Meiotic maturation of mouse oocytes in vitro: association of newly synthesized proteins with condensing chromosomes

1976 ◽  
Vol 20 (3) ◽  
pp. 549-568 ◽  
Author(s):  
P.M. Wassarman ◽  
G.E. Letourneau
Keyword(s):  
Protein Synthesis ◽  
Mammalian Species ◽  
Germinal Vesicle ◽  
Chromosome Condensation ◽  
Intracellular Distribution ◽  
Meiotic Maturation ◽  
Meiotic Progression ◽  
Mouse Oocytes

The nature, intracellular distribution, and role of proteins synthesized during meiotic maturation of mouse oocytes in vitro have been examined. Proteins synthesized during the initial stages of maturation are concentrated within the nucleus (germinal vesicle) and become intimately associated with the condensing chromosomes. Inhibition of protein synthesis during this period does not prevent germinal vesicle dissolution or chromosome condensation, but meiotic progression is blocked reversibly at the circular bivalent stage. A protein is synthesized during meiotic maturation of the mouse oocyte which exhibits several of the characteristics of the very lysine-rich histone, FI; this and other histones are phosphorylated during the initial stages of maturation. These results are discussed in relation to studies of meiotic maturation of oocytes from non-mammalian species and chromosome condensation in both oocytes and mitotic cells.

245 EXPRESSION AND FUNCTIONAL ROLE OF CELL DIVISION CYCLE 42 IN MOUSE OOCYTES MATURATION AND PRE-IMPLANTATION DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 230
Author(s):  
X.-S. Cui ◽  
X.-Y. Li ◽  
N.-H. Kim
Keyword(s):  
Protein Synthesis ◽  
Cell Division ◽  
Mrna Expression ◽  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Cell Division Cycle ◽  
Mrna Levels ◽  
Mouse Oocytes

Cell division cycle 42 (Cdc42), a member of the Rho family of small guanosine triphosphatase (GTPase) proteins, regulates multiple cell functions, including motility, proliferation, apoptosis, and cell morphology. In order to gain insight into the role of Cdc42 in embryo development, we first characterized mRNA and protein levels of Cdc42 in mouse oocytes and early embryogenesis. We then examined the possible role of the gene in oocyte maturation and pre-implantation development using RNA interference analysis. The relative abundance of Cdc42 transcripts were measured by real time RT-PCR. After normalization with histone H2a mRNA levels, the mRNA expression of Cdc42 was abundant in immature oocytes and reduced slightly in zygotes and 2- to 8-cell stage embryos. The expression levels were significantly increased during the morula and blastocyst stages. Indirect immunocytochemistry showed protein synthesis of Cdc42 in oocytes and embryos of all stages. Introducing small interference RNA (siRNA) of Cdc42 into germinal vesicle stage oocytes or zygotes specifically reduce both mRNA expression and protein synthesis of Cdc42 in metaphase II stage oocytes and early embryos developing in vitro. Meiotic maturation was significantly reduced following siRNA injection into germinal vesicle stage oocytes. It is evident that actin distribution in siRNA treated blastocysts is morphologically abnormal following injection of siRNA for Cdc42. Injection of siRNA into zygotes did not influence cleavage, but significantly decreased in vitro development to morulae and blastocysts. While housekeeping genes such as tissue plasminogen activator were not altered by siRNA, wiskott-aldrich syndrome protein family 1 (WASP1) mRNA was down-regulated in the morula. Interestingly, mRNA of WASP1, tubulin alpha 1 (Tuba1), and actin-related protein 2/3 complex subunit V (Arpc5) increased at the blastocyst stage following siRNA injection. These results suggest that Cdc42 plays an important role during oocyte maturation and early pre-implantation development, likely through linkage with several other genes. This work was funded by a grant from National Research Laboratory Program in Korea.

Suppression of chromosome condensation during meiotic maturation induces parthenogenetic development of mouse oocytes

Development ◽  
1988 ◽  
Vol 104 (1) ◽  
pp. 97-103 ◽  
Author(s):  
H.J. Clarke ◽  
J. Rossant ◽  
Y. Masui
Keyword(s):  
Protein Synthesis ◽  
Blastocyst Stage ◽  
Chromosome Condensation ◽  
Meiotic Maturation ◽  
Dibutyryl Cyclic Amp ◽  
Developmental Arrest ◽  
Mouse Oocytes ◽  
Parthenogenetic Development ◽  
No Activation ◽  
Metaphase I

Mouse oocytes at metaphase I were treated with puromycin, which caused the chromosomes to become decondensed within an interphase nucleus. When the oocytes were allowed to resume protein synthesis, they returned to metaphase within 8–10 h and neither synthesized DNA nor cleaved, indicating that they had not been parthenogenetically activated by the puromycin treatment. However, when dibutyryl cyclic AMP was added to the medium after protein synthesis resumed, the oocytes remained in interphase. These oocytes maintained in interphase began DNA synthesis beginning 20 h after puromycin withdrawal, even though no activation stimulus had been given to them. After transfer to the oviducts of foster mothers, the oocytes could develop to the blastocyst stage. These results indicate that oocytes whose chromosomes were decondensed by puromycin treatment at metaphase I could begin parthenogenetic development in the absence of an activating stimulus, provided that they were prevented from returning to metaphase. In contrast, when the puromycin-treated oocytes were allowed to return to metaphase, they became developmentally arrested at the end of maturation. This suggests that the mechanism responsible for the developmental arrest of mature oocytes at metaphase II depends on cytoplasmic conditions that cause chromosome condensation to the metaphase state.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro

1997 ◽  
Vol 9 (6) ◽  
pp. 603 ◽  
Author(s):  
J. C. Bell ◽  
L. C. Smith ◽  
R. Rumpf ◽  
A. K. Goff
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Uv Exposure ◽  
One Dimensional ◽  
Repair Mechanisms ◽  
Bovine Oocytes

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulusoocyte complexes (COCs) were denuded before or after maturationin vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high-possibly due to nuclear repair mechanisms. Protein proles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specic proteins are synthesized in the nuclear region.

Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation

1996 ◽  
Vol 43 (2) ◽  
pp. 228-235 ◽  
Author(s):  
Li Meng ◽  
Jean Rutledge ◽  
Ying Zhu ◽  
Gerald M. Kidder ◽  
Firouz Khamsi ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
In Vitro Maturation ◽  
Germinal Vesicle

Meiotic maturation of mouse oocytes in vitro: protein synthesis in nucleate and anucleate oocyte fragments

1978 ◽  
Vol 30 (1) ◽  
pp. 251-264
Author(s):  
R.M. Schultz ◽  
G.E. Letourneau ◽  
P.M. Wassarman
Keyword(s):  
Protein Synthesis ◽  
Meiotic Prophase ◽  
Cytochalasin B ◽  
Meiotic Maturation ◽  
In Vitro Protein Synthesis ◽  
Mammalian Oocyte ◽  
Mouse Oocytes ◽  
Meiotic Competence ◽  
Vitro Protein

Nucleate and anucleate fragments of mouse oocytes have been isolated following treatment of fully grown oocytes with cytochalasin B. The nucleate oocyte fragments resume meiosis in vitro, progressing from dictyate of the first meiotic prophase to metaphase II (‘meiotic maturation’), and exhibit all of the changes in protein synthesis normally associated with meiotic maturation of mouse oocytes. The anucleate oocyte fragments also undergo certain of the changes in protein synthesis associated with meiotic maturation, despite the absence of nuclear progression. These results suggest that the acquisition of meiotic competence (i.e. the ability to undergo meiotic maturation) during growth of the mammalian oocyte is due to changes in the quality, rather than the quantity, of cytoplasm and that the reprogramming of protein synthesis during meiotic maturation is directed by RNA templates already present in the cytoplasm. The behaviour of anucleate oocyte fragments is discussed in terms of the proposed role for nucleoplasm in the initiation of changes in protein synthesis during meiotic maturation of mouse oocytes.

scholarly journals Spindle assembly in the absence of chromosomes in mouse oocytes

Reproduction ◽  
2007 ◽  
Vol 134 (6) ◽  
pp. 731-738 ◽  
Author(s):  
Ji-Wen Yang ◽  
Zi-Li Lei ◽  
Yi-Liang Miao ◽  
Jun-Cheng Huang ◽  
Li-Hong Shi ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Spindle Assembly ◽  
Meiotic Maturation ◽  
Bipolar Spindle ◽  
Microtubule Organizing Centers ◽  
Mouse Oocytes ◽  
Inactive Form ◽  
Different Shapes ◽  
Metaphase I

This study was carried out to investigate the contributions of chromosomes to spindle assembly in mouse oocytes. We generated two groups of cytoplasts (holo- and hemi-cytoplasts) by enucleation of germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) oocytes using micromanipulation technology. After in vitro culture for 18 h, spindles with different shapes (bi-, mono-, or multipolar) formed in most of these cytoplasts except in hemi-GV cytoplasts. Two or more spindles were observed in most of holo-GV, holo-MI, and holo-MII cytoplasts (76.1, 77.0, and 83.7% respectively). However, the proportions of hemi-MI and hemi-MII cytoplasts with multiple sets of spindles decreased to 17.6 and 20.7% respectively. A single bipolar spindle was observed in each sham-operated oocyte generated by removing different volumes of cytoplasm from the oocytes and keeping nuclei intact. Localization of γ-tubulin showed that microtubule organizing centers (MTOCs) were dispersed at each pole of the multiple sets of spindles formed in holo-cytoplasts. However, most of the MTOCs aggregated at the two poles of the bipolar spindle in sham-operated oocytes. Our results demonstrate that chromosomes are not essential for initiating spindle assembly but for directing distinct MTOCs to aggregate to form a bipolar spindle. Some factors of undetermined nature may pre-exist in an inactive form in GV-stage ooplasm, serving as initiators of spindle assembly upon their activation. Moreover, GV materials released into the cytoplasm may facilitate spindle assembly in normal meiotic maturation.

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.

231 STAGE-DEPENDENT CHANGES OF IP3R1 PHOSPHORYLATION DURING IN VITRO MATURATION AND DEVELOPMENT IN PIG OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 195
Author(s):  
J. Ito ◽  
C. Suzukamo ◽  
T. Mochida ◽  
A. Furugaichi ◽  
N. Nakajima ◽  
...  
Keyword(s):  
Cell Cycle ◽  
In Vitro Maturation ◽  
Mammalian Species ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Cell Stage ◽  
Mouse Oocytes ◽  
M Phase ◽  
Pn Stage

During fertilization in mammalian species, a sperm-induced intracellular Ca2+ signal [Ca2+] is well suited to mediate the highly specialized spatiotemporal patterns of [Ca2+]i responses that underlie fertilization. Recently, we demonstrated that the expression pattern of inositol 1,4,5- triphosphate receptor type 1 (IP3R1) did not change during in vitro maturation and parthenogenetic activation in mouse oocytes; however, the phosphorylation status of IP3R1 depended on the cell cycle during meiosis. Moreover, it was shown that IP3R1 phosphorylation played a crucial role in the induction of [Ca2+]i oscillations (Lee et al. 2006 Development 133, 4355–4365). In other species, expression of IP3R1, especially phosphorylation levels of IP3R1 during meiosis, has not been examined. The aim of this study was to examine the kinetics of IP3R1 expression and phosphorylation during in vitro maturation and activation in pig oocytes. Immature oocytes at the germinal vesicle (GV) stage were collected from ovaries and cultured in modified NCSU37 up to 48 h. After culture, cumulus cells were removed and oocytes were parthenogenetically activated by 25 µm Ca2+ ionophore for 3 min and 2 mm 6-DMAP for 6 h. After activation, oocytes were further cultured up to the 2-cell stage. Groups of 30 oocytes were collected at each culture period for detection of IP3R1. According to our previous report in the mouse, IP3R1s were detected by western blotting using MPM-2 and Rbt03 antibody for detecting IP3R1 phosphorylation and total IP3R1 expression, respectively (Lee et al. 2006). In pig oocytes, IP3R1 was abundantly expressed at the GV stage. The total level of IP3R1 expression did not change during in vitro maturation or after activation. However, phosphorylated IP3R1 levels increased by 24 h although they were undetectable at the start of culture. Phosphorylation of IP3R1 reached maximal levels at 36 h. After activation, phosphorylation levels decreased progressively until the pronuclear (PN) stage. Phosphorylation of IP3R1 was observed at mitosis I to some extent. From these results, we detected for the first time IP3R1 expression and phosphorylation in pig oocytes. Moreover, our data suggest that phosphorylation of IP3R1 is dependent on cell cycle at least during meiosis, especially M-phase, as already shown for mouse oocytes. In vitro kinase assays for p34cdc2 kinase and MAPK will be carried out to clarify the relationship between IP3R1 phosphorylation and M-phase kinase(s).

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