Early grey crescent formation experimentally induced by cycloheximide in the axolotl oocyte

Summary The effects of cycloheximide (CH) on grey crescent formation in artificially maturedAmbystoma mexicanum oocytes were determined. CH induced grey crescent formation after a few hours, especially after a 45° to 90° rotation from the vertical animal-vegetal axis. With low concentrations of CH (about 0.5 ng/oocyte), meiosis was still able to proceed normally to the stable second metaphase stage, but higher concentrations blocked it after 1st polar body extrusion and an interphasic nucleus appeared. Such effects were compared to those of inactone, an analogue of cycloheximide, which as a pure substance does not inhibit protein synthesis, but still contained a small amount of CH in the available samples. It is concluded that grey crescent formation can occur in non-activated oocytes. The effects of cycloheximide might be due to partial inhibition of protein synthesis and the presence of a proteinic inhibitor of the symmetry reaction in the normal oocyte is suggested.     

Immunocytochemical studies of hamster oocyte activation

By indirect immunofluorescence, using rabbit anti-heparin-binding placental protein (HBPP) antiserum, we studied HBPP expression by physiologically and non-physiologically (microsurgically) activated hamster gametes. Whereas mature gametes (sperm, metaphase II oocytes) were negative, in vivo conceived preimplantation embryos, from pronuclear to two- and four-cell stages, were HBPP positive. No HBPP was demonstrated in the zona pellucida, but HBPP-dependent immunofluorescence was localized in the perivitelline space. Oocytes incubated with hyaluronidase demonstrated variable responses from negative to positive. (Diluent or sperm) microinjected oocytes were all activated and HBPP positive within 4 h after stimulation. Thus neither activation by microinjection nor HBPP expression required paternal gametes. These kinetics suggest that HBPP may be a cortical granule secretogogue which can be applied to monitor oocyte responses during in vitro manipulations.     

Age dependence of bovine oocyte activation

The ability of bovine oocytes to undergo parthenogenetic activation using either a Ca⁺⁺-Mg⁺⁺-H⁺ ionophore (A23187) or electric shock was investigated, as a prelude to understanding activation potential following nuclear transfer into ooplasm. Oocytes were collected from slaughterhouse ovaries by aspiration of 1–5-mm follicles. The time of placement into maturation medium was noted, and maturational age (time in culture) measured from that point. After exposure to activating conditions eggs were cultured for a further 12–16 hours, fixed, and stained with aceto-orcein. Oocytes that progressed to telophase or pronuclear formation were considered activated. Concentrations of A23187 ranging from 100 pM to 100 μM showed that 1–100 μM levels resulted in 94–100% activation at 30 hours maturation. Frequency of activation differed from controls (no ionophore) at 100 nM (49%; P < 0.05). With A23187 maximum response occurred between 26 and 30 hours of maturation (77% and 92%, respectively). A short pulse electric shock, capable of causing oocyte membrane fusion, gave similar results relative to maturational age (82% and 90% activation for 26 and 30 hours, respectively). Therefore, maximum response to the two activating stimuli occurred in oocytes at similar maturational ages. Exposure to activating conditions prior to onset of activating ability (18 hours) followed by another exposure at 26 hours showed that the oocytes were still fully able to activate upon reaching maturational activation competence. Because cytochalasin B is present in the medium used for nuclear transfer, oocytes were incubated with cytochalasin B prior to exposure to an activating stimulus. Frequency of activation was similar to the control treatment (61% and 73%). The effect of mechanical stress of cytoplasm removal and replacement by electrofusion on activation was also not significant. Overall, maturational age of the oocyte was the main determinant of activation ability.     

Normal and mutant rhodopsin activation measured with the early receptor current in a unicellular expression system.

The early receptor current (ERC) represents molecular charge movement during rhodopsin conformational dynamics. To determine whether this time-resolved assay can probe various aspects of structure-function relationships in rhodopsin, we first measured properties of expressed normal human rhodopsin with ERC recordings. These studies were conducted in single fused giant cells containing on the order of a picogram of regenerated pigment. The action spectrum of the ERC of normal human opsin regenerated with 11-cis-retinal was fit by the human rhodopsin absorbance spectrum. Successive flashes extinguished ERC signals consistent with bleaching of a rhodopsin photopigment with a normal range of photosensitivity. ERC signals followed the univariance principle since millisecond-order relaxation kinetics were independent of the wavelength of the flash stimulus. After signal extinction, dark adaptation without added 11-cis-retinal resulted in spontaneous pigment regeneration from an intracellular store of chromophore remaining from earlier loading. After the ERC was extinguished, 350-nm flashes overlapping metarhodopsin-II absorption promoted immediate recovery of ERC charge motions identified by subsequent 500-nm flashes. Small inverted R(2) signals were seen in response to some 350-nm flashes. These results indicate that the ERC can be photoregenerated from the metarhodopsin-II state. Regeneration with 9-cis-retinal permits recording of ERC signals consistent with flash activation of isorhodopsin. We initiated structure-function studies by measuring ERC signals in cells expressing the D83N and E134Q mutant human rhodopsin pigments. D83N ERCs were simplified in comparison with normal rhodopsin, while E134Q ERCs had only the early phase of charge motion. This study demonstrates that properties of normal rhodopsin can be accurately measured with the ERC assay and that a structure-function investigation of rapid activation processes in analogue and mutant visual pigments is feasible in a live unicellular environment.     

Platelet activating factor-induced increase in cytosolic calcium and transmembrane current in human macrophages

Platelet-activating factor (PAF) is synthesized and secreted by macrophages in response to inflammatory stimuli. When exogenously applied to human monocyte derived macrophages (HMDMs), PAF induces a rapid rise in cytosolic free calcium (Ca _i ) believed to be an early triggering event in macrophage activation. We investigated PAF-induced Ca²⁺ signaling in HMDMs using the calcium indicator Fura-2, combining single cell ratio fluorimetry and digital video imaging with whole-cell recording techniques. Application of PAF (20 ng/ml) to adherent macrophages induced transient increases in Ca, that were biphasic, consisting of an initial phase that could be observed in Ca²⁺-free solutions and a second phase that was critically dependent upon Ca²⁺ entry. When Mn²⁺ was applied to cells in the presence and absence of Ca²⁺, PAF increased the rate of Mn²⁺ entry rate only when Ca²⁺ was absent. PAF increased the rate of Ba²⁺ entry even when measured in the presence of external Ca²⁺. Ca²⁺ entry was reversibly inhibited in the presence of external La³⁺ (1 mm). Data obtained from simultaneous voltage-clamp/microfluorimetry experiments demonstrated the activation of a nonselective cation current which closely paralleled the rising phase of the Ca _i transient. We investigated whether the non-selective cation conductance provided for the bulk of the agonist-induced Ca²⁺ influx. Changes in Ca _i following removal of extracellular Ca²⁺ (Ca _o ) during the agonist-induced Ca _i response were not associated with changes in whole-cell current. The inability to detect whole-cell current changes correlated with a decrease in Ca _o suggests that the bulk of the Ca²⁺ influx was not through the nonselective conductance and either does not occur through a conductance pathway or occurs via a parallel pathway consisting of channels which are both low conductance and highly Ca²⁺ selective.     

Gating charge movement precedes ionic current activation in hERG channels

We recently reported gating currents recorded from hERG channels expressed in mammalian TSA cells and assessed the kinetics at different voltages. We detected 2 distinct components of charge movement with the bulk of the charge being carried by a slower component. Here we compare our findings in TSA cells with recordings made from oocytes using the Cut Open Vaseline Gap clamp (COVG) and go on to directly compare activation of gating charge and ionic currents at 0 and +60 mV. The data show that gating charge saturates and moves more rapidly than ionic current activates suggesting a transition downstream from the movement of the bulk of gating charge is rate limiting for channel opening.     

Follicular factors influence oocyte fertilizability by modulating the intercellular cooperation between cumulus cells and oocyte

In order to investigate whether the follicular tissue influences cumulus-oocyte interaction and, consequently, the fertilizability of the egg, four experiments were carried out. In the first, cumulus-enclosed pig oocytes were cultured for 44 h in control medium (modified TCM-199) or in follicle-conditioned medium, and the intercellular coupling was studied by measuring ³H-uridine uptake. In control medium the intercellular cooperation started to decline immediately, and at 24–32 h the uncoupling was almost complete. By contrast, in follicle, conditioned medium, it remained at high levels until 24?32 h. In the second experiment protein synthesis patterns of oocytes were studied. Oocytes cultured in conditioned medium were characterized by a 45-kD protein band, while those maturing in control medium were identifiable by a marked 56-kD band. In the third experiment mature oocytes were fertilized in vitro. The percentage of penetrated egg was higher in oocytes matured in conditioned medium than in control medium. In addition, only oocytes matured in conditioned medium could consistently decondense spermatozoa and form male pronuclei. Metabolic cooperation, protein synthesis patterns, and fertilizability were also studied in oocytes matured in control medium supplemented with either 17β-estradiol or progesterone or testosterone or dihydrotestosterone or androstenedione or ether extract of conditioned medium. Only ether extract and progesterone stimulated cumulus oocyte interaction and sperm decondensation. In the last experiment oocytes denuded at different stage of their maturation in conditioned medium were fertilized in vitro. The longer the eggs were cultured with the cumulus, the higher was their penetrability. Moreover, only oocytes denuded after 40 h of culture could, once fertilized, promote the formation of male pronuclei. These data demonstrate that follicular secretions are fundamental for the maintenance in vitro of a functional intercellular coupling between cumulus and oocyte, which is necessary for the egg to become penetrable by spermatozoa and to acquire the conditions required for the formation of male pronuclei.     

Intrafollicular control of oocyte maturation in the PIG

Summary The mammalian oocyte becomes arrested at the diplotene stage of the first meiotic division during prenatal or early postnatal life. It remains arrested in meiosis until shortly before ovulation when the surge of gonadotropin induces resumption and completion of meiosis to the metaphase II stage. When oocytes are harvested from medium-sized or large follicles of pig and other species and cultured, they resume meiosis spontaneously indicating that the follicles exert an inhibitory influence on meiosis. To analyze the control of meiosis by follicular components, culture of isolated pig oocytes in the presence of follicular cells or follicular fluid (FF1) has been used as a model in this laboratory. An oocyte maturation inhibitor (OMI) has been isolated and partially purified by ultrafiltration and gel chromatography of FF1 and shown to be a polypetide with a molecular weight in the order of 2000 daltons. Physiological characterization has shown that the effect of OMI in vitro is reversible and that it can be overcome by luteinizing hormone (LH). The action of OMI requires the presence of cumulus cells surrounding the oocyte since it was found that denuded oocytes, stripped of cumulus cells, do not respond to OMI. Furthermore, when cumulus-enclosed oocytes were cultured, OMI inhibited the differentiation of the cumulus cells in terms of morphology and progesterone secretion in a dose-related manner. The inhibition of cumulus differentiation by OMI was reversible and could be overcome by LH. The results indicate that the effect of partially purified OMI upon meiosis may be mediated by the cumulus cells. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. This study was supported by Grants 760–0530 from the Ford Foundation (to C.P.C.), and Grant B78-14F-5158-01 from the Swedish Medical Research Council (to T.H.).     

A remarkable increase in the pHi sensitivity of voltage-dependent calcium channels occurs in human sperm incubated in capacitating conditions

Human sperm are endowed with voltage-dependent calcium channels (VDCC) that produce increases in [Ca2+]i in response to depolarization with KCl. These channels are stimulated during "capacitation", a complex biochemical process, accompanied by a slight pHi alkalization, that sperm must accomplish to acquire the ability to fertilize the egg. The stimulation can be explained in part by the fact that in non-capacitated sperm, calcium influx through VDCC is stimulated by pHi alkalization in the range of pHi observed during capacitation. In this work, we explored the effect of pHi on VDCC in capacitated sperm loaded with fura ff. Strikingly, the pHi sensitivity of VDCC increased approximately 7-fold when sperm was capacitated, as compared with non-capacitated sperm. This finding suggests that the pHi sensitivity of VDCC can be modulated during capacitation so that a combined effect of pHi alkalization and biochemical regulation enhances calcium influx through these channels.     

A voltage-activated proton current in human cardiac fibroblasts

A voltage-activated proton current in human cardiac fibroblasts, measured using the whole-cell recording configuration of the patch-clamp technique, is reported. Increasing the pH of the bathing solution shifted the current activation threshold to more negative potentials and increased both the current amplitude and its rate of activation. Changing the pH gradient by one unit caused a 51mV shift in the reversal potential of the current, demonstrating a high selectivity for protons of the channel carrying the current. Extracellularly applied Zn(2+) reversibly inhibited the current. Activation of the current contributes to the resting membrane conductance under conditions of intracellular acidosis. It is proposed that this current in cardiac fibroblasts is involved in the regulation of the intracellular pH and the membrane potential under physiological conditions as well as in response to pathological conditions such as ischemia.     

Store-operated calcium channels trigger exocytosis of the sea urchin sperm acrosomal vesicle

The acrosome reaction (AR) of sperm is a prerequisite for fusion with the egg. In sea urchins, the complete AR (CAR) consists of exocytosis of the acrosomal vesicle (AV) and polymerization of acrosomal actin to form the approximately 1 micro m long acrosomal process. The fucose sulfate polymer (FSP) of egg jelly stimulates Ca(2+) entry through two distinct Ca(2+) channels and induces the CAR. Here we report that the second channel is blocked by SKF96365 (SKF), an inhibitor of store-operated channels. SKF also blocks the thapsigargin (TG), trifluoperazine (TFP), and calmidizolium (CMZ) stimulated Ca(2+) entry into sperm. These data indicate that the second Ca(2+) channel is a store-operated channel (SOC) that may be regulated by calmodulin. The TG, TFP, and CMZ-induced intracellular Ca(2+) elevations are similar to those induced by FSP, but the sperm acrosomal process does not polymerize. An antibody to bindin, the major protein of the AV, showed that in a significant percentage of these drug-treated sperm, the AV had undergone exocytosis. When NH(4)Cl was added to increase intracellular pH, the TG-treated sperm polymerized actin to form the acrosomal process. We conclude that the second Ca(2+) channel of sea urchin sperm is a SOC that triggers AV exocytosis.     

Pathway and kinetics of vitellogenin-gold internalization in the Xenopus oocyte

After in vitro incubation of Xenopus oocytes with vitellogenin (VTG)-gold conjugate, the gold particles are distributed on the whole plasma membrane. Their concentration in coated pits still occurs at 0 degrees C. At +20 degrees C the label quickly (30 sec) appears in multi-vesicular endosomes (MVE) which segregate together with primary endocytic vesicles into distinct clusters below the plasma membrane. From this step up to crystallization of the yolk platelets, the gold particles stay in the same compartment. During 5.5 h the label progressively increases along the MVE membrane, first (1.5 h) by fusion of primary endocytic vesicles with consecutively enlarging endosomes, then (4 h) by decreasing of the MVE membrane. As concerns the yolk platelet formation, concentration of primordial yolk platelets (PYP) occurs at 5.5 h from the incubation onset, the labeling of preexisting yolk platelets starts at 7 h, while crystallization of PYP begins only after 12-13 h. Our results indicate that VTG receptors are not preclustered in coated pits and their lateral translation is not inhibited at 0 degrees C. The yolk protein processing takes place within one compartment only. The VTG condensation begins with a long concentration phase of receptor-VTG complexes still integrated in the endosome membrane. It occurs in MVE by: i) a repeated fusion of primary endocytic vesicles; ii) removing part of the endosome membrane by internal vesiculation. Fusion between endosomes occurs only after VTG has dissociated from its receptors and VTG dissociates only when when the density of the VTG-receptor complexes in the endosome membrane is sufficient. Crystallization begins after a 7-8 h delay. The endosome migration into the oocyte is also controlled by the binding of VTG to its receptors. Our results also demonstrate that binding of VTG colloidal gold modifies neither the vitellogenic pathway nor the duration of the vitellogenin internalization. However when vitellogenin is bound to colloidal gold, dissociation of ligand-receptor complexes is delayed because the amount of ligand in the incubation medium is necessarily low.     

Polarization of mitochondria in the unfertilized mouse oocyte

Maturation of an immature oocyte into one capable of being fertilized involves tightly choreographed movements of chromosomes and organelles. The localizaton of mitochondria during maturation was studied in live mouse oocytes by confocal laser scanning microscopy (CLSM). Mitochondria were labeled with rhodamine 123 or Mitotracker (Molecular Probes, Eugene, OR) both of which are cell permeant and accumulate in mitochondria; acridine orange was used to mark chromatin. Prior to maturation, oocytes appeared to be radially symmetrical with no evident polarity; fully mature oocytes exhibited obvious polarity marked by the position of the metaphase II spindle in the cortex. CLSM revealed several interesting features of mitochondrial distribution: (1) A cortical clump of mitochondria was seen approximately 30-45to one side of the metaphase II spindle and marked the region of polar body I extrusion. (2) Large foci of mitochondria (7–14μM) were frequently found around the central region of the mature oocyte, while the central region often exhibited markedly fewer mitochondria. (3) Small mitochondrial foci (3μM) in the cortex and near the GV characterized several oocytes which failed to mature. (4) Non-spindle-associated mitochondria were not uniformly distributed in the mature oocyte but were concentrated in the hemisphere containing the metaphase II spindle. (5) The distal margins of this mitochondrial hemisphere were sharply demarcated at the cortex. These findings should help us understand organelle localization during mammalian oocyte maturation, and may give insights into possible causes of infertility and into early events of preimplantation development. © 1995 Wiley-Liss, Inc.     

Mitochondrial function in the human oocyte and embryo and their role in developmental competence

The role of mitochondria as a nexus of developmental regulation in mammalian oogenesis and early embryogenesis is emerging from basic research in model species and from clinical studies in infertility treatments that require in vitro fertilization and embryo culture. Here, mitochondrial bioenergetic activities and roles in calcium homeostasis, regulation of cytoplasmic redox state, and signal transduction are discussed with respect to outcome in general, and as possible etiologies of chromosomal defects, maturation and fertilization failure in human oocytes, and as causative factors in early human embryo demise. At present, the ability of mitochondria to balance ATP supply and demand is considered the most critical factor with respect to fertilization competence for the oocyte and developmental competence for the embryo. mtDNA copy number, the timing of mtDNA replication during oocyte maturation, and the numerical size of the mitochondrial complement in the oocyte are evaluated with respect to their relative contribution to the establishment of developmental competence. Rather than net cytoplasmic bioenergetic capacity, the notion of functional compartmentalization of mitochondria is presented as a means by which ATP may be differentially supplied and localized within the cytoplasm by virtue of stage-specific changes in mitochondrial density and potential (ΔΨm). Abnormal patterns of calcium release and sequestration detected at fertilization in the human appear to have coincident effects on levels of mitochondrial ATP generation. These aberrations are not uncommon in oocytes obtained after ovarian hyperstimulation for in vitro fertilization. The possibility that defects in mitochondrial calcium regulation or bioenergetic homeostasis could have negative downstream development consequences, including imprinting disorders, is discussed in the context of signaling pathways and cytoplasmic redox state.     

Model systems for the discovery and development of antiarrhythmic drugs

Cardiovascular diseases are the leading cause of mortality worldwide and about 25% of cardiovascular deaths are due to disturbances in cardiac rhythm or “arrhythmias”. Arrhythmias were traditionally treated with antiarrhythmic drugs, but increasing awareness of the risks of presently available antiarrhythmic agents has greatly limited their usefulness. Most common treatment algorithms still involve small molecule drugs, and antiarrhythmic agents with improved efficacy and safety are sorely needed. This paper reviews the model systems that are available for discovery and development of new antiarrhythmic drugs. We begin with a presentation of screening methods used to identify specific channel-interacting agents, with a particular emphasis on high-throughput screens. Traditional manual electrophysiological methods, automated electrophysiology, fluorescent dye methods, flux assays and radioligand binding assays are reviewed. We then discuss a variety of relevant arrhythmia models. Two models are widely used in testing for arrhythmogenic actions related to excess action potential prolongation, an important potential adverse effect of chemical entities affecting cardiac rhythm: the methoxamine-sensitized rabbit and the dog with chronic atrioventricular block. We then go on to review models used to assess potential antiarrhythmic actions. For ventricular arrhythmias, chemical induction methods, cardiac or neural electrical stimulation, ischaemic heart models and models of cardiac channelopathies can be used to identify effective antiarrhythmic agents. For atrial arrhythmias, potentially useful models include vagally-maintained atrial fibrillation, acute asphyxia with atrial burst-pacing, sterile pericarditis, Y-shaped atria surgical incisions, chronic atrial dilation models, atrial electrical remodelling due to sustained atrial tachycardia, heart failure-related atrial remodelling, and acute atrial ischaemia. It is hoped that the new technologies now available and the recently-developed models for arrhythmia-response assessment will permit the introduction of newer and more effective antiarrhythmic therapies in the near future.     

Protein phosphorylation patterns during in vitro maturation of the goat oocyte

Protein phosphorylation patterns were studied by radiolabelling goat cumulus oocyte complexes with [³²P]orthophosphate for various periods of time. The radiolabelled denuded oocytes were assessed for nuclear status and were used individually for gel electrophoresis. This study demonstrated that specific changes in protein phosphorylations were programmed during goat oocyte maturation. One of the most prominent changes was a general increase in the phosphorylation rate at germinal vesicle breakdown (GVBD). From 8 hr of culture, dominant phosphoprotein bands with apparent molecular weights of 27, 31, 40, and 50 kD were observed; they remained at this level until the metaphase II stage. In the molecular weight range of 65–80 kD, the protein phosphorylation pattern exhibited characteristic differences, with a complex series of phosphoproteins appearing and disappearing, during maturation. Addition of 6-dimethylaminopurine (6-DMAP) at the onset of culture blocked the maturation process after GVBD and induced a dramatic condensation of chromatin. When added at different times after GVBD, 6-DMAP invariably induced chromosome condensation. This inhibition was partly reversible; i.e., after removal of the drug, oocytes were able to progress only until metaphase l. © 1993 Wiley-Liss, Inc.     

卵母细胞成熟发生机制的研究进展

卵母细胞体外成熟培养已成为现代胚胎生物技术的重要内容之一,是体外受精、核移植等生物技术的重要环节。卵母细胞体外成熟受到众多因素的调控,其调控机制十分复杂。本文主要针对卵母细胞成熟过程中卵母细胞胞质成熟、核成熟及其主要调控因子等方面的发生发展机制进行总结。     

Changes in histone acetylation during mouse oocyte meiosis

We examined global changes in the acetylation of histones in mouse oocytes during meiosis. Immunocytochemistry with specific antibodies against various acetylated lysine residues on histones H3 and H4 showed that acetylation of all the lysines decreased to undetectable or negligible levels in the oocytes during meiosis, whereas most of these lysines were acetylated during mitosis in preimplantation embryos and somatic cells. When the somatic cell nuclei were transferred into enucleated oocytes, the acetylation of lysines decreased markedly. This type of deacetylation was inhibited by trichostatin A, a specific inhibitor of histone deacetylase (HDAC), thereby indicating that HDAC is able to deacetylate histones during meiosis but not during mitosis. Meiosis-specific deacetylation may be a consequence of the accessibility of HDAC1 to the chromosome, because HDAC1 colocalized with the chromosome during meiosis but not during mitosis. As histone acetylation is thought to play a role in propagating the gene expression pattern to the descendent generation during mitosis, and the gene expression pattern of differentiated oocytes is reprogrammed during meiosis to allow the initiation of a new program by totipotent zygotes of the next generation, our results suggest that the oocyte cytoplasm initializes a program of gene expression by deacetylating histones.