Cortical granules behave differently in mouse oocytes matured under different conditions

BACKGROUND: To better understand the differences between in vivo (IVO) and in vitro (IVM) matured oocytes, we studied the chronological changes in cortical granule (CG) distribution and nuclear progression during maturation, and the competence of CG release and embryo development of mouse oocytes matured under different conditions. METHODS: Oocytes matured in vivo or in different culture media were used and CG distribution and release were assessed by fluorescein isothiocyanate-labelled Lens culinaris agglutinin and laser confocal microscopy. RESULTS: Tempos of nuclear maturation and CG redistribution were slower, and competence for CG exocytosis, cleavage and blastulation were lower in the IVM oocytes than in the IVO oocytes. These parameters also differed among oocytes matured in different culture media. Hypoxanthine (HX, 4 mM) blocked germinal vesicle breakdown (GVBD), postponed CG migration and prevented CG-free domain (CGFD) formation. Cycloheximide (CHX) facilitated both GVBD and CG migration, but inhibited CGFD formation. The presence of serum in maturation media enhanced CG release after aging or activation of oocytes. Maintenance of germinal vesicle intact for some time by a trace amount (0.18 mM) of HX was beneficial to oocyte cytoplasmic maturation. CONCLUSION: CGs behaved differently in mouse oocytes matured under different conditions, and cytoplasmic maturity was not fully achieved in the IVM oocytes.     

Mitochondrial signaling and fertilization

The magnitude of the potential difference (polarity) across the inner mitochondrial membrane (DeltaPsim) determines levels of several mitochondrial activities, including ATP generation, focal regulate calcium homeostasis and organelle volume homeostasis. We investigated whether a domain of mitochondria in the mouse oocyte, characterized by high DeltaPsim and a unique location in the subplasmalemmal cytoplasm, is involved in the earliest events of fertilization: sperm attachment, penetration and cortical granule exocytosis. Experimental manipulations of the magnitude of DeltaPsim and the distribution of mitochondria in zona-free MII oocytes, followed by insemination and culture, indicate that high-polarized mitochondria (HPM) are required for penetration and cortical granule exocytosis, but not for persistent attachment to the oolemma. The capacity of subplasmalemmal mitochondria to undergo transient reductions (dissipations) of DeltaPsim appears necessary for penetration and cortical granule exocytosis. We suggest that the HPM normally establish a continuous circumferential circuit of 'reactive' organelles capable of responding to and propagating, triggering or activating signals across the subplasmalemmal cytoplasm, such as those initiated by the fertilizing sperm at the site of penetration. The HPM in the oocyte and early embryo may have functions similar to those of their somatic cell counterparts and promote the focal regulation of developmental activities that are themselves spatially localized. The establishment of high DeltaPsim in the subplasmalemmal cytoplasm may be among the first steps in the preovulatory maturation of the oocyte and defects in this domain may result in fertilization failure or abnormality.     

Is the human oocyte plasma membrane polarized?

Using scanning electron microscopy, we have shown that the plasma membrane of the human metaphase II oocyte is organized in evenly spaced, short microvilli of 1-3 microns in length. In contrast to other mammals studied to date, there is no microvillus-free area overlying the metaphase spindle and there were no other indications of polarization at this level of organization. Functional polarity of the plasma membrane, studied using localized microsurgery of the zona pellucida followed by insemination, suggests that sperm fusion and entry in the human may occur anywhere over the oocyte surface. Aged oocytes and those exposed to acidic Tyrode's solution had surfaces which were not homogeneously covered by microvilli. Oocytes exposed to a sucrose solution and subzonally injected with spermatozoa showed evidence of partial cortical granule exocytosis.     

The fertilization and development of mouse oocytes following cortical granule discharge in the presence of a protease inhibitor.

The effect of leupeptin, a serine protease inhibitor, on the fertilization and development potential of oocytes stimulated to undergo cortical granule exocytosis has been investigated. An in-vitro bioassay system was used in which mouse oocytes were exposed to calcium ionophore, A23187, in the presence and absence of leupeptin, before their fertilization and development to the blastocyst stage was assessed. We have demonstrated that the presence of leupeptin in the incubation medium, at concentrations of 1 micrograms/ml and 10 micrograms/ml during the first 10 min of cortical granule exocytosis, reversed the ionophore-induced decrease in the capacity of oocytes to fertilize and develop to blastocysts. The induction of exocytosis of cortical granules by calcium ionophore was confirmed using fluorescence microscopy. Using this technique, we also confirmed that leupeptin did not inhibit ionophore-induced cortical granule exocytosis, thus supporting the contention that leupeptin acted upon released cortical exudate. It was concluded that leupeptin acted by inhibiting proteases released into the perivitelline space during the early stages of cortical granule exocytosis. Based on these results it was proposed that leupeptin could be used to prevent premature loss of fertility of human oocytes which are inadvertently activated under in-vitro conditions.     

Multiple activation currents can be evoked inXenopus laevis eggs when cortical granule exocytosis is inhibited by weak bases

The fertilization potential inXenopus eggs under normal circumstances is considered to be a unique event. It is associated with a concomitantly occurring cortical granule exocytosis. If eggs were exposed to weak bases, exocytosis was inhibited but the fertilization potential could still be evoked. After recovery from this first transient increase in membrane conductance, a second could be elicited by a further stimulus. A fertilization potential could be triggered either before or after the egg had undergone an electrically induced activation potential. This suggests that sperm receptors and sperm activated ionic channels in the egg membrane remain functional following the conductance change, at least when the exocytotic event was prevented.A transient conductance increase could only be induced by NH ₄ ⁺ (pH 9.0) in unactivated eggs that had not undergone cortical granule exocytosis.Tremendous variation was noticed between successive activation currents elicited in the same egg. Under voltageclamp at 0 mV holding potential, the current often changed from inward to outward. Although cortical granule exocytosis may only play a minor role in the transient conductance change triggered at fertilization, it may well be involved in subsequent modifications of membrane conductance.     

ATP signaling and NTPDase in Systemic Lupus Erythematosus (SLE)

Systemic lupus erythematosus (SLE) is an autoimmune and inflammatory disease with periods of exacerbation and remission. SLE is characterized by the irreversible breakdown of immunological self-tolerance, where there is deregulation of multiple aspects of the immune system. SLE immune dysfunction is characterized by activation of autoreactive T lymphocytes, and hyperactivity of B lymphocytes with consequent production of several autoantibodies. ATP is a purinergic mediator released into the extracellular space in response to cell and tissue damage which operates as a danger signal to modulate immune and inflammatory responses. ATP binds to P2 receptors and its levels are regulated by NTPDase (CD39). SLE patients exhibit increased levels of ATP which binds to P2X receptors resulting in activation of the inflammasome and consequent release of IL-1β and IL-18, cytokines associated with disease pathogenesis. CD39 is upregulated in SLE representing an important immunoregulatory mechanism by controlling inflammation and favoring the production of adenosine. The aim of this review is to clarify the effects of ATP on the modulation of the inflammatory process and immune responses via P2 receptors as well as the role of NTPDase in the immunopathogenesis of SLE.     

Involvement of FK506-sensitive and insensitive granule exocytosis pathways in perforin-dependent target cell lysis mediated by a CD8+ CTL clone

The release of granzyme A and B through granule exocytosis by CD8+ CTL clone OE4 upon T cell receptor (TCR) activation was blocked by FK506 in a dose-dependent manner (IC50 = 3 nM), whereas a significant granzyme release was still detectable even in the presence of excess FK506. In contrast, the production of IFN-gamma was highly sensitive to FK506 (IC50 = 0.01 nM) and could be completely blocked by FK506. Both FK506-sensitive and insensitive granule exocytosis pathways were involved in the actual perforin-dependent killing toward different target cells. The combination of ionomycin and phorbol ester was able to mimic TCR stimulation to induce IFN-gamma production, although the same treatment triggered granule exocytosis inefficiently. Granule exocytosis and IFN-gamma production following TCR activation were profoundly prevented by calphostin C. Thus, these results demonstrate that the granule exocytosis pathway in this CD8+ CTL clone depends on the activation of protein kinase C, and requires either calcineurin-dependent or independent additional signals downstream of TCR activation.     

NTPDase1 governs P2X7‐dependent functions in murine macrophages

P2X₇ receptor is an adenosine triphosphate (ATP)‐gated ion channel within the multiprotein inflammasome complex. Until now, little is known about regulation of P2X₇ effector functions in macrophages. In this study, we show that nucleoside triphosphate diphosphohydrolase 1 (NTPDase1)/CD39 is the dominant ectonucleotidase expressed by murine peritoneal macrophages and that it regulates P2X₇‐dependent responses in these cells. Macrophages isolated from NTPDase1‐null mice (Entpd1^?/?) were devoid of all ADPase and most ATPase activities when compared with WT macrophages (Entpd1^+/+). Entpd1^?/? macrophages exposed to millimolar concentrations of ATP were more susceptible to cell death, released more IL‐1β and IL‐18 after TLR2 or TLR4 priming, and incorporated the fluorescent dye Yo‐Pro‐1 more efficiently (suggestive of increased pore formation) than Entpd1^+/+ cells. Consistent with these observations, NTPDase1 regulated P2X₇‐associated IL‐1β release after synthesis, and this process occurred independently of, and prior to, cytokine maturation by caspase‐1. NTPDase1 also inhibited IL‐1β release in vivo in the air pouch inflammatory model. Exudates of LPS‐injected Entpd1^?/? mice had significantly higher IL‐1β levels when compared with Entpd1^+/+ mice. Altogether, our studies suggest that NTPDase1/CD39 plays a key role in the control of P2X₇‐dependent macrophage responses.     

T-cell receptor signaling events triggering granule exocytosis

T-cell receptor (TCR) engagement by antigen results in proliferation, differentiation and cytokine secretion. In the CD8+ T-cell subset, TCR triggering also induces granule exocytosis, the directional release of contents of lysosome-like granules toward the target cell presenting the antigen. This process is responsible for immediate death of target cells. The intracellular events required for granule exocytosis are distinct from those of proliferation and cytokine secretion, as the former do not require de novo protein synthesis. Consequently, the key TCR signaling events required for granule exocytosis may be distinct. In this article, we review present knowledge of regulation of granule exocytosis by molecules of the TCR signaling cascade.     

Studies on oogenesis and oviposition in the brown spider Loxosceles intermedia (Araneae: Sicariidae)

Loxosceles intermedia is a poisonous spider that has a wide distribution in southern Brazil, and constitutes a public health problem. In this study, the ovaries of mature females were examined by light and electron microscopy, and by histochemistry. Oocytes at all stages of development were observed in the same area of the mature ovarian wall, surrounded by a basement membrane and a proteic band. They were supported by a group of pedicular cells, which may function as follicular cells. No follicular cell was seen around the oocyte. Mature oocytes pass through the ovarian epithelial wall and are released into the ovarian lumen, covered by a vitelline membrane. The basement membrane and proteic band remain in the ovarian wall. On its way out, the oocyte is coated by proteins that will form the chorion. The presence of different coats throughout oogenesis, and at the time of egg release, is correlated with conditions that indicate fertilization occurs in the uterus lumen during oviposition.     

Enhancement of developmental capacity of meiotically inhibited bovine oocytes by retinoic acid

BACKGROUND: Although high vitamin A may be teratogenic to the embryo, retinol has been shown to support oocyte developmental potential in vivo. Similarly, addition of retinol metabolite 9-cis-retinoic acid to in-vitro cultured oocytes could promote cytoplasmic maturation and subsequent early embryonic development. The objective of this study was to evaluate the effects of 5 nmol/l retinoic acid during in-vitro pre-maturation and maturation of bovine oocyte-cumulus complexes. METHODS AND RESULTS: Oocytes were aspirated from cows and either kept under meiotic arrest with 25 micro mol/l roscovitine and matured, or allowed to mature in permissive conditions (control). Cortical granule migration was analysed both after pre-maturation and maturation by fluorescent labelling of oocytes and subsequent laser confocal and fluorescence microscopy. Variables studied after in-vitro fertilization and culture in modified synthetic oviduct fluid were: (i) in-vitro embryonic development; (ii) ability of blastocysts to survive vitrification and warming; and (iii) differential cell counts measured by fluorescence microscopy. Although the presence of 5 nmol/l retinoic acid throughout in-vitro maturation was harmful, its presence during pre-maturation alone improved cytoplasmic granular migration, embryonic development, cryopreservation tolerance, total cell numbers and, as a consequence, embryonic quality. CONCLUSIONS: Pre-maturation in the presence of retinoic acid improves cytoplasmic competence of in-vitro matured bovine oocytes. Until more is known of the molecular mechanisms it would be irresponsible to use retinoic acid in maturation of human oocytes, especially in view of the narrow time window and possible species-specific differences in susceptibility and protection of the oocyte from epigenetic influences of retinol.     

Volume changes of mature human oocytes on exposure to cryoprotectant solutions used in slow cooling procedures

BACKGROUND: Despite the recent increase in pregnancies from cryopreserved human oocytes, success in terms of births per thawed oocyte is still poor. Modifications to cryopreservation protocols have not been based on measurement of the osmotic response of oocytes, and methodologies are often poorly described or protocols not strictly adhered to, inevitably resulting in variability. METHODS: Volume change of mature human oocytes was measured on exposure to cryoprotectant. Oocytes were exposed to either 0.75 mol/l propane-1,2-diol (PrOH) for 10 min; 1.5 mol/l PrOH for 10 min, having been exposed to 0.75 mol/l PrOH for 7.5 min; or 1.5 mol/l PrOH plus 0.2 or 0.3 mol/l sucrose for 10 min, having been exposed to 1.5 mol/l PrOH for 10 min. RESULTS: On exposure to PrOH alone, oocytes shrank and then re-expanded, having reached 75 and 84% of their starting volume in 0.75 and 1.5 mol/l, respectively. Oocytes shrank continuously in PrOH plus sucrose, reaching 67 or 55% of their initial volume in 0.2 or 0.3 mol/l sucrose, respectively. CONCLUSIONS: To improve consistency following cryopreservation, protocols must be strictly adhered to; small changes in duration of exposure to cryoprotectant can result in drastic changes in cellular hydration and thus the fate of the cell during freezing/thawing.     

Morphological diversity of oocytes released from the adult mouse ovary

This study was designed to classify and differentiate the population of oocytes released from the mouse ovary by mechanical means. Liberated oocytes were classified on the basis of their number, size and by the nature of the attachment of follicle cells to these oocytes. Microscopical examination of oocytes mechanically released from the ovaries revealed three distinct morphological classifications of oocytes: (1) those completely devoid of follicle cells, (2) those encased in follicle cells and (3) those in the process of degeneration. Oocytes ranged in size from approximately 30μ to 119μ. Those oocytes surrounded by follicle cells could be further subdivided into two groups depending on whether the follicle cells could be mechanically removed from the oocyte. These data demonstrate that the adult ovary contains a variety of classes of oocytes which differ in size and in the extent to which the follicle cells are attached to the oocytes. It is suggested that the metabolic activities and meiotic potentials of the various oocyte populations differ and as a result, care should be employed to insure a uniform population of oocytes when conducting further studies with mechanically liberated oocytes.     

Extracellular ATP regulates exocytosis by inhibiting multiple Ca2+ channel types in bovine chromaffin cells

Feedback modulation of voltage-dependent Ca2+ channels by ATP is a well documented phenomenon in bovine chromaffin cells. However, its influence in the control of hormone release is at present poorly understood. By using combined patch-clamp and fura-2 fluorescence measurements we provide evidence that the three Ca2+ channel types (L, N and P/Q) expressed in bovine chromaffin cells are inhibited by ATP (30 microM), and that their involvement in the secretory response, as assayed by capacitance measurements, is roughly proportional to their contribution to the whole-cell Ca2+ current (ICa) both in the absence and presence of ATP. ATP did not modify the capacitance increase observed in cells dialyzed with Ca(2+)-EGTA buffers (1.5 microM free Ca2+), thus excluding a direct effect of ATP on the secretory machinery. Voltage predepolarizations or long chemical (2 s, 70 mM KCl) depolarizations attenuate the effect of ATP on exocytosis by partially relieving the inhibition of ICa Likewise, a strong stimulation that depletes the readily releasable pool of vesicles prevents an inhibitory effect of ATP on the secretory response. While these results lend support to the hypothesis of autocrine modulation of exocytosis by endogenously released ATP acting on P2y-purinoceptors to inhibit ICa, feedback regulation of the rate of release will be a complex function of the occupancy of those receptors and of the electrical and secretory activity of the cell.     

Possible role for Ca(2+) calmodulin-dependent protein kinase II as an effector of the fertilization Ca(2+) signal in mouse oocyte activation

The present study shows that Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) is physiologically activated in fertilized mouse oocytes and is involved in the Ca(2+) response pathways that link the fertilization Ca(2+) signal to meiosis resumption and cortical granule (CG) exocytosis. After 10 min of insemination, CaM kinase II activity increased transiently, then peaked at 1 h and remained elevated 30 min later when most of the oocytes had completed the emission of the second polar body. In contrast, in ethanol-activated oocytes the early transient activation of CaM kinase II in response to a monotonic Ca(2+) rise was not followed by any subsequent increase. Inhibition of CaM kinase II by 20 micromol/l myristoylated-AIP (autocamtide-2-related inhibitory peptide) negatively affected MPF (maturing promoting factor) inactivation, cell cycle resumption and CG exocytosis in both fertilized and ethanol-activated oocytes. These results indicate that the activation of CaM kinase II in mouse oocytes is differently modulated by a monotonic or repetitive Ca(2+) rise and that it is essential for triggering regular oocyte activation.     

A cytosolic factor that inhibits KATP channels expressed in Xenopus oocytes by impairing Mg-nucleotide activation by SUR1

ATP-sensitive K⁺ (K_ATP) channels couple cell metabolism to cell electrical activity. Wild-type (Kir6.2/SUR1) K_ATP channels heterologously expressed in Xenopus oocytes give rise to very small inward currents in cell-attached patches. A large increase in the current is observed on patch excision into zero ATP solution. This is presumably due to loss of intracellular ATP leading to unblock of K_ATP channels. In contrast, channels containing Kir6.2 mutations associated with reduced ATP-sensitivity display non-zero cell-attached currents. Unexpectedly, these cell-attached currents are significantly smaller (by ∼40%) than those observed when excised patches are exposed to physiological ATP concentrations (1–10 m m ). Cramming the patch back into the oocyte cytoplasm restores mutant K_ATP current amplitude to that measured in the cell-attached mode. This implies that the magnitude of the cell-attached current is regulated not only by intracellular ATP but also by another cytoplasmic factor/s. This factor seems to require the nucleotide-binding domains of SUR1 to be effective. Thus a mutant Kir6.2 (Kir6.2ΔC-I296L) expressed in the absence of SUR1 exhibited currents of similar magnitude in cell-attached patches as in inside-out patches exposed to 10 m m MgATP. Similar results were found when Kir6.2-I296L was coexpressed with an SUR1 mutant that is insensitive to MgADP or MgATP activation. This suggests the oocyte contains a cytoplasmic factor that reduces nucleotide binding/hydrolysis at the NBDs of SUR1. In conclusion, our results reveal a novel regulatory mechanism for the K_ATP channel. This was not evident for wild-type channels because of their high sensitivity to block by ATP.     

血管内皮细胞与血小板聚集、释放和超微结构变化的相互关系研究

实验用培养的牛主动脉内皮细胞(EC)在体外条件下与采自兔血的富含血小板血浆(PRP)孵育,观察EC对血小板聚集的影响,用生物发光法测定血小板聚集时ATP的释放量,并观察这一反应过程中血小板的超微结构变化。结果表明血小板聚集强度与加入的EC量呈负相关性,且血小板ATP释放量和形态学变化与聚集程度相符合,即随血小板聚集受抑,其变形减轻,颗粒物质及ATP释放减少。形态观察中发现血小板对EC的粘附及颗粒释放现象,即使在血小板聚集完全为EC抑制时仍可见这一现象,其机理尚不明了。     

Exocytosis of azurophil and arginase 1-containing granules by activated polymorphonuclear neutrophils is required to inhibit T lymphocyte proliferation

ARG1, expressed by human PMNs, inhibits T cell proliferation by depleting extracellular L-arginine. Here, we report that ARG1, released from gelatinase granules by PMNs, is inactive at physiological pH unless activated by factor(s) stored in azurophil granules. Whereas ARG1 exocytosis was induced by TNF-α or ionomycin, only the latter mediated the release of both granules, resulting in extracellular ARG enzyme activity at physiological pH. Furthermore, after fractionation of the different classes of granules, only the mixture of gelatinase and azurophil granules resulted in ARG1 activity at physiological pH. The use of protease inhibitors indicated the involvement of a PMSF- and leupeptin-susceptible serine protease in ARG1 processing and activation. Finally, the supernatant of viable PMNs undergoing frustrated phagocytosis, which mediates gelatinase and azurophil granule release, inhibited T cell proliferation through ARG-dependent mechanisms. In vivo, high ARG1 concentrations and increased ARG enzyme activity, sufficient to inhibit T cell proliferation, were observed in synovial fluids from RA. These findings suggest that PMNs, recruited at sites of immune complex deposition, induce ARG1-dependent immune suppression through concomitant exocytosis of gelatinase and azurophil granules.     

Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation

BACKGROUND: To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and P(EG) (cryoprotectant permeability) of mature human oocytes after exposure to ethylene glycol (EG) and tested the efficiency of a multi-step slow cooling protocol based on this cryoprotectant. METHODS: Oocytes were perfused with 1.5 mol/l EG for 10 min. Oocyte volume at each time point was calculated and normalized to the original volume. Slow cooling was conducted by exposing oocytes to increasing EG concentrations (0.5, 1.0 and 1.5 mol/l n = 155) or 1.5 mol/l of propane-1,2-diol (PrOH) n = 102. Oocytes which survived cryopreservation n = 80 and fresh oocytes n = 73 were prepared for confocal microscopy analysis of the meiotic spindle. RESULTS: During EG exposure, oocytes underwent an abrupt 50% volume reduction. Complete recovery of the initial volume was not observed. From the values of a best fit plot, the coefficients Lp = 0.82 +/- 0.29 microm min(-1) atm(-1) (mean +/- SD) and P(EG) 0.10 +/- 0.01 microm s(-1) were generated. Survival rates after freezing with EG were lower than with PrOH (51.6 versus 71.5%, respectively, P < 0.05). The frequencies of normal spindle configuration were lower in frozen EG and frozen PrOH oocytes compared with fresh oocytes (53.8, 50.9 and 66.7%, respectively, P < 0.05). CONCLUSIONS: The oocyte plasmalemma possesses limited permeability to EG and EG exposure causes considerable osmotic stress. However, post-thaw rates of survival and normal meiotic spindle organization may be preserved by protocols which are designed in order to minimize osmotic stress.     

Cell cycle regulation: Egg activation induced by osmotic pressure change and the effects of amiloride on the cryopreservation of mouse oocytes

Activation of oocytes is caused by osmotic pressure change insome species. However, cryopreservation of oocytes occurs inthe presence of osmotic pressure change induced by cryoprotectants.We investigated the effect of 5-(N,N,-dimethyl)-amiloride (NNDMA),a selective inhibitor of Na⁺/H⁺ exchange, on the cryopreservationand osmotic activation of mouse oocytes. The percentage (23.2%)of degenerate oocytes after cryopreservation in the presenceof NNDMA was found to be lower than that (39.5%) of untreatedoocytes. After thawing, the percentage (23.6%) of oocytes whichcould be fertilized following cryopreservation in the presenceof NNDMA was significantly higher than that of untreated (18.0%)oocytes. These results suggest that amiloride increased thesurvival rate after thawing following cryopreservation. To investigatethe effect of NNDMA on oocyte activation caused by the cryoprotectant,dimethyl sulphoxide (DMSO) was used to induce osmotic pressurechange. NNDMA was found to inhibit cortical granule exocytosis,the second polar body emission and pronuclear formation whichoccurs upon activation due to osmotic pressure change. It alsoinhibited the increase in phosphorylation of many proteins including33 and 45 kDa proteins, which occurs during fertilization andchemical oocyte activation. In contrast, protein phosphorylationwas not inhibited by W7, a calmodulin inhibitor. The actionsof these inhibitors suggest that oocyte activation induced byosmotic pressure change involves a pathway mediated by Na⁺/H⁺exchange which may be distinct from the Ca-calmodulin pathway.Amiloride may be a useful drug for increasing the rate of survivalof cryopreserved oocytes. amiloride/cryopreservation/oocyte activation/osmotic pressure change