Mechanistic studies of the plasma membrane block to polyspermy in mouse eggs

The mechanisms responsible for the plasma membrane associated block to polyspermy in mouse eggs were studied. Reinsemination experiments using zona-free eggs indicated that, after fertilization, the egg plasma membrane is altered such that sperm binding to the egg plasma membrane is blocked, except in the region of the second polar body. Activation of the egg with either ethanol or strontium chloride did not result in a block to polyspermic penetration, as artificially activated eggs displayed identical penetration levels as to nonactivated control eggs. The penetrability of activated eggs was not altered by the presence or absence of the zona pellucida during activation. Lectin staining for egg cortical granule material indicated that activation did cause cortical granule exocytosis; however, activated eggs remained penetrable. These data support the following conclusions: (1) an alteration in the ability of the egg plasma membrane to allow sperm adherence accounts for the block to polyspermy; (2) establishment of the plasma membrane block to polyspermy is sperm dependent, since artificial egg activation does not result in a block response; (3) the contents of the egg's cortical granules do not play a role in the establishment of the plasmalemma block response. © 1993 Wiley-Liss, Inc.     

None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm-egg binding and fusion

Antibody inhibition and alpha6beta1 ligand binding experiments indicate that the egg integrin alpha6beta1 functions as a receptor for sperm during gamete fusion; yet, eggs null for the alpha6 integrin exhibit normal fertilization. Alternative integrins may be involved in sperm-egg binding and fusion and could compensate for the absence of alpha6beta1. Various beta1 integrins and alphav integrins are present on mouse eggs. Some of these integrins are also reported to be receptors for ADAMs, which are expressed on sperm. Using alpha3 integrin null eggs, we found that the alpha3beta1 integrin was not essential for sperm-egg binding and fusion. Oocyte-specific, beta1 integrin conditional knockout mice allowed us to obtain mature eggs lacking all beta1 integrins. We found that the beta1 integrin null eggs were fully functional in fertilization both in vivo and in vitro. Furthermore, neither anti-mouse beta3 integrin function-blocking monoclonal antibody (mAb) nor alphav integrin function-blocking mAb inhibited sperm binding to or fusion with beta1 integrin null eggs. Thus, function of beta3 or alphav integrins does not seem to be involved in compensating for the absence of beta1 integrins. These results indicate that none of the integrins known to be present on mouse eggs or to be ADAM receptors are essential for sperm-egg binding/fusion, and thus, egg integrins may not play the role in gamete fusion previously attributed to them.     

A sperm factor as the counterpart to the sperm-binding factor of the homologous eggs

A substance was isolated from sperm of the sea urchin, $\text{Hemicentrotus pulcherrimus}$. When unfertilized eggs of homologous species were pre-treated with this substance, they rapidly lost the fertilizability due to loss of the sperm-binding capacity. Such an effect was not exerted upon eggs either in CaMg free sea water or of heterologous species. This substance caused neither iso-agglutination of eggs nor precipitation of jelly coat. When it was pre-incubated with the sperm-binding factor purified from eggs of homologous species, it lost the fertilization-inhibiting effect on eggs. It seems very likely that a complementary relationship exists between the present substance and the sperm-binding factor.     

Role of the vitellus in the block to polyspermy in golden hamster eggs

The block to polyspermy in golden hamster eggs is believed to operate only at the zona pellucida. However, changes in the egg vitellus also prevent further entry of capacitated sperm. When zona-free hamster eggs spontaneously activated in vitro, and in vivo fertilized eggs at pronuclear stage were inseminated with capacitated human sperm, penetration did not occur. In the case of a homologous system using hamster sperm and in vivo fertilized hamster eggs, slight attachment of sperm was observed but no penetration. The cortical granules were found to be released in spontaneously activated and in fertilized eggs as observed by phase contrast microscopy. These observations suggest that the egg vitellus plays a role in the block to poiyspermy in addition to that of the zona block.     

Response of the sandfly Lutzomyia longipalpis to an oviposition pheromone associated with conspecific eggs

Response of female Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) to an oviposition attractant and/or stimulant associated with conspecific eggs was investigated in the laboratory. Females of two populations laid significantly higher mean number of eggs on sites with 160 eggs already present than on bare control sites. This response was lost when eggs placed on test sites were previously washed in organic solvents and distilled water. Age of eggs placed on test sites, 1-6 days after being laid, did not seem to affect the oviposition response of the females. Comparing effects of different numbers of eggs, positive responses were obtained when 80, 160 or 320 eggs were placed on test sites. No significant differences between mean egg numbers laid on test sites and controls were detected when batches of only 20 or 40 eggs were used.     

Adaptation of the Avian Egg to High Altitude

Theoretical predictions and experiments on eggs of domesticatedbirds indicate that the diffusion coefficient of gases is inverselyproportional to barometric pressure. Therefore potentially lethallosses of CO₂ and water vapor from eggs laid at high altitudemight result if the increased tendency of gases to diffuse atreduced barometric pressure were not counteracted in some manner.Limited data from two wild populations indicate that water lossfrom eggs is independent of altitude over a 3000 m elevationalgradient. Four different possibilities are discussed by whichcompensation for increased diffusion of water vapor might beachieved at high elevations 1) a reduction in eggshell conductance(GH₂O) 2) an increase in the initial water content of the eggs3) an increase in shell thickness, and 4) alteration of watervapor pressure in the nest microenvironment or incubation temperatureby variation in parental behavior. Mean GH₂O of eggs of twoprecocial and four altricial species breeding above 2800 m issignificantly reduced below values of related birds breedingat lower elevations, but no change in initial water contentor shell thickness has been observed in such eggs. Observationsof parental behavior in species breeding over wide elevationalgradients have not yet been made. Identification of the mechanismsby which eggshell structure is modified to achieve a reducedGH₂O the environmental cues used by females to determine theelevation of the breeding location and the rapidity with whichshell structuie can be modified awaits further research.     

Zona pellucida-mediated acrosomal exocytosis in mouse spermatozoa: characterization of an intermediate stage prior to the completion of the acrosome reaction

The zona pellucida (ZP)-induced acrosome reaction in mouse sperm proceeds in two steps, identified by three sperm fluorescence patterns observed sequentially with the fluorescent probe chlortetracycline. Capacitated, acrosome-intact sperm displaying a B pattern proceed to an intermediate S pattern, and then progress from the S pattern to the fully acrosome-reacted AR pattern. Previously, it was not feasible to characterize the nature of the transient intermediate S pattern. Recently, it was demonstrated that sperm bind to the ZP of eggs treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and undergo a B to S transition, but do not complete the acrosome reaction. These cells accumulate in the S pattern and fail to undergo the S to AR transition (Endo, Y., Schultz, R. M., and Kopf, G. S. 1987a. Dev. Biol. 119, 119-209). The present study utilized ZP from TPA-treated eggs to assess the state of S pattern sperm. The kinetics of the B to S transition of sperm incubated with either structurally intact or solubilized ZP from untreated or TPA-treated eggs are identical. Addition of either solubilized ZP from untreated eggs or A-23187 to S pattern sperm bound to intact or solubilized ZP from TPA-treated eggs induces the S to AR transition, while ZP from TPA-treated or fertilized eggs does not. Loss of the transmembrane pH gradient in the anterior portion of the sperm head, monitored by the fluorescent pH probe 9-N-dodecyl aminoacridine, follows the B to S transition in sperm incubated with ZP from unfertilized eggs, but no loss is observed when the B to S transition is induced using ZP from TPA-treated eggs. Subsequent addition of solubilized ZP from untreated eggs or A-23187 results in the loss of the transmembrane pH gradient of these S pattern sperm. Addition of nigericin to S pattern sperm bound to ZP from TPA-treated eggs discharges the transmembrane pH gradient and causes the S to AR transition. In contrast, nigericin added to B pattern sperm discharges the pH gradient but does not induce a B to S transition. Electron microscopic evaluation of S pattern-arrested sperm using ZP from TPA-treated eggs reveals intact plasma and outer acrosomal membranes. These results suggest that ZP from TPA-treated and fertilized eggs are modified such that the ZP ligands inducing the S to AR transition are lost or are inactivated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resistance of Apanteles eggs to the haemocytic encapsulation by their habitual host, Pieris

The calyx fluid in the lateral oviduct of a gregarious parasitoid, Apanteles glomeratus contained ellipsoid particles of ca. 130 × 200 nm. These calyx fluid particles did not appear to be embedded in a fibrous outer layer on the surface of eggs in the lateral oviduct. They were not observed on the surfaces of the eggs 3 to 4 hr after being deposited into the host haemocoele. Oviposition experiments indicated that the occurrence of haemocytic defence reactions of the late 2nd instar larvae of the Pieris rapae crucivora against 1 st instar larvae of the parasitoid increased with a decreasing number of the parasitoid eggs introduced into a host, and that more than 5 to 9 parasitoid eggs were needed for suppressing the ability of the host to encapsulate its parasitoid larvae immediately after hatching. When eggs with calyx fluid obtained from egg reservoir were injected into the host, they were found to be encapsulated 1 to 2 days after the injection. They could not start their embryonic development. When calyx fluid-free 3-hr-old eggs were injected in a number of more than 5 eggs into a 5th instar larva of Pieris, 58% of 31 eggs injected had normally hatched without evoking encapsulation reactions by the host. Both electron microscopic observations of parasitoid eggs in the host haemocoele and the experimental results suggested that calyx fluid or calyx fluid particles of the parasitoid might not be involved in the encapsulation-inhibiting activity of the parasitoid eggs. Rather it was anticipated that a substance (or substances) might be secreted by the parasitoid eggs into the haemocoele of the host, which suppressed defence reactions of the host.     

Human sperm do not bind to rat zonae pellucidae despite the presence of four homologous glycoproteins

The specificity of sperm-egg recognition in mammals is mediated primarily by the zona pellucida surrounding ovulated eggs. Mouse sperm are quite promiscuous and bind to human eggs, but human spermatozoa will not bind to mouse eggs. The mouse zona pellucida contains three glycoproteins, ZP1, ZP2, and ZP3, which are conserved in rat and human. The recent observation that human zonae pellucidae contain a fourth protein raises the possibility that the presence of four zona proteins will support human sperm binding. Using mass spectrometry, four proteins that are similar in size and share 62-70% amino acid identity with human ZP1, ZP2, ZP3, and ZP4/ZPB were detected in rat zonae pellucidae. However, although mouse and rat spermatozoa bind to eggs from each rodent, human sperm bind to neither, and the presence of human follicular fluid did not alter the specificity of sperm binding. In addition, mutant mouse eggs lacking hybrid/complex N-glycans or deficient in Core 2 O-glycans were no more able to support human sperm binding than normal mouse eggs. These data suggest that the presence of four zona proteins are not sufficient to support human sperm binding to rodent eggs and that additional determinants must be responsible for taxon-specific fertilization among mammals.     

Egg transport in the coelom of the newt cynops pyrrhogaster. I. The ovulated egg is transported to the ostium by the ciliary movement of coelomic epithelial cells

We investigated the mechanism of egg transport in the newt not only by inserting various conditioned eggs into the recipient's body but also by placing them on the coelomic epithelia of the opened body cavity in the adult female newt. Most of the inserted coelomic eggs were oviposited, while 4 of 14 inserted de-jellied uterine eggs and 3 of 10 inserted de-jellied fertilized eggs were oviposited. The coelomic eggs placed on the coelomic epithelia were transported toward the ostium and entered the ostium. The de-jellied uterine eggs and the de-jellied fertilized eggs were transported to the ostium as well. Of all the eggs examined, the coelomic egg was transported the fastest. The transport speeds of coelomic eggs treated with periodic acid and the speed of boiled coelomic eggs were less than those of untreated coelomic eggs. In contrast, the transport speeds of coelomic eggs treated with trypsin and the speed of coelomic eggs removed from their vitelline envelopes (naked eggs) were faster than those of untreated coelomic eggs. Other experiments were carried out in order to ascertain the dependence of sexual activity on egg transport. The speed of coelomic egg transport in artificially sexually activated females was faster than in sexually inactive females, although the ciliary movement could always be observed in both sexually active females and sexually inactive females. This suggests that the speed of egg transport on the coelomic epithelia is controlled by the sexual activity of the female.     

Tolerance Limit of the Sugarbeet to Heterodera schachtii

Field and greenhouse experiments showed that yield losses of sugarbeet, Beta vulgaris, did not occur in soil infested with fewer than eight Heterodera schachtii eggs/g soil. However, larger population densities greatly reduced sugarbeet yield. In the field experiment, the yield in microplots inoculated with more than 64 eggs/g soil was less than 20% of yields in uninoculated microplots. Nevertheless, tolerance limits of 4 and 1.8 eggs/g soil, in greenhouse and field microplots, respectively, were derived by fitting the data with the equation y =m + (l - m)z^P-T. Maximum rates of multiplication of 55 and more than 300, and equilibrium densities of 340 and 130 eggs/g soil, were estimated in greenhouse and field microplot tests, respectively.     

The Chaetopterus vitelline envelope is not necessary for the gamete interactions that lead to fertilization

It has been recently shown that, in several genera of annelids, including Chaetopterus, fertilizing sperm attach to and fuse with egg microvilli which penetrate the vitelline envelope. This suggests that the annelid vitelline envelope may have no direct or obligatory role in normal fertilization. The present study was undertaken to investigate the involvement of the vitelline envelope in fertilization in Chaetopterus experimentally, by examining the fertilization of vitelline envelope-free eggs quantitatively and qualitatively. Brief exposure of the eggs to isotonic sucrose-EDTA removed the vitelline envelope as determined by both phase-contrast and electron microscopy, rendered the eggs more sensitive to polyspermy and substantially reduced the binding of supernumerary sperm to eggs but did not decrease fertilizability as determined by sperm dilution assay and did not make the eggs more sensitive to cross-fertilization. The events of fertilization were examined by electron microscopy and found to be very similar in vitelline envelope-free eggs to those in intact eggs. We conclude that the vitelline envelope in Chaetopterus has binding sites for sperm but that it has no obligatory role in fertilization and is primarily involved in the prevention of polyspermy.     

Two pathways from glycogen to glucose-6-phosphate in sea urchin eggs with special reference to the difference between the species in the contribution ratio of each pathway

In the unfertilized eggs of the sea urchins, Anthocidaris crassispina, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, Mespilia globulus, Temnopleurus toreumaticus, Toxopeneustes pileolus, and Clypeaster japonicus, the activities of phosphorylase [EC 2.4.1.1], phosphoglucomutase [EC 2.7.5.1], exo-l,4-α-glucosidase [EC 3.2.1.3], and hexokinase [EC 2.7.1.1] are very similar. In all species, only phosphorylase activity is higher in fertilized eggs than in unfertilized eggs. The concentrations of glycogen, glucose, GIP, G6P, ATP, ADP, and Pi; the products and substrates in reactions catalyzed by these enzymes, were measured in these eggs. Based on the concentrations of these compounds in the eggs, it is assumed that G6P is produced by the combined action of glucosidase and hexokinase in all species examined, and that it is also produced in the reaction catalyzed by phosphorylase and phosphoglucomutase in all species except A crassispina and P depressus. Glycogen was found both in supernatant and in precipitate fractions, which were obtained by adding perchloric acid. Glycogen in the precipitate seems to be protein-bound. Whole glycogen level in the eggs is almost the same in all species examined, but the level of acid-soluble glycogen, as well as GIP, is markedly lower in the eggs of A crassispina and P depressus than in the eggs of other species examined. Protein-bound glycogen is utilized by glucosidase activity but not by phosphorylase activity, in contrast to acid-soluble glycogen, which is utilized by both enzyme activities. Hence, it is assumed that the failure of G6P production by phosphorylase and phosphoglucomutase-in A crassispina and P depressus eggs is due to a low level of acid-soluble glycogen in these 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).     

Female Pied Flycatchers fail to respond to variations in nest humidity

We examined the possibility that a cavity-nesting passerine, the Pied Flycatcher (Ficedula hypoleuca), can modify the permeability of its eggs to water vapor facultatively in response to variations in nest humidity (P(N)). We found no significant relationship between the water-vapor conductance (G(E)) of flycatcher eggs and P(N). In addition, incubating female flycatchers failed to change their patterns of incubation when we modified P(N) experimentally. These data, coupled with large variations that occur in the G(E) of the eggs within individual clutches, suggest that Pied Flycatchers do not alter the structure of their eggs as they lay them and may not be aware of differences in P(N). It is more likely that G(E) is determined genetically and has been established by natural selection for eggs with high degrees of hatchability.     

A comparison of in vitro and in vivo estimates of the viability of Taenia pisiformis eggs aged under controlled conditions, and their ability to immunise against a challenge infection

Storing the eggs of T. pisiformis for periods of 0 to 9 days at a temperature of 36–38°C and relative humidity of 87–93 % revealed at least 4 stages in the ageing process. Firstly, a stage in which eggs hatched and activated in vitro and were also fully infective for rabbits. Secondly, a stage in which eggs hatched and activated in vitro but underwent only partial development in rabbits. Thirdly, a stage in which eggs hatched and activated in vitro, and presumably therefore in vivo, but no evidence of infection could be found when they were fed to rabbits. Fourthly, a stage in which eggs either did not hatch, or if they did hatch, the oncosphere was rapidly digested. In vitro techniques for assessing viability of T. pisiformis eggs were not a reliable guide to their infectivity for rabbits. ‘Senescent’ eggs, that is eggs which produced evidence of early infection in rabbits but did not complete their development to cysticerci, failed to produce immunity to challenge infection with T. pisiformis eggs.     

A saltwater flotation technique to identify unincubated eggs

ABSTRACT.   Field studies on nesting birds sometimes involve questions related to nest initiation dates, length of the incubation period, or changes in parental incubation behavior during various stages of incubation. Some of this information can be best assessed when a nest is discovered before the eggs have undergone any incubation, and this has traditionally been assessed by floating eggs in freshwater. However, because the freshwater method is not particularly accurate in identifying unincubated eggs, we developed a more reliable saltwater flotation method. The saltwater method involves diluting a saturated saltwater solution with freshwater until a salt concentration is reached where unincubated eggs sink to the bottom and incubated eggs float to the surface. For Laughing Gulls ( Leucophaeus atricilla ), floating eggs in freshwater failed to identify 39.0% ( N = 251) of eggs that were subsequently found by candling to have undergone incubation prior to collection. By contrast, in a separate collection of gull eggs, no eggs that passed the saltwater test ( N = 225) were found by a later candling to have been incubated prior to collection. For Double-crested Cormorants ( Phalacrocorax auritus ), floating eggs in freshwater failed to identify 15.6% ( N = 250) of eggs that had undergone incubation prior to collection, whereas in a separate collection, none of the eggs that passed the saltwater test ( N = 85) were found by a later candling to have been incubated prior to collection. Immersion of eggs in saltwater did not affect embryo survival. Although use of the saltwater method is likely limited to colonial species and requires calibrating a saltwater solution, it is a faster and more accurate method of identifying unincubated eggs than the traditional method of floating eggs in freshwater.     

Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro

The question of whether the acrosome reaction, which leads to fertilization, occurs in intact sperm bound to the zona pellucida of the egg or in intact sperm before contact with the egg, was addressed by assessing the effect of 3-quinuclidinyl benzilate (QNB) on the two types of acrosome reaction. QNB is a specific inhibitor of the fertilization of zona-intact mouse eggs by mouse sperm. Mouse spermatozoa in suspension underwent acrosome reactions at a low rate, which could be accelerated by addition of 5 μM divalent cation ionophore A23187; the occurrence of such acrosome reactions was not inhibited by QNB. The rate at which acrosome reactions occurred in sperm bound to the zona pellucida of cumulus-free eggs, bound to isolated zonae, or exposed to acid-solubilized zona components, was greatly accelerated relative to that observed in the absence of zonae. These acrosome reactions were strongly inhibited by QNB at concentrations which inhibit the fertilization of zona-intact mouse eggs in vitro. These data suggest that the zona pellucida can induce acrosome reactions in mouse spermatozoa and that these acrosome reactions are the ones which lead to the fertilization of zona-intact eggs. In contrast, the acrosome rection in sperm which are not in contact with the zona is not associated with fertilization of zona-intact eggs.     

Bacterial Infection of Washed and Unwashed Eggs with Reference to Salmonellae

A survey of eggs in the Fraser Valley area of British Columbia was conducted to determine the level of bacterial contamination on washed and unwashed shell eggs and the incidence of Salmonella infection in shell eggs. Samples of eggs were taken from 15 grading stations. Determinations of eggs from 87 shipments showed that a higher proportion of eggs was heavily contaminated (>5-million organisms per egg) after washing than as received at the grading stations. This finding suggests the need for the development of more satisfactory washing procedures. No salmonellae were detected in the 180 samples (3,995 eggs) which were examined. It was concluded that Salmonella contamination of intact shell eggs does not constitute a serious health hazard in eggs from this area.