Progesterone-induced down-regulation of an electrogenic Na+, K+-ATPase during the first meiotic division in amphibian oocytes

Summary Progesterone initiates the resumption of the meiotic divisions in the amphibian oocyte. Depolarization of theRana pipiens oocyte plasma membrane begins 6–10 hr after exposure to progesterone (1–2 hr before nuclear breakdown). The oocyte cytoplasm becomes essentially isopotential with the medium by the end of the first meiotic division (20–22 hr). Voltage-clamp studies indicate that the depolarization coincides with the disappearance of an electrogenic Na⁺, K⁺-pump, and other electrophysiological studies indicate a decrease in both K⁺ and Cl^– conductances of the oocyte plasma membrane. Measurement of [³H]-ouabain binding to the plasma-vitelline membrane complex indicates that there are high-affinity (K _d-4.2×10^–8 m), K⁺-sensitive ouabain-binding sites on the unstimulated (prophase-arrest) oocyte and that ouabain binding virtually disappears during membrane depolarization. [³H]-Leucine incorporation into the plasma-vitelline membrane complex increased ninefold during depolarization with no significant change in uptake or incorporation into cytoplasmic proteins or acid soluble pool(s). This together with previous findings suggests that progesterone acts at a translational level to produce a cytoplasmic factor(s) that down-regulates the membrane Na⁺, K⁺-ATPase and alters the ion permeability and transport properties of both nuclear and plasma membranes.     

Regulation of the amphibian oocyte plasma membrane ion permeability by cytoplasmic factors during the first meiotic division.

The denuded Rana pipiens oocyte depolarizes from 80–90 mV inside negative to 3–5 mV? inside positive during progesterone-induced meiotic maturation, apparently due to decreased K⁺ permeability. Depolarization is dependent upon protein synthesis and coincides with breakdown of the oocyte nucleus, but occurs even in the absence of the nucleus, suggesting cytoplasmic regulation of cation selectivity of the oocyte plasma membrane.     

Regulation of Ca2+ and cyclic AMP during the first meiotic division in amphibian oocytes by progesterone

Progesterone appears to be the physiological inducer of meiosis in amphibian oocytes. In Rana pipiens, dl-propranolol mimics the action of progesterone and both agents have a common action in producing a rapid [45Ca] efflux and a fall in intracellular cAMP followed by nuclear breakdown. Comparison of the rate of hydrolysis of injected [3H]-cAMP and of the conversion of injected [3H]-ATP to [3H]-cAMP followed exposure to meiotic inducers and inhibitors indicates that adenylate cyclase and not phosphodiesterase is the rate-limiting step in regulating [cAMP]i in the oocyte. The results suggest that progesterone initiates the resumption of the meiotic divisions by down-regulation of membrane adenylate cyclase, possibly via Ca2+ release from specific membrane sites.     

Progesterone induces transient changes in plasma membrane fluidity of amphibian oocytes during the first meiotic division

Progesterone acts at the surface of the amphibian oocyte to induce resumption of the meiotic divisions. Progesterone binding leads to a transient dose-dependent decrease in the fluidity (increase in order parameter) of the Rana oocyte plasma membrane, which was detected by electron spin resonance in isolated plasma membranes using either 5- or 16-DOXYL stearic acid probes. The 5-DOXYL probe, which inserts into the membrane with the spin label nearest the surface, showed an increase in the order parameter within minutes, a maximum change by 2 h, and a return to control levels by 6 h. The order parameter for the 16-DOXYL probe, which reflects the fluidity deeper within the plasma membrane, increased slowly and remained elevated during the first meiotic division. RU 38486, a synthetic steroid that blocks progesterone receptors, prevents progesterone-induced fluidity changes. These findings indicate that the binding of progesterone to its receptor changes the oocyte plasma membrane structure resulting in a differential decrease in mobility near the membrane surface compared to that deeper in the membrane.     

Progesterone induction of phospholipid methylation and arachidonic acid turnover during the first meiotic division in amphibian oocytes

Progesterone is the physiological stimulus that acts at the amphibian oocyte plasma membrane to induce the meiotic divisions. Rana oocytes were preincubated with [3H]-arachidonic acid, [3H]-methionine and/or [14C]choline. Total and plasma membrane phospholipids were monitored during the first 2 h after induction with progesterone. A transient increase in methylation of phosphatidylethanolamine during the first 10 minutes coincided with an increased Ca2+ efflux and was followed by increased arachidonic acid incorporation into phosphatidylcholine during a period of increasing membrane conductance. The labeled phospholipids disappeared sequentially 5-90 min after the hormone stimulus, suggesting that activation of phospholipases A2 and/or C occur as part of a cascade of membrane events.     

Progesterone induces meiotic division in the amphibian oocyte by releasing lipid second messengers from the plasma membrane.

Meiosis in the amphibian oocyte is normally initiated by gonadotropins, which stimulate follicle cells to secret progesterone. The progesterone-induced G2/M transition in the amphibian oocyte was the first well-defined example of a steroid effect at the plasma membrane, since it could be shown that exogenous, but not injected, progesterone induced meiosis and that many of the progesterone-induced changes associated with meiosis occurred in enucleated oocytes. We find that [3H]progesterone binding to isolated plasma membranes of Rana pipiens oocytes is saturable, specific and temperature-dependent. Photoaffinity labeling with the synthetic progestin [3H]R5020 followed by gel electrophoresis demonstrated progestin binding to both 80 and 110 kDa proteins in the oocyte cytosol, whereas only the 110 kDa R5020 binding protein was present in the oocyte plasma membrane. We have shown that progesterone acts at Rana oocyte plasma membrane receptors within seconds to release a cascade of lipid messengers. Membrane-receptor binding causes the successive activation of: 1) N-methyltransferases, which convert phosphatidylethanolamine to phosphatidylcholine (PC); 2) an exchange reaction between PC and ceramide to form sphingomyelin (SM) and 1,2-diacylglycerol (DAG); 3) phospholipase D/phosphatidate phosphohydrolase, releasing a second DAG transient; and 4) phosphatidylinositol-specific phospholipase C, generating inositol trisphosphate and a third DAG transient. Within minutes, diglyceride kinase converts newly formed DAG species to phosphatidic acid, turning off the successive DAG signals. A transient fall (0-30 s) in intracellular ceramide is followed (within 1-2 min) by a sustained rise in intracellular ceramide lasting 3-4 h. This ceramide may be significant in later cyclin-dependent steps. We conclude that the initial action of progesterone at its plasma membrane receptor triggers a series of enzyme activations that modify the membrane and release multiple DAG species.     

Route,mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

A single Na⁺/K⁺-ATPase pumps three Na⁺ outwards and two K⁺ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na⁺ than K⁺ generates outward current across the cell membrane. Less well understood is the ability of Na⁺/K⁺ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K⁺ and Na⁺ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K⁺ and Na⁺ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na⁺ release from phosphorylated Na⁺/K⁺ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na⁺/K⁺ pumps that enables proton import is not required for completion of the 3 Na⁺/2 K⁺ transport cycle. However, the back-step occurs readily during Na⁺/K⁺ transport when external K⁺ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na⁺-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na⁺ and K⁺ ions that passes through binding site II. The inferred occurrence of Na⁺/K⁺ exchange and H⁺ import during the same conformational cycle of a single molecule identifies the Na⁺/K⁺ pump as a hybrid transporter. Whether Na⁺/K⁺ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.     

ATP-driven exchange of Na+ and K+ ions by Streptococcus faecalis

We describe the characterization of KtrII, a novel potassium transport system of Streptococcus faecalis, first discovered by H. Kobayashi [1982) J. Bacteriol. 150, 506-511). KtrII requires sodium ions and mediates the stoichiometric exchange of internal Na+ for external K+. Potassium accumulation is not energized by the electrochemical potentials of either H+ or Na+; the energy source is probably ATP. Two lines of evidence indicate that KtrII is a manifestation of the sodium-stimulated ATPase reported earlier (Heefner, D. L., and Harold, F. M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2798-2802). (i) Mutants that lack the ATPase also lack KtrII, and revertants recover both in parallel. (ii) KtrII and the Na+-ATPase are induced in parallel when cells are grown on media rich in sodium, particularly under conditions that limit the generation of a proton potential. KtrII is not induced in response to K+ deprivation. We propose that the Na+-ATPase exchanges Na+ for K+ ions.     

Evidence for mediated protein uptake by amphibian oocyte nuclei

The objective of this investigation was to determine whether there is mediated transport of endogenous proteins across the nuclear envelope. For this purpose, we studied the nuclear uptake of a 148,000-dalton Rana oocyte polypeptide (RN1) and compared its actual uptake rate with the rate that would be expected if RN1 crossed the envelope by simple diffusion through the nuclear pores. Nuclear uptake was studied in two ways: first, oocytes were incubated in L-[3H]leucine for 1 h and, at various intervals after labeling, the amount of 3H-RN1 present in the nucleoplasm was determined. Second, L-[3H]leucine-labeled nuclear extracts, containing RN1, were microinjected into the cytoplasm of nonlabeled cells, and the proportion of 3H-RN1 that subsequently entered the nucleus was measured. It was found that RN1 can readily penetrate the nuclear envelope; for example, after 6 h, approximately 36% of the newly synthesized RN1 and 17% of the injected RN1 had entered the nucleus. The diffusion rate through pores having a radius of 45 A was calculated for several possible molecular configurations of RN1. Using axial ratios of 34, 7.5, 2, and 1, the estimated times required to reach 63% of diffusion equilibrium are 757, 468, 6,940 h, and infinity, respectively. Even assuming an axial ratio of 7.5 (the most diffusive configuration) and an equilibrium distribution of 45, simple diffusion through the pores could account for only approximately 1/20 the observed nuclear uptake of RN1. This and other comparisons indicate that some form of mediated transport is involved in the nucleocytoplasmic exchange of this polypeptide.     

Charge translocation by the Na+/K+ pump under Na+/Na+ exchange conditions: intracellular Na+ dependence

The effect of intracellular (i) and extracellular (o) Na+ on pre-steady-state transient current associated with Na+/Na+ exchange by the Na+/K+ pump was investigated in the vegetal pole of Xenopus oocytes. Current records in response to 40-ms voltage pulses from -180 to +100 mV in the absence of external Na+ were subtracted from current records obtained under Na+/Na+ exchange conditions. Na+-sensitive transient current and dihydroouabain-sensitive current were equivalent. The quantity of charge moved (Q) and the relaxation rate coefficient (ktot) of the slow component of the Nao+-sensitive transient current were measured for steps to various voltages (V). The data were analyzed using a four-state kinetic model describing the Na+ binding, occlusion, conformational change, and release steps of the transport cycle. The apparent valence of the Q vs. V relationship was near 1.0 for all experimental conditions. When extracellular Na+ was halved, the midpoint voltage of the charge distribution (Vq) shifted -25.3+/-0.4 mV, which can be accounted for by the presence of an extracellular ion-well having a dielectric distance delta=0.69+/-0.01. The effect of changes of Nai+ on Nao+-sensitive transient current was investigated. The midpoint voltage (Vq) of the charge distribution curve was not affected over the Nao+ concentration range 3.13-50 mM. As Nai+ was decreased, the amount of charge measured and its relaxation rate coefficient decreased with an apparent Km of 3.2+/-0.2 mM. The effects of lowering Nai+ on pre-steady-state transient current can be accounted for by decreasing the charge available to participate in the fast extracellular Na+ release steps, by a slowly equilibrating (phosphorylation/occlusion) step intervening between intracellular Na+ binding and extracellular Na+ release.     

C9 heterochromatin during the first meiotic prophase of human foetal oocyte

During the first meiotic prophase in human oocyte the C₉ heterochromatin shows changes resulting in the formation of small bodies similar to the parameres described in male pachytene. At diplotene, this segment is often associated to nucleolar-like material. Even though there is no direct evidence of synthetic activity on a morphological basis alone, the observed phenomena could be related to specific stimulation, since they are observed at the same stage in both male and female.     

Amiloride-inhibited Na+ uptake into toad bladder microsomes is Na+-H+ exchange

Amiloride-inhibited Na+ transport into toad urinary bladder microsomes is sensitive to a pH gradient across the vesicular membrane. The magnitude of the gradient was measured directly with acridine orange. Also Na+ could stimulate amiloride-sensitive proton efflux from the microsomes. These results indicated that the transport process was Na+-H+ exchange.     

Monensin blocks the first meiotic cell division of the Xenopus oocyte: Effect at the nuclear membrane level

The carboxylic ionophore monensin inhibits the meiotic maturation of the Xenopus oocyte. When oocytes are exposed to high concentrations of monensin (10 μM), both progesterone and MPF-induced (maturation-promoting factor-induced) maturations are blocked. Lower doses of monensin (1–10 μM) do not inhibit the formation or amplification of MPF activity in the oocyte cytoplasm; however, breakdown of the nuclear envelope does not occur. These observations show that monensin, which is known to abolish intracellular proton gradients, interferes with the mechanism of the breakdown of the nuclear envelope induced by MPF.     

Role of Na+/K+ exchange and organic acids in base induced hyperpolarization of renal proximal amphibian tubule.

Fast peritubular alkaline perturbations in Necturus renal proximal tubule evoke hyperpolarizations of the basolateral membrane. These voltage changes are partly due to an increase in basolateral K(+)-permeability. Additional role of the Na(+)/K(+)-ATPase and organic acids in generating these base induced hyperpolarizations (BIH) can be deduced from the reduction in BIH during low K+, high amiloride or omission of organic acids.     

The modulation of rat brain Na(+)-Ca2+ exchange by K+

The involvement of potassium ions in the Na(+)-Ca2+ exchange process was studied in rat brain synaptic plasma membrane (SPM) vesicles. Addition of equimolar [K+] to the intravesicular and the extravesicular medium led to a stimulation of the Na+ gradient-dependent Ca2+ influx; this stimulation was noticeable already at 0.5 mM and reached its maximum at 2 mM K+. The magnitude of the K+ stimulation was between 1.3-2.5-fold in different SPM preparations. K+ ions also stimulated the Na(+)-dependent Ca2+ efflux. K+ stimulation of Na(+)-Ca2+ exchange is of considerable specificity, since it is not mimicked by either Li+ or H+. The following lines of evidence suggest that K+ modulation of Na(+)-Ca2+ exchange involves the catalytic moiety of the transporter itself and not an unrelated K+ channel which modulates the membrane potential. 1) K+ stimulation of the transport process was conserved following reconstitution of the transporter into phospholipid-rich liposomes, an experimental condition which presumably separates the native membrane proteins among different vesicular structures. 2) K+ stimulation of Na+ gradient-dependent Ca2+ influx persists also when the build up of negative inside membrane potential is prevented by addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone which renders the membrane highly permeable to protons both in the native and the reconstituted preparation. 3) K+ stimulation of Na+ gradient-dependent Ca2+ influx is obtained also when tetraethylammonium chloride, 2,3-diaminopyridine and Cs+ are added to the Ca2+ uptake medium. Reconstituted SPM vesicles take up 86Rb+ in response to activation of Na+ gradient-dependent Ca2+ influx. The ratio of Ca2+ taken up by SPM vesicles in a Na+ gradient-dependent manner to the corresponding amounts of Rb+ taken up varies between 8-5 in different SPM preparations. If the stoichiometry of the process is 1 Rb+/1 Ca2+, then Rb+ cotransport is mediated by 10-20% of the transporters present in the preparation.     

Changes in the patterns of birefringence and filament deployment in the meiotic spindle ofNephrotoma suturalis during the first meiotic division

Summary A study combining polarizing and Nomarski differential interference microscopes was carried out onNephrotoma suturalis spermatocytes in order to correlate changes in the distribution of spindle birefringence with both the fibrillar organization of the spindle and movement of chromosomes within it. Records of fluctuations in birefringence obtained from measurements of retardation and analyses of ciné films show that there are significant transient fluctuations in birefringence during prometaphase (the so-called northern lights phenomenon) and gradual changes in the magnitude and distribution of birefringence from prometaphase through telophase. A negative correlation was found between the distribution of birefringence at the various stages of division and the amount of fibrillar organization in the spindle observed with the Nomarski system. Neither the transient nor gradual birefringence changes in theNephrotoma spindle can be explained adequately in terms of the number or distribution of spindle filaments. The possibility that these changes are associated with the forces developed within the spindle is discussed.     

6-Dimethylaminopurine (6-DMAP), a reversible inhibitor of the transition to metaphase during the first meiotic cell division of the mouse oocyte

The first meiotic cell division (meiotic maturation) of dictyate stage mouse oocytes removed from the follicle resumes spontaneously in vitro. We used the puromycin analog 6-dimethylaminopurine (6-DMAP) to test the respective roles of protein synthesis and protein phosphorylation in driving this process. While protein synthesis inhibitors do not block meiosis resumption, 6-DMAP was found to inhibit germinal vesicle breakdown (GVBD), by inhibiting the burst of protein phosphorylation without changing the rate of incorporation of [35S]methionine into proteins. This effect is reversible; it depends both upon drug concentration and the particular female. When added after GVBD and before the emission of the first polar body, 6-DMAP decreases the level of protein phosphorylation and induces decondensation of the chromosomes and reformation of the nuclear envelope. In contrast, 6-DMAP did not trigger these processes in metaphase II oocytes which only produce resting nuclei when treated by protein synthesis inhibitors. From these data, we conclude that (1) the early appearance and stability of mouse MPF in Metaphase I oocytes depend on protein phosphorylation rather than on protein synthesis, and (2) protein synthesis is necessary to maintain the condensation of the chromosomes in metaphase II oocytes.