IMMUNO-ELECTRON MICROSCOPE ANALYSIS OF THE SURFACE LAYERS OF THE UNFERTILISED SEA URCHIN EGG : I. Effects of the Antisera on the Cell Ultrastructure

The response of unfertilised Paracentrotus lividus eggs to γ-globulin fractions of antisera against isolated homologous jelly coat substance or homologous homogenates of jellyless eggs has been studied at the ultrastructural level. The antijelly γ-globulin caused precipitation of the jelly layer, the density of precipitation varying between different eggs and being proportional to the γ-globulin concentration. Agglutination of the jelly substance of adjacent eggs, which is species specific, occurred frequently with higher γ-globulin concentrations. Antiegg γ-globulins (from antiserum against total homogenates of jelly-free eggs or the heat-stable fraction thereof) did not produce these effects. Instead, these γ-globulins caused various structural alterations mostly representing stages in parthenogenetic activation. This species-specific activation was induced by the reaction of antibodies with some heat-stable egg antigens different from those involved in jelly precipitation. Surface alterations included the formation of small papillae, membrane blisters, hyaline layer, and activation membrane, the release of material from the cell surface, and the breakdown of cortical granules. These alterations were dependent on both γ-globulin concentration and the variable reactivity among different females. Aster formation, found intracellularly, verified that the surface responses represented real parthenogenetic activation and were not the result of immune lysis. No such alterations appeared in the controls.     

Action of trypsin on the eggs of Dendraster excentricus

Crystalline trypsin in 3 × 10^?8 M concentration and higher, elicits fertilization membranes in the unfertilized eggs of Dendraster excentricus. These membranes are adequate in artificial parthenogenesis. If the action of trypsin on these eggs is continued for two or three hours the result is first, digestion of the membranes, followed later by reduction of the egg to amoeboid form. When fertilized, some of the partially digested eggs segment and form irregular cell masses, thus demonstrating that, in response to trypsin, there is first the cortical reaction giving rise to the fertilization membrane, and second, the progressive digestion and disintegration of the cytoplasm.Chymotrypsin causes rounding of the unfertilized eggs and, in rare instances, a few membranes, but the enzyme is not an adequate parthenogenetic agent.Fertilization of the egg renders the cytoplasm resistant to trypsin. The facts lead to the suggestion that fertilization liberates trypsin inhibitors in the cytoplasm.     

Endoplasmic reticulum whorls as a source of membranes for early cytaster formation in parthenogenetically stimulated sea urchin eggs

Summary Sea urchin eggs exposed to a continuous hypertonic treatment rapidly form many concentric whorls of endoplasmic reticulum (ER) during the pre-activation period of the parthenogenetic development. These whorls, however, are only a temporary configurational alteration of ER which begin to break up just prior to egg activation. The conversion back to normal vesicles and lamellae occurs not only concurrently with the appearance of early cytastral areas, but also frequently in close association with the formation of these membranous areas. It is revealed here that membrane elements from disrupting whorls may become incorporated into adjacent, developing clear areas, early cytastral areas, and that this ER constitutes an initial major source of membranes for these early astral areas. Having previously suggested that the actual formation of ER whorls occurs in direct response to released intracellular calcium in hypertonic stressed eggs, the new findings, along with other related data and correlations, further suggest that whorl disruption and the formation of associated astral areas can be correlated with a corresponding decrease in the concentration of this released calcium in the cytoplasm.     

Regulation of cleavage by protein kinase C in Chaetopterus

We report that protein kinase C (PKC) plays a regulatory role in early cleavage in Chaetopterus eggs. Using Western blotting, we assayed the expression patterns of conventional PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC). During early development after fertilization, PKC protein levels varied independently by isoform. PKC protein expression during differentiation, without cleavage and after parthenogenetic activation, was very similar to that during normal development indicating that PKC gene expression does not require cellularization. Since PKC has been shown to regulate meiosis in this organism, we also assayed the membrane association of these isoforms as an indicator of their activation during meiosis and early cleavage. PKC-gamma transiently associated with membranes and therefore became activated before meiotic division and cleavage, whereas PKC-alpha and -beta transiently dissociated from membranes and therefore became inactivated at these times. Inhibition of these PKC isoforms by bisindolylmaleimide I had no effect on cleavage or early development to the trochophore larva, indicating that PKC-gamma activation is not essential for cleavage or early development. However, their persistent activation by thymeleatoxin blocked cleavage. The results indicate that the dissociation of PKC-alpha and/or -beta from the membrane fraction, and therefore their inactivation, is essential for normal cleavage. Elevated PKC activity is essential for nuclear envelope breakdown and spindle formation at meiosis I. By contrast, down-regulation of this activity is essential for cleavage after fertilization.     

Body length, size of resting eggs and identification of the ephippia of Ceriodaphnia megalops and Ceriodaphnia pulchella

The ephippia of Ceriodaphnia megalops and Ceriodaphnia pulchella are described. Their ephippia may be identified by their size and shape, supplemented by measurement of the length of the resting egg. The ephippia are shed free in the water and float by means of a special area of gas-filled tissue. Each ephippium contained one ellipsoid resting egg. The length, width and shape of the resting eggs of both species were investigated, and comparisons made with published data of their parthenogenetic eggs. For both species the volume of a resting egg was virtually the same as the volume of a parthenogenetic egg, but the resting egg was more elongated. The shape of resting eggs was linearly related to egg volume on logarithmic axes in a manner similar to that reported for the parthenogenetic eggs of these two species. The linear relationship between total parthenogenetic egg volume and body length reported for these two species did not apply to their resting eggs.     

Females from resting eggs and parthenogenetic eggs in the rotifer Brachionus calyciflorus: lipid droplets, starvation resistance and reproduction

1. In the heterogonic life cycle of monogonont rotifers, amictic (female‐producing) females develop from two types of eggs: fertilised resting (diapausing) eggs and parthenogenetic subitaneous eggs. Females hatched from resting eggs initiate clonal populations by female parthenogenesis and are called stem females. This study compares females from resting and parthenogenetic eggs that were produced under identical culture conditions and were of similar birth order. 2. Newborn stem females had many more lipid droplets in their tissues than similar‐sized, newborn females from parthenogenetic eggs. When neonates were stained with Nile Red and viewed under epifluorescent illumination, these droplets were shown to be sites of neutral‐lipid storage products. 3. Stem females had no posterolateral spines and short anterior spines, while their mothers and offspring in subsequent, parthenogenetic generations typically had long posterolateral spines and elongated anterior spines. 4. Newborn stem females survived starvation significantly longer than newborn females from parthenogenetic eggs. 5. When females from resting and parthenogenetic eggs were cultured from birth to death at a high food concentration, the reproductive potential (r day^?1) of the stem females was significantly higher (0.82–0.88 versus 0.70), primarily because of egg production at an earlier age. The mean lifetime fecundity (R_o) of stem females was significantly greater than that of females from parthenogenetic eggs. 6. Extensive lipid reserves should increase the ability of stem females to colonise new habitats. Firstly, compared with females from parthenogenetic eggs, stem females are more likely to experience starvation or food limitation. Resting eggs hatch in response to physical and chemical factors that are not directly related to food availability, and from sediments that may be far from food‐rich surface waters. Secondly, when food is abundant, stem females have a greater reproductive potential.     

Nitric Oxide-Donor SNAP Induces Xenopus Eggs Activation

Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions including meiotic maturation and parthenogenetic activation of mammalian oocytes. We observed that nitric oxide donor SNAP was potent to induce parthenogenetic activation in Xenopus eggs. NO-scavenger CPTIO impaired the effects of SNAP, providing evidence for the effects of the latter to be specific upon NO release. In Xenopus eggs, SNAP treatment induced pigment rearrangement, pronucleus formation and exocytosis of cortical granules. At a biochemical level, SNAP exposure lead to MAPK and Rsk inactivation within 30 minutes whereas MPF remained active, in contrast to calcium ionophore control where MPF activity dropped rapidly. MAPK inactivation could be correlated to pronuclear envelope reformation observed. In SNAP-treated eggs, a strong increase in intracellular calcium level was observed. NO effects were impaired in calcium-free or calcium limited medium, suggesting that that parthenogenetic activation of Xenopus oocytes with a NO donor was mainly calcium-dependent.     

Influence of microbe-associated parthenogenesis on the fecundity ofTrichogramma deion andT. pretiosum

Microbe-associated parthenogenesis (thelytoky) has been discovered in nineTrichogramma species, parasitoids of mainly lepidopteran eggs. Parthenogenetic and bisexual conspecifics co-occur in many field populations. As an initial step to understand the dynamics of these two reproductive strategies we studied the effect of microbe-associated parthenogenesis on fecundity. The fecundity of two parthenogenetic isofemale lines ofT. pretiosum and one ofT. deion was compared with bisexual lines derived from them by antibiotic treatment. In all three cases parthenogenetic females were less fecund over their lifetime than bisexual females. Also, parthenogenetic females produced fewer daughters in two cases and in one case a similar number of daughters as their respective bisexual counterparts. The lack of mating and insemination was excluded as an explanation for the reduced fecundity of parthenogenetic females, because mated and virgin parthenogenetic females produce the same number of offspring. Antibiotic treatment can also be excluded because females of field-collected bisexual line treated with antibiotics produced the same number of offspring as untreated females. The reduced fecundity of parthenogenetic females was caused by a lower number of eggs being laid rather than by a greater developmental mortality. Parthenogenetic females produced less daughters than bisexual females when host availability was not limiting, but when host availability was severely limited, parthenogenetic females produced more daughters than the bisexual females.     

Cytological investigation of the mechanism of parthenogenesis in Drosophila mercatorum

Thelytokous parthenogenesis (female progeny only) in animals is believed to arise initially in unfertilized eggs produced by bisexual females via the fusion of two haploid nuclei following meiosis, to produce diploid female progeny. The transition from sexual to parthenogenetic mechanisms of reproduction requires that the egg replace the paternal contributions of a haploid genetic complement and the basal body, which is thought to be essential for centrosome formation. The transitional facultative parthenogenetic stage is usually associated with a high rate of failed or abortive development, but the molecular and mechanistic reasons for this failure remain unclear. We show that a facultative parthenogenetic strain of Drosophila mercatorum produces a high percentage of unfertilized eggs competent to restore diploidy and form centrosomes de novo following meiosis. The female meiotic products replicate and divide by an acentrosomal mechanism in most oocytes and cytoplasmic centrosomes form in 35% of the oocytes. However, after pronuclear replication the cytoplasmic centrosomes must "capture" two haploid nuclei in order to restore diploidy. In practice, this process frequently fails due to centrosome-mediated capture events of single or more than two haploid nuclei, as well as multiple nuclear capture events in a single embryo when excess free centrosomes are not inactivated following formation of the first zygotic nucleus. Additionally, as development proceeds, many of the centrosomes that initiate syncytial development do not remain functional, possibly due to centrosome maturation defects, and later stages of syncytial development fail. The combined effect of the high error rate associated with nuclear capture and the failure of centrosome maturation during later developmental prevents successful parthenogenesis in most of the eggs that initiate development. This shows that the high rate of failed development associated with the transition from sexual to parthenogenetic reproduction is limited by the low probability of the formation of a diploid zygotic nucleus with the correct complement of centrosomes in D. mercatorum.     

Membrane fusion during secretion: cortical granule exocytosis in sex urchin eggs as studied by quick-freezing and freeze-fracture

Exocytosis of cortical granules was observed in sea urchin eggs, either quick-frozen or chemically fixed after exposure to sperm. Fertilization produced a wave of exocytosis that began within 20 s and swept across the egg surface in the following 30 s. The front of this wave was marked by fusion of single granules at well-separated sites. Toward the rear of the wave, granule fusion became so abundant that the egg surface left with confluent patches of granule membrane. The resulting redundancy of the egg surface was accommodated by elaboration of characteristic branching microvilli, and by an intense burst of coated vesicle formation at approximately 2 min after insemination. Freeze-fracture replicas of eggs fixed with glutaraldehyde and soaked in glycerol before freezing displayed forms of granule membrane interaction with the plasma membrane which looked like what other investigators have considered to be intermediates in exocytosis. These were small disks of membrane contact or membrane fusion, which often occurred in multiple sites on one granule and also between adjacent granules. However, such membrane interactions were never found in eggs that were quick-frozen fixation, or in eggs fixed and frozen without exposure to glycerol. Glycerination of fixed material appeared to be the important variable; more concentrated glycerol produced a greater abundance of such "intermediates." Thus, these structures may be artifacts produced by dehydrating chemically fixed membranes, and may not be directly relevant to the mechanism by which membranes naturally fuse.     

Evolutionary implications of developmental instability in parthenogenetic Drosophila mercatorum. II. Comparison of two strains with identical genotypes,but different modes of reproduction

Developmental instability is particularly pronounced in parthenogenetic strains of Drosophila mercatorum. All parthenogenetically produced eggs in a given strain have the same genotype, but even when reared in the same environment, only approximately 5% of the eggs initiating development ever reach adulthood. A sexual analogue of a parthenogenetic strain was created to investigate the basis of this developmental instability. The two strains have identical genotypes (except for the Y chromosome in males of the sexual strain) and differ only in mode of reproduction. The sexual strain had a much lower rate of developmental instability than the parthenogenetic strain, suggesting that the instability is caused by the mode of reproduction per se and is not due to homozygosity, disruption of coadapted gene complexes, or any other feature of the parthenogenetic genome. The increased rate of abortion with parthenogenetic reproduction is caused by a proportional increase in the normal panoply of errors that occur in sexual reproduction but at a much lower rate. Attempts to establish other sexual analogues of laboratory parthenogenetic strains revealed different male sterility factors within several parthenogenetic genomes that could potentially act to prevent hybridization with sexually reproducing ancestors during the incipient stages in the evolution of an entirely parthenogenetic lineage.     

Cytological Investigation of the Mechanism of Parthenogenesis in Drosophila mercatorum

Thelytokous parthenogenesis (female progeny only) in animals is believed to arise initially in unfertilized eggs produced by bisexual females via the fusion of two haploid nuclei following meiosis, to produce diploid female progeny. The transition from sexual to parthenogenetic mechanisms of reproduction requires that the egg replace the paternal contributions of a haploid genetic complement and the basal body, which is thought to be essential for centrosome formation. The transitional facultative parthenogenetic stage is usually associated with a high rate of failed or abortive development, but the molecular and mechanistic reasons for this failure remain unclear. We show that a facultatively parthenogenetic strain of Drosophila mercatorum produces a high percentage of unfertilized eggs competent to restore diploidy and form centrosomes de novo following meiosis. The female meiotic products replicate and divide by an acentrosomal mechanism in most oocytes and cytoplasmic centrosomes form in 35% of the oocytes. However, after pronuclear replication the cytoplasmic centrosomes must "capture" two haploid nuclei in order to restore diploidy. In practice, this process frequently fails due to centrosome-mediated capture events of single or more than two haploid nuclei, as well as multiple nuclear capture events in a single embryo when excess free centrosomes are not inactivated following formation of the first zygotic nucleus. Additionally, as development proceeds, many of the centrosomes that initiate syncytial development do not remain functional, possibly due to centrosome maturation defects, and later stages of syncytial development fail. The combined effect of the high error rate associated with nuclear capture and the failure of centrosome maturation during later developmental prevents successful parthenogenesis in most of the eggs that initiate development. This shows that the high rate of failed development associated with the transition from sexual to parthenogenetic reproduction is limited by the low probability of the formation of a diploid zygotic nucleus with the correct complement of centrosomes in D. mercatorum.     

Resource Availability as Driving Factor of the Reproductive Mode in Soil Microarthropods (Acari,Oribatida)

The availability of high quality resources is an important factor driving community structure and reproductive mode of animals. Parthenogenetic reproduction prevails when resources are available in excess, whereas sexuality correlates with resource shortage. We investigated the effect of resource availability on the community structure of oribatid mites in a laboratory experiment. Availability of food resources was increased by addition of glucose to leaf litter and reduced by leaching of nutrients from leaf litter. Experimental systems were incubated at three different temperatures to establish different regimes of resource exploitation. Community structure of oribatids and numbers of eggs per female were measured over a period of ten months. We expected the density of oribatid mites to decline in the reduced litter quality treatment but to increase in the glucose treatment. Both effects were assumed to be more pronounced at higher temperatures. We hypothesized sexual species to be less affected than parthenogenetic species by reduced resource quality due to higher genetic diversity allowing more efficient exploitation of limited resources, but to be outnumbered by parthenogenetic species in case of resource addition due to faster reproduction. In contrast to our hypotheses, both sexual and parthenogenetic oribatid mite species responded similarly with their densities declining uniformly during incubation. The parthenogenetic Brachychthoniidae and Tectocepheus dominated early in the experiment but were replaced later by parthenogenetic Desmonomata and Rhysotritia. In parthenogenetic species the number of eggs per female increased during the experiment while the number of eggs in sexual females remained constant or decreased slightly; in general, egg numbers were higher in sexual than in parthenogenetic species. The results indicate that for sustaining oribatid mite populations other resources than litter and associated saprotrophic microorganisms are needed. They also indicate that there are two groups of parthenogenetically reproducing species: exploiters of easily available resources and consumers of leaf litter associated resources.     

Starvation tolerance of rotifers produced from parthenogenetic eggs and from diapausing eggs: a life table approach

Monogonont rotifers have to cope with environmental variationby producing diapausing stages. Cyclically, parthenogeneticrotifers produce females by (i) parthenogenetic eggs and (ii)sexually produced eggs. Parthenogenetic eggs hatch shortly afterovoposition, frequently while they are carried by their mothers.Sexual eggs go into dormancy and hatch in the sediment, in responseto specific environmental cues. Therefore, it is expected thatpost-diapause and parthenogenetic offspring would face differentenvironments. Moreover, resource allocation is higher in diapausingeggs than in parthenogenetic eggs. In this study, the responseto starvation of females obtained from parthenogenetic eggsand from diapausing eggs of the rotifer Brachionus plicatiliswas compared. Starved individuals showed unexpectedly long maximumlongevity (12 days). The average lifespan and the lifespan varianceof individuals hatched from parthenogenetic eggs were higherthan those of individuals hatched from diapausing eggs. Contrastingwith a previous experimental finding, our results do not supportthe hypothesis that high resource allocation in diapausing eggscauses high starvation tolerance. Lower survival of individualshatched from diapausing eggs could be caused by the diversionof energy to other functions (i.e. maintenance during diapauseor the hatching event), or the result of the variance in theallocation of resources in parthenogenetic eggs. We hypothesizethat resource allocation in diapausing eggs follows a conservativepattern, while it is more opportunistic in parthenogenetic eggs. This paper was presented at Plankton Symposium III, held atFiguera da Foz, Portugal between 17 and 20 March 2005, underthe auspices of the University of Coimbra and the Universityof Aveiro, and coordinated by Mário Jorge Pereira andUlisses M. Azeiteiro.     

A novel procedure for obtaining denuded sea urchin eggs and observations on the role of the vitelline layer in sperm reception and egg activation

A procedure is described for the complete removal of the vitelline layer of the eggs of the sea urchin, Strongylocentrotus purpuratus. The method involves treatment of unfertilized eggs with an S. purpuratus cortical granule protease preparation followed by incubation in an alkaline dithiothreitol seawater solution. Eggs denuded of their vitelline layers react metabolically to parthenogenetic agents and sperm like unfertilized eggs, whereas the fertilizability of denuded eggs and receptivity to sperm is much less than controls. The present method is superior to previous methods using mercaptans in that all of the vitelline layer is removed and to procedures using other proteolytic enzymes in that no ¹²⁵I-labelled plasma membrane proteins are extensively modified. Thus the cortical granule protease dithiothreitol procedure is ideal for studies of the plasma membrane of the unfertilized egg and for studies on the role of the vitelline layer in normal fertilization and development.     

Physiological responses of sea urchin eggs to stimulation by calcium ionophore A23187 analysed with protease inhibitors

Protease inhibitors were used to study certain physiological responses (secretion of the cortical granule protease, altered resceptively to sperm penetration, initiation of cell division and embryogenesis) of sea urchin eggs to stimulation by calcium ionophore A23187. Protease activity in the secretory product released from the eggs 5 min after insemination or parthenogenetic activation with ionophore was completely inhibited by soybean trypsin inhibitor (SBTI), antipain (Ap), and leupeptin (Lp). A barrier was established to prevent subsequently added sperm from penetrating (fertilizing) ionophore-activated eggs, co-incident with the elevation of the fertilization membrane. These processes were retarded by inhibitors of the cortical granule protease in ionophore-activated eggs, just as they are when eggs are initially stimulated by sperm at fertilization. A23187-activated eggs did not divide unless they had been secondarily fertilized by sperm, even if the ionophore was subsequently removed by extensive washing. However, ionophore-activated eggs that were penetrated by a single spermatozoan in SBTI developed into normal larvae under similar conditions. These results suggest that A23187 may be an incomplete parthenogenetic agent because it cannot stimulate eggs to assemble centrioles required to organize the mitotic apparatus. The centrioles are normally provided by the sperm during fertilization. A23187 may also be toxic to the eggs. Furthermore, since cortical granules are secretory organelles, the data suggest a possible functional relationship between calcium ions and protease activation in stimulus-secretion coupling in sea urchin eggs at fertilization.     

The induction of de novo centrioles in sea urchin eggs: a possible common mechanism for centriolar activation among parthenogenetic procedures

Eggs from two species of sea urchins were subjected to a variety of novel two-step parthenogenetic activation procedures. These treatments readily resulted in the formation of cytasters and centrioles. Centrioles are restricted to the cytasters, as well as to the broad perinuclear zone, and it appears that all cytasters contain one or more centrioles. The mode of centriolar origin and maturation, as revealed by these new procedures, is identical to that of centrioles induced by other procedures. Both centriolar and cytastral development are retarded by the parthenogenetic treatments; also, mitosis is generally delayed. These results, along with those of others, reveal that parthenogenetic agents have opposing effects on eggs. The agents are stimulatory in that they activate latent centrioles and the cell cycle, in general. They are inhibitory in that they suppress a variety of physiochemical events. Of these events, only one appears to be common to all parthenogenetic agents: the inhibition of protein synthesis. Egg centrioles may be kept in a latent state by repressor proteins. If these proteins normally cycle rapidly, then an inhibition of their continuous synthesis will disrupt their normal replacement rate. As such, a delay in replacement will allow functioning repressor proteins, associated with latent centrioles, to become nonfunctional, which in turn allows latent centrioles to become derepressed. This deblocking reaction would then activate the appearance of de novo centrioles in eggs.     

CaMKII can participate in but is not sufficient for the establishment of the membrane block to polyspermy in mouse eggs

Fertilization triggers initiation of development and establishment of blocks on the egg coat and plasma membrane to prevent fertilization by multiple sperm (polyspermy). The mechanism(s) by which mammalian eggs establish the membrane block to polyspermy is largely unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) appears to be the key regulator of several egg activation events (completion of meiosis, progression to embryonic interphase, recruitment of maternal mRNAs). Since sperm-induced increases in cytosolic Ca(2+) play a role in establishment of the membrane block to polyspermy in mouse eggs, we hypothesized that CaMKII was a Ca(2+)-dependent effector leading to this change in egg membrane function. To test this hypothesis, we modulated CaMKII activity in two ways: activating eggs parthenogenetically by introducing constitutively active CaMKIIalpha (CA-CaMKII) into unfertilized eggs, and inhibiting endogenous CaMKII in fertilized eggs with myristoylated autocamtide 2-related inhibitory peptide (myrAIP). We find that eggs treated with myrAIP establish a less effective membrane block to polyspermy than do control eggs, but that CA-CaMKII is not sufficient for membrane block establishment, despite the fact that CA-CaMKII-activated eggs undergo other egg activation events. This suggests that: (1) CaMKII activity contributes to the membrane block, but this not faithfully mimicked by CA-CaMKII and furthermore, other pathways, in addition to those activated by Ca(2+) and CaMKII, also participate in membrane block establishment; (2) CA-CaMKII has a range of effects as a parthenogenetic trigger of egg activation (high levels of cell cycle resumption, modest levels of cortical granule exocytosis, and no membrane block establishment).     

Factors determining the frequency and pathways of the parthenogenetic development of ethanol-activated mouse oocytes

Ethanol activates the eggs inside the mother upon intraperitoneal, rather than intragastric, injection. The eggs are also activated and engaged into parthenogenetic development when the eggs or the whole oviducts with the ovulated eggs are placed in a culture medium with ethanol. The intensity of the activating effect of ethanol in vitro and ways of parthenogenetic development depend both on the ethanol concentration and temperature. At a temperature below 17 degrees ethanol did not activate the mouse eggs. There is a temperature optimum for each ethanol concentration studied (from 2 to 6.6%), at and ways of parthenogenetic development depend both on the ethanol concentration and the ability of parthenogenetic embryos to develop until the blastocyst stage are determined by the efficiency of activation and depend on the selection of optimal conditions for the action of ethanol. Cytochalasin B or D did not enhance the activating action of ethanol on the mouse eggs. The mechanisms of ethanol action on the eggs are discussed.     

Regulation of parthenogenetic activation of metaphase II mouse oocytes by pyruvate

We report that parthenogenetic activation (pronuclear formation) is induced during in vitro culture of recently ovulated (13-14 hr post-hCG) mouse oocytes in pyruvate deficient medium. Pronuclear formation occurred when oocytes were cultured in medium containing 1/10X (Pyr-) or lower concentrations of pyruvate but failed to occur either in oocytes cultured in the presence of 0.47 mM (1X, Pyr+) or 1/2X pyruvate or in oocytes cultured in the absence of pyruvate but with cumulus cells. Pronuclear formation was evident within 8 hr of culture and completed by 16 hr and remained intact during continuous culture in Pyr- medium. Transfer of pronuclear oocytes to Pyr+ medium resulted in pronuclear membrane disassembly and further parthenogenetic development. A similar incidence of parthenogenetic activation occurred when recently ovulated oocytes were cultured in the presence of cycloheximide but not following ethanol or hyaluronidase treatment. However, both ethanol and hyaluronidase induced pronuclear formation in in vivo aged oocytes. Results suggest that the type of activation induced varies with the age of the oocyte and the nature of the stimulus. Amino acid uptake ([35S]methionine) by oocytes was unaffected by Pyr- culture whereas incorporation into protein was markedly inhibited. Gel electrophoretic analysis of labeled egg extracts revealed a marked inhibition of egg protein synthesis after 4 hr of culture in Pyr-. The occurrence of a cortical reaction was monitored by binding of fluorescent labeled lectin to the oocyte surface. A cortical reaction occurred in response to ethanol treatment of freshly ovulated and in vivo aged oocytes cultured in Pyr+ medium but not in pronucleate oocytes induced by Pyr- culture. Suppression of ethanol-induced cortical reaction by Pyr- culture was restored following transfer of oocytes to Pyr+ medium. Results demonstrate that nuclear events as well as plasma membrane events can be simply regulated by controlling the amount of energy substrate available to the germ cell. Effects of Pyr- culture in inducing pronuclear formation appear to be mediated in a large part via inhibition of protein synthesis.