Immunofluorescent anti-tubulin staining of spindles during meiotic maturation of mouse oocytes in vitro

1978 ◽  
Vol 29 (1) ◽  
pp. 171-188
Author(s):  
P.M. Wassarman ◽  
K. Fujiwara
Keyword(s):  
Polar Body ◽  
Immunofluorescent Staining ◽  
Meiotic Maturation ◽  
Cytoplasmic Bridge ◽  
Specific Staining ◽  
Mouse Oocytes ◽  
Maturation In Vitro ◽  
Reproducible Manner ◽  
Metaphase I

Immunofluorescent anti-tubulin staining has been used to follow nuclear progression from dictyate to metaphase II during meiotic maturation of mouse oocytes in vitro. Antibody directed against tubulin isolated from sea-urchin eggs decorates the metaphase I and metaphase II spindles, as well as the cytoplasmic bridge, midbody, and polar body of the maturing mouse oocytes. Changes in the tubulin-specific staining pattern during meiotic maturation in vitro take place in a highly reproducible manner. Oocytes exposed continuously to cytochalasin B arrest at metaphase I and display a spindle which by immunofluorescent staining is virtually indistinguishable from the spindle of untreated oocytes.

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

2014 ◽  
Vol 26 (8) ◽  
pp. 1084 ◽  
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.

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.

scholarly journals Effects of sperm insemination on the final meiotic maturation of mouse oocytes arrested at metaphase I after in vitro maturation

2017 ◽  
Vol 44 (1) ◽  
pp. 15
Author(s):  
Jeong Yoon ◽  
Kyoung-Mi Juhn ◽  
San-Hyun Yoon ◽  
Yong Ko ◽  
Jin-Ho Lim
Keyword(s):  
In Vitro Maturation ◽  
Meiotic Maturation ◽  
Mouse Oocytes ◽  
Metaphase I

Reversal of postmortem degeneration of mouse oocytes during meiotic maturation in vitro

1991 ◽  
Vol 258 (2) ◽  
pp. 240-245 ◽  
Author(s):  
Allen C. Schroeder ◽  
Daniel Johnston ◽  
John J. Eppig
Keyword(s):  
Meiotic Maturation ◽  
Mouse Oocytes ◽  
Maturation In Vitro

348 INACTIVATION OF MAPK AFFECTS CENTROSOME ASSEMBLY, BUT NOT ACTIN FILAMENT ASSEMBLY, IN MOUSE OOCYTES MATURING IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 289
Author(s):  
S. E. Lee ◽  
J. H. Kim ◽  
Y. K. Ji ◽  
N. H. Kim
Keyword(s):  
Actin Filament ◽  
Mapk Pathway ◽  
Polar Body ◽  
Metaphase Plate ◽  
Specific Inhibitor ◽  
Mapk Signaling ◽  
Meiotic Maturation ◽  
Mouse Oocytes ◽  
Filament Assembly

Mitogen-activated protein kinase (MAPK) plays a crucial role in meiotic maturation of mouse oocytes. In order to understand the mechanism by which MAPK regulates meiotic maturation, we examined the effects of the MAPK pathway inhibitor U0126 on microtubule organization, γ-tubulin and NuMA distribution, and actin filament assembly in mouse oocytes maturing in vitro. Significant differences between control and treatment were determined using Tukey&apos;s multiple range test; P &lt; 0.05 was considered significant. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK was inactive in fully grown germinal vesicle (GV) oocytes. Phosphorylated MAPK was first detected 3 h after the initiation of maturation cultures, was fully active at 6 h, and remained active until metaphase II (MII). When GV oocytes were cultured in medium containing 20 �M U0126 for 9 h, the oocytes underwent geminal vesicle breakdown (GVBD), but were significantly inhibited in progression to the MI stage as compared to controls (control: 76.7 � 5.66%; 10 �M U0126: 29.0 � 7.35%; 20 �M U0126: 26.4 � 5.74%). Polar body extrusion was observed in most oocytes when cultured in the presence of 20 �M U0126 from 4 h after initiation of in vitro oocyte maturation to 14 h into the in vitro maturation incubation (control: 76.6 � 4.21%; 20 �M U0126: 70.7 � 4.61%). However, there was a decrease in normal MII-stage oocytes when cultured for 4 to 14 h (control: 87.7 � 5.12%; 20 �M U0126: 32.8 � 3.79%); abnormality included, for example, a polar body lacking a nucleus, as well as an abnormal spindle or metaphase plate. The inhibition of MAPK resulted in abnormal spindles and abnormal distributions of γ-tubulin and NuMA, but did not affect actin filament assembly. In conclusion, the meiotic abnormalities caused by U0126, a specific inhibitor of MAPK signaling, indicate that MAPK plays an important regulatory role in microtubule and centrosome assembly, but not actin filament assembly.

H1foo is essential for in vitro meiotic maturation of bovine oocytes

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Linker Histone ◽  
Meiotic Maturation ◽  
Control Group ◽  
Bovine Oocyte ◽  
First Polar Body ◽  
Effective Sirnas ◽  
Bovine Oocytes

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

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.

Diethylstilbestrol exposure disrupts mouse oocyte meiotic maturation in vitro through affecting spindle assembly and chromosome alignment

Chemosphere ◽  
2020 ◽  
Vol 249 ◽  
pp. 126182 ◽  
Author(s):  
Zhi-Ming Ding ◽  
Li-Ping Hua ◽  
Muhammad Jamil Ahmad ◽  
Muhammad Safdar ◽  
Fan Chen ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Mouse Oocyte ◽  
Meiotic Maturation ◽  
Maturation In Vitro ◽  
Chromosome Alignment ◽  
Oocyte Meiotic Maturation

287 RESCUE AND IN VITRO MATURATION OF FOLLICULAR OOCYTES IN CHINCHILLA LANIGER

2007 ◽  
Vol 19 (1) ◽  
pp. 259 ◽  
Author(s):  
G. Aiudi ◽  
M. Cinone ◽  
F. Maritato ◽  
A. De Sandro Salvati ◽  
M. E. Dell'Aquila
Keyword(s):  
Breeding Season ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Metaphase Plate ◽  
Meiotic Maturation ◽  
Current Therapy ◽  
Embryo Cryopreservation ◽  
Vaginal Smear ◽  
First Polar Body

The chinchilla is a hystricomorph rodent with a natural habitat in the Andes mountains of Chile (see review by Boussarie 2002 Proc. 27th WSAVA Congr.). For most of the chinchilla subspecies, the decline in the natural population can be attributed to human destruction of the native ecosystems and hunting for fur. Chinchillas are listed as a protected endangered species, at immediate risk of extinction. In Europe, chinchillas are reared for pets and fur production. The female has a seasonal polyestrous reproductive activity with a breeding season from November to May. The estrous cycle length is variable (28–41 days), with an estrous duration of 2 days. After a gestation of about 112 days, a litter of 1 to 6 young is born (see reviews by Morrow 1986 in Current Therapy in Theriogenology 2, W.B. Saunders; and Collot 1998 in Proc. I EVSSAR Congr.). Reproductive biotechniques in this species could play an important role in managing both captive and natural populations as well as in sustaining and improving genetic and global biodiversity. The specific aim of this preliminary work was to standardize an efficient in vitro maturation (IVM) procedure for Chinchilla laniger oocytes so that it will be possible, in the future, to perform IVF and embryo cryopreservation and transfer. Oocytes from 4 cyclic breeding females were recovered by slicing ovaries, obtained by ovariohysterectomy, and were matured in vitro according to the procedure described for bovine oocytes by Dell'Aquila et al. (2002 Mol. Reprod. Dev. 63, 210–222). Two trials of 2 estrous subjects each were performed, on the basis of behavioral signs of estrous and vaginal cytology (Harris-Schorr staining), in the early and late breeding seasons. During estrus, the vaginal smear consisted of superficial cells, further neutrophils, and small and large intermediates, whereas parabasal cells were not found. At the end of the culture time, oocytes were stained with Hoechst 33258 and evaluated for the stage of meiotic maturation. Three out of 4 oocytes recovered in November (75%) reached full meiotic maturation, showing the second metaphase plate and the first polar body (PB) extruded. The fourth oocyte, showing the first PB together with multiple pronuclear structures, was classified as activated. On the contrary, none out of 12 oocytes recovered in May reached full maturation. Of them, 7 (58%) remained at the germinal vesicle stage, 2 (17%) reached metaphase I, and 3 (25%) showed abnormally dispersed chromatin configuration. To our knowledge, this is the first study reporting that chinchilla oocytes can be matured in vitro by bovine IVM procedures. Even though the number of oocytes was poor, we can hypothesize that oocytes from C. laniger are best collected in the breeding season when subjected to an IVM technique.

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