Ribosomal preparations may induce maturation in Xenopus laevis oocytes

Xenopus laevis oocytes undergo maturation when they are injected with large quantities of crude ribosomes from various origins: X laevis full-grown or matured oocytes, Xenopus ovaries and embryos, Xenopus liver or mouse liver. All have the same efficiency, whatever their origin: they include 50-90% maturation in the injected oocytes at about the same speed as progesterone treatment. The ribosomal preparations are inactive wen injected into recipient oocytes pretreated with cholera toxin or cycloheximide. After dissociation with the high salt extract, but not with the subunits. Hypotheses concernning the mode action of this ribosomal extract are disussed.     

Endogenous phosphorylated proteins during maturation of Xenopus laevis oocytes

During the course of maturation of Xenopus laevis oocyte a burst of phosphorylation occurs around germinal vesicle breakdown. At the same time a relative drop in a unique phosphoprotein (protein I; mot wt ～40,000) is observed. Enucleation of [³²P] labeled oocytes has shown the cytoplasmic localization of protein I. Methylxanthines and cholera toxin, which inhibit progesterone-induced maturation, block the burst of phosphorylation and do not change the amount or the distribution of [³²P] phosphoproteins.     

Cholinergic modulation of progesterone-induced maturation of Xenopus oocytes in vitro

Mixed and muscarinic cholinergic agonists (acetylcholine, carbamylcholine, methacholine, oxotremorine, and pilocarpine) accelerated in a dose-dependent manner the progesterone-induced maturation of Xenopus laevis oocytes. None of these agonists induced oocyte maturation in the absence of progesterone. The accelerating effect of cholinergic agonists was blocked in a dose-dependent manner by specific muscarinic antagonists (atropine and scopolamine) but not by specific nicotinic antagonists (d-tubocurarine and hexamethonium). The specific nicotinic agonist, dimethylphenylpiperazine, alone induced maturation in the absence of progesterone. The optimal promoting effect of acetylcholine was observed when oocytes were exposed to acetylcholine for 30 min, 5 min after the addition of progesterone, and was markedly better than when oocytes were exposed to acetylcholine throughout their incubation with progesterone. The effect of acetylcholine was observed in both follicle-enclosed and in defolliculated oocytes, indicating that follicular cells were not the target of the cholinergic drugs.     

Boron deficiency disables Xenopus laevis oocyte maturation events

Processes of oocyte maturation that may be affected by boron (B) deficiency were studied to potentially determine a possible biochemical role of B in the Xenopus laevis oocyte. More specifically, the Xenopus oocyte membrane progesterone receptor (OMPR) in B-deficient oocytes was characterized by evaluating progesterone affinity for the OMPR and OMPR responsiveness to progesterone stimulation. The responsiveness of B-deficient oocytes to microinjection of a purified oocyte cytoplasmic fraction (OCF) from B-adequate oocytes was also studied to evaluate which aspects of the maturation process were affected by B deficiency. Results suggested that B deficiency resulted in incomplete oocyte maturation and that maturation could not be induced by the administration of exogenous progesterone. Progesterone successfully induced germinal vesicle breakdown (GVBD) in oocytes from females fed a B-supplemented diet (+B) and females administered a traditional diet of beef liver and lung (B adequate). Addition of exogenous B to the -B oocytes increased the rate of progesterone-induced GVBD slightly. The B-deficient X. laevis oocytes were capable of undergoing GVBD when endogenously stimulated by microinjected purified B-adequate OCF. These results indicated that the inability of the B-deficient oocytes to undergo GVBD was not associated with the cytoplasmic induction process specifically, but possibly in the progesterone receptor or signal transduction pathways. Radio-binding studies found that progesterone binding to the B-deficient OPMR was greatly reduced compared to B-adequate or B-supplemented OMPR. Moreover, washout studies determined that progesterone binding to the OMPR in B-deficient oocytes was more transient than the B adequate or +B oocytes.     

ras-p21 Activates phospholipase D and A2, but not phospholipase C or PKC,in Xenopus laevis Oocytes

Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Since activation of PLA2, PLC, or PLD has been postulated as a mediator of ras function, we have used the oocyte system to study the putative functional relationship between ras-p21 and these phospholipases. A rapid generation of PA and DAG was observed after ras-p21 microinjection, suggesting the activation of both PLC and PLD enzymes. However, production of DAG was sensitive to inhibition of the PA-hydrolase by propranolol, indicating that PLD is the enzyme responsible for the generation of both PA and DAG. Microinjection of PLD or ras-p21 induced the late production of lysophosphatidylcholine on a p42^MAPK-dependent manner, an indication of the activation of a PLA2. Inhibition of this enzyme by quinacrine does not inhibit PLD- or ras-induced GVBD, suggesting that PLA2 activation is not needed for ras or PLD function. Contrary to 3T3 fibroblasts, where ras-p21 is functionally dependent for its mitogenic activity on TPA- and staurosporine-sensitive PKC isoforms, in Xenopus oocytes, induction of GVBD by ras-p21 was independent of PKC, while PLC-induced GVBD was sensitive to PKC inhibition. Thus, our results demonstrate the activation of PLD and PLA2 by ras-p21 proteins, while no effect on PLC was observed.     

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.     

Freeze-fracture analysis of structural reorganization during meiotic maturation in oocytes of Xenopus laevis

Summary During meiotic maturation, the cortex of oocytes of Xenopus laevis undergoes structural reorganization, visualized in this study by freeze-fracture electron microscopy. In the full-grown but immature oocyte, annulate lamellae are dispersed throughout the subcortex of the egg, 5 to 20 m from the plasma membrane. The annulate lamellae consist of well-organized stacks of membrane with visible pores. Stimulation of meiotic maturation by progesterone leads to disruption of the annulate lamellae and formation of an elaborate cortical endoplasmic reticulum which surrounds the cortical granules and intertwines throughout the cortex of the mature egg. Pore-like structures similar to those previously observed in the subcortical annulate lamellae are observed in the mature cortical endoplasmic reticulum. The cortical endoplasmic reticulum is often in close apposition with the plasma membrane and with membranes of cortical granules, but no junctions are visualized. This study provides further evidence that the cortical endoplasmic reticulum develops during progesterone-stimulated meiotic maturation in vitro, and that the annulate lamellae are precursors to the cortical endoplasmic reticulum.     

Induction of meiosis of Xenopus laevis oocytes by mianserine

Maturation of Xenopus laevis oocytes can be induced by mianserine, a tricyclic antidepressant. K⁺-free medium facilitates this maturation process. Mianserine must be kept in contact with the oocytes during the whole process of maturation for maximal efficiency. It is inactive after injection into the oocytes. Mianserine induces the formation of maturation-promoting factor (MPF) in the treated oocytes. Mianserine-induced maturation is strongly inhibited by theophylline, even in K⁺-free medium. Progesterone displays synergistic effects with mianserine for the induction of maturation. Likewise, oestradiol shows cooperative maturing effects with progesterone as well as with mianserine. It is suggested that mianserine exerts its primary effects on oocyte maturation by inhibiting a membrane adenylate cyclase.     

Analysis of farnesyl transferase activity during hormone-induced maturation of Xenopus laevis oocytes

Preincubation of Xenopus laevis oocytes with insulin or insulin-like growth factor 1 (IGF-1) resulted in inhibition of farnesyl transferase (FTase) activity measured both in vivo (after microinjection of tritiated farnesyl pyrophosphate and Ras-CVIM into oocytes) and in extracts using a filtration assay. FTase activity measured in oocyte extracts was inhibited 55% after a 20 min treatment of oocytes with 1 microM insulin or 10 nM IGF-1. The apparent IC(50) for inhibition of oocyte FTase by IGF-1 is 0.3 nM. The observed decrease in FTase activity was apparently not due to translocation of enzyme from cytosol to membrane, since activities measured both in soluble extracts and resuspended crude pellets displayed comparable levels of inhibition following hormone treatment. Using a hexapeptide (TKCVIM) as substrate, FTase activity was also inhibited 65% when oocytes were pretreated with 10 nM IGF-1. Two FTase inhibitors [(alpha-hydroxyfarnesyl) phosphonic acid (HFPA) and chaetomellic acid A (CA)] effectively inhibited Xenopus oocyte FTase by 80-90% when added to assay mixtures (IC(50) values of 338 +/- 96 nM HFPA and 232 +/- 80 nM CA) or after incubation of oocytes in drug before preparation of soluble extracts for assay (IC(50) values of 7 +/- 6 nM HFPA and 328 +/- 128 nM CA). The farnesyl transferase inhibitors were observed to slow the time course of oocyte maturation but did not block the IGF-1-induced maturation response. J. Exp. Zool. 286:193-203, 2000.Copyright 2000 Wiley-Liss, Inc.     

Ultrastructure of canine oocytes during in vivo maturation

The aim of the present study was to describe the canine oocyte ultrastructural modifications during in vivo maturation, with precise reference to the timing of the LH surge and of ovulation. Twenty-five bitches were ovariectomized at specific stages between the onset of proestrus and the fifth day post-ovulation: 65 oocytes were observed by transmission electron microscopy (TEM), either before the LH surge (n = 10), between the LH surge and ovulation (n = 12) or after ovulation (n = 43). Prior to the LH surge, the oocyte nucleus had already begun its displacement to the vicinity of the oolemma and reticulated nucleoli were infrequent. The cytoplasm showed signs of immaturity (few organelles preferentially located in the cortical zone, "mitochondrial cloud", scarce cortical granules). The LH surge was immediately followed by cumulus expansion but the ovulation occurred 2 days later. Retraction of the transzonal projections and the meiotic resumption occurred after another 3 days (5 days after the LH peak). The ovulation was then followed by gradual cytoplasmic modifications. Nucleoli re-assumed a reticulated aspect around 24 hr post-ovulation. From 48 hr post-ovulation mitochondria and SER were very numerous and evenly distributed. In conclusion canine oocyte maturation began prior to the LH surge and no cytoplasmic or nuclear modifications followed immediately the LH surge and ovulation. This study suggests that two distinct signals are needed for the final in vivo maturation: one prior to the LH surge (to induce maturation) and another one, around 3 days post-ovulation (to induce meiotic resumption).     

Nucleoli from growing oocytes inhibit the maturation of enucleolated, full-grown oocytes in the pig

In mammals, the nucleolus of full‐grown oocyte is essential for embryonic development but not for oocyte maturation. In our study, the role of the growing oocyte nucleolus in oocyte maturation was examined by nucleolus removal and/or transfer into previously enucleolated, growing (around 100 µm in diameter) or full‐grown (120 µm) pig oocytes. In the first experiment, the nucleoli were aspirated from growing oocytes whose nucleoli had been compacted by actinomycin D treatment, and the enucleolated oocytes were matured in vitro. Most of non‐treated or actinomycin D‐treated oocytes did not undergo germinal vesicle breakdown (GVBD; 13% and 12%, respectively). However, the GVBD rate of enucleolated, growing oocytes significantly increased to 46%. The low GVBD rate of enucleolated, growing oocytes was restored again by the re‐injection of nucleoli from growing oocytes (23%), but not when nucleoli from full‐grown oocytes were re‐injected into enucleolated, growing oocytes (49%). When enucleolated, full‐grown oocytes were injected with nucleoli from growing or full‐grown oocytes, the nucleolus in the germinal vesicle was reassembled (73% and 60%, respectively). After maturation, the enucleolated, full‐grown oocytes injected with nucleoli from full‐grown oocytes matured to metaphase II (56%), whereas injection with growing‐oocyte nucleoli reduced this maturation to 21%. These results suggest that the growing‐oocyte nucleolus is involved in the oocyte's meiotic arrest, and that the full‐grown oocyte nucleolus has lost the ability. Mol. Reprod. Dev. 78:426–435, 2011. © 2011 Wiley‐Liss, Inc.     

Nuclear maturation and embryo development of porcine oocytes vitrified by cryotop: Effect of different stages of in vitro maturation

The present study was designed to evaluate the viability, meiotic competence and subsequent development of porcine oocytes vitrified using the cryotop method at different stages of in vitro maturation (IVM). Cumulus–oocyte complexes (COCs) were cultured in IVM medium supplemented with 1 mM dibutyryl cAMP (dbcAMP) for 22 h and then for an additional 22 h without dbcAMP in the medium. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase I (AI/TI) and metaphase II (MII) were found to occur predominantly at 0–22, 26, 32, 38 and 44 h of IVM, respectively. Oocytes were exposed to cryoprotectant (CPA) or vitrified after different durations of IVM (0, 22, 26, 32, 38 and 44 h). After CPA exposure and vitrification, surviving oocytes that were treated before completion of the 44 h maturation period were placed back into IVM medium for the remaining maturation period, and matured oocytes were incubated for 2 h. CPA treatment did not affect the viability of oocytes matured for 26, 32, 38 or 44 h, but significantly decreased survival rate of oocytes matured for 0 or 22 h. CPA treatment had no effect on the ability of surviving oocytes to develop to the MII stage regardless of the stage during IVM; however, blastocyst formation following PA was severely lower (P < 0.05) than that in the control. At 2 h post-warming, the survival rates of oocytes vitrified at 26, 32, 38 and 44 h of IVM were similar but were higher (P < 0.05) than those of oocytes vitrified at 0 or 22 h of IVM. The MII rates of surviving oocytes vitrified at 0 and 38 h of IVM did not differ from the control and were higher (P < 0.05) than those of oocytes vitrified at 22, 26 or 32 h of IVM. After parthenogenetic activation (PA), both cleavage and blastocyst rates of vitrified oocytes matured for 22, 26, 32, 38 and 44 h did not differ, but all were lower (P < 0.05) than those matured 0 h. In conclusion, our data indicate that survival, nuclear maturation and subsequent development of porcine oocytes may be affected by their stage of maturation at the time of vitrification; a higher percentage of blastocyst formation can be obtained from GV oocytes vitrified before the onset of maturation.     

EGF-like peptides mediate FSH-induced maturation of cumulus cell-enclosed mouse oocytes

This study was carried out to examine the participation of epidermal growth factor (EGF)-like peptides in the induction of germinal vesicle breakdown (GVB) in mouse cumulus cell-enclosed oocytes (CEO). The EGF-like peptide, amphiregulin (AR), dose-dependently stimulated meiotic resumption in CEO, but not denuded oocytes (DO) maintained in meiotic arrest with 300 microM dbcAMP. The EGF receptor (EGFR) kinase inhibitor, AG1478, blocked meiotic resumption induced by FSH and AR in CEO, but had no effect in DO. FSH-induced maturation was also suppressed by antisera to both EGFR and EGF. Maturation occurred with slightly faster kinetics in AR-stimulated CEO when compared to FSH-stimulated CEO. When CEO were maintained in meiotic arrest with a low level of dbcAMP, FSH was initially inhibitory to maturation and later stimulatory; the stimulatory phase was prevented by AG1478, indicating mediation by EGF-like peptides. Pulsing CEO with high levels of dbcAMP also stimulated GVB and could be blocked by AG1478. Treatment of arrested CEO with PKC agonists stimulated maturation and this was prevented with AG1478 as well as antibodies to EGFR. FSH-induced maturation of dbcAMP-arrested CEO was blocked by bisindolylmaleimide I (BIM-I), an inhibitor of PKC, implicating PKC in FSH action. EGF-stimulated CEO failed to resume maturation in the presence of glycerrhetinic acid, a gap junction inhibitor, suggesting transfer of positive signal through the cell-cell coupling pathway. These data support the idea that EGF-like peptides provide a common pathway mediating the meiosis-inducing influence of FSH, cAMP pulsing, and PKC activation in mouse CEO by a gap junction-dependent process.     

In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. ¹⁵N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional ¹H–¹⁵N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein–protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Induction of meiotic maturation in mouse oocytes by adenosine analogs

In this study we have examined the meiosis-inducing influence of adenosine analogs in mouse oocytes. When a varied group of nucleosides and nucleotides were tested on overnight cultures of hypoxanthine-arrested, cumulus cell-enclosed oocytes (CEO), halogenated adenosine nucleosides, but not native adenosine, exhibited a significant meiosis-inducing capability. When tested under a variety of conditions, meiotic induction by 8-bromo-adenosine (8-Br-Ado) and a second adenosine analog, methylmercaptopurine riboside (MMPR), was especially potent in denuded oocytes (DO) compared to CEO and was not dependent on the type of inhibitor chosen to maintain meiotic arrest. Germinal vesicle breakdown (GVB) was stimulated with rapid kinetics and was preceded by an increase in AMP-activated protein kinase (AMPK) activity. Moreover, compound C, an inhibitor of AMPK, blocked the meiosis-inducing activities of both adenosine analogs. When tested for an effect on meiotic progression to metaphase II (MII) in spontaneously maturing CEO, 8-Br-Ado and the AMPK activator, 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), increased the percentage of MII-stage oocytes, but MMPR decreased this number. Adenosine and inhibitors of de novo purine synthesis had no effect on the completion of maturation, while compound C suppressed this process. These results support the proposition that oocyte AMPK mediates the positive influence of AICAR and 8-Br-Ado on both the initiation and completion of meiotic maturation. The role of AMPK in MMPR action is less clear.     

Effects of NO-synthase inhibitors on maturation mouse oocytes in cumulus-oocyte complexes

We studied the effects of various NO-synthase inhibitors on meiotic maturation of mouse oocytes in vitro in cumulus-oocyte complexes isolated from follicles of varying sizes. Selective and nonselective inhibitors of NO-synthase isoforms suppressed meiotic maturation of oocytes to varying degrees, which was expressed in a decreased number of oocytes at metaphase II. The results obtained suggest that the role of inducible form of NO-synthase (iNOS) increases with the development of follicles and oocytes.     

5-羟色胺转运体基因异体表达模型的建立

目的:探讨建立5-羟色胺转运体(5-HTT)基因异体表达模型的可行性,且为进一步探讨5-羟色胺重摄取的动力过程和条件以及其功能的调控机制奠定基础.方法:利用体外转录cRNA技术将克隆至pOTV中的5-HTT的cDNA转录、合成5HTT的cRNA,通过显微注射技术将该cRNA注入成熟雌性非洲爪蟾卵母细胞的胞质中,使其表达以建立5-羟色胺转运体(SERT)的异体表达模型,并用电压钳技术检测其转运功能.结果:爪蟾卵母细胞可被用做5-HTT的异体表达模型,其转运功能呈浓度依赖性并具饱和现象,转运过程可被特异阻断剂Desipramine阻断.结论:非洲爪蟾卵母细胞可作为5-羟色胺等单胺类神经递质转运体的异体表达系统,为进一步研究转运体蛋白的功能和调控提供了有效工具.     

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certain maturation-linked changes were noted. Sperm apposition and sperm-oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrounded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck-piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.