Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes

The effect of the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the maturation promoting factor (MPF) activity, pronuclear formation, and parthenogenetic development of electrically activated in vitro matured (IVM) porcine oocytes was investigated. Oocytes were activated by exposure to two DC pulses, each of 1.5 kV/cm field strength and 60 microsec duration, applied 1 sec apart. In the first experiment, subsequent incubation with 2 or 5 mM 6-DMAP for 3 hr increased the incidence of blastocyst formation compared with no treatment, whereas incubation with 2 or 5 mM 6-DMAP for 5 hr did not. In the proceeding experiments, oocytes exposed to 6-DMAP were incubated with 2 mM of the reagent for 3 hr. Assaying histone H1 kinase activity in the second experiment revealed that the levels of active MPF in electrically activated oocytes treated with 6-DMAP were depleted more rapidly and remained depleted for longer compared with electrical activation alone. The kinetics of MPF activity following 6-DMAP treatment were similar to that found in inseminated oocytes in the third experiment. The effect of 6-DMAP was correlated with an increased incidence of parthenogenetic blastocyst formation. A fourth experiment was undertaken to examine the diploidizing effect of 6-DMAP. Electrically activated oocytes treated with 6-DMAP and cytochalasin B, either alone or in combination, displayed a higher incidence of second polar body retention compared with those that were untreated or treated with cycloheximide alone. After 6 days of culture in vitro, parthenotes exposed to 6-DMAP, either alone or in combination with cytochalasin B, formed blastocysts at a greater rate compared with those exposed to cytochalasin B alone, cycloheximide alone or no treatment. The combined 6-DMAP and cytochalasin B treatment induced the highest rate of blastocyst formation (47%), but the numbers of trophectoderm and total cells in these blastocysts were lower compared with those obtained following exposure to 6-DMAP alone. These results suggest that the increased developmental potential of 6-DMAP-treated parthenotes may be attributable to the MPF-inactivating effect of 6-DMAP, rather than the diploidizing effect of 6-DMAP.     

Injection of frozen-thawed porcine first polar bodies into enucleated oocytes results in fertilization and embryonic development

The objective was to determine whether enucleated oocytes injected with frozen porcine first polar bodies (pPB1s) could be fertilized and developed into viable embryos in vitro. Metaphase II (MII) oocytes with pPB1s were frozen (vitrified) and stored for 2 mo. The pPB1s were isolated from thawed MII oocytes and injected into enucleated recipient oocytes by micromanipulation. All recipients injected with thawed pPB1s were fertilized by intracytoplasmic sperm injection (ICSI), and the resulting recombinant zygotes were incubated to assess their developmental competence in vitro. Furthermore, double-antibody immunohistochemistry was used to verify that the nucleus of the pPB1 participated in fertilization and supported embryonic development. Porcine embryos (2- to 8-cell stage) were obtained from the recombinants. The average in vitro cleavage rate of 2-, 4-, and 8-cell stage recombinant embryos was 25.3, 17.7, and 9.3% (P < 0.05), respectively. Chromosomes in the labeled pPB1 participated in the formation of the two blastomere nuclei of 2-cell stage embryos derived from recombinant oocytes. In conclusion, nuclear materials of frozen-thawed pPB1 supported oocyte fertilization and subsequent embryonic development, thereby providing a new way to use frozen PB1s for preservation and reproduction of mammals.     

Inactivation of histone H1 kinase by Ca2+ in rabbit oocytes

The present study investigated the role of intracellular Ca²⁺ (Ca²⁺_i) elevation on the inactivation of maturation promoting factor (MPF) in rabbit oocytes. The effects of the number of Ca²⁺ stimulations and of the amplitude of Ca²⁺_i elevation on the profile of histone H1 kinase activity were determined. A Ca²⁺ stimulation consisted of transferring mature oocytes from culture medium to 0.3 M mannitol containing 0.1–1.0 mM CaCl₂, and pulsing them at 1.25 kV/cm for 10 μsec, or microinjecting 2–8 mM CaCl₂ into the oocyte cytoplasm. The number of electrically-induced Ca²⁺ stimulations was varied, and amplitude of the Ca²⁺_i rise was controlled by altering Ca²⁺ concentration in the pulsing medium or the injection pipette. Ca²⁺_i concentration was determined with fura-2 dextran; oocytes were snap-frozen at indicated time points and assayed for H1 kinase activity. The activity was quantified by densitometry and expressed as a fraction of activity in nonstimulated oocytes. Electrically-mediated Ca²⁺_i rises inactivated H1 kinase in a manner dependent on the number of Ca²⁺ stimulations. A single Ca²⁺ stimulation inactivated H1 kinase to 30–40% of its initial activity. However, H1 kinase inactivation was only transient, regardless of the amplitude of the electrically- or injection-mediated Ca²⁺_i elevation. Increasing the number of Ca²⁺ stimulations helped to maintain H1 kinase activity at basal (pronuclear) levels. The results show the necessity of a threshold of Ca²⁺_i concentration to trigger MPF inactivation, and suggest a role for the extended period of time over which Ca²⁺_i oscillates at fertilization. © 1995 Wiley-Liss, Inc.     

Alterations in the protein kinase C signaling activated by a parthenogenetic agent in oocytes from reproductively old mice

To investigate the effect of female age on oocyte developmental competence, we focused on protein kinase C (PKC), a major component of the signalling pathway involved in oocyte activation, and put forward the hypothesis that, as it occurs in many organs and tissues, aging affects PKC function in mouse oocytes. Biochemical activity of PKC along with the expression and subcellular distribution of some PKC isoforms were monitored in young and old mouse oocytes parthenogenetically activated by SrCl(2). We found that PKC activity increased reaching a level that was lower in old compared to young oocytes in association with an incomplete translocation of PKCbetaI to the plasma membrane. Moreover, old oocytes exhibited a reduced expression of PKCbeta1 and PKCalpha at the protein level, without significant effects on the expression of the Ca(2+)-independent PKCdelta. Detectable amounts of PKCbeta1 mRNA were observed in young and old oocytes at GV stage with no difference between the two groups of age. When meiotic progression to anaphase II up to first cleavage were analyzed, a delay in meiosis resumption and significantly lower rates of pronuclei formation and first cleavage were observed in old compared to young oocytes. Moreover, we found that, in contrast to SrCl(2), PMA (12-O-tetradecanoyl phorbol-13-acetate), a PKC agonist, was ineffective in activating old oocytes. Present findings provide evidence that aging affects the correct storage and activation of some PKCs, functional components of the machinery involved in oocyte activation, and suggest that these changes may negatively influence the activation competence of old oocytes.     

Role of AMPK throughout meiotic maturation in the mouse oocyte: Evidence for promotion of polar body formation and suppression of premature activation

This study was conducted to assess the role of AMPK in regulating meiosis in mouse oocytes from the germinal vesicle stage to metaphase II. Exposure of mouse cumulus cell‐enclosed oocytes (CEO) and denuded oocytes (DO) during spontaneous maturation in vitro to AMPK‐activating agents resulted in augmentation of the rate and frequency of polar body formation. Inhibitors of AMPK had an opposite, inhibitory effect. In addition, the AMPK inhibitor, compound C (Cmpd C) increased the frequency of oocyte activation. The stimulatory action of the AMPK‐activating agent, AICAR, and the inhibitory action of Cmpd C were diminished if exposure was delayed, indicating an early action of AMPK on polar body formation. The frequency of spontaneous and Cmpd C‐induced activation in CEO was reduced as the period of hormonal priming was increased, and AMPK stimulation eliminated the activation response. Immunostaining of oocytes with antibody to active AMPK revealed an association of active kinase with chromatin, spindle poles, and midbody during maturation. Immunolocalization of the α1 catalytic subunit of AMPK showed an association with condensed chromatin and the meiotic spindle but not in the spindle poles or midbody; α2 stained only diffusely throughout the oocyte. These data suggest that AMPK is involved in a regulatory capacity throughout maturation and helps promote the completion of meiosis while suppressing premature activation. Mol. Reprod. Dev. 77:888–899, 2010. © 2010 Wiley‐Liss, Inc.     

Fate of the first polar bodies in mouse oocytes

Both nuclear transfer and intracytoplasmic sperm injection (ICSI) practice necessitates studies on the spatial relationship between the MII spindle and the first polar bodies (FPB). Although recent observations have shown that the FPB position does not predict accurately the location of the meiotic spindle in metaphase II oocytes of monkey, hamster, and human, detailed studies on FPB deviation and its affecting factors are lacking. Since polar bodies can be used for genetic testing and oocyte quality grading, their life span under different conditions should be studied. The timing of formation and degeneration and the position relative to the MII spindle of the FPB and the factors affecting FPB deviation and degeneration during in vivo and in vitro aging of both in vivo and in vitro matured mouse oocytes were investigated in this study. Mice of the Kun-ming breed were used, and the intact and degenerated FPB were identified through microscopic morphology in combination with propidium iodide (PI) exclusion test and the chromosomes visualized by Hoechst staining. Results are summarized as follows: (i) oocytes started FPB extrusion at 8 hr after the onset of in vivo or in vitro maturation, but the number of FPB reached maximum much later in vitro (14 hr of culture) than in vivo (10 hr post hCG). (ii) Some FPB began to degenerate before ovulation and around 70% became degenerated within 6 hr after maximal nuclear maturation both in vivo and in vitro; they disappeared faster during in vivo than in vitro aging but turned from intact to degenerated at a similar tempo. (iii) Some FPB began to deviate from the MII spindle 10 hr after hCG injection or in vitro culture and the distance between FPB and the spindle increased with time during both in vivo and in vitro aging. (iv) FPB deviated more slowly in the in vitro matured oocytes than in in vivo matured. (v) Denudation performed after FPB extrusion markedly enhanced its deviation. (vi) The perivitelline space (PVS) increased with time during maturation and aging in vivo and in vitro and the values of PVS and the percentages of FPB adjacent to the spindle were significantly negatively correlated. (vii) Cytochalasin B and colchicine had no effect on FPB deviation. (viii) None of the more than 3,500 FPBs observed was found to be dividing or have divided into two cells at any time points before or after ovulation or in vitro maturation. Our results were consistent with the possibility that the displacement of the FPB was a time- and PVS-dependent process, indicating that PVS would increase with time and its formation and enlargement would facilitate the lateral displacement of the degenerating FPB.     

Activation at the germinal vesicle stage of starfish oocytes produces parthenogenetic development through the failure of polar body extrusion

Starfish oocytes can be fertilized after germinal vesicle breakdown (GVBD) and artificial parthenogenesis can be induced by activating the oocytes after GVBD (post-GVBD activation). In the present study, parthenogenotes were obtained by the activation of immature oocytes with caffeine before treatment with 1-methyladenine (1-MeAde) to induce oocyte maturation. Most of the caffeine-treated eggs developed as tetraploids, as parthenogenotes produced by the post-GVBD activation. The parthenogenotes were derived only from eggs that failed to extrude polar bodies, mostly from eggs failing to extrude a second polar body. Eggs derived from immature oocytes activated by A23187, treated with 1-MeAde and post-treated with cytochalasin B failed to extrude polar bodies, and eventually developed into parthenogenetic embryos. These results indicate that the present parthenogenesis mechanism shares the same characteristics as that achieved by post-GVBD activation in the suppression of polar body formation as a key means for successful starfish parthenogenesis.     

Quality of vitrified porcine immature oocytes is improved by coculture with fresh oocytes during in vitro maturation

It is essential to enhance the in vitro maturation (IVM) condition for immature oocytes after cryopreservation, particularly if limited numbers of oocytes collected from specific donors. The objective of this study was to determine if quality of vitrified porcine immature oocytes was enhanced by coculturing with fresh oocytes during IVM. To distinguish fresh versus vitrified oocytes, we used two types of coculture systems: (a) transwell two‐chamber coculture; (b) labeling and tracing fresh oocytes with CellTracker? Green CMFDA during conventional culture. Coculture systems significantly accelerated meiotic progression of vitrified oocytes and significantly increased blastocyst formation rates following parthenogenetic activation and somatic cell nuclear transfer. Reactive oxygen species generation in vitrified oocytes was ameliorated by the coculture conditions, with no significant difference between fresh and vitrified oocytes for intracellular glutathione level. Both coculture systems significantly increased rate of normal mitochondrial distribution in vitrified oocytes, but did not affect fluorescence intensity of mitochondria. The percentage of oocytes with normal endoplasmic reticulum (ER) distribution and ER fluorescence intensity were significantly higher in vitrified oocytes cocultured with fresh oocytes. After 20 hr of IVM, mRNA expression of COX2, HAS2, PTX3, and TNFAIP6 remained significantly higher in cumulus cells derived from vitrified oocytes and coculture systems significantly decreased the expression of these genes. Additionally, coculture methods prevented the reduction of mRNA expression for BMP15, ZAR1, POU5F1, and DNMT3A in vitrified oocytes. In conclusion, oocyte quality and subsequent embryo development of vitrified porcine immature oocytes were significantly improved by fresh oocyte coculture during IVM.     

Mitogen-activated protein kinase kinase inhibitor suppresses cyclin B1 synthesis and reactivation of p34cdc2 kinase, which improves pronuclear formation rate in matured porcine oocytes activated by Ca2+ ionophore

To investigate the role of mitogen-activated protein (MAP) kinase kinase (MEK)/MAP kinase cascade on p34cdc2 kinase activity and cyclin B1 levels during parthenogenetic activation of porcine oocytes, MEK activity, MAP kinase activity, p34cdc2 kinase activity, and cyclin B1 levels were assayed in mature porcine oocytes after treatment with different concentrations of Ca2+ ionophore. A high concentration of Ca2+ ionophore (50 microM) rapidly reduced MEK activity in oocytes for up to 8 h of culture. MEK activity in the 10-microM treatment group was significantly higher. The low concentration treatment transiently decreased p34cdc2 kinase activity but did not affect MAP kinase activity and ultimately induced reactivation of p34cdc2 kinase via the synthesis of cyclin B1. On the other hand, treatments of a high concentration of Ca2+ ionophore or a low concentration of Ca2+ ionophore plus MEK inhibitor, U0126, linearly decreased MAP kinase activity following the decrease of p34cdc2 kinase activity; most of these oocytes formed pronuclei. These results suggest that decreasing MAP kinase activity is essential to maintaining low p34cdc2 kinase activity resulting from the degradation of cyclin B via a Ca(2+)-dependent pathway; lower activities of both MAP kinase and p34cdc2 kinase induce normal meiotic completion and pronuclear formation of parthenogenetically activated porcine oocytes.     

Stage‐dependent effects of epidermal growth factor on Ca2+ efflux in mouse oocytes

Epidermal growth factor (EGF) has received much attention recently for its positive effects on mammalian oocyte maturation and embryo development and its potential importance in cytoplasmic maturation of oocytes. Calcium (Ca²⁺) homeostasis in germinal vesicle stage oocytes has also been suggested to play a role in cytoplasmic maturation. This study examined the effects of EGF on Ca²⁺ mobilization as measured by its efflux from mouse oocytes at three time periods throughout maturation (0–4 hr, 4–8 hr, and 12 hr). Immature cumulus oocyte complexes (COCs) removed from the ovary for less than 4 hr exhibit oscillations in Ca²⁺ efflux that initiated 5–30 min following EGF stimulation. This response was not observed in COCs matured for 4–8 hr or 12 hr or in unstimulated 0–4 hr COCs. Denuded oocytes and cumulus cells did not show the same response to EGF (8.2 nM and 16.4 nM). Immunohistochemistry for detection of the EGF receptor along with EGF internalization studies showed that receptors are present both on cumulus cells and the oocyte but EGF appears to be internalized mainly by the cumulus cells. These data demonstrate that EGF induces oscillations in Ca²⁺ efflux in COCs 0–4 hr old and this response is mediated by the cumulus cells. Mol. Reprod. Dev. 53:244–253, 1999. © 1999 Wiley‐Liss, Inc.     

Inactivation of MPF and MAP kinase by single electrical stimulus for parthenogenetic development of porcine oocytes

This study was conducted to examine the activities of maturation-promoting factor (MPF) and mitogen-activated protein (MAP) kinase in the porcine oocytes after artificial activation. To determine optimal electrical activation condition, oocytes were exposed to single DC pulse in a variety of electric field strengths (120, 150, 180, and 210 V/mm) and pulse durations (15, 30, 45, and 60 microsec). After the artificial activation, 40-50 oocytes were cultured in a 50 microl drop of NCSU23 medium supplemented with 0.4% BSA at 39 degrees C, 5% CO2 in air for 6 days. No difference was detected in the preimplantation development of pocine oocytes and the mean nuclei number of blastocysts between electric field strengths. Under the 180 V/mm electric field strength, short pulse durations (15 and 30 microsec) showed a higher preimplantation developmental rate of the oocytes and mean nuclei number of blastocysts than an extended electric pulse (60 microsec) (P < 0.05). Single electrical stimulus (180 V/mm, 15 microsec) resulted in higher preimplantation development of porcine oocytes as compared to other chemical stimulators (P < 0.01). Western blot analyses showed the decrease of MPF and MAP kinase in the electrically-activated oocytes. After single electrical stimulus, the amounts of both cdc2 and ERK in porcine oocytes were remarkably reduced by 4 hr and then further decreased by 8 hr. However, the chemically-stimulated oocytes did not show any significant change at the levels of MPF and MAP kinase. Our results indicate that the optimal single electrical pulse is effective on the inactivation of MPF and MAP kinase, eventually leading to the parthenogenetic development of porcine oocytes.     

SKAP2 regulates Arp2/3 complex for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes

SKAP2 (Src kinase-associated phosphoprotein 2), a substrate of Src family kinases, has been suggested to be involved in actin-mediated cellular processes. However, little is known about its role in mouse oocyte maturation. In this study, we thus investigated the expression, localization, and functions of SKAP2 during mouse oocyte asymmetric division. SKAP2 protein expression was detected at all developmental stages in mouse oocytes. Immunofluorescent staining showed that SKAP2 was mainly distributed at the cortex of the oocytes during maturation. Treatment with cytochalasin B in oocytes confirmed that SKAP2 was co-localized with actin. Depletion of SKAP2 by injection with specific short interfering RNA caused failure of spindle migration, polar body extrusion, and cytokinesis defects. Meanwhile, the staining of actin filaments at the oocyte membrane and in the cytoplasm was significantly reduced after these treatments. SKAP2 depletion also disrupted actin cap and cortical granule-free domain formation, and arrested a large proportion of oocytes at the telophase stage. Moreover, Arp2/3 complex and WAVE2 expression was decreased after the depletion of SKAP2 activity. Our results indicate that SKAP2 regulates the Arp2/3 complex and is essential for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes.     

Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes

Meiotic spindle structure and chromosome alignment were examined after porcine oocytes were cooled at metaphase II (M II) stage. Cumulus-oocyte complexes (COCs) collected from medium size follicles were cultured in an oocyte maturation medium at 39 degrees C, 5% CO(2) in air for 44 hr. At the end of culture, oocytes were removed from cumulus cells and cooled to 24 or 4 degrees C for 5, 30, or 120 min in a solution with or without 1.5 M dimethyl sulfoxide (DMSO). After being cooled, oocytes were either fixed immediately for examination of the meiotic spindle and chromosome alignment or returned to maturation medium at 39 degrees C for 2 hr for examination of spindle recovery. Most oocytes (65-71%) cooled to 24 degrees C showed partially depolymerized spindles but 81-92% of oocytes cooled at 4 degrees C did not have a spindle after cooling for 120 min. Quicker disassembly of spindles in the oocytes was observed at 4 degrees C than at 24 degrees C. Cooling also induced chromosome abnormality, which was indicated by dispersed chromosomes in the cytoplasm. Limited spindle recovery was observed in the oocytes cooled to both 4 and 24 degrees C regardless of cooling time. The effect of cooling on the spindle organization and chromosome alignment was not influenced by the presence of DMSO. These results indicate that the meiotic spindles in porcine M II oocytes are very sensitive to a drop in the temperature. Both spindle and chromosomes were damaged during cooling, and such damage was not reversible by incubating the oocytes after they had been cooled.     

The effect of protein kinase C activator and nitric oxide donor on oocyte activation and cortical granule exocytosis in porcine eggs

Nitric oxide (NO) and protein kinase C (PKC) are involved in the activation of mammalian oocytes, although their role in the exit from the metaphase II stage and cortical granule (CG) exocytosis is still not fully understood. The aim of this study was to verify whether the NO-donor together with specific PKC-activators induce the complete activation of porcine oocytes assessed as meiosis resumption and a cortical reaction. Pig maturated oocytes were treated with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or PKC-activators such as phorbol-12-myristate-13-acetate (PMA, 100 nM), 1-oleoyl-2-acetyl-sn-glycerol (OAG, 400 μM) and l-α-phosphatidylinositol-3,4,5-trisphosphate dipalmitoyl heptaammonium salt (DPAM, 2 μM). To study the combined effect of NO-donor and PKC-activators, aliquots of oocytes were also incubated with SNAP (0.5 mM) together with PKC-activators at the same concentration as above (SNAP–DPAM, SNAP–OAG and SNAP–PMA groups). After in vitro maturation, an aliquot of oocytes was placed in a fresh medium without NO-donor or PKC-activators (Control group). Another aliquot of oocytes was activated by calcium ionophore A23187 (25 μM, 5 min). The results showed that 0% of the control oocytes reassumed meiosis. However, both the PKC-activators (DPAM 44.0 ± 10.0%, OAG 63.3 ± 1.0% and PMA 45.0 ± 16.5%) as well as the NO-donor alone (48.7 ± 21.0%) significantly induced exit from MII. Interestingly, the combination of PKC-activators and SNAP mainly restrained to the meiosis resumption (SNAP–OAG 0, SNAP–DPAM 17.4 ± 2.5% and SNAP–PMA 38.4 ± 8.5%). Control oocytes did not show a cortical reaction and the area occupied by CG reached 25.9 ± 1.7%, whereas CGs were partially released after Ca²⁺ ionophore treatment (13.0 ± 3.2%). Treatment with PKC-activators induced a cortical reaction compared with the control group (8.6 ± 2.5, 6.7 ± 1.9 and 0.7 ± 0.4%, respectively, for DPAM, OAG and PMA groups). However, treatment with the NO-donor alone (SNAP group 17.2 ± 2.2%) or combined with any PKC-activator prevented cortical reaction (SNAP–DPAM 20.7 ± 2.6%, SNAP–OAG 16.7 ± 2.9% or SNAP–PMA 20.0 ± 2.4%). Besides, meiosis resumption was not always accompanied by a cortical reaction, indicating that these two activation events are independent. In conclusion, PKC-activators alone induce CG exocytosis to the same degree as calcium ionophore. However, an NO-donor alone or combined with PKC-activators is not able to induce a cortical reaction in pig oocytes.     

Extracellular pH modulates the Ca2+ current activated by depletion of intracellular Ca2+ stores in human macrophages

Intracellular Ca²⁺ (Ca_i) signaling following the binding of surface receptors activates a Ca²⁺ permeable plasma membrane conductance which has been shown to be associated with store depletion in a number of cell types. We examined the activation of this conductance in human monocyte-derived macrophages (HMDMs) using whole-cell voltage-clamp techniques coupled with fura-2 microfluorimetry and characterized the importance of external pH (pH_o) as a modulator of current amplitude. Current activation was observed following experimental maneuvers designed to deplete intracellular Ca²⁺-stores including: (i) dialysis of the cell with 100 m inositol 1,4,5-triphosphate (IP₃), (ii) intracellular dialysis with high concentrations of the Ca²⁺ buffers EGTA and BAPTA, or (iii) exposure of the cell to the Ca²⁺-ATPase inhibitor thapsigargin (1 m). Currents associated with store depletion were inwardly rectifying with kinetics, inactivation, and selectivity that appeared similar irrespective of the mode of activation. Currents were Ca²⁺ selective with a selectivity sequence of Ca²⁺ > Sr²⁺ Mg²⁺ = Mn²⁺ = Ni²⁺. The Ca²⁺ influx current was modulated by changes in pH_o; modulation was not produced as a consequence of changes in internal pH (pH_i). External acidification led to a reversible reduction in current amplitude with a pKa at pH 8.2. Changes in pH_o alone failed to induce current activation. These observations are consistent with a scheme by which changes in pH_o, as would be encountered by macrophages at sites of inflammation, could change the time course and magnitude of the Ca_i transient associated with receptor activation by regulating the influx of Ca²⁺ ions.The authors wish to gratefully acknowledge the expert technical assistance of Weiwen Xie without whom the study could not have been completed. This work was supported by National Institutes of Health GM36823.     

Mitochondrial distribution, ATP-GSH contents, calcium [Ca2+] oscillation during in vitro maturation of dromedary camel oocytes

Dromedary camel oocytes have the ability to spontaneous parthenogenetic activation and development in vivo and in vitro. The present study was conducted to investigate changes in mitochondrial distribution, adenosine triphosphate (ATP), and glutathione (GSH) contents and [Ca²⁺] oscillation during in vitro maturation and spontaneous parthenogentic activation of dromedary camel oocytes. Dromedary camel cumulus-oocyte complexes (COCs) were matured in TCM199 medium supplemented with 10% FCS + 10 μg/mL FSH + 10 IU hCG + 10 IU eCG + 10 ng/mL EGF and 50 μg/mL gentamycine. Maturation was performed at 38.5 °C under 5% CO₂ in humidified air for 40 h. After maturation and removal of cumulus cells, oocytes were classified into: immature cultured (Group 1); metaphase II (M II, Group 2); and spontaneously parthenogenetically activated (with 2 polar bodies, Group 3); cleaved embryos (Group 4); and immature oocytes served as a control (Group 5). Cytoplasmic mitochondrial distribution, ATP-GSH contents, calcium [Ca²⁺] oscillation were determined. Results indicated that M II and spontaneously parthenogenetically activated oocytes represent 37.53% and 32.67% of the cultured oocytes, respectively, and 3.3% cleaved and developed to 2-16-cell stage embryos. Mitochondrial distribution, ATP-GSH contents and [Ca²⁺] oscillation were significantly (P < 0.01) differ between immature and matured dromedary camel oocytes. Mitochondrial distribution showed clustering form in matured oocytes without polar body. High polarized mitochondrial distribution (HPM) was detected in M II and spontaneously parthenogenetically activated oocytes, and the intensity of MitoTracker Red was higher in spontaneously parthenogenetically activated than M II. ATP-GSH contents and the duration of [Ca²⁺] oscillation were significantly (P < 0.01) higher in spontaneously parthenogenetically activated than M II oocytes or that matured without polar body. In conclusion, the higher incidence of spontaneously parthenogenetically activated in vitro matured dromedary camel oocytes could be attributed to the high polarized mitochondrial distribution associated with significantly higher ATP-GSH contents and duration of [Ca²⁺] oscillation.     

Effects of spindle removal on MPF and MAP kinase activities in porcine matured oocytes

Intracellular localization of maturation/M-phase promoting factor (MPF) and mitogen activated protein (MAP) kinase in mature oocytes has been examined by immunocytochemical methods and the authors of these studies have reported that they are localized on spindles during M-phase. Although these reports showed the relative localization of MPF and MAPK on spindles, it has never been shown whether these kinases are present in the cytoplasm and, if they are present, how many parts of the kinases are localized on the metaphase spindle. In the present study, we made quantitative analyses of MPF and MAP kinase localized on oocyte spindles by kinase assays and immunoblotting after removal of the spindles from porcine mature oocytes. First, we certified their intracellular distribution by immunocytochemical methods and observed sharp signals of cyclin B1 on spindle poles and MAP kinase signals on the microtubule of metaphase spindles. In contrast to these results by immunostaining, the amounts of MPF and MAP kinase localized on spindles examined by immunoblotting and kinase assays were undetectable and less than 20%, respectively. These results indicate that the immunocytochemical technique is a powerful method for showing relative localization, but it is not suitable for quantitative analysis, and that the removal of metaphase spindles from mature oocytes does not have a severe negative impact on the subsequent MPF and MAP kinase activity and on the cell cycle progression in early embryo development.