Induction of meiotic maturation in Xenopus oocytes by 12-O-tetradecanoylphorbol 13-acetate

Fully grown Xenopus oocytes are physiologically arrested at the G2/prophase border of the first meiotic division. Addition in vitro of progesterone or insulin causes release of the G2/prophase block and stimulates meiotic cell division of the oocyte, leading to maturation of the oocyte into an unfertilized egg. The possibility that the products of polyphosphoinositide breakdown, diacylglycerol and inositol-1,4,5-trisphosphate (IP3-, are involved in oocyte maturation was investigated. Microinjection of IP3 into oocytes just prior to addition of progesterone or insulin accelerated the rate of germinal vesicle breakdown (GVBD) by up to 25%. Half-maximal acceleration occurred at an intracellular IP3 concentration of 1 microM. Treatment of oocytes with the diacylglycerol analog and tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced GVBD in the absence of hormone. Half-maximal induction of GVBD occurred with 150 nM TPA and was blocked by pretreatment of oocytes with 10 nM cholera toxin. Microinjection of highly purified protein kinase C from rat brain into oocytes did not induce maturation but markedly accelerated the rate of insulin-induced oocyte maturation. However, injection of the enzyme had no effect on progesterone action. In oocytes with a basal intracellular pH below 7.6, TPA increased intracellular pH, but GVBD occurred with TPA in Na-substituted medium. Neomycin, a putative inhibitor of polyphosphoinositide breakdown, reversibly inhibited insulin- but not progesterone-induced maturation. Half-maximal inhibition occurred at 1.6 mM neomycin. These results indicate that protein kinase C is capable of regulating oocyte maturation in Xenopus.     

Is a decrease in cyclic AMP a necessary and sufficient signal for maturation of amphibian oocytes?

Acetylcholine rapidly lowered the intracellular levels of cyclic AMP in stage 5 and 6 Xenopus laevis oocytes. Acetylcholine alone did not induce oocyte maturation, though it did accelerate maturation induced by progesterone. The effect of acetylcholine on oocyte maturation was independent of extracellular calcium concentration. Adenosine increased cyclic AMP and abolished the progesterone-induced decrease in cyclic AMP levels in follicles and in denuded oocytes. This effect of adenosine was blocked by the Ra purinergic receptor antagonist, theophylline. Despite those effects, adenosine alone induced maturation in stage 6 oocytes and accelerated progesterone-induced maturation in both stage 5 and 6 cells. Adenosine also induced a significant increase in the rate of 45Ca efflux from oocytes in the presence and the absence of external calcium. We suggest that the activation of cell surface receptors involved in the release of calcium from cellular stores may induce or accelerate oocyte maturation independently of small changes in intracellular cyclic AMP concentration.     

The role of the germinal vesicle in producing maturation-promoting factor (MPF) as revealed by the removal and transplantation of nuclear material in starfish oocytes

In starfish, oocytes are released from prophase block by a hormone, which has been identified as 1-methyladenine. The action of 1-methyladenine is indirect in inducing oocyte maturation: it acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), the direct trigger of germinal vesicle breakdown (GVBD). Less than 5 min after hormone addition, thus about 10 min before appearance of the cytoplasmic maturation-promoting factor, a factor appears in the germinal vesicle, which triggers the production of cytoplasmic MPF, GVBD, and the subsequent events of meiotic maturation when transferred in the cytoplasm of any fully grown oocyte of the starfishes Marthasterias glacialis and Asterias rubens. Before hormone action, the germinal vesicle also contains a factor capable of inducing meiosis reinitiation in recipient oocytes, but in contrast with nuclear MPF, this factor acts exclusively when transferred in the cytoplasm of a special category of oocytes (the “competent” oocytes). In contrast to other oocytes (the “incompetent” oocytes) the competent oocytes are capable of producing MPF to some extent after enucleation, upon hormonal stimulation. Transfer of either nuclear or cytoplasmic MPF initially produced in hormone-treated maturing oocytes triggers the production of both cytoplasmic and nuclear MPF in non-hormone-treated recipient oocytes of both categories.     

Intracellular pH plays a role in regulating protein synthesis in Xenopus oocytes

Full-grown Xenopus oocytes undergo meiotic maturation in response to progesterone stimulation. Using [¹⁴C]dimethyloxazolidine dione (DMO), we have measured a cytoplasmic alkalization in these oocytes starting at pH 7.14 ± 0.17 during the germinal vesicle (GV) stage, and increasing to 7.56 ± 0.14 at the time of germinal vesicle breakdown (GVBD). During this period, the rate of protein synthesis increases 2-fold from 18.9 ± 3.1 to 37.7 ± 8.8 ng/hr/oocyte. Artificial alkalization of GV stage oocytes to pH_i 7.68 ± 0.16, by exposure to the weak bases trimethylamine, methylamine, procaine, or imidazole, led to a 1.8-fold increase in the synthetic rate. Intracellular acidification from 7.5 back to 7.0 had no apparent effect on the elevated rate of protein synthesis following GVBD. Therefore, a cytoplasmic alkalization in the range of 7.5 to 7.6 seems to be one of the events that is necessary for initiating the increase in protein synthesis in maturing Xenopus oocytes; however, it does not appear that an elevated pH_i is necessary to maintain the increased synthetic rate following GVBD.     

Maturation and fertilization in Lottia gigantea oocytes: intracellular pH, Ca(2+), and electrophysiology

Intracellular pH and Ca(2+) were measured with BCECF- and Calcium Green-dextran during maturation and fertilization of oocytes of the limpet Lottia gigantea. Maturation of oocytes from prophase to metaphase I of meiosis was induced in seawater adjusted to pH 9 with NH(4)OH. Intracellular pH rose during maturation induction, and maturation was also induced by microinjecting pH 8, but not pH 7, HEPES buffer. Intracellular Ca(2+) rose during NH(4)OH-induced maturation, but maturation was not inhibited when the increase was blocked by microinjection of BAPTA. When the metaphase I oocytes were fertilized(), there was an abrupt increase in intracellular Ca(2+), and activation (polar body formation) failed to occur in BAPTA-injected oocytes. Intracellular pH did not rise during fertilization. These observations show that maturation from prophase to metaphase I of meiosis is pH-dependent and activation of the metaphase I oocytes is Ca(2+)-dependent. A Ca(2+) action potential was present in both immature and mature oocytes but was more prominent in mature oocytes whose input resistance was higher. Fertilization produced a long-lasting (17-20 min) Na(+)-dependent fertilization potential with superimposed oscillations resembling Ca(2+) action potentials.     

Larvicidal toxin from Bacillus sphaericus spores: Isolation of toxic components

Meiotic maturation of amphibian oocytes induced by progesterone is known to be regulated by protein phosphorylation. To investigate a possible role for protein phosphatase-1 in this process, the effect of phosphatase inhibitor-2 was determined on the in vivo rate of dephosphorylation of phosphorylase a and on the rate of oocyte maturation. Dephosphorylation of microinjected phosphorylase a was inhibited up to 40% in the presence of inhibitor-2, with half-maximal inhibition at an intracellular concentration of 0.6 μM. Inhibitor-2 also caused over a 3-fold increase in the half-time for maturation, suggesting a possible role for protein phosphatase-1 in the regulation of meiosis.     

ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

A possible role for Mg2+ ions in the induction of meiotic maturation of Xenopus oocyte

Progesterone induces in vitro the meiotic cell division of Xenopus full-grown oocytes. Microinjection into oocyte of a solution containing Mg2+ (20 mM) facilitates by one order of magnitude the dose of progesterone which induces 50% of germinal vesicle breakdown. Microinjected in the absence of hormone, Mg2+ and also Mn2+ can induce maturation with efficiencies of, respectively, 24% (SEM = 8; n = 13) and 70% (SEM = 6; n = 23). The dose-response curves of cation-induction of maturation show an optimum of 20 mM for Mg2+ and 15 mM for Mn2+ (pipet concentration); higher doses were less active. Cation-induction of maturation is inhibited when oocytes are preincubated with cholera toxin (500 ng/ml); nevertheless, it cannot be interpreted at the level of cAMP, since both Mg2+ and Mn2+ microinjections provoke an increase in the oocyte cAMP content. Mg2+ induction of maturation is more efficient when oocytes are incubated in trimethylamine at pH 8.2, which is known to increase intracellular pH suggesting an action at the level of alkali pH-sensitive enzymes. Altogether, our results indicate a positive role for Mg2+ ions in the induction of oocyte maturation and raise an attractive hypothesis about the respective roles of cAMP and Mg2+ changes involved in the mechanism of progesterone action. Our results also show that co-injection of 2-glycerophosphate and Mg2+ ions, which are both commonly used in the preparation of the MPF mitotic factors from dividing cells, induces oocyte maturation more efficiently than Mg2+ alone and drastically shortens the kinetics of germinal vesicle breakdown to 1 h 30 min to 2 h 30 min.     

Role of germinal vesicle material in producing maturation-promoting factor in starfish oocyte

In starfish, oocyte maturation is induced by 1-methyladenine (1-MeAde). 1-MeAde acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which in turn brings about germinal vesicle breakdown and subsequent process of oocyte maturation. The participation of germinal vesicle material in the production of MPF was investigated with oocytes of the starfish, Asterina pectinifera. When enucleated oocytes or oocyte fragments without germinal vesicles were treated with 1-MeAde, MPF was found to be produced. However, the amount of MPF produced was small as compared with that in the case of intact oocytes with germinal vesicles. The capacity of the enucleated oocytes to produce MPF was restored when germinal vesicle material was injected. On the other hand, it has been known that the amount of MPF increases when MPF is injected into intact oocytes (amplification of MPF). However, in the case of enucleated oocytes such increase of MPF was no longer observed, suggesting that germinal vesicle material is required for MPF amplification.     

G beta gamma signaling reduces intracellular cAMP to promote meiotic progression in mouse oocytes

In nearly every vertebrate species, elevated intracellular cAMP maintains oocytes in prophase I of meiosis. Prior to ovulation, gonadotropins trigger various intra-ovarian processes, including the breakdown of gap junctions, the activation of EGF receptors, and the secretion of steroids. These events in turn decrease intracellular cAMP levels in select oocytes to allow meiotic progression, or maturation, to resume. Studies suggest that cAMP levels are kept elevated in resting oocytes by constitutive G protein signaling, and that the drop in intracellular cAMP that accompanies maturation may be due in part to attenuation of this inhibitory G protein-mediated signaling. Interestingly, one of these G protein regulators of meiotic arrest is the Galpha(s) protein, which stimulates adenylyl cyclase to raise intracellular cAMP in two important animal models of oocyte development: Xenopus leavis frogs and mice. In addition to G(alpha)(s), constitutive Gbetagamma activity similarly stimulates adenylyl cyclase to raise cAMP and prevent maturation in Xenopus oocytes; however, the role of Gbetagamma in regulating meiosis in mouse oocytes has not been examined. Here we show that Gbetagamma does not contribute to the maintenance of murine oocyte meiotic arrest. In fact, contrary to observations in frog oocytes, Gbetagamma signaling in mouse oocytes reduces cAMP and promotes oocyte maturation, suggesting that Gbetagamma might in fact play a positive role in promoting oocyte maturation. These observations emphasize that, while many general concepts and components of meiotic regulation are conserved from frogs to mice, specific differences exist that may lead to important insights regarding ovarian development in vertebrates.     

The regulation of ribosomal protein S-6 phosphorylation in Xenopus oocytes: A potential role for intracellular pH

The intracellular pH of full-grown Xenopus oocytes increases from 7.2 to 7.6 in response to progesterone stimulation. Phosphorylation of the 40 S ribosomal protein S-6 increases six- to eightfold in these stimulated cells and this phosphorylation coincides with the intracellular alkalization. This is followed by a two- to threefold increase in the protein synthetic rate. Progesterone-treated cells cultured in choline chloride substituted Na-free medium fail to alkalize and S-6 is not phosphorylated. Weak bases, such as trimethylamine, methylamine, and procaine, artificially alkalize the cell cytoplasm and stimulate S-6 phosphorylation in medium containing or lacking sodium. The methylxanthine theophylline, suppresses S-6 phosphorylation and inhibits protein synthesis. This inhibition does not appear to involve cAMP. Rather, theophylline acidifies the oocyte cytoplasm. Thus, S-6 phosphorylation appears to be regulated by the intracellular pH of the cell. In addition, the level of protein synthesis in the oocyte seems to be correlated with the level of S-6 phosphorylation.     

Alkaline pH favors microtubule self-assembly in surf clam, Spisula solidissima, oocyte extracts

Microtubule assembly in surf clam oocytes is dependent upon events that occur during fertilization. Prior to fertilization there are few, if any microtubules, but within minutes after fertilization microtubules assemble to form the meiotic apparatus. This study demonstrates that the assembly of microtubules after fertilization may be dependent on the fertilization-induced pH change of the cytoplasm. Since the magnitude of the intracellular pH (pHi) change in Spisula oocytes has not been determined, surf clam microtubule assembly was examined at pH values that reflect the pHi change that occurs during sea urchin fertilization. The results indicate that microtubule assembly in crude oocyte extracts is favored at alkaline pH. In contrast, purified surf clam tubulin assembles to a greater extent at pH 6.6 than at pH 7.2. These results reveal that the tubulin in unfertilized oocytes can assemble into microtubules at pH 6.6 but that they are prevented from doing so by pH-dependent cytoplasmic regulatory factors in the oocyte.     

Functional reconstitutional of the human epidermal growth factor receptor system in Xenopus oocytes

We have expressed the human EGF receptor (hEGF-R) in Xenopus oocytes by injecting mRNA synthesized in vitro using SP6 vectors containing receptor cDNAs. Each oocyte could express over 1 x 10(10) receptors of a single affinity class and these were able to bind and rapidly internalize EGF. Occupancy resulted in receptor tyrosine autophosphorylation, downregulation, and release of intracellular calcium. Occupied receptors also rapidly induced meiotic maturation in stage VI oocytes. Receptors lacking tyrosine kinase activity bound EGF normally, but did not downregulate or induce any biological responses. The rate of oocyte maturation was proportional to hEGF-R occupancy and was significantly faster than progesterone-induced maturation at nanomolar EGF concentrations. Mutant hEGF-R truncated at residue 973 displayed identical phenotypes in both mammalian cells and oocytes in that they were defective in their ability to release intracellular calcium, undergo ligand induced internalization and receptor downregulation. However, these receptors were fully capable of inducing oocyte maturation. The remarkable retention of specific biological activities of different hEGF-R in the context of oocytes suggests that this receptor system interacts with generally available cellular components that have been conserved during evolution. In addition, it suggests that cell surface tyrosine kinase activity may play an important role in regulating resumption of the cell cycle.     

Intracellular signals trigger ultrastructural events characteristic of meiotic maturation in oocytes of Xenopus laevis

Summary Oocytes of Xenopus laevis were treated with agents which induce individual intracellular signals normally evoked during the process of meiotic maturation. Ultrastructural analysis of these oocytes allowed identification of specific second messengers that individually trigger single ultrastructural changes characteristic of the meiotic maturation process: Manipulation of intracellular cAMP levels induced changes in cortical granule position. Cytoplasmic alkalinization triggered a disruption of the annulate lamellae, a specialized organelle in the periphery of oocytes. Activation of protein kinase C caused rapid formation of a cortical endoplasmic reticulum and subsequent disruption of cortical granules. Manipulation of transmembrane calcium flux had varied results dependent upon the agent employed. Two of the treatments, Verapamil and zero external calcium, induced a reorganization in the oocyte periphery. The results indicate that these ultrastructural events are under the control of specific intracellular signals known to be elicited during meiotic maturation.     

Effects of polyunsaturated fatty acids and prostaglandins on oocyte maturation in a marine teleost, the European sea bass (Dicentrarchus labrax)

The effects of the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and prostaglandins (PGs) on oocyte maturation were investigated in a marine teleost, the sea bass (Dicentrarchus labrax). Follicle-enclosed postvitellogenic, preovulatory oocytes were cultured in vitro and maturation was verified by assessing volume increase, lipid droplet coalescence, yolk clarification, and germinal vesicle migration and breakdown. Human chorionic gonadotropin was administered as the maturation-inducing gonadotropin (GTH) and was capable of inducing maturation in a time- and dose-dependent manner. Free AA induced maturation in a dose- and time-dependent manner and enhanced GTH-induced maturation, while EPA, DHA, and oleic acid were ineffective. Maturation induced by GTH was significantly suppressed by a phospholipase A(2) blocker, suggesting that mobilization of AA was involved in GTH-induced maturation. Moreover, EPA and DHA exhibited a significant, dose-dependent attenuation of GTH-induced maturation. Maturation induced by GTH was inhibited in the presence of a cyclooxygenase inhibitor, indomethacin, and this inhibition was reversed by addition of AA, PGE(2), or PGF(2alpha). PGE(2) and PGF(2alpha) alone were both effective stimulators of maturation, while PGE(1) and PGE(3) were ineffective. The effect of PUFAs on oocyte maturation in vitro were corroborated with studies in vivo. Oocytes were obtained from females fed a commercial, PUFA-enriched diet (RD) and maturational behavior was compared with oocytes from females fed a natural diet (ND) with a higher EPA content and n-3:n-6 ratio. Although no significant difference was observed in the rate of spontaneous oocyte maturation, a higher percentage of GTH-induced maturation and lower percentage of atresia were observed in RD oocytes. Moreover, while basal PGE production from oocytes from both groups was the same, RD oocytes produced significantly higher levels of PGE in the presence of hCG. The results from this study provide evidence for the participation of AA metabolism in GTH-induced oocyte maturation, and suggest that other PUFAs and PGs may play important roles in the induction of maturation in a marine teleost.     

Nonhormonal stimulation in vitro of oocyte maturation in sturgeons

Incubation of sturgeon full-grown ovarian follicles in amphibian Ringer solution with increased sodium bicarbonate concentration results in “spontaneous” oocyte maturation. Addition of sodium bicarbonate to diluted Leibovitz medium also induces maturation of follicle-enclosed oocytes. Effective threshold concentration of sodium bicarbonate depends on the composition of culture medium and, especially, on the physiological state of follicle-enclosed oocytes. As evidenced by experiments with actinomycin D, oocyte maturation induced by bicarbonate ions does not depend on RNA synthesis. An attempt was made to elucidate the involvement of steroidogenesis in bicarbonate ions-induced oocyte maturation. Surprisingly, the inhibitors used, such as aminoglutethimide, diltiazem, and estradiol-17β, not only did not inhibit but also enhanced oocyte maturation. Manual removal of follicle envelopes demonstrated that denuded oocytes retained the ability to mature in a culture medium with increased sodium bicarbonate concentration. However, the range of effective bicarbonate ion concentrations for denuded oocytes is more restricted than for the follicle-enclosed oocytes. A hypothesis of competition of different processes occurring in the ovarian follicle for energy resources is proposed to explain the revealed paradoxical effect of substances affecting steroidogenesis.     

鸟氨酸脱羧酶参与孕酮诱导的卵成熟过程吗?

在孕酮诱导的蟾蜍卵母细胞成熟过程中,ODC活性约增加2倍。佛波酯PMA能促进孕酮诱导的成熟速度,而对ODC的活性无明显影响。精胺对ODC活性有显著的作用:当卵母细胞培养在含5mmol/L精胺的碱性任氏生理盐水(pH11.6)中时,ODC活性下降17%,而孕酮诱导的成熟速度却大为增加;当精胺注入卵母细胞后。ODC活性明显下降,而且即使孕酮诱导的ODC活性增加完全被抑制,仍有80%以上的卵母细胞发生GVBD。上述结果充分表明,ODC活性的变化不参与孕酮诱导蟾蜍卵母细胞成熟的调控过程。由于在孕酮处理的卵母细胞中,ODC活性的增加发生在精胺水平下降之后,又外源精胺能大幅度抑制卵内ODC活性,故精胺很可能是卵母细胞ODC活性的调节物。     

The role of cAMP in oocyte maturation and the role of the germinal vesicle contents in mediating maturation and subsequent developmental events in hydrozoans

Summary Externally applied membrane permeable cAMP derivatives and the injection of cAMP induce oocyte maturation in several species of hydrozoans. This technique for inducing oocyte maturation has been used to study ion permeability changes, maturation promoting factor activity and surface tension changes during maturation. Oocyte membrane potential remains constant during maturation. Cyclic AMP induced maturation proceeds in the absence of external Ca²⁺, K⁻, Mg²⁺ or Na⁺. Cytoplasm from maturing oocytes that induces oocyte maturation when it is injected into untreated oocytes is produced during cAMP induced maturation. Surface tension, as measured by the application of a standardized force that mechanically deforms individual oocytes, declines during the first part of maturation. This is followed by a sharp rise and fall of surface tension at first and second polar body formation that accompanies a slow rise in the resistance of oocytes to deformation during the last part of maturation. The production of maturation promoting factor activity and some of the changes in surface tension during maturation can occur in the absence of germinal vesicle material. Two early developmental events that follow oocyte maturation are the production of sperm chemoattractant and calcium channel function. Neither of these events occurs in eggs that have undergone maturation in the absence of germinal vesicle material. The addition of germinal vesicle contents from oocytes to eggs that have undergone maturation in the absence of germinal vesicle material initiates calcium channel function. This experiment indicates that the germinal vesicle contains factors that are necessary for post-maturation developmental events.     

Maturation of pig oocytes: observations on membrane potential

The membrane-potential changes of pig oocytes during maturation are described. Cumulus-enclosed oocytes have a resting potential of -41.81 +/- 0.60 mV; the removal of cumulus cells caused this potential to drop to -30.95 +/- 0.43 mV. Adding LH to the culture medium did not influence the potential of denuded oocytes but depolarized the potential of cumulus-enclosed oocytes to -32.90 +/- 0.43 mV. FSH did not affect the membrane potential of denuded or cumulus-enclosed oocytes, but significantly reduced the amplitude of the depolarization induced by LH. The effect of gonadotropins on cultured granulosa cells was also investigated. Plated granulosa cells have a resting potential of -45.21 +/- 0.72 mV, similar to that of cumulus-enclosed oocytes. As recorded in cumulus-enclosed oocytes, LH depolarized granulosa cell membrane potential (-30.33 +/- 0.69 mV) and FSH reduced this effect. To evaluate if oocyte maturation in vivo is accompanied by membrane-potential depolarization, follicular growth and oocyte maturation were induced in 6 prepubertal gilts by using an eCG-hCG treatment. Twenty hours after the beginning of oocyte maturation in vivo (induced by hCG), the membrane potential of the oocyte was depolarized to -28.84 +/- 1.01 mV, a value similar to that observed in vitro. These data indicate that both LH and FSH can influence the membrane potential of follicular somatic cells and, consequently, that of the oocyte. The electrical coupling between somatic cell and oocyte may represent a means by which the gonadotropin message is passed to the germinal cell by the somatic compartment.