Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.     

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit,Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

Background/Aims

The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium.

Methodology and Principal Findings

Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2^nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization.

Conclusions

This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.     

Possible role of calcium on oocyte development after intracytoplasmic sperm injection in quail (Coturnix japonica)

Although a rise in intracellular calcium concentration of vertebrate oocytes plays a pivotal role for the initiation of fertilization or oocyte activation, no study on this subject has been reported in birds. This study was conducted to study the role of intracellular calcium in relation to fertilization in avian oocytes. First, immediately after a quail oocyte was injected with a sperm, it was treated with strontium chloride as an inducer for intracellular calcium rise at doses of 0, 2.5, 5, 7.5, 10 mM for 4 hr in the culture medium and was followed by 20-hr culture. Treatment with 5 mM of strontium chloride induced blastodermal development in 24.2% of injected eggs, although no oocytes developed without strontium treatment. Second, quail oocytes were injected with a sperm and 0.1 M calcium chloride or a sperm and saline solution, cultured without calcium for 4 hr and was followed by 20-hr culture without strontium. The calcium solution induced blastodermal development in 20.5% of the oocytes, although no oocytes developed without calcium treatment. Third, quail oocytes were injected with 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) as a calcium chelator, cultured with strontium (5 mM) for 4 hr followed by 20-hr culture without strontium. Only one oocyte developed after BAPTA and strontium treatment of 36 oocytes examined. Developmental stages of all the oocytes ranged from IV to VII. These results suggest that intracellular calcium rise may participate in quail oocyte activation and allow fertilization and blastodermal development.     

Spatial and temporal aspects of cell signalling

As new techniques are developed to measure intracellular messengers it becomes increasingly apparent that there is a remarkable spatial and temporal organization of cell signalling. Cells possess a small discrete hormone-sensitive pool of inositol lipid. In some cells such as Xenopus oocytes and Limulus photoreceptors this phosphoinositide signalling system is highly concentrated in one region of the cell, so establishing localized calcium gradients. Another example is the hydrolysis of inositol lipids in eggs at the point of sperm entry resulting in a localized increase in Ins(1,4,5)P3 and calcium which spreads like a wave throughout the egg. In hamster eggs this burst of calcium at fertilization recurs at 1-3 min intervals for over 100 min, a particularly dramatic example of spontaneous activity. Spontaneous oscillations in intracellular calcium exist in many different cell types and are often induced by agonists that hydrolyse inositol lipids. We have made a distinction between oscillations that are approximately sinusoidal and occur at a higher frequency where free calcium is probably continuously involved in the oscillatory cycle and those where calcium falls to resting levels for many seconds between transients. In the former case, the oscillations are thought to be induced through a cytoplasmic oscillator based on the phenomenon of calcium-induced calcium release. Such oscillations can be induced in Xenopus oocytes after injection with Ins(1,4,5)P3. A receptor-controlled oscillator based on the periodic formation of Ins(1,4,5)P3 is probably responsible for the generation of the widely spaced calcium transients. The function of such calcium oscillations is currently unknown. They may be a reflection of the feedback interactions that operate to control intracellular calcium. Another possibility emerged from observations that in some cells the frequency of calcium oscillations varied with agonist concentration, suggesting that cells might employ these oscillations as a way of encoding information. One advantage of using such a frequency-dependent mechanism may lie in an increase in fidelity, especially at low agonist concentrations. Whatever these functions might be, it is clear that uncovering the mechanisms responsible for such oscillatory activity will greatly enhance our understanding of the relation between the phosphoinositides and calcium signalling.     

Meiosis-associated calcium waves in ascidian oocytes are correlated with the position of the male centrosome

We have used confocal microscopy to measure calcium waves and examine the distribution of tubulin in oocytes of the ascidian Ciona intestinalis during meiosis. We show that the fertilisation calcium wave in these oocytes originates in the vegetal pole. The sperm penetration site and female meiotic apparatus are found at opposite poles of the oocyte at fertilisation, confirming that C. intestinalis sperm enter in the vegetal pole of the oocyte. Following fertilisation, ascidian oocytes are characterised by repetitive calcium waves. Meiosis I-associated waves originate at the vegetal pole of the oocyte, and travel towards the animal pole. In contrast, the calcium waves during meiosis II initiate at the oocyte equator, and cross the oocyte cytoplasm perpendicular to the point of emission of the polar body. Immunolocalisation of tubulin during meiosis II reveals that the male centrosome is also located between animal and vegetal poles prior to initiation of the meiosis II-associated calcium waves, suggesting that the male centrosome influences the origin of these calcium transients. Ascidians are also characterised by an increase in sensitivity to intracellular calcium release after fertilisation. We show that this is not simply an effect of oocyte activation. The data strongly suggest a role for the male centrosome in controlling the mechanism and localisation of post-fertilisation intracellular calcium waves.     

Mammalian oocyte activation by the synergistic action of discrete sperm head components: induction of calcium transients and involvement of proteolysis

Sperm-borne oocyte-activating factor (SOAF) elicits activation sufficient for full development and originates from sperm head submembrane matrices. SOAF comprises discrete, heat-sensitive and -stable components (referred to here respectively as SOAF-I and -II) which are each necessary but not sufficient to activate oocytes. The heat-sensitive SOAF component, SOAF-I(m), becomes solubilized from the perinuclear matrix under reducing conditions (the SOAF transition) to generate SOAF-I(s). Although calcium transients likely play an important role in oocyte activation at fertilization, the question is open as to whether demembranated heads or SOAF-I(s) and/or SOAF-II can induce calcium transients. We now report that injection of demembranated sperm heads into mouse oocytes efficiently induced Ca(2+) oscillations. When injected independently, SOAF-I(s) and demembranated heads heated to 48 degrees C failed to generate Ca(2+) oscillations. However, co-injection of SOAF-I(s) and 48 degrees C-heated heads induced oscillations, mirroring their synergistic ability to activate oocytes. This suggests that SOAF-mediated activation proceeds via pathways resembling those at fertilization and provides the first direct evidence that multiple sperm components are required to induce Ca(2+) oscillations. We probed the SOAF-I(s) liberation at the center of this activation and show that in vitro it was sensitive to a profile of serine protease inhibitors. These findings support a model in which mammalian oocyte activation, including the induction of calcium transients, involves proteolytic processing of SOAF from sperm head submembrane compartments.     

A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization

Fertilization, a critical step in animal reproduction, is triggered by a series of specialized sperm-egg interactions. However, the molecular mechanisms underlying fertilization are not well understood. Here, we identify a sperm-enriched C. elegans TRPC homolog, TRP-3. Mutations in trp-3 lead to sterility in both hermaphrodites and males due to a defect in their sperm. trp-3 mutant sperm are motile, but fail to fertilize oocytes after gamete contact. TRP-3 is initially localized in intracellular vesicles, and then translocates to the plasma membrane during sperm activation. This translocation coincides with a marked increase in store-operated calcium entry, providing an in vivo mechanism for the regulation of TRP-3 activity. As C. elegans oocytes lack egg coats, our data suggest that some TRPC family channels might function to mediate calcium influx during sperm-egg plasma membrane interactions leading to fertilization.     

Comparative biology of calcium signaling during fertilization and egg activation in animals.

During animal fertilizations, each oocyte or egg must produce a proper intracellular calcium signal for development to proceed normally. As a supplement to recent synopses of fertilization-induced calcium responses in mammals, this paper reviews the spatiotemporal properties of calcium signaling during fertilization and egg activation in marine invertebrates and compares these patterns with what has been reported for other animals. Based on the current database, fertilization causes most oocytes or eggs to generate multiple wavelike calcium oscillations that arise at least in part from the release of internal calcium stores sensitive to inositol 1,4,5-trisphosphate (IP3). Such calcium waves are modulated by upstream pathways involving oolemmal receptors and/or soluble sperm factors and in turn regulate calcium-sensitive targets required for subsequent development. Both "protostome" animals (e.g., mollusks, annelids, and arthropods) and "deuterostomes" (e.g., echinoderms and chordates) display fertilization-induced calcium waves, IP3-mediated calcium signaling, and the ability to use a combination of external calcium influx and internal calcium release. Such findings fail to support the dichotomy in calcium signaling modes that had previously been proposed for protostomes vs deuterostomes and instead suggest that various features of fertilization-induced calcium signals are widely shared throughout the animal kingdom.     

Distribution of 5-chloromethylfluorescein diacetate staining during meiotic maturation and fertilization in vitro of mouse oocytes

The aim of this confocal microscopy study was to determine whether the pattern of CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) staining changes during meiotic maturation and fertilization in vitro of mouse oocytes. At different times during meiotic maturation and fertilization, oocytes, zygotes and two-cell embryos were stained with CMFDA to demonstrate intracellular glutathione S-transferase activity. After washing in CMFDA-free medium, most oocytes, zygotes and embryos were stained with dihydroethidium (HE) to visualize DNA structures. Meiotic maturation and fertilization in vitro of mouse oocytes were associated with changes in the pattern of intracellular CMFDA staining. In particular, accumulations of CMFDA-positive membranes were observed around the nucleus of germinal vesicle (GV) oocytes, overlaying the sperm nucleus as well as overlaying the first mitotic spindle if this approached the plasma membrane. Staining of oocytes and zygotes with the probes 3,3'-dihexyloxacarbocyanine iodine [DiOC6(3)], which stains all the intracellular membranes, and rhodamine 123, which stains active mitochondria, demonstrated that the intracellular structures evidenced by CMFDA staining did not correspond to accumulations of mitochondria. Exposure of oocytes and zygotes to the microtubule-disrupting agent nocodazole or the actin-depolymerizing drug cytochalasin D revealed an autonomous microfilament-dependent transport and relocation of CMFDA-positive membranes during meiotic maturation and fertilization. Such a transport of CMFDA-positive membranes may be envisaged as a protective shield built to prevent damage to DNA from endogenous and exogenous mutagen metabolites.     

Time-Lapse Confocal Imaging of Calcium Dynamics in Starfish Embryos

During fertilization and cleavage, embryos undergo transient rises in their intracellular free calcium levels that are postulated to provide essential signals enabling normal development to proceed. In order to analyze the spatiotemporal patterns and possible biological significance of these calcium transients, time-lapse confocal microscopy was used to monitor starfish embryos during normal development and following experimental manipulations that disrupted cleavage and/or the release of calcium ions from internal stores in the embryo. For such analyses, oocytes were co-injected with dextran-conjugated forms of the calcium-sensitive fluorochrome calcium green (CG) and the calcium-insensitive dye rhodamine (Rh) for dual-channel confocal ratioing. Based on CG/Rh ratioed images obtained every 15 sec far the first few hours of development, no prominent calcium spikes were typically evident at the onset of the first cell cycle as hormone-treated oocytes resumed maturation and underwent germinal vesicle breakdown (GVBD). Subsequently, fertilizations of post-GVBD oocytes caused a single prolonged calcium wave that reached relatively uniform amplitudes throughout the ooplasm. Within 90 min after fertilization, most starfish zygotes began to display clusters of repetitive calcium oscillations that typically—but not invariably—preceded nuclear envelope breakdown, anaphase onset, and the formation of the first cleavage furrow. Rapidly decaying calcium oscillations continued through at least the first five cleavages in specimens that developed into normal blastulae, and unlike fertilization-induced calcium waves, such spikes tended to be more pronounced in the cortical cytoplasm during early cleavages. Thus, three different types of calcium dynamics—no marked transients, a single nonoscillating wave, and repetitive oscillations—were observed as normally developing starfish underwent prefertilization maturation, fertilization, and cleavage, respectively, suggesting that the spatiotemporal patterns of calcium spiking can change during starfish early development. In specimens microinjected with colchicine, calcium transients were also visible in the absence of cell divisions, indicating that calcium spiking can be uncoupled from cytokinesis. To assess whether calcium fluxes are required for normal development, oocytes were also treated with haparia to black calcium release mediated by inositol 1,4,5-trisphosphate (IP₃). Injections of heparin, but not the control molecule de-N-sulfated heparin, caused abnormal fertilization-induced calcium dynamics in a does-dependent fashion and typically abolished marked postfertilization calcium oscillations and normal cleavage. Based on correlative studies using caged IP₃, heparin interfered with IP₃-mediated calcium release, suggesting that such release is involved in the production of the free calcium elevations that occur in normally developing starfish embryos.     

Time course of cortical and zona reactions of pig oocytes upon intracellular calcium increase induced by thimerosal

Our previous study indicated that thimerosal is one of the most effective artificial activators to mimic sperm-induced increases in the intracellular free calcium concentration ([Ca2+]i) and other activation events in pig oocytes (Macháty et al., 1997). The present study was conducted to examine the temporal relationship between intracellular calcium transients, cortical granule (CG) exocytosis and the zona reaction induced by thimerosal. When pig oocytes matured in vitro were exposed to 200 microM thimerosal the first intracellular calcium transient, with a mean peak ratio of 4.97 +/- 1.14, was observed 509.64 +/- 122.03 s after addition of thimerosal. The density of CGs fell significantly from 63.3 +/- 11.7 CGs/100 micron 2 of cortex in control oocytes to 25.7 +/- 19.2 CGs/100 micron 2 of cortex (59.4% release) at 2 min after the first intracellular calcium transient. At 5 min after the calcium transient the residual CG density had been reduced to 10.7 +/- 10.4 CGs/100 micron 2 of cortex (83.1% release). This degree of CG exocytosis was the same as that in oocytes penetrated by sperm (9.5 +/- 5.1 CGs/100 micron 2 of cortex). No further decrease in residual CG density was observed at 10 min (10.3 +/- 14.8 CGs/100 micron 2 of cortex). Whereas 77.4% (120/155) of control oocytes were penetrated by spermatozoa only 1.4% (2/144) of thimerosal-treated oocytes were penetrated. Further experimental results obtained by in vitro fertilisation of oocytes with preincubated (capacitated) spermatozoa suggested that the zona block to sperm penetration in thimerosal-treated oocytes occurred within 35 min after CG exocytosis and 40 min after the first calcium transient. These results indicate that polyspermic penetration of pig oocytes inseminated in vitro is not due to delayed or incomplete CG exocytosis but more likely to a delayed zona reaction and/or simultaneous sperm penetration.     

Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.     

A comparison of intracellular changes in porcine eggs after fertilization and electroactivation.

The experiments compare intracellular changes in porcine eggs induced by electrical activation with those induced by sperm penetration. Adequate electrostimulation induces changes in both cortical granule exocytosis and protein synthesis similar to those induced by sperm during fertilization. However, ionic changes induced by electrostimulation differ markedly from those initiated at fertilization. Thus, dynamic video imaging using Fura-2 as a Ca2+ probe provides evidence that parthenogenetic activation induced by electrostimulation is initiated by a single sharp rise in the concentration of intracellular free calcium ([Ca2+]i) in the egg. The intracellular Ca2+ transient increase is triggered by an influx of extracellular Ca2+ immediately after electrostimulation. The amplitude of the intracellular Ca2+ transient increase is a function both of the extracellular Ca2+ concentration and of electric field parameters (field strength and pulse duration). Imaging demonstrates further that a single electrical pulse can only induce a single Ca2+ transient which usually lasts three to five minutes; no further Ca2+ transients are observed unless additional electrical stimuli are applied. By contrast, sperm-induced activation is characterised by a series of Ca2+ spikes which continue for at least 3 hours after sperm-egg fusion. The pattern of Ca2+ spiking after fertilization is not consistent during this period but changes both in frequency and amplitude. Overall, the results demonstrate that, although electrostimulation induces both cortical granule exocytosis and protein reprogramming in porcine eggs, it does not reproduce the pattern of [Ca2+]i changes induced by sperm entry at fertilization.     

Ability of integrins to mediate fertilization, intracellular calcium release, and parthenogenetic development in bovine oocytes

The ability of arginine-glycine-aspartic acid (RGD; a sequence recognized by integrins) or non-RGD-containing peptides to block fertilization, induce intracellular Ca(2+) oscillations, and initiate parthenogenetic development in bovine oocytes was investigated. Addition of a soluble RGD peptide during fertilization at concentrations ranging from 10 to 1000 microg/ml significantly decreased (P<0.05) fertilization as compared to the in vitro-fertilized controls. The addition of non-RGD peptide had no effect on fertilization. Two intracellular Ca(2+) transients 21.5+/- 1.9 min apart were observed in 56 of 60 oocytes incubated in RGD peptide concentrations ranging from 20 to 1000 microg/ml. No intracellular Ca(2+) transients were observed in medium alone, non-RGD treatment groups or in the RGD peptide at 10 microg/ml. The percentage of oocytes activated with ionomycin and 6-dimethylaminopurine (63% cleavage and 34% blastocyst development) was significantly higher (P<0.05) than those activated with the RGD peptide and 6-dimethylaminopurine (35% cleavage and 19% blastocyst development). These groups were significantly higher (P<0.05) than either peptide alone, 6-dimethylaminopurine alone, or the non-RGD peptide and 6-dimethylaminopurine treatment groups. These data provide evidence that ligation of an integrin on bovine oocytes with a soluble RGD peptide is capable of blocking fertilization, inducing intracellular Ca(2+) transients, and initiating parthenogenetic development.     

A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in hamster eggs

Microinjection of cytosolic sperm extracts into unfertilized golden hamster eggs caused a series of increases in cytoplasmic free calcium, Ca2+i, and membrane hyperpolarizing responses, HRs. These HRs and Ca2+i transients are similar to those seen during in vitro fertilization of hamster eggs. The sperm factor that is responsible for causing these effects appears to be of high molecular weight and protein based. Injection of sperm factor activated eggs and mimicked fertilization in causing repetitive HRs in the presence of phorbol esters and in sensitizing the egg to calcium-induced calcium release. Since these effects cannot be mimicked by injecting G-protein agonists or calcium-containing solutions, it seems unlikely that a receptor-G-protein signalling system is involved at fertilization. These data instead suggest a novel signal transduction system operates during mammalian fertilization in which a protein factor is transferred from the sperm into the egg cytoplasm after gamete membrane fusion.     

Porcine oocyte activation induced by a cytosolic sperm factor

It is not known how the fertilizing sperm elicits the release of Ca(2+) from the oocyte's intracellular stores. We investigated whether a crude extract isolated from boar sperm could induce the Ca(2+) release and trigger subsequent early and late activation events upon injection into matured porcine oocytes. The sperm extract induced an immediate rise in the intracellular free Ca(2+) concentration in all oocytes tested, which was followed by repetitive Ca(2+) transients in 11 out of 14 oocytes. Heat or trypsin treatment of the extract totally abolished the Ca(2+) releasing activity of the sperm factor. The injected oocytes showed cortical granule exocytosis, they resumed meiosis and entered first interphase: pronuclei were formed in 89.2% (132/148) of the cases. Pronuclear formation was accompanied by the appearance of a new 22 kDa protein as normally seen at fertilization. Of the successfully injected oocytes 51.7% (105/203) cleaved and 2.0% (4/203) developed to the blastocyst stage after being cultured for 7 days in NCSU 23 medium. Injection of the carrier medium could not trigger these changes. The results indicate that the sperm might activate porcine oocytes by introducing a soluble factor into the oocyte's cytoplasm after gamete fusion.     

Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes

Background  

Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC) and the exocytosis of cortical granules in mouse oocytes.     

Role of dihydropyridine-sensitive calcium channels in meiosis and fertilization in the bivalve molluscs Ruditapes philippinarum and Crassostrea gigas

Prophase-arrested oocytes of Ruditapes philippinarum can not be fertilized or stimulated by a depolarizing agent such as an excess of KCl, in contrast to the situation found in Crassostrea gigas. We have performed a comparative study between the two situations found in these species. In vitro, both of these oocytes can be triggered to reinitiate meiosis following a treatment by serotonin which promotes an intracellular calcium surge. Ruditapes and Crassostrea oocytes further arrest in metaphase I, at which stage they can be either activated by sperm or by excess KCl. These treatments trigger an intracellular calcium increase. This suggests that functional voltage-operated Ca2+ channels are expressed in Ruditapes during the course of maturation between prophase and metaphase I. Results obtained using pharmacological tools and direct binding of specific dihydropyridines, strongly suggest that these channels are dihydropyridine-sensitive calcium channels. In Ruditapes they become functional after 5-HT stimulation, their number increasing before GVBD. In Crassostrea the dihydropyridine-sensitive Ca2+ channels are already present at prophase stage and their density is constant from prophase to metaphase I. Moreover, we have shown for Ruditapes and Crassostrea that: 1) the addition of 10 microM of S(-)BayK8644, an agonist of dihydropyridine-sensitive calcium channels to metaphase-arrested oocytes releases them from metaphase block; and 2) incubating these oocytes with nicardipine, a potent blocker of dihydropyridine-sensitive Ca2+ channels, inhibits both their activation by excess KCl or fertilization. Taken together these data suggest that the absence of dihydropyridine-sensitive Ca2+ channels in the membrane of prophase-arrested oocytes of Ruditapes may account for their inability to be fertilized at this stage, while the presence of dihydropyridine-sensitive Ca2+ channels in prophase-arrested oocytes of Crassostrea may explain their fertilizability at this stage.     

Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta

Background  

During natural fertilization, sperm fusion with the oocyte induces long lasting intracellular calcium oscillations which in turn are responsible for oocyte activation. PLCZ1 has been identified as the factor that the sperm delivers into the egg to induce such a response. We tested the hypothesis that PLCZ1 cRNA injection can be used to activate bovine oocytes.     

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium,duration of maturation,and sperm calcium ionophore treatment,and comparison with rates of fertilization in vivo after oviductal transfer

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.