Microinjection of transforming p21 ras protein induces maturation of Xenopus laevis oocytes, and the induction is blocked by coinjection of monoclonal antibody (Y13-259) against p21 ras proteins. Similar to other inducing agents, the effect of p21 ras protein is mediated via the appearance of maturation or meiosispromoting factor activity. In addition, the neutralizing antibody markedly reduces oocyte maturation after insulin induction, whereas it fails to inhibit progesterone induction. Our results suggest that insulin induces maturation of oocytes via a different pathway than that of steroidal agents. The induction by insulin is ras dependent, and the action of ras may be directed at the steps before meiosis-promoting factor autocatalytic activation. These results suggest a role of p21 ras protein in the events associated with amphibian oocyte maturation.The ras family forms a group of closely related transforming genes that are highly conserved in eucaryotes (5,11,18,28,34,48). Normal (cellular) ras genes acquire transforming properties by single point mutations within their coding sequences (4,32,33,41,(45)(46)(47)54), and these mutated ras genes have been detected in a significant fraction of human cancers as well as in experimentally induced animal tumors (45,48,54). It has been speculated that p21 ras proteins are essential components of normal cells that are involved in cell division and differentiation (30,31). Yet, little is known regarding the mechanisms by which p21 proteins induce malignant transformation, and even less is known regarding their role in normal cells.It has been demonstrated that p21 proteins can induce transformation of NIH 3T3 cells when introduced by microinjection (13,44) and that the transforming activity of the ras proteins can be blocked by coinjection with monoclonal antibody Y13-259 (19). Recently, Birchmeier et al. (3) have shown that ras proteins can induce meiosis in Xenopus laevis oocytes. Fully grown oocytes can be easily injected and analyzed biochemically. Oocytes surgically removed from adult Xenopus ovaries are arrested in the prophase of meiosis and can be triggered to undergo meiosis by treatment with progesterone, insulin, and a variety of other agents. In the present study, we determined the effect of injected neutralizing antibody (Y13-259) on the maturation of oocytes.Gravid X. laevis females were obtained from Nasco Co. (Fort Atkinson, Wis.) and maintained under laboratory conditions (9). Ovarian fragments were surgically removed from frogs that were anesthetized by hypothermia. Fully grown stage VI oocytes were manually dissected, and follicle cells were removed after collagenase treatment of ovarian fragments at 21°C following the procedure of Eppig and Dumont (12) in modified Barth medium (MBSH) buffered with 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.6 (22). The selected stage VI oocytes were thoroughly washed in MBSH and equilibrated for at least 2 h before use. Oocytes were cultured in 30-mm Falcon * Corresponding author. ster...

Plasma membrane fractions from Xenopus laevis oocytes were incorporated into planar lipid bilayers. We show the existence of numerous Ca2+-activated nonspecific channels that are more permeable to anions. These Ca2+ (13,14) trigger membrane depolarization and oocyte and egg activation.To understand the molecular basis of the fertilization potential and its possible association with intracellular calcium changes, we recently described a simple procedure for enriching plasma membranes of Xenopus oocytes (unpublished data). Here, we have incorporated purified plasma membrane fractions of X. laevis oocytes into planar lipid bilayers. The high impedance of the planar bilayer system allows measurements of small conductance fluctuations with improved time resolution. Furthermore, the voltage across the membrane can be clamped to mimic cell plasma membrane potential. Here, the properties of a Ca '-activated nonspecific channel found abundantly in X. Iaevis oocyte membranes are described. This Ca2+-activated channel may be associated directly with the fertilization potential in X. laevis. MATERIALS AND METHODSOocyte Membranes. Ovaries were surgically removed from gravid X. laevis females immersed in water containing 0.15% tricaine (Calbiochem-Behring), and the tissues were placed in ice-cold modified Barth's medium (MBSH medium) containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2 4H2O, 0.41 mM CaC12, 10 mM Hepes (pH 7.2), and 10 mg of benzylpenicillin and streptomycin per liter. After several washes in MBSH medium, oocytes (Dumont stage IV-VI, with an average diameter of 1.0-1.4 mm) were manually isolated from ovary fragments by dissection with fine forceps. The oocytes were exposed to Pronase (75 ,g/ml, from Streptomyces griseus; Calbiochem-Behring) for 10 min at 22-24°C to remove adhering follicular cells. The oocytes were allowed to sediment with six changes of MBSH medium. Oocytes were pooled and lysed by three freeze-thaw cycles in a sorbitol/sucrose medium (175 mM D-sorbitol/0.3 M NaCl/75 mM sucrose/30 mM KCl/10 mM MgCl2/5 mM EDTA/1 mM NaHCO3, pH 7.5, with 1 mM phenylmethylsulfonyl fluoride (Sigma) and aprotinin (FBA Pharmaceuticals, New York) at 0.5 units/ ml). As a second method, oocyte extracts in sorbitol/ sucrose medium were prepared by homogenizing ovary fragments (<2-mm fragments) in a Potter-Elvehjem homogenizer with gentle strokes (pestle clearance, >0.5 mm), which ensured breakage mainly of Dumont stage IV-VI oocytes (unpublished data).The oocyte homogenate was centrifuged at 350 x g for 5 min. The postnuclear supernatant was then centrifuged at 1,500 x g for 10 min. The sediment, after two more cycles of centrifugation (1,500 x g for 10 min at 4°C) (fraction P3), was placed in cellulose nitrate tubes, adjusted to density 1.22 with sucrose, overlayered with discontinuous sucrose layers of densities 1.18 and 1.16, and centrifuged with a SW-27 rotor at 85,000 x g for 90 min at 4°C (unpublished data). Fractions were collected from the top in three main bands (I, II, and ...

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