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Gamete Biology |
a State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
b College of Life Science, Suzhou University, Suzhou 215006, China
c College of Life Science, Agricultural University of China, Beijing 100094, China
d College of Animal Science and Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, in vitro fertilization, meiosis, oocyte development, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The completion of two meiotic divisions is a result of the coordination of meiotic spindle assembly and function with meiotic cell-cycle progression [3]. The dynamic changes of assembly and disassembly of microtubules (MTs) and microfilaments (MFs) in meiosis have been studied in Xenopus [4, 5], Drosophila [6], yeast [7], and many mammals [8–14]. Nevertheless, little is known about MT and MF functions or about their interaction in meiotic spindle formation and rotation, chromosomal movement, and polar body extrusion.
Polymerization of G-actin into F-actin results in MF assembly. Cytochalasin B (CB), an inhibitor of MF polymerization, is widely used in animal cloning [15, 16] and polyploid embryo or cell induction [17–19]. It is generally accepted that CB inhibits the polymerization of MFs by blocking monomer addition at the fast-growing end of F-actin.
In the present study, we observed meiotic progress in oocytes treated with CB to block F-actin polymerization during in vitro fertilization (IVF) or parthenogenetic activation to analyze the role of MFs in processes accompanying meiosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal care and handling were conducted in accordance with policies on the care and use of animals promulgated by the ethical committee of the State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences.
Female Kunming White mice (age, 6–8 wk) were injected with 7.5 IU of eCG (Hua Fu Biotechnology Company, Tianjin, China), followed 48 h later by 7.5 IU of hCG (Institute of Zoology, Chinese Academy of Sciences, Beijing). The females were killed 14–17 h post-hCG administration, and the ovulated oocytes were released from the ampullae of oviducts into pre-equilibrated M2 medium (Sigma, St. Louis, MO). Cumulus-oocyte complexes (COCs) were used directly for IVF. Alternatively, the cumulus cells removed by brief exposure to M2 containing 300 IU/ml of hyaluronidase (Sigma), and then the cumulus-free, MII-stage oocytes were used for parthenogenetic activation after being washed at least three times with M2.
In Vitro Fertilization
Cauda epididymides from male Kunming mice (age, 8–12 wk) were immersed in a 200-µl drop of M16 medium (Sigma). Sperm were released by cutting cauda epididymides with a pair of scissors. For capacitation, the sperm were cultured for 1–1.5 h in M16 medium under liquid paraffin in a humidified atmosphere of 5% CO2 at 37°C. Five 10-µl drops of sperm suspension was added to a 50-µl drop of M16 medium containing COCs, giving a final concentration of 106–7 sperm/ml. After coculturing sperm and oocytes for 6 h, sperm were removed by washing oocytes in M2 medium. The fertilized oocytes were further cultured until they were used.
Parthenogenetic Activation
Just before use, SrCl2 was added to Ca2+-free M16 medium. Cumulus-free MII oocytes were incubated in M16 medium containing 10 mM SrCl2 for 4–6 h. After washing with M2, the oocytes were further cultured in CZB (Chatot-Ziomek-Bavister) medium.
Treatment with CB
Cytochalasin B (Sigma) was dissolved as a stock solution (1 mg/ml) in dimethyl sulfoxide (DMSO; Sigma) and stored at -20°C. It was later diluted to a final concentration of 10 µg/ml in insemination medium or activation medium. An equivalent dilution of DMSO was used for controls. The IVF oocytes were treated with CB for 6 h, and the parthenogenetic oocytes were treated for 4 h. Both the IVF oocytes and the parthenogenetic oocytes were removed from the medium containing CB, washed at least three times in M2, and then cultured in CB-free medium. In addition, the effects of diverse CB-treatment periods (0–5 h) on polar body extrusion of parthenogenetic oocytes were analyzed. Data were analyzed using the chi-square test, with significance determined at P < 0.05.
Immunofluorescent Staining and Confocal Microscopy
Samples were taken at 1-h intervals until clear pronuclei were observed. Oocytes were fixed with 3.7% (w/v) paraformaldehyde in PBS for 40 min at room temperature. Fixed oocytes were stored in PBS containing 0.3% (w/v) BSA for up to 1 wk at 4°C. Fixed oocytes were permeabilized by transferring into PBS containing 0.1% (w/v) Triton X-100 and 0.3% BSA and then incubating them for 30–40 min at 37°C. After washing twice with PBS containing 0.01% Triton X-100, oocytes were incubated in block solution (PBS containing 150 mM glycine and 0.3% BSA) for 30 min at 37°C. The MTs were localized with a mouse monoclonal antibody against ß-tubulin (Sigma), which was diluted in the blocking solution (1:160 [w/v]) before use. Oocytes were incubated for 30–40 min at 37°C or overnight at 4°C, followed by three washes of 5 min each. Oocytes were incubated with fluorescein isothiocyanate-labeled goat-anti-mouse IgG (Sigma) at 1:80 (w/v) final dilution for 30–40 min at 37°C, followed by three washes of 5 min each. Chromatin was stained with 10 µg/ml of propidium iodide (Sigma). Finally, oocytes were mounted on slides with antifluorescence-fade medium (DABCO). The samples were examined with a laser-scanning confocal microscope (Leica TCS-4D, Bensheim, Germany). Images were processed with Photoshop 6.0 software (Adobe Systems, Inc., San Jose, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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By immunofluorescent staining, MTs were found mainly in the well-organized spindle in MII oocytes. The spindle was symmetrical, bipolar, barrel-shaped, and located near the cortex of the oocyte (Fig. 1A). From 1 to 3 h postinsemination, a period during which oocytes were at the anaphase of meiosis II, the spindle migrated further into the cortex, and daughter chromatids separated toward the two poles, followed by the spindle rotating from parallel to vertical with respect to the surface of the oocyte (Fig. 1, B and C). From 3 to 6 h (i.e., at the telophase stage), the spindle was oriented vertically with respect to the oocyte's surface. It then elongated and formed the second polar body, which was extruded into the perivitelline space. A female pronucleus and a male pronucleus were observed in the cytoplasm before long (Fig. 1D).
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Effect of CB on Spindle During IVF-Induced Meiosis
Sperm and oocytes were cocultured in the insemination medium containing 10 µg/ml of CB for 6 h. The oocytes were washed to remove sperm and transferred into a CB-free M16 medium. Neither the resumption of meiosis nor the separation of daughter chromatids (i.e., nuclear division) was disturbed. However, in CB-treated IVF oocytes, spindle rotation was inhibited. At 1 to 4 h post-CB treatment, 35 of 56 spindles (62.5%) of fertilized oocytes were still parallel to the plasmalemma, whereas only 7 of 56 spindles (12.5%) were vertical with the oocyte's surface (Fig. 1E). At 4–6 h, meiosis was terminated. Chromosomes that had reached spindle poles decondensed and, therefore, formed two female pronuclei that were connected by a midbody. As a consequence of this treatment, these fertilized oocytes were triploid, containing one male pronucleus and two female pronuclei (Fig. 1F).
Dynamic Changes of Spindle During Meiosis Induced by Parthenogenetic Activation
Dynamic changes of spindle during meiosis induced by parthenogenetic activation were similar to those of IVF-induced meiosis. Spindle rotation and second polar body extrusion occurred mainly within 2 and 3 h, respectively, which was slightly earlier than in IVF oocytes. However, a female pronucleus was formed at 4–6 h, which was not earlier than in IVF oocytes (Fig. 2, A–D).
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Effect of CB on Spindle During Parthenogenetic Activation of Oocytes
Addition of CB to the activation medium inhibited spindle rotation, but nuclear division did not appear to be disturbed. Fifty-one of 75 spindles (68%) of activated oocytes were still parallel to the plasmalemma, whereas only 8 of 75 (10.7%) were vertical to the oocyte's surface (Fig. 2E). The maternal chromatin normally released into the forming second polar body remained within the oocyte's cytoplasm. Therefore, two haploid female pronuclei connected by a midbody were observed in the cytoplasm (Fig. 2F).
Influence of CB Treatment Time on Second Polar Body Extrusion
Oocytes were treated with CB for various times (0–5 h) during activation. As shown in Table 1, 88.7% of activated oocytes in the control group extruded the second polar body at 3 h postactivation. When the CB treatment time was less than 2 h, polar body extrusion and spindle rotation occurred on time. When the CB treatment time was 3 h, 55.9% of activated oocytes failed to extrude the second polar body, which transformed into an extra female pronucleus that remained within the cytoplasm. The rates of second polar body extrusion were 19.8% and 7.6% when oocytes were treated for 4 and 5 h, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytochalasin B, an MF depolymerization drug widely used in animal cloning [15, 16] and polyploid induction [17–19], can inhibit polar body extrusion and cytokinesis, but its mechanism has not been clearly understood. Longo and Chen [21] reported that germinal vesicle breakdown could occur normally when germinal vesicle-stage oocytes were treated with CB but that spindles could not migrate to cortex region, so that the extrusion of the first polar body was inhibited. Our results showed that CB did not affect chromosomal movement and nuclear division but did inhibit spindle rotation and, thus, cytokinesis. As a result, the chromatin normally partitioned to the second polar body remained within the oocyte cytoplasm and was transformed into an extra pronucleus. These results illustrate that the spindle rotation is essential for polar body extrusion, and MFs are instrumental in controlling rotation of the meiotic spindle.
Changes in oocyte cytoplasmic organization are executed with great temporal and spatial precision to ensure that peri- and postfertilization events of embryogenesis proceed on schedule and without error [22]. When the time of CB treatment was beyond 3 h during activation, irreversible inhibition of spindle rotation was observed. This is probably because CB depolymerizes MFs during the key period of spindle rotation and polar body extrusion. Following washing to remove CB, both spindle rotation and polar body extrusion failed to occur, indicating that temporal and spatial factors associated with these procedures were no longer satisfied. We also showed that 2-h CB treatment did not inhibit second polar body extrusion, which is consistent with previous reports [1, 8].
Recent studies have demonstrated that jasplakinolide, a drug promoting MF polymerization and stabilization, in contrast to CB, also inhibited oocyte maturation and polar body extrusion [23]. It is suggested that the dynamic balance between assembly and disassembly of MFs is disordered by the drug either promoting or inhibiting MF polymerization and, thus, inhibiting spindle rotation and polar body extrusion.
In conclusion, the present results indicate that MFs play a crucial role in controlling spindle rotation in mouse oocytes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Da-Yuan Chen, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. FAX: 010 62565689; e-mail: chendy{at}panda.ioz.ac.cn
Received: 1 August 2002.
First decision: 21 August 2002.
Accepted: 28 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes. Biol Reprod 2000 62:1184-1192-tubulin. Dev Biol 2001 239:281-294[CrossRef][Medline]This article has been cited by other articles:
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  S. Hou, L Zhang, K Wu, and L Xia Thioglycolic acid inhibits mouse oocyte maturation and affects chromosomal arrangement and spindle configuration Toxicology and Industrial Health, May 1, 2008; 24(4): 227 - 234. [Abstract] [PDF]
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 Q.-Y. Sun and H. Schatten Regulation of dynamic events by microfilaments during oocyte maturation and fertilization Reproduction, February 1, 2006; 131(2): 193 - 205. [Abstract] [Full Text] [PDF]
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H.-Y. Fan and Q.-Y. Sun Involvement of Mitogen-Activated Protein Kinase Cascade During Oocyte Maturation and Fertilization in Mammals Biol Reprod, March 1, 2004; 70(3): 535 - 547. [Abstract] [Full Text] [PDF]
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Y.-H. Choi, L. B. Love, M. E. Westhusin, and K. Hinrichs Activation of Equine Nuclear Transfer Oocytes: Methods and Timing of Treatment in Relation to Nuclear Remodeling Biol Reprod, January 1, 2004; 70(1): 46 - 53. [Abstract] [Full Text] [PDF]
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