Hostname: page-component-77c89778f8-vpsfw Total loading time: 0 Render date: 2024-07-20T18:14:48.511Z Has data issue: false hasContentIssue false
  • English
  • Français

Microinjection of anti-α-tubulin antibody (DM1A) inhibits progesterone-induced meiotic maturation and deranges the microtubule array in follicle-enclosed oocytes of the frog, Rana pipiens

Published online by Cambridge University Press:  26 September 2008

Charles A. Lessman ,
Tao Wang ,
David L. Gard  and
Catherine W. Woods
Charles A. Lessman*
Affiliation:
Department of Microbiology & Molecular Cell Sciences, University of Memphis, Tennessee and Department of Biology, University of Utah, Salt Lake City, Utah, USA.
Tao Wang
Affiliation:
Department of Microbiology & Molecular Cell Sciences, University of Memphis, Tennessee and Department of Biology, University of Utah, Salt Lake City, Utah, USA.
David L. Gard
Affiliation:
Department of Microbiology & Molecular Cell Sciences, University of Memphis, Tennessee and Department of Biology, University of Utah, Salt Lake City, Utah, USA.
Catherine W. Woods
Affiliation:
Department of Microbiology & Molecular Cell Sciences, University of Memphis, Tennessee and Department of Biology, University of Utah, Salt Lake City, Utah, USA.
*
Charles A. Lessman, PhD, Department of Microbiology & Molecular Cell Sciences, Campus Box 526041, University of Memphis, Memphis, TN 381526041, USA. Tel: +1 (901) 678-2963. Fax: +1 (901) 678-4457. e-mail: lessmance@msuvxl.memphis.edu.
Get access Rights & Permissions [Opens in a new window]

Summary

Microinjection of anti-α-tubulin (Dm1A) inhibited progesterone-induced meiotic maturation in large follicle-enclosed oocytes of the frog, Rana pipiens. DM1A (46nl; 10mg/ml) injection significantly increased the ED50 value for progesterone as determined by germinal vesicle dissolution (GVD) bioassay. By contrast, low doses of microinjected DM1A (46nl; 2.5mg/ml), anti-actin (clone KJ43A), anti-cytokeratin (C-11), anti-intermediate filament antibody (IFA), generic IgG (46 nl; 20 mg/ml) or sodium azide (46 nl; 1 mg/ml), an antibody preservative, were without inhibitory effect in this bioassay. Microinjected, affinity-purified DM1A (46n1; 7.5mg/ml) was also inhibitory, but preabsorption with pure tubulin prior to injection significantly reduced the inhibitory effect. DM1A injection had no effect on centrifugation-induced germinal vesicle migration (GVM). Previous work indicated that drugs (e.g. demecolcine and nocodazole), which destabilise microtubules, enhance both centrifugation-induced GVM and progesterone-induced GVD in Rana oocytes. Taking these results together, it is suggested that DM1A injection may have differential effects on microtubules in this cell. Thus, while the majority of microtubules were apparentl depolymerised by DM1A (46nl; 10mg/ml) injection, a small subpopulation appeared to be stabilised as bundles. Confocal immunofluorescence microscopy of follicle-enclosed oocytes after DM1A injection revealed a major loss of microtubules throughout the cell however, apparent sparse bundles of nucrotubules arranged in an approximately 600 μm shell were associated with the injectate region 24h post-injection. By contrast, control follicle-enclosed oocytes topically labelled with DM1A post-fixation had extensive microtubule arrays similar to those previously reported in Xenopus oocytes. Intracellular recording after DM1A injection and progesterone treatment yielded an intermediate membrane potential (Vm= −31.8mV) compared with control (immature) DMIA-injected cells (Vm= −44.7 mV) or potassium balanced salt solution (KBS)-injected cells matured with progesterone (Vm= −13.9mV). These results suggest that DM1A injection does not completely inhibit electrophysiological changes initiated by progesterone. Working hypotheses are proposed that suggest a role for microtubules in the action of progesterone which normally lifts the prophase I block in the Rana follicle-enclosed oocyte.

Keywords

Confocal immunofluorescence microscopyGerminal versicleα-tubulinMeiosisElectro-physiologyFollicle
Type
Article
Information
Zygote , Volume 5 , Issue 1 , February 1997 , pp. 83 - 95
Copyright
Copyright © Cambridge University Press 1997

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Albertini, D.F.. (1992). Regulation of meiotic maturation in the mammalian oocyte: interplay between exogenous cues and the microtubule cytoskeleton. BioEssays 14, 97–103.Google Scholar
Blose, S.H., Meltzer, D.I. & Feramisco, J.R.. (1984). 10nm filaments are induced to collapse in living cells micro-injected with monoclonal and polyclonal antibodies against tubulin. J. Cell Biol. 98, 847–58.CrossRefGoogle Scholar
Breitling, F. & Little, M.. (1986). Carboxy-terminal regions on the surface of tubulins and microtubules: epitope locations of YOL1/34, DM1A and DM1B. J. Mol. Biol. 189, 367–70.CrossRefGoogle ScholarPubMed
Brown, B.W.. (1970). Quantal response assays. InStatistics in Endocrinology, McArthur, J.W. & Colton, T. pp. 129–43. Cambridge, Mass.: MIT Press.Google Scholar
Deshpande, A.K. & Kung, H.-F (1987). Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras protein. Mol. Cell. Biol. 7, 1285–8.Google Scholar
Downie, N.M. & Heath, R.W.. (1965). Basic Statistical Methods, 2nd edn, pp. 146–53. New York: Harper & Row.Google Scholar
Draber, P., Vater, W., Bohm, K.J., Kuklova, E. & Unger, E. (1990). Inhibition of microtubule assembly in vitro by antitubulin monoclonal antibodies. FEBS Lett. 262, 209–11.CrossRefGoogle ScholarPubMed
Elinson, R.P. & Houliston, E. (1990). Cytoskeleton in Xenopus oocytes and eggs. Semin. Cell Biol. 1,349–57.Google ScholarPubMed
Fosket, D.E. & Morejohn, L.C. (1992). Structural and functional organization of tubulin. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 201–40.CrossRefGoogle Scholar
Gard, D.L. (1991). Organization, nucleation, and acetylation of microtubules in Xenopus loevis oocytes: a study by confocal immunofluorescence microscopy. Dev. Biol. 143, 346–62.CrossRefGoogle Scholar
Gard, D.L. (1992). Microtubule organization during maturation of Xenopus oocytes: assembly and rotation of the meiotic spindles. Dev. Biol. 151, 516–30.CrossRefGoogle ScholarPubMed
Gard, D.L. (1993). Ectopic spindle assembly during maturation of Xenopus oocytes: evidence for functional polarization of the oocyte cortex. Dev. Biol. 159. 298–310.CrossRefGoogle ScholarPubMed
Han, J.K., Fukami, K. & Nuccitelli, R. (1992). Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres. J. Cell Biol. 116, 147–56.CrossRefGoogle ScholarPubMed
Harlow, E. & Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.Google Scholar
Huchon, D., Crozet, N., Cantenot, N. & Ozon, R. (1981). Germinal vesicle breakdown in the Xenopus laevis oocyte: description of a transient microtubular structure. Reprod. Nutr. Dev. 21,135–48.Google Scholar
Huchon, D., Jessus, C., Thibier, C. & Ozon, R. (1988). Presence of microtubules in isolated cortices of prophase I and metaphase II oocytes in Xenopus laevis. Cell Tissue Res. 254, 415–20.Google Scholar
Jessus, C., Huchon, D. & Ozon, R. (1986). Distribution of microtubules during the breakdown of the nuclear envelope of the Xenopus oocyte: an immunocytochemical study. Biol. Cell 56, 113–20.CrossRefGoogle ScholarPubMed
Jessus, C., Thibier, C. & Ozon, R. (1987). Levels of micro-tubules during the meiotic maturation of the Xenopus oocyte. J. Cell Sci. 87, 705–12.CrossRefGoogle Scholar
Jessus, C., Thibier, C., Huchon, D. & Ozon, R. (1988). Taxol reveals cortical sites of microtubule assembly in Xenopus oocyte: role of the nucleus. Cell Differ. Dev. 25, 57–64.CrossRefGoogle ScholarPubMed
Jha, M.N.. Bamburg, J.R. & Bedford, J.S. (1994). Cell cycle arrest by colcemid differs in human normal and tumor cells. Cancer Res. 54, 5011–15.Google Scholar
Jockusch, B.M. & Temm-Crove, C.J. (1993). Microinjection of antibodies in the analysis of cellular architecture and motility. Methods Cell Biol. 37, 343–59.CrossRefGoogle ScholarPubMed
Kubiak, J.Z., Weber, M., Pennart, H.D., Winston, N.J. & Maro, B. (1993). The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of csf. EMBO J. 12, 3373–8.Google Scholar
Laemmli, E.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–5.CrossRefGoogle ScholarPubMed
Lessman, C.A. (1987). Germinal vesicle migration and dissolution in Rena pipiens oocytes: effect of steroids and microtubule poisons. Cell Differ. 20, 239–51.CrossRefGoogle Scholar
Lessman, C.A. (1993). Taxol-induced assembly of brain and testis tubulins, and ovarian tubulin dynamics in the frog (genus Rena) in vitro. Comp. Biochem. Physiol. 104, 155–62.Google Scholar
Lessman, C.A. & Kessel, R.C. (1992). Effects of acrylamide on germinal vesicle migration and dissolution in oocytes of the frog, Rana pipiens. Exp. Cell Res. 202, 151–60.CrossRefGoogle ScholarPubMed
Lessman, C.A. & Marshall, W.S. (1984). Electrophysiology of in vitro insulin- and progesterone-induced reinitiation of oocyte meiosis in Rana pipiens. J. Exp. Zool. 231, 257–66.CrossRefGoogle ScholarPubMed
Lessman, C.A. & Ventura, A. (1994). Effects of soluble acrylamide and cold (4°C) on centrifugation-induced germinal vesicle positioning in Rana pipiens oocytes subjected to centrifugal force. Adv. Sci. Res. 1, 25–30.Google Scholar
Lessrnan, C.A., Marshall, W.S. & Habibi, H.R. (1986). Movement and dissolution of the nucleus (germinal vesicle) during Rena oocyte meiosis: effect of demecolcine (Colcemid) and centrifugation. Gamete Res. 14, 11–23.CrossRefGoogle Scholar
Lieuvin, A., Labbe, J.C., Doree, M. & Job, D. (1994). Intrinsic microtubule stability in interphase cells. J. Cell Biol. 124, 985–96.CrossRefGoogle ScholarPubMed
McKinnell, R.G., DeBruyne, G.K., Marcel, M.M., Tarin, D. & Tweedell, K.S. (1984). Cytoplasmic microtubules of normal and tumor cells of the leopard frog. Differentiation 26, 231–4.Google Scholar
Meyer, T., Weber, K. & Osbom, M. (1992). Microinjection of IFA antibody induces intermediate filament aggregates in epithelial cell lines but perinuclear coils in fibroblast-like lines. Eur. J. Cell Biol. 57, 75–87.Google Scholar
Moreau, N., Laine, M., Billoud, B. & Angelier, N. (1994). Transcription of amphibian lampbrush chromosomes is disturbed by microinjection of HSP7O monoclonal antibodies. Exp. Cell Res. 211. 108–14.Google Scholar
Morgan, D.O., Ho, L., Korn, L.J. & Roth, R.A. (1986). Insulin action is blocked by a monoclonal antibody that inhibits the insulin receptor kinase. Proc. Natl. Acad. Sci. USA 83, 328–32.Google Scholar
Ookata, K., Hisanaga, S., Bulinski, J.C., Murofushi, H., Aizawa, H., Itoh, T.J., Hotani, H., Okumura, E., Tachibana, K. & Kishimoto, T. (1995). Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics. J. Cell Biol. 128, 849–62.CrossRefGoogle ScholarPubMed
Pruss, R.M., Mirsky, R. & Raft, M.C. (1981). All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell 27, 419–28.Google Scholar
Redeker, V., Melki, R., Prome, D., Lecaer, J.P. & Rossier, J. (1992). Structure of tubulin C-terminal domain obtained by subtilisin treatment: the major à-tubulin and à-tubulin isotypes from pig brain are glutamylated. FEBS Lett. 313. 185–92.Google Scholar
Scheer, U. (1986). Injection of antibodies into the nucleus of amphibian oocytes: an experimental means of interfering with gene expression in the living cell. J. Embryol. Exp. Morphol. 97, 223–42.Google Scholar
Schuetz, A.W. & Lessman, C.A. (1982). Role of the surface epitheliuni and follicle wall in ovulation and progesterone production by frog (Rena pipiens) follicles. Differentiation 22, 79–84.CrossRefGoogle Scholar
Soreq, H. & Seidman, S. (1992). Xenopus oocyte micro- injection: from gene to protein. Methods Enzymol. 207, 225–65.Google Scholar
Tang, W-JY (1993). Blot-affinity purification of antibodies. Methods Cell Biol. 37, 95–104.Google Scholar
Thibier, C., Denoulet, P., Jessus, C. & Ozon, R. (1992). A predominant basic à-tubulin isoform present in prophase Xenopus oocyte decreases during meiotic maturation. Biol. Cell 75, 173–80.CrossRefGoogle Scholar
Wang, T. & Lessman, C.A. (1994). Tubulin heterogeneity in brains and oocytes of southern and northern frogs of the genus Rena. Am. Zool. 34, 67a.Google Scholar
Wang, T., Lessman, C.A. & Gard, D.L. (1993). Developmental regulation of cold-stable microtubules (MTs) during oogenesis in Rana pipiens. Mol. Biol. Cell 4, 26a.Google Scholar
Warn, R.M., Fleg, L. & Warn, A. (1987). An investigation of microtubule organization and functions in living Drosophila embiyos by injection of a fluorescently labeled antibody against tyrosinated α-tubulin. J. Cell Blol. 105, 1721–30.Google Scholar
Wehland, J., Willingham, M.C. & Sandoval, I.V. (1983). A rat monoclonal antibody reacting specifically with the tyrosylated form of α-Tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and micro-Tubule polymerization and organization in viva. J. Cell Biol. 97, 1467–75.CrossRefGoogle Scholar
Wright, B.D., Terasaki, M. & Scholey, J.M. (1993). Roles of kinesin and kinesin-like proteins in sea urchin embryonic cell divisioit evaluation using antibody microinjection. J. Cell Biol. 123, 681–9.CrossRefGoogle ScholarPubMed
Wu, W.-L. & viorgan, G.T. (1994). Ovary-specific expression of a gene encoding a divergent à-tubulin isotype in Xenopus. Differentiation 58,9–18.Google Scholar
Ziegler, D. & Morrill, G.A. (1977). Regulation of the amphibian oocyte plasma membrane ion permeability by cytoplasmic factors during the first meiotic division. Dev. Biol. 60, 318–25.Google Scholar
Zhou, R., Oskasson, M., Paules, R.S., Schultz, N., Cleveland, D. & Woude, G.F.V. (1991 a). Ability of the c-mos product to associate with and phosphorylate tubulin. Science 251, 671–5.Google Scholar
Zhou, R., Rulong, S., Pinto da Silva, P. & Vande Woude, G.F. (1991 b). in vitro and in vivo characterization of pp39mos association with tubulin. Cell Growth Differ. 2 257–65.Google Scholar