine oocytes by laser scanning confocal microscopy, and determined requirements of their assembly during in vitro maturation. After germinal vesicle breakdown, small microtubular asters were observed near the condensed chromatin. The asters appeared to elongate and encompass condensed chromatin particles. At the metaphase stage, microtubules were observed in the second meiotic spindle at the metaphase stage. The meiotic spindle was a symmetrical, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Treatment with nocodazole did not inhibit germinal vesicle breakdown. However, progression to metaphase failed to occur in oocytes treated with nocodazole. In contrast, microfilaments were observed as a relatively thick uniform area around the cell cortex and overlying chromatin following germinal vesicle breakdown. Treatment with cytochalasin B inhibited microfilament polymerisation but did not prevent either germinal vesicle breakdown or metaphase formation. However, movement of chromatin to the proper position was inhibited in oocytes treated with cytochalasin B. These results suggest that both microtubules and microfilaments are closely associated with reconstruction and proper positioning of chromatin during meiotic maturation in bovine oocytes.

Microfilament assembly and cortical granule distribution during maturation, parthenogenetic activation and fertilisation in the porcine oocyte

Zygote ◽  
1996 ◽  
Vol 4 (2) ◽  
pp. 145-149 ◽  
Author(s):  
Nam-Hyung Kim ◽  
Billy N. Day ◽  
Hoon Taek Lee ◽  
Kil-Saeng Chung
Keyword(s):  
Laser Scanning ◽  
Germinal Vesicle ◽  
Laser Scanning Confocal Microscopy ◽  
Cell Cortex ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown ◽  
Parthenogenetic Activation ◽  
Scanning Confocal Microscopy

SummaryIn this study we imaged integral changes in microfilament assembly and cortical granule distribution, and examined effects of microfilament inhibitor on the cortical granule distribution during oocyte maturation, parthenogenetic activation and in vitro fertilisation in the pig. The microfilament assembly and cortical granule distribution were imaged with fluorescent-labelled lectin and rhodamine-labelled phalloidin under laser scanning confocal microscopy. At the germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively wide area on the oolemma. Microfilaments were also observed in a wide uniform area around the cell cortex. Following germinal vesicle breakdown, microfilaments concentrated in the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerisation and prevented movement of cortical granules to the cortex. Cortical granule exudation following sperm penetration was evenly distributed in the entire perivitelline space. These results suggest that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilisation.

scholarly journals Proline-rich tyrosine kinase2 is involved in F-actin organization during in vitro maturation of rat oocyte

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 859-867 ◽  
Author(s):  
Xiao-Qian Meng ◽  
Ke-Gang Zheng ◽  
Yong Yang ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Actin Filaments ◽  
Laser Scanning ◽  
Polar Body ◽  
Laser Scanning Microscopy ◽  
Meiotic Spindle ◽  
Confocal Laser ◽  
Cell Cortex ◽  
First Polar Body

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.

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

Zygote ◽  
2003 ◽  
Vol 11 (2) ◽  
pp. 119-129 ◽  
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.

scholarly journals Aspects of cytoplasmic maturation of bovine oocytes: Interplay between mapk, mRNA-cap binding complex and cytoplasmic mRNA metabolism in regulation of translation

2003 ◽  
Vol 19 (3-4) ◽  
pp. 1-8 ◽  
Author(s):  
Tatjana Smiljakovic ◽  
Melo Sterza ◽  
M. Kubelka ◽  
Z. Vohnikova ◽  
W. Tomek
Keyword(s):  
Binding Protein ◽  
Germinal Vesicle ◽  
Initiation Factor ◽  
Meiotic Maturation ◽  
Detailed Knowledge ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Growth ◽  
Stage Of Development ◽  
Bovine Oocytes

Bovine oocytes are arrested in the germinal vesicle stage (GV stage)and mature spontaneously when they are removed from their follicles and transferred to a suitable culture medium. This process, known as meiotic maturation is characterized among others, by germinal vesicle breakdown followed by metaphase I (MI) stage and further development to metaphase II (MII), where they become arrested again. During GVBD to MI transition, the overall protein synthesis reaches the highest level and it rapidly declines in MII. We have previously shown that transcription completely declines during meiotic maturation. Therefore we suppose that gene expression is exclusively regulated on translational level at this stage of development. This means that mRNAs, which were stored in repressed form during oocyte growth, were actively translated during meiotic maturation. Therefore we have investigated specific regulators of translation, namely the eukaryotic initiation factor of translation eIF4E (cap binding protein) and a specific repressor of eIF4E function, the 4E-binding protein 4E-BP1. Furthermore, we have elucidated pathways, which lead to eIF4E and 4E-BP1 phosphorylation by using specific M-phase kinase inhibitors, and we compare these results with transcription and cytoplasmic polyadenylation events during the course of meiotic maturation. The detailed knowledge of such regulatory processes can help to improve in vitro bio-techniques and to estimate the risk of these techniques.

Microtubule and microfilament organization in immature, in vitro matured and in vitro fertilized prepubertal goat oocytes

Zygote ◽  
2005 ◽  
Vol 13 (2) ◽  
pp. 155-165 ◽  
Author(s):  
Esther Velilla ◽  
Elisabet Rodríguez-Gonzalez ◽  
Francesca Vidal ◽  
Maria-Teresa Paramio
Keyword(s):  
Laser Scanning ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Confocal Laser Scanning Microscope ◽  
Meiotic Spindle ◽  
Confocal Laser ◽  
Cortical Region ◽  
Microtubule Networks

The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured–in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed spermatozoa. Oocytes and presumptive zygotes were treated with anti-α-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 μg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.

161 USE OF FORSKOLIN TO DELAY MEIOSIS AND PRODUCE IN VITRO BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 194
Author(s):  
D. Paschoal ◽  
R. Maziero ◽  
M. Sudano ◽  
M. Guastali ◽  
L. Vergara ◽  
...  
Keyword(s):  
Rate Control ◽  
Germinal Vesicle ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Bovine Embryos ◽  
Hoechst 33342 ◽  
Germinal Vesicle Breakdown ◽  
Tunel Reaction ◽  
Level Of Significance ◽  
Bovine Oocytes

The maintenance of oocytes in germinal vesicle (GV) stage for a few hours could result in more competent oocytes for use in biotechnology. This study aimed to show if the use of forskolin is able to inhibit and reverse the maturation in bovine oocytes, producing a higher rate of in vitro embryos without apoptosis rates. Eight replicates in total were performed. Nellore oocytes were matured in TCM-199 and to delay meiosis, the oocytes (n = 584) were maintained for 6 h in medium in presence of 0.025, 0.05, or 0.1 mM Forskolin. Then, the oocytes were cultured for 18 h in agent-free medium to resume meiosis. After resumption of meiosis, the oocytes (n = 336) were stained with Hoechst 33342 to evaluate the state of the nucleus: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), or degenerated or unidentified (D/U). Then (Day 0) oocytes were fertilized in human tubal fluid (Irvine, New Zealand) and the presumed zygotes were culture in SOFaa + 0.6% BSA + 2.5% FCS until Day 7, when the blastocyst (n = 177) rate was evaluated. Apoptosis in blastocysts was assessed by terminal deoxynucleotidyl transferase uracil nick-end labeling (TUNEL) reaction. Data were analysed by ANOVA, followed by Tukey test using the general linear model (PROC GLM) of SAS (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. There were no statistical differences in state of the nucleus, only in MI (Control = GV: 0.0, GVBD: 0.8, MI: 8.3a, MII: 67.7, D/U: 7.3; F 0.025 mM = GV: 2.8, GVBD: 0.7, MI: 20.8ab, MII: 67.7, D/U: 8.9; F 0.05 mM = GV: 0.0, GVBD: 4.4, MI: 15.8ab, MII: 65.9, D/U: 13.7; and F 0.1 mM = GV: 0.0, GVBD: 1.0, MI: 34.1b, MII: 50.2, D/U: 14.6; P < 0.05). There were no statistical differences in blastocyst rate (Control: 36.7, F 0.025 mM: 32.6, F 0.05 mM: 29.2 and F 0.1 mM: 25.1 – P > 0.05). But when we analysed the apoptosis rate, differences were found among groups: (Control: 12.1a, F 0.025 mM: 12.9a, F 0.05 mM: 13.5a and F 0.1 mM: 30b; P < 0.05). Although Forskolin was able to inhibit meiosis and produce embryos at the same rates as controls, the higher dosage of this drug damaged the embryos. The authors acknowledge FAPESP 10/50410-2 for support.

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.

250 CYTOPLASMIC DYNEIN INTERMEDIATE CHAIN AND DYNACTIN p150Glued EXHIBIT DISTINCT SPATIAL AND TEMPORAL MICROTUBULE ASSOCIATIONS DURING BOVINE IN VITRO MATURATION AND ARE AFFECTED BY FOLLICLE SIZE

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
S. E. Racedo ◽  
M. C. Branzini ◽  
D. Salamone ◽  
V. Y. Rawe ◽  
H. Niemann
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Chromatin Condensation ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Meiotic Spindle ◽  
Germinal Vesicle Breakdown ◽  
Intermediate Chain

Microtubule molecular motors are critically involved in transporting vesicles during interphase, in building and maintaining spindles during mitosis and meiosis, and also in the localization of various organelles. DYNC1I1 (cytoplasmic dynein 1 intermediate chain) and its cofactor DCTN1 (dynactin p150Glued) are crucial for oocyte maturation but their role during mammalian female meiosis is not yet known. The goal of this study was to analyze the dynamics of these proteins in oocytes collected from different-size follicles at different stages of in vitro maturation (IVM), i.e., germinal vesicle stage (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII), and their association with microtubules. Ovaries were collected at a local abattoir. Cumulus–oocyte complexes (COCs) were aspirated from follicles either <2 mm or 2–8 mm in size and matured in M199, supplemented with 1% fatty acid-free BSA, 10 UI pregnant mare serum gonadotropin (PMSG)/5 UI HCG, and 100 µm cysteamine, at 39�C and 5% CO2. Follicle sizes and time points for fixation were: GV-0 h; GVBD-8 h for oocytes <2 mm and 9 h for oocytes 2–8 mm; MI-15 h; MII-24 h (Racedo et al. 2007, pub. online: 10.1002/mrd.20770). The distribution of the proteins was assessed by immunocytochemistry and laser confocal microscopy. The attached cumulus cells and zona pellucida of oocytes were removed in TALP-HEPES medium containing 1 mg mL–1 hyaluronidase and 2 mg mL–1 pronase, respectively. The oocytes were then incubated in a fixation–permeabilization solution containing 2% formaldehyde and 0.1%Triton X-100 for 1 h. Samples were then blocked for 1 h in 10 mm PBS + 0.3% BSA + 1% fetal calf serum (ICC blocking solution). The primary antibody was applied over night at 4�C, followed by treatment with fluorochrome-conjugated secondary antibodies for 1 h at 37�C in the dark. After RNase treatment, oocytes were incubated with TOTO-3 (Invitrogen, Carlsbad, CA, USA) to visualize the DNA. The material was mounted in an anti-fade medium (Vectashield�, Vector Laboratories, Burlingame, CA, USA) and imaged with a Zeiss laser scanning microscope. Immediately after chromatin condensation (GVBD), dynactin was in close association with the DNA and interacting with the spindles in MI and MII oocytes recovered from large follicles. No clear association with the DNA was observed in GVBD oocytes obtained from small follicles; little dynactin was found in MI and MII spindles. Dynein localization did not differ from dynactin in GVs and was homogeneously distributed in the cytoplasm of both groups of follicles. Dynein was not associated with the DNA in the GVBD stage while at MI and MII it was associated with the meiotic spindle. The association of dynein with microtubules was weak at the MI stage in oocytes from small follicles. Results provide insight into the regulatory mechanisms of oocyte maturation and a possible relationship with oocyte competence.

Sign in / Sign up

Export Citation Format

Share Document