Cytochalasin B-induced pseudo-cleavage of mouse oocytes in vitro: asymmetric localization of mitochondria and microvilli associated with a stage-specific response

1976 ◽  
Vol 21 (3) ◽  
pp. 523-535
Author(s):  
P.M. Wassarman ◽  
D.F. Albertini ◽  
W.J. Josefowicz ◽  
G.E. Letourneau
Keyword(s):  
Zona Pellucida ◽  
Cytochalasin B ◽  
Germinal Vesicle ◽  
Structural Features ◽  
Meiotic Maturation ◽  
Specific Response ◽  
Mouse Oocytes ◽  
Structural Differences ◽  
Cellular Compartments

Mouse oocytes are induced by cytochalasin B to undergo ‘pseudo-cleavage’ in vitro into 2 equally sized and separable compartments. This response to the drug is dependent upon the meiotic state of the oocytes, as well as upon the presence of an intact zona pellucida. The resulting 2 cellular compartments can be completely separated from another and cultured in vitro. Each of the compartments possesses characteristic structural features. The most pronounced structural differences include: (i) the presence of a nucleus (germinal vesicle) and nucleolus in one compartment; (ii) the presence of microvilli on the surface of the anucleate, but not the nucleate, compartment; and (iii) the localization (segregation) of mitochondria at the periphery of the anucleate, but not the nucleate, compartment. The results presented suggest that pseudo-cleavage induced by cytochalasin B arises as a consequence of a limited interaction of the drug with the oocyte surface and/or cortex and that it may represent a topographical dissociation of transporting and non-transporting regions of the membrane. These and other features of mouse oocytes treated with cytochalasin B are of interest in view of the involvement of the oocyte zona pellucida and plasma membrane during meiotic maturation, fertilization, and early embryogenesis.

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.

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: 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.

scholarly journals Luteinizing hormone-accelerated redistribution of lysosome-like organelles preceding dissolution of the nuclear envelope in rat oocytes maturing in vitro.

1979 ◽  
Vol 82 (1) ◽  
pp. 264-277 ◽  
Author(s):  
R M Ezzell ◽  
C M Szego
Keyword(s):  
Luteinizing Hormone ◽  
Nuclear Envelope ◽  
Germinal Vesicle ◽  
Defined Medium ◽  
Meiotic Maturation ◽  
Homogeneous Distribution ◽  
Target Cells ◽  
Specific Response ◽  
Germinal Vesicle Breakdown

Maturation of the mammalian oocyte is characterized in part by dissolution of the nuclear envelope, or germinal vesicle breakdown (GVB). By fluorescence microscopy after vital uptake of acridine orange (AO), redistribution and perinuclear accumulation of organelles corresponding to lysosomes occur before GVB in rat oocytes undergoing meiotic maturation in vitro. In follicle-enclosed oocytes explanted during the preovulatory gonadotropin surge (GS) and individually cultured as such in chemically defined medium at approximately 22 degrees C, lysosomes aggregated into disperse clusters after 30 min; by 60 min, perinuclear concentration of lysosomes and their essential disappearance from the cortical ooplasm were observed. GVB occurred within 120 min. In contrast, follicle-enclosed oocytes explanted before the GS displayed a generally homogeneous distribution of lysosomes and intact GV for up to 5 h in culture. In oocytes aspirated from follicles before the GS, partially denuded of granulosa cells, and cultivated without added hormone, most lysosomes concentrated around the GV within 60 min, with GVB occurring generally by 120 min. Luteinizing hormone (LH) added in vitro to the isolated preparation at 3 or 30 x 10(-8) M sharply accelerated these events. The effects of LH, not seen with 1.5 x 10(-8) M hormone, were blocked by anti-LH IgG. Up to 60 x 10(-8) M follicle-stimulating hormone or 80 x 10(-8) M prolactin were ineffective in accelerating lysosome redistribution or GVB. After GVB, lysosomes became once again uniformly dispersed and unresponsive, even to 60 x 10(-8) M added LH, a finding consistent with tachyphylaxis of target cells by independent criteria. The present data, all statistically significant at P less than 0.05, demonstrate that mobilization of lysosomes before GVB is a specific response to factors that promote resumption of meiotic maturation of rat 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 Cytochalasin B-induced triploidy in mouse oocytes fertilized in vitro

Reproduction ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 279-284 ◽  
Author(s):  
A. Niemierko ◽  
A. Komar
Keyword(s):  
Cytochalasin B ◽  
Mouse Oocytes

Differential cytolocalization of prosomes in axolotl during oogenesis and meiotic maturation

1988 ◽  
Vol 90 (4) ◽  
pp. 543-553 ◽  
Author(s):  
J. Gautier ◽  
J.K. Pal ◽  
M.F. Grossi de Sa ◽  
J.C. Beetschen ◽  
K. Scherrer
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Germinal Vesicle ◽  
Immunoblot Analysis ◽  
Ambystoma Mexicanum ◽  
Meiotic Maturation ◽  
Crescent Formation ◽  
Oocyte Growth ◽  
Oocyte Cytoplasm

The prosomes, a novel type of small RNA-protein complex previously characterized in avian and mammalian cells, were isolated from axolotl (Ambystoma mexicanum) oocytes and identified by sedimentation analysis and protein composition. The prosomal nature of these particles was further ascertained by immunoblot analysis with anti-duck prosome monoclonal antibodies. By in vitro [35S]methionine labelling, de novo synthesis of prosomal proteins could be detected neither during oogenesis nor meiotic maturation. The results obtained by both indirect immunofluorescence and immunoblot analyses demonstrated a dramatic change in the localization of prosomal antigens during oocyte development. They were initially detected in the oocyte cytoplasm, during oocyte growth. At the end of vitellogenesis (stages V-VI), they entered the nucleus (germinal vesicle) and were accumulated there to the highest concentration. During oocyte maturation, after nuclear envelope breakdown, prosomal antigens were found to be localized again in the cytoplasm, until fertilization. No specific localization of prosomal antigens in mature oocytes, unfertilized and fertilized eggs was observed within the oocyte cytoplasm in relation to the cytoplasmic rearrangements leading to grey crescent formation.

Cytochalasin B-induced pseudo-cleavage of mouse oocytes in vitro. II. Studies of the mechanism and morphological consequences of pseudocleavage

1977 ◽  
Vol 26 (1) ◽  
pp. 323-337
Author(s):  
P.M. Wassarman ◽  
T.E. Ukena ◽  
W.J. Josefowicz ◽  
G.E. Letourneau ◽  
M.J. Karnovsky
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Cytochalasin B ◽  
Oocyte Size ◽  
Contractile Ring ◽  
Subsequent Formation ◽  
Mouse Oocytes ◽  
Meiotic Competence ◽  
Scanning Electron

Mouse oocytes are induced by cytochalasin B to undergo ‘pseudocleavage’ in vitro into 2 compartments, only one of which possesses microvilli. It has been found that this particular response to cytochalasin B is related to oocyte size and, possibly, to the acquisition of meiotic competence by the oocyte during its growth phase. Certain of the morphological events which characterize pseudocleavage have been determined using transmission and scanning electron microscopy. These events include: (i) an initial withdrawal of microvilli from the surface of the oocyte, together with the concomitant disappearance of microfilaments normally associated with the microvilli; (ii) the subsequent formation of a pseudocleavage furrow and contractile ring; and (iii) the reappearance of microvilli and associated microfilaments in one of the two resulting oocyte compartments. These changes in surface architecture are reflected in the distribution of fluorescein-conjugated lectins bound to the oocyte surface during pseudocleavage.

Sign in / Sign up

Export Citation Format

Share Document