Biochemical correlates of progesterone-induced plasma membrane depolarization during the first meiotic division inRana oocytes

Summary Changes in protein synthesis, protein phosphorylation and lipid phosphorylation in the amphibian oocyte plasma membrane have been correlated with electrical changes following steroid induction of the completion of the first meiotic division. The oocyte first depolarizes from about –60 mV (inside negative) to about –25 mV 1 to 2 hr before breakdown of the large nucleus followed by a further depolarization beginning 3 to 6 hr after nuclear breakdown. The initial depolarization is associated with appearance of previously described cycloheximide-sensitive cytoplasmic factor(s) which induce both nuclear breakdown and plasma membrane depolarization. We found a similar ED₅₀ (0.4 m) for cycloheximide inhibition of nuclear breakdown, membrane depolarization, and [³H]-leucine incorporation. Emetine (1nm to 1mm) was inactive. The period of cycloheximide sensitivity (first 5 hr) is essentially the same for plasma membrane depolarization phase following nuclear breakdown is associated with a marked increase in the rate of [³H]-leucine and [³²PO₄] incorporation into membrane protein and lipid. Polyacrylamide gel electrophoresis of membrane protein and lipoprotein indicated that a major newly synthesized membrane component is proteolipid. An increase in [³²PO₄] incorporation into membrane phosphatidylserine and phosphatidylethanolamine (with a decrease in phosphatidylcholine [³²PO₄] begins during the second depolarization phase and coincides with the appearance of excitability in the oocyte plasma membrane. In toto, the bulk of the biochemical changes (proteins, phosphoproteins, proteolipids, phospholipids) appear to be associated with plasma membrane components and coincide with stepwise changes in membrane permeability to specific ions (e.g. Cl^–).     

H-ras(val12) induces cytoplasmic but not nuclear events of the cell cycle in small Xenopus oocytes.

Microinjection of H-ras(val12) protein into fully grown Xenopus oocytes has been shown to induce meiotic maturation. In the present study, mRNA encoding the mutant ras protein was injected into both fully grown (stage 6) and growing (stage 4) oocytes. The mRNA induced nuclear breakdown in stage 6 oocytes, as expected. However, the mRNA induced neither nuclear breakdown nor maturation promoting factor when injected into stage 4 oocytes. Instead, the response in stage 4 oocytes included an activation pulse of calcium, cortical granule breakdown, elevation of the vitelline envelope, and abortive cleavage furrows, all of which are characteristics of the activation response in mature eggs. In addition, the injected mRNA led to increased rates of endogenous protein synthesis and the migration of subcortical organelles into the oocyte interior. These observations are discussed relative to the suggestion that oncogenic ras protein leads to an increase in both diacylglycerol and inositol trisphosphate, which then regulate the various cytoplasmic events described.     

Alkaline phosphatase activity in the membrane of Xenopus laevis oocytes: effects of steroids, insulin, and inhibitors during meiosis reinitiation

The mechanism of steroid hormone-induced reinitiation of meiosis in Xenopus laevis oocytes in vitro involves interaction of the hormone with an ooplasma membrane receptor and early changes of enzymatic activities (adenylate cyclase, p48 protein kinase). In full-grown (stage 6) oocytes, we have observed cytochemically, at the ultrastructural level, alkaline phosphatase activity in the ooplasma membrane of microvilli, its decrease by 2 hr of progesterone action, and its complete disappearance at the time of germinal vesicle breakdown (GVBD). Insulin (30 micrograms/ml) also provoked a decrease of phosphatase activity, although it did not promote GVBD under these circumstances. When oocytes were exposed simultaneously to progesterone (1 microM) and insulin (30 micrograms/ml), the enzymatic activity disappeared earlier than with any one of them, correlating with the faster occurrence of GVBD. Inhibitors of alkaline phosphatase activity and competitive substrates potentiated progesterone action on GVBD. Insulin and beta-glycerophosphate potentiating activities were additive. These results suggest that the ooplasma membrane alkaline phosphatase may be implicated in the course of reinitiation of meiosis in X. laevis oocytes.     

Na+ and K+ uptake and exchange by the amphibian oocyte during the first meiotic division

The uptake and efflux of ²²Na and ⁴²K were studied in denuded Rana pipiens oocytes following progesterone induction of the resumption of meiotic maturation. Coincident with the breakdown of the large nucleus, or germinal vesicle, there is a virtual disappearance of K⁺ permeability of the oocyte plasma membrane. Only about 1–2% of the total [K⁺]_i is exchanged by completion of nuclear breakdown (8–10 hr) and accounts for the finding that there is no detectable change in total [K⁺]_i during the first meiotic division (20–24 hr). In the case of Na⁺, influx, exchange, and efflux kinetics were unchanged during the first meiotic division, with 20 and 35% of the total oocyte Na⁺ exchanging by the completion of nuclear breakdown and first meiotic division, respectively. Removal of Na⁺ from the incubation medium produced and earlier nuclear breakdown, whereas a K-free medium delayed breakdown. There was no effect of 10 μm/ml tetrodotoxin or 10^?5M strophanthidin on the time course of nuclear breakdown. Thus one action of progesterone appears to be a selective turning off of “K channels” in the oocyte plasma membrane. The disappearance of K selectivity of the oocyte plasma membrane coincides with plasma membrane depolarization, as well as nuclear swelling and breakdown.     

The cyclic behavior of a cytoplasmic factor controlling nuclear membrane breakdown

The activity of a cytoplasmic factor (MPF), capable of inducing nuclear membrane breakdown (germinal vesicle breakdown) when injected into amphibian oocytes, has been studied during the course of early cleavage in amphibian embryos. Mature egg cytoplasm was found to contain high levels of this activity, but this was quickly lost after fertilization or artificial activation. MPF activity later reappeared in the egg cytoplasm and started to cycle with time. The peak of embryonic MPF activity during each cycle coincided with the time the embryonic nuclei were entering the G2-M transition, i.e., mitosis. However, in colchicine-arrested embryos, this activity remained at an elevated level and no longer oscillated. The timing of the appearance and disappearance of this activity appeared to be under the control of the cytoplasm because such behavior was still observed in enucleated eggs. Continued protein synthesis in the embryo was required for the reappearance, but not for the disappearance, of this activity. MPF, previously thought to be restricted to oocyte maturation, may play a more general role in controlling nuclear membrane breakdown during mitosis as well as meiosis.     

Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

Cytoplasmic targeting signals mediate delivery of phospholemman to the plasma membrane

The FXYD protein family consists of several small, single-span membrane proteins that exhibit a high degree of homology. The best-known members of the family include the -subunit of the Na⁺-K⁺-ATPase and phospholemman (PLM), a phosphoprotein of cardiac sarcolemma. Other members of the family include corticosteroid hormone-induced factor (CHIF), mammary tumor protein of 8 kDa (Mat-8), and related to ion channels (RIC). The exact physiological roles of the FXYD proteins remain unknown. To better characterize the function of the members of the FXYD protein family, we expressed several members of the family in Madin-Darby canine kidney (MDCK) cells. All of the FXYD proteins, with the exception of PLM, were primarily found in the basolateral plasma membrane. Surprisingly, PLM, a previously characterized plasma membrane protein, was found to colocalize with the endoplasmic reticulum marker protein disulfide isomerase. Treatment of MDCK cells expressing PLM with an agonist of PKC caused some of the PLM to be redistributed to the plasma membrane. Site-directed mutagenesis of residues within the cytoplasmic domain of PLM indicated that a negative charge at Ser69 is necessary to shift the localization of PLM to the plasma membrane. In addition, other regions of PLM necessary for either its endoplasmic reticulum or plasma membrane localization have been elucidated. In contrast to PLM, the plasma membrane localization of CHIF and RIC was not altered by mutation of potential cytoplasmic phosphorylation sites. Overall, these results suggest that phosphorylation of specific residues of PLM may direct PLM from an intracellular compartment to the plasma membrane. protein trafficking; FXYD proteins     

HORMONAL CONTROL OF OOCYTE MATURATION IN ARENICOLA MARINA L. (ANNELIDA, POLYCHAETA) I. MORPHOLOGICAL STUDY OF OOCYTE MATURATION

In Arenicola marina (Annelida, Polychaeta) the oocytes are arrested in the first prophase stage of mciosis until spawning. Oocyte maturation is under hormonal control: when incubated in vitro in a brain extract oocytes reach the first metaphase at which they remain arrested until fertilization. The meiosis reinitiating substance induces numerous morphological changes in the oocytes: general (shape), cortical (microvilli retraction, plasma membrane flattening), cytoplasmic (cortical granules repartition) and nuclear modifications (germinal vesicle breakdown, chromosome condensation, formation of a meiotic maturation spindle). A kinetic study of these morphological modifications has been performed.     

Effect of 2,4,5-trichlorophenol on hyphal membrane potentials in rhythmic mutants of Neurospora crassa

The uncoupler 2,4,5-trichlorophenol (TCP) was used to test for differences in maintaining the hyphal membrane potentials in the wild type and rhythmic mutants of Neurospora crassa. Trichlorophenol (0.1 mmol·1^–1) resulted in a depolarization of 93 mV (wild type) and 144 mV in the mutant clock. A total recovery was achieved in both strains after washing out the uncoupler. The circadian conidiation mutant band was more sensitive than the two other strains and showed two different reaction patterns: one with delayed reaction, total breakdown and without recovery; the other one with nearly immediate reaction, slow but not entirely total decline and partial recovery. These differences are discussed in their relation to the circadian rhythm of conidiation.Abbreviations PD potential difference - TCP trichlorophenol     

Trafficking and proteolytic processing of RNF13, a model PA-TM-RING family endosomal membrane ubiquitin ligase

RING finger protein 13 (RNF13) is a ubiquitously expressed, highly regulated ubiquitin ligase anchored in endosome membranes. A RING domain located in the cytoplasmic half of this type 1 membrane protein mediates ubiquitination in vitro but physiological substrates have not yet been identified. The protein localized in endosomal membranes undergoes extensive proteolysis in a proteasome-dependent manner, but the mRNA level can be increased and the encoded protein stabilized under specific physiological conditions. The cytoplasmic half of RNF13 is released from the membrane by regulatory proteases and therefore has the potential to mediate ubiquitination at distant sites independent of the full-length protein. In response to protein kinase C activation, the full-length protein is stabilized and moves to recycling endosomes and to the inner nuclear membrane, which exposes the RING domain to the nucleoplasm. Thus RNF13 is a ubiquitin ligase that can potentially mediate ubiquitination in endosomes, on the plasma membrane, in the cytoplasm, in the nucleoplasm or on the inner nuclear membrane, with the site(s) regulated by signaling events that modulate protein targeting and proteolysis.     

Maturation-specific deadenylation in Xenopus oocytes requires nuclear and cytoplasmic factors.

During the meiotic maturation of Xenopus oocytes, maternal mRNAs that lack a cytoplasmic polyadenylation element are deadenylated and translationally inactivated. In this report, we have characterized the regulation of poly(A) removal during maturation. Deadenylation in vivo is detected only after germinal vesicle breakdown and does not require de novo protein synthesis. Enucleated oocytes do not deadenylate either endogenous or microinjected RNAs upon maturation, indicating that a nuclear component is required for poly(A) removal. Whole cell extracts prepared from both immature and mature oocytes deadenylate exogenous RNA substrates in vitro. Deadenylation activity is not detected in isolated nuclear or cytoplasmic extracts obtained from immature oocytes, but is reconstituted when these fractions are combined in vitro. These results indicate that the factors required for deadenylation activity are present in immature oocytes, but that poly(A) removal is prevented by the sequestration of one or more of these components within the nucleus. Maturation-specific deadenylation of maternal mRNAs occurs upon the release of nuclear factors into the cytoplasm at germinal vesicle breakdown.     

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.     

Regulation of nuclear membrane assembly and maintenance during in vitro maturation of mouse oocytes: role of pyruvate and protein synthesis

Summary In the absence of a suitable energy source, mouse oocytes cultured in vitro resume, but fail to complete, meiotic maturation. However, little is known about the underlying mechanisms leading to this meiotic failure. We utilized pyruvate-deficient medium to test for the role of pyruvate throughout the meiotic maturation process. Germinal vesicle-stage (GV) oocytes underwent germinal vesicle breakdown (GVBD), but failed to form a polar body when cultured continuously in pyruvate-free medium. However, when GV oocytes were preincubated for 4 h in pyruvate-free medium containing dibutyryl cyclic adenosine monophosphate (dbcAMP) and then cultured in pyruvate-free medium, GVBD was markedly inhibited. Preincubation of GV oocytes in dbcAMP and cycloheximide, followed by culture in cycloheximide only, also inhibited GVBD. A longer preincubation period was required in the cycloheximide-dbcAMP case (12 h) than in pyruvate-free-dbcAMP medium situation (4 h). Strikingly, reassembly of the nuclear membrane without polar body formation was observed following GVBD in oocytes continuously cultured in pyruvate-free medium. The reassembled nuclear membrane increased in size with continued culture, and it surrounded partially-decondensed chromatin. Nuclear membrane reassembly also occurred in oocytes which had undergone GVBD during continuous culture in medium containing only cycloheximide. Reformation of nuclear membranes after GVBD was confirmed by electron-microscopic analyses of oocytes cultured in pyruvate-free medium or in the presence of cycloheximide. We conclude that both pyruvate and protein synthesis are required for nuclear membrane disassembly, whereas lack of pyruvate or protein synthesis is associated with interruption of the metaphase state and reassembly of the nuclear membrane. The evidence suggests that assembly and maintenance of an intact nucleus and its disintegration are all amenable to regulation by pyruvate, possibly via mechanism(s) involving protein synthesis.     

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.     

Protein synthesis requirements during resumption of meiosis in the hamster oocyte: early nuclear and microtubule configurations.

The organization of chromatin and cytoplasmic microtubules changes abruptly at M-phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus-enclosed hamster oocytes were cultured in the presence of 100 micrograms/ml puromycin or cycloheximide for 5 hr. Both control (untreated) and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4-4.5 hr in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5-5.5 hr in culture, microtubule asters are no longer detected in most cells, and the spindle is the only tubulin-positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation is inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug-free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus-enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption.     

Maturation of Xenopus oocytes: II. Observations on membrane potential

Large, progesterone-responsive oocytes within their follicles have an average resting potential of about ?25 mV. When manually dissected out of their follicles, most of these oocytes undergo a hyperpolarization over the next 30–60 min to values of about ?60 to ?80 mV. The relatively high negative membrane potentials previously recorded on dissected amphibian oocytes may thus be an artifact in the sense that such measurements do not reflect the electrical characteristics of the oocyte within the follicle. The available evidence indicates that the hyperpolarization reflects the activation of an electrogenic Na⁺,K⁺-transport process. One of the terminal events of oogenesis appears to be a suppression of the generation of the Na⁺,K⁺-transport process when oocytes are ovulated artificially (by dissection) or naturally. Continuous, long-term recordings on dissected oocytes reveal that a rather pronounced depolarization of the membrane potential together with an inflection in the recorded potential around the time of germinal vesicle breakdown takes place in the presence of progesterone. Recordings of oocytes within the follicle reveal similar changes, although reduced in absolute magnitude. In both cases, final membrane potentials of ?10 to ?15 mV are achieved. The electrophysiological changes which accompany the normal maturation process thus do not appear to be as pronounced as previously indicated.     

中华大蟾蜍卵母细胞成熟过程中膜电位变化的实验分析

The full-grown oocytes obtained from toad (bufo bufo gargarizans) submitted in hibernation state or reared at 25-30 degrees C for several months, named hibernation oocyte or high temperature oocyte, had a membrane potential of -41.51 +/- 0.77 mV and -43.83 +/- 1.39 mV in Ringer's solution respectively. The hibernation oocytes underwent GVBD (germinal vesicle breakdown) and membrane depolarization at 19 +/- 1 degree C after progesterone stimulation. The membrane potential was about -20 mV at the period of GVBD, and -10 mV or so at 20 hours after the hormone treatment. However, the high temperature oocytes did not undergo GVBD, their membrane potential decreased before the fourth hour after treatment with progesterone and then recovered. If the hibernation oocytes were preincubated at 37-38 degrees C for 13 hours prior to the culture in the medium containing progesterone (10(-6)M, 37-38 degrees C), no GVBD was observed and the membrane depolarized before the fourth hour after treatment with progesterone then recovered, but MPF was detectable in the cytoplasm (unpublished). Both GVBD and membrane depolarization appeared in the hibernation oocytes and high temperature oocytes after injection of MPF. The time required for the hibernation oocytes injected MPF to attain the membrane potential about -20 mV was 4 hours earlier than that of progesterone treatment. It was just the time required for the appearance of MPF in the cytoplasm of oocytes treated with the hormone. It was noticed in our precedent article that a factor which appeared in the cytoplasm of high temperature oocytes differed from MPF. The factor was called Hibernation Oocyte Mature Promoting Factor (HOMPF).(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of porcine sperm plasma membrane proteins to sheep, hamster and mouse oocyte plasma membrane

Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders.     

Protein modification by phosphorylation during the process of nuclear membrane dissolution in puromycin-treated mouse oocytes.

The present study was undertaken to elucidate the mechanism of nuclear membrane dissolution (NMD) in puromycin-treated mouse oocytes. Treatment of germinal vesicle breakdown (GVBD) oocytes with puromycin (50 micrograms/ml) induced chromosome decondensation with formation of a polar body; these are designated nuclear membrane (NM) oocytes. After withdrawal of puromycin, NM oocytes underwent NMD (approximately 70%) during a 12-h culture period. Either dibutyryl cyclic AMP (dbcAMP, 25-100 micrograms/ml) or isobutylmethylxanthine (IBMX, 0.1-1.0 mM) inhibited the process of NMD in a dose-dependent manner, suggesting the involvement of cAMP in the process of NMD. To determine which protein(s) participated in the transition from interphase to metaphase II during NMD, NM oocytes were labeled with [35S]methionine, and one- and two-dimensional gel electrophoresis were performed. Although the synthesis of stage-specific proteins during NMD was not found, two specific proteins of Mr 27,000 and 46,000, which were synthesized at interphase following removal of puromycin, were modified during NMD. Phosphatase treatment and 32PO4-labeling experiments indicated that phosphorylation was responsible for these modifications, which were inhibited by either dbcAMP or IBMX. Therefore, it appears that phosphorylation of specific proteins may play an important role in the transition from interphase to metaphase II.     

Cytoplasmic changes in relation to nuclear maturation and early embryo developmental potential of porcine oocytes: effects of gonadotropins, cumulus cells, follicular size, and protein synthesis inhibition

Morphological and biochemical changes indicative of cytoplasmic maturation in relation to nuclear maturation progression and early embryo developmental potential was studied. Fluorescently labeled microfilaments and cortical granules were visualized by using laser scanning confocal microscopy. The mitogen-activated protein (MAP) kinase phosphorylation and cyclin B1 levels were revealed by Western blot. With the maturation of oocytes, cortical granules and microfilaments were localized at the cell cortex. A cortical granule-free domain (CGFD) and an actin-thickening area were observed over both the MII spindle of a mature oocyte and chromosomes of a nocodazole-treated oocyte, suggesting that chromosomes, but not the spindle, determined the localization of CGFD and actin-thickening area. In oocytes that are incompetent to resume meiosis, as indicated by the failure of germinal vesicle breakdown (GVBD), peripheral localization of cortical granules and microfilaments, phosphorylation of MAP kinase and synthesis of cyclin B1 did not occur after 44 hr in vitro. These cytoplasmic changes were also blocked when GVBD of meiotically competent oocytes was inhibited by cycloheximide. Culture of oocytes in a chemically defined medium showed that biological factors such as gonadotropins, cumulus cells and follicle size affected both nuclear and cytoplasmic maturation as well as embryo developmental potential. Absence of gonadotropins or removal of cumulus cells alone did not significantly influence GVBD or cyclin B1 levels, but decreased the final maturation and developmental ability of oocytes. A combination of gonadotropin absence and cumulus removal decreased GVBD, MAP kinase phosphorylation and embryo development. A high proportion of oocytes derived from small follicles were able to resume meiosis, synthesize cyclin B(1), phosphorylate MAP kinase and translocate CGs, but their maturation and embryo developmental ability were limited. Removal of cumulus cells from small follicle-derived oocytes severely affected their ability to undergo cytoplasmic and nuclear maturation.