Role of Protein Phosphorylation in Meiotic Maturation of Mouse Oocytes in Vitro

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.

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Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes

Reproduction Fertility and Development ◽

10.1071/rd12332 ◽

2014 ◽

Vol 26 (8) ◽

pp. 1084 ◽

Cited By ~ 2

Author(s):

Yu-Ting Shen ◽

Yue-Qiang Song ◽

Xiao-Qin He ◽

Fei Zhang ◽

Xin Huang ◽

...

Keyword(s):

Polar Body ◽

Meiotic Maturation ◽

Dependent Manner ◽

Germinal Vesicle Breakdown ◽

Mouse Oocytes ◽

First Polar Body ◽

Triphenyltin Chloride ◽

Dose Dependent

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

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In Vitro Culture of Mouse Oocytes for Meiotic Maturation

Methods in Molecular Biology - Mouse Oocyte Development ◽

10.1007/978-1-4939-8603-3_2 ◽

2018 ◽

pp. 13-21

Author(s):

Ewa Borsuk ◽

Jacek Z. Kubiak

Keyword(s):

In Vitro Culture ◽

Meiotic Maturation ◽

Mouse Oocytes

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

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab245 ◽

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.

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

Journal of Cell Science ◽

10.1242/jcs.30.1.251 ◽

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.

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Spatiotemporal dynamics of intracellular [Ca2+]i oscillations during the growth and meiotic maturation of mouse oocytes

Development ◽

10.1242/dev.120.12.3507 ◽

1994 ◽

Vol 120 (12) ◽

pp. 3507-3517 ◽

Cited By ~ 3

Author(s):

J. Carroll ◽

K. Swann ◽

D. Whittingham ◽

M. Whitaker

Keyword(s):

Spatial Organization ◽

Calcium Release ◽

Calcium Oscillations ◽

Meiotic Maturation ◽

Similar Proportion ◽

Calcium Waves ◽

Apparent Difference ◽

Immature Oocytes ◽

Mouse Oocytes

Calcium oscillations occur during meiotic maturation of mouse oocytes. They also trigger activation at fertilization. We have monitored [Ca2+]i in oocytes at different stages of growth and maturation to examine how the calcium release mechanisms alter during oogenesis. Spontaneous calcium oscillations occur every 2–3 minutes in the majority of fully grown (but immature) mouse oocytes released from antral follicles and resuming meiosis. The oscillations last for 2–4 hours after release from the follicle and take the form of global synchronous [Ca2+]i increases throughout the cell. Rapid image acquisition or cooling the bath temperature from 28 degrees C to 16 degrees C did not reveal any wave-like spatial heterogeneity in the [Ca2+]i signal. Calcium appears to reach highest levels in the germinal vesicle but this apparent difference of [Ca2+] in nucleus and cytoplasm is an artifact of dye loading. Smaller, growing immature oocytes are less competent: about 40% are able to resume meiosis and a similar proportion of these oocytes show spontaneous calcium oscillations. [Ca2+]i transients are not seen in oocytes that do not resume meiosis spontaneously in vitro. Nonetheless, these oocytes are capable of [Ca2+]i oscillations since they show them in response to the addition of carbachol or thimerosal. To examine how the properties of calcium release change during meiotic maturation, a calcium-releasing factor from sperm was microinjected into fully grown immature and mature oocytes. The sperm-factor-induced oscillations were about two-fold larger and longer in mature oocytes compared to immature oocytes. Calcium waves travelling at 40–60 microns/second were generated in mature oocytes, but not in immature oocytes. In some mature oocytes, successive calcium waves had different sites of origin. The modifications in the size and spatial organization of calcium transients during oocyte maturation may be a necessary prerequisite for normal fertilization.

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Spindle assembly in the absence of chromosomes in mouse oocytes

Reproduction ◽

10.1530/rep-07-0149 ◽

2007 ◽

Vol 134 (6) ◽

pp. 731-738 ◽

Cited By ~ 11

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.

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