The distribution of polymerized actin in the rat egg and its sensitivity to cytochalasin B during fertilization

The distribution of polymerized actin in rat eggs fertilized in vitro was determined using NBD-phallacidin (NBD-ph). Unfertilized and fertilized eggs exhibited a 3-5-micron-thick band of fluorescence that encompassed the entire cortical cytoplasm. There was no dramatic increase in the staining of the cortex in association with any component of the fertilizing sperm during its incorporation into the egg. Unfertilized eggs and fertilized eggs obtained at intervals after sperm-egg fusion were treated with cytochalasin B (CB; 5 micrograms/ml) and subsequently stained with NBD-ph. Unfertilized eggs treated with CB exhibited a continuous ring of cortical staining identical to that seen in untreated eggs. Eggs treated with CB 15 min after sperm-egg fusion exhibited small gaps in the cortical staining pattern, whereas those exposed to CB 1 hr after fusion exhibited larger gaps and the staining pattern appeared punctate. This pattern could be seen throughout the remainder of the 7 hr period of sperm incorporation and for at least 13 hr thereafter. CB-treated fertilized eggs that were washed to remove the drug again exhibited uninterrupted cortical staining on treatment with NBD-ph. CB also induced the resorption of surface elevations that are normally seen on the eggs during sperm incorporation, but it did not affect the morphology of unfertilized eggs. The sensitivity to CB during fertilization coincides with the onset of a variety of egg shape changes that occur during the period of sperm incorporation (Battaglia and Gaddum-Rosse, Gamete Res., 10:107-118, 1984a).(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and role of microfilaments during early events of sheep fertilization

The distribution of actin was studied during early events of sheep fertilization by fluorescence microscopy after staining with 7-nitrobenz-2-oxal-1.3 diazole (NBD)-phallacidin and anti-actin antibody and by electron microscopy after heavy meromyosin labelling. Unfertilized and fertilized eggs exhibited a continuous band of fluorescence with both NBD-phallacidin and anti-actin antibody. Unlike in mice, no high concentration of actin overlying the spindle was detected in ovulated sheep oocytes. At the site of sperm head incorporation, the fertilization cone developed above the decondensing male chromatin and was underlined by a submembranous area rich in microfilaments. A similar actin network was observed in the cortex of the second polar body. Cytochalasin D was used to investigate the role of actin during the fertilization process. This drug did not prevent sperm fusion and incorporation but inhibited polar body abstriction and fertilization cone development and retarded sperm tail incorporation. Moreover, in the presence of the drug, the anchorage of the metaphase II spindle at the surface of the egg was destroyed. The role of microfilaments in these early events is discussed.     

Effects of motility inhibitors during sea urchin fertilization : Microfilament inhibitors prevent sperm incorporation and restructuring of fertilized egg cortex, whereas microtubule inhibitors prevent pronuclear migrations

The sensitivity of specific stages of fertilization to microfilament inhibitors (cytochalasins B (CB), D (CD), and E (CE) and phalloidin) and to inhibitors of microtubule assembly (colcemid (CMD), colchicine (CLC), griseofulvin (GSF), maytansine (MAY), nocodazole (NCD), podophyllotoxin (PDP), and vinblastine (VB)) was investigated using differential interference contrast, time-lapse video microscopy of the sea urchin Lytechinus variegatus. Cytochalasins (CDCE>CB) will prevent sperm incorporation if added prior to or simultaneous with insemination. Sperm-egg fusion and the cortical reaction appear normal, but then the subsequent elevation of the fertilization coat lifts and eventually detaches the ‘fertilizing’ sperm from the egg plasma membrane. When the cytochalasins are added after fusion, the forming fertilization cone is rapidly resorbed, and the lateral displacement of the sperm along the egg cortex is terminated; the pronuclear migrations and mitoses occur normally though cytokinesis is never observed. Cytochalasin treatment before or within 2 min of insemination results in the development of aberrant egg cortices, whereas cytochalasin treatments after 2 min post-fusion have little effect. Phalloidin results in large and long-lasting fertilization cones and a retardation of the rate of sperm incorporation. Eggs exposed to any of the microtubule inhibitors 15 min prior to insemination will incorporate the spermatozoon, though the formation of the sperm aster and the accompanying pronuclear migrations are prevented. Interestingly, the final stage of sperm incorporation involving a lateral displacement of the sperm along the egg cortex is greater (27.1 vs 12.4 μm in controls) and faster (5.4 vs 3.5 μm/min in controls) in microtubule-inhibited eggs. GSF and VB, which readily permeate fertilized eggs, will prevent the formation of the sperm aster if added 3 min after sperm-egg fusion, they will prevent the migration of the female pronucleus if added 5 or 7 min after sperm-egg fusion, pronuclear centration if added 10 min post-fusion, and syngamy if added 12 min post-fusion. CLC- or CMD- treated eggs will develop normally if these drugs are photochemically inactivated with 366 nm light within 4 min post-fusion, arguing that sperm incorporation is completely independent of assembling microtubules. These results indicate that microfilament inhibitors will prevent sperm incorporation and the restructuring of the fertilized egg cortex, and that microtubule inhibitors will prevent the formation and functioning of the sperm aster during the pronuclear migrations; an interplay between cortical microfilaments and cytoplasmic microtubules appears required for the successful completion of fertilization.     

Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342

When unfertilized sea urchin eggs are pretreated with the bisbenzimide DNA-specific fluorochrome Hoechst 33342, then washed and fertilized, a single sperm bound to the egg surface becomes intensely fluorescent. The location of the fluorescent sperm on the egg surface coincides exactly with the epicenter of the cortical reaction and the site at which the insemination cone subsequently appears. These observations, coupled with studies of eggs treated with quercetin to prevent fusion, as well as eggs made polyspermic by halothane exposure, indicate that the sperm acquires fluorescence as a consequence of fusion with the fluorochrome preloaded egg. Using a modification of this technique, we have found that cytoplasmic continuity between the sperm and egg is established at 4-8 sec after the onset of the sperm-induced conductance increase in the egg.     

A cell surface block to polyspermy occurs in golden hamster eggs

We have examined the frequency and fate of supernumerary sperm in the perivitelline space (PVS) of in vitro fertilized hamster eggs to determine if there is a cell surface block to polyspermy. The zona pellucida block to polyspermy is very effective since only one sperm penetrated the zona pellucida in 72.8% of the 876 fertilized eggs examined. Of the polypenetrated eggs, 41.6% had a supernumerary sperm within the PVS. The proportion of polypenetrated eggs with PVS sperm did not change when the duration of coincubation was increased from 3 to 6 hr. PVS sperm were found in 67% of the inseminations. From these data we conclude that there is a cell surface block to polyspermy in the hamster. To investigate the mechanism of the cell surface block, we used the Hoechst-transfer technique (R. Hinkley, B. Wright, and J. Lynn, 1986, Dev. Biol. 118, 148-154) to monitor sperm-egg fusion. We first demonstrated that dye transfer from zona pellucida-free eggs to sperm only occurred when fusion was possible, i.e., in the presence of calcium, and that dye was transferred to all fused sperm. When cumulus-free, zona-intact eggs were preloaded with Hoechst dye and viewed 3 hr postinsemination, three classes of eggs with supernumerary sperm in the PVS were observed: eggs with only Hoechst-positive sperm (62%), eggs with only Hoechst-negative sperm (27%), and eggs with both a Hoechst-positive and a Hoechst-negative sperm (11%). Because of the limited time resolution of the Hoechst-transfer technique, the cell surface block could operate by preventing sperm fusion (Hoechst-negative), by the failure of the eggs to incorporate fused sperm (Hoechst-positive), and/or by the "unfusing" of fused sperm (Hoechst-positive and Hoechst-negative). We are unable at this time to differentiate between these mechanisms.     

Observation on the incorporation cone in the rat

At the time of in vivo sperm–egg fusion in the rat, a small region of the oolemma under the head of the fertilizing sperm is observed to be free of microvilli. The microvilli-free region increases in area, and by one hour after sperm–egg contact extends over an area 20–30 μ in circumference and bulges out to form an “incorporation cone” visible by light microscopy. The microvilli-free incorporation cone reaches its maximum size at about two hours after sperm–egg interaction. It soon becomes smaller and has disappeared three to four hours after sperm–oocyte fusion. The cone cytoplasm is characterized by a 0.1 μ zone of thin filaments below the plasma membrane. Cytochalasin-B, 2.5 μg/ml, prevents formation of the cone or destroys the intact cone. It is suggested that micro filaments may be involved in the formation of the incorporation cone.     

Mammalian sperm-egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion.

Rat epididymal protein DE is localized on the fusogenic region of the acrosome-reacted spermatozoa and has a potential role in sperm-egg fusion. We investigated the presence of DE binding sites on the egg surface by co-incubating zona-free eggs and capacitated sperm in different concentrations of pure DE. Results indicate that DE produced a concentration-dependent decrease in egg penetration by sperm (fusion), with almost complete inhibition at 200 micrograms/ml. This inhibition was not due to an effect of DE on initial sperm binding to the egg membrane, since the presence of this protein did not affect the percentage of oocytes with bound sperm nor the number of bound sperm per egg. Those sperm that failed to penetrate the egg in the presence of DE became able to do so after transfer of the eggs to protein- and sperm-free medium, indicating a role for DE in an event subsequent to binding and leading to fusion. Indirect immunofluorescence using a polyclonal antibody against DE revealed a patchy labeling over the entire egg surface, with the exception of the area overlying the second metaphase spindle. This conclusion was supported by the disappearance of the DE-negative area on the fertilized egg. Zona-free eggs, incubated with DE at 4 degrees C or fixed before exposure to DE, displayed a uniform staining, suggesting that the patchy labeling resulted from aggregation of DE binding sites by the purified protein. The aggregation of these egg components may represent a necessary step of the fusion process. To our knowledge, this is the first study reporting the existence and localization of complementary sites to a specific sperm protein on the plasma membrane of the mammalian egg.     

Scanning electron-microscopical and other observations of sperm fertilization reactions in Limulus polyphemus L. (Merostomata: Xiphosura).

Sperm fertilization reactions of Limulus polyphemus were examined by scanning electron and/or light microscopy. The following were considered: sperm motility, attachment of sperm to egg, acrosome reaction, and penetration of the acrosomal filament. The spermatozoa after semination are non-motile and become active only in close proximity to a defined region surrounding the egg. Egg materials diffusing into this region induce sperm motility and stimulate large numbers of spermatozoa to move towards the egg surface. Each sperm initially attaches by the apical tip and undergoes the acrosome reaction which causes a more permanent secondary attachment by the adhesion of acrosomal contents to the egg surface. The acrosome reaction also initiates the penetration of the acrosomal filament through the egg envelope, an event occurring in 70-80% of the attached spermatozoa (about 10(6). Shortly after this penetration, a secondary reaction occurs which involves a spiralling of the flagellum and an incorporation into the sperm body of the flagellar fibrous components, which then become closely apposed to the sperm nucleus. These sperm fertilization reactions were performed or initiated with 0-34 M CaCl2 in whole eggs, egg sections, excised egg envelopes and/or the outer basement lamina of the egg envelope. The Limulus fertilization system is very valuable since sperm reactions can be examined biochemically, which may lead to a better understanding of the chemical mechanisms involved in sperm-egg interactions in all animal species.     

Fusion of hamster and pig zona-free eggs stimulated by boar and guinea pig sperm at fertilization in vitro

In the course of in vitro fertilization of zona-free hamster and pig eggs by boar and guinea-pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4–4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 10⁴ per ml and an optimum was attained at a concentration of 5.0 × 10⁵ per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm-egg fusion, or by products released during the sperm acrosome reaction.     

Electron microscopic observations on sperm entry and pronuclear formation in naked eggs of the rose bitterling in polyspermic fertilization

Unfertilized eggs of the rose bitterling (Rhodeus ocellatus ocellatus) were squeezed out of females that had an elongated ovipositor and were dechorionated mechanically with fine forceps in physiological saline. The dechorionated eggs were transferred into fresh water then inseminated at once by spermatozoa of the same species. A large number of spermatozoa was found on the surface of eggs that had not yet had cortical reaction following insemination. The surface of the naked eggs responded by formation of many small cytoplasmic protrusions (viz., fertilization cones) at sperm attachment sites. The formed fertilization cones were rosettelike structures formed by the aggregation of some bleblike swellings devoid of microvilli and microplicae. About 10 min after insemination, the fertilization cones retracted, but marks of their presence characterized by less microvilli and microplicae remained in the eggs 15 min after insemination. Many spermatozoa penetrated into the cytoplasm of each naked egg. The sperm nuclear envelope disappeared by means of vesiculation resulting from fusion of the inner and outer membranes. The sperm nucleus decondensed and developed into a larger male pronucleus. Smooth-surfaced vesicles surrounded the decondensing sperm nucleus and formed the new male pronuclear envelope. Sperm mitochondria and flagella were found in the egg 15 min after insemination. The response of the egg surface to sperm entry and pronucleus formation are discussed.     

Scanning electron microscope studies of sperm incorporation into the zebrafish (Brachydanio) egg

Morphological studies on the gametes and entry of the spermatozoan into the egg of the zebra danio, Brachydanio rerio, were conducted primarily with scanning electron microscopy. The spermatozoan showed a spherical head, which lacked an acrosome, a midpiece containing several mitochondria, and a flagellum. Observations of the unfertilized egg confirmed and extended prior studies showing a distinct cluster of microvilli on the plasma membrane, identified as the sperm entry site, beneath the inner micropylar aperture (Hart and Donovan, '83). The fertilizing spermatozoan attached to the sperm entry site within 5 seconds of the mixing of a gamete suspension. Binding to the egg microvilli appeared restricted to the equatorial surface of the spermatozoan. Fusion between the plasma membranes of the interacting gametes was followed by the formation of a distinct, nipple-shaped fertilization cone. The sperm head was partially incorporated into the fertilization cone cytoplasm by 60 seconds postinsemination. The incorporation of the entire sperm head, midpiece, and a portion of the flagellum occurred between 1 and 2 minutes. During this time, the fertilization cone shortened and was transformed into a massive, blister-like cytoplasmic swelling. Concurrently, upward movements of the ooplasm resulted in the gradual disappearance of the original depression in the egg surface containing the sperm entry site. The second polar body, fully developed by 10 minutes postinsemination, formed approximately 10-15 microns from the site of sperm penetration. Development of the fertilization cone, formation of the second polar body and exocytosis of cortical granules at the sperm entry site readily occurred in parthenogenetically activated eggs, indicating that these surface rearrangements do not require sperm binding and/or fusion.     

CHANGES IN THE SPERMATOZOON DURING FERTILIZATION IN HYDROIDES HEXAGONUS (ANNELIDA) : II. Incorporation with the Egg

This, the last of a series of three papers, deals with the final events which lead to the incorporation of the spermatozoon with the egg. The material used consisted of moderately polyspermic eggs of Hydroides hexagonus, osmium-fixed at various times up to five minutes after insemination. The first direct contact of sperm head with egg proper is by means of the acrosomal tubules. These deeply indent the egg plasma membrane, and consequently at the apex of the sperm head the surfaces of the two gametes become interdigitated. But at first the sperm and egg plasma membranes maintain their identity and a cross-section through the region of interdigitation shows these two membranes as a number of sets of two closely concentric rings. The egg plasma membrane rises to form a cone which starts to project into the hole which the spermatozoon earlier had produced in the vitelline membrane by means of lysis. But the cone does not literally engulf the sperm head. Instead, where they come into contact, sperm plasma membrane and egg plasma membrane fuse to form one continuous membranous sheet. At this juncture the two gametes have in effect become mutually incorporated and have formed a single fertilized cell with one continuous bounding membrane. At this time, at least, the membrane is a mosaic of mostly egg plasma membrane and a patch of sperm plasma membrane. The evidence indicates that the fusion of the two membranes results from vesiculation of the sperm and egg plasma membranes in the region at which they come to adjoin. Once this fusion of membranes is accomplished, the egg cytoplasm intrudes between the now common membrane and the internal sperm structures, such as the nucleus, and even extends into the flagellum; finally these sperm structures come to lie in the main body of the egg. The vesiculation suggested above appears possibly to resemble pinocytosis, with the difference that the vesicles are formed from the plasma membranes of two cells. At no time, however, is the sperm as a whole engulfed and brought to the interior of the egg within a large vesicle.     

Analysis of the role of egg integrins in sperm-egg binding and fusion

Sperm-egg fusion is believed to be mediated via specific molecular interactions. Integrin alpha6beta1 is a strong candidate for a sperm receptor on the egg plasma membrane. However, the ability of the egg integrin alpha6beta1 to interact with molecules on intact sperm has not yet been proven. In this report, possible involvement of integrin alpha6beta1 in sperm-egg interactions was examined by biochemical and immunocytochemical analyses. To identify egg molecules that specifically interact with sperm, we first incubated sperm with biotin-labeled egg surface proteins. Under this condition, solubilized proteins from eggs inhibited sperm-egg fusion. Western blot analysis under reducing conditions indicated that a major-labeled band of 135 kDa bound to sperm. An immunodepletion experiment using the anti-integrin alpha6 antibody GoH3 indicated that the 135 kDa egg surface molecule that bound to sperm was the integrin alpha6 subunit. To investigate the potential involvement of integrin alpha6beta1 in sperm-egg fusion, we next examined the localization of integrin alpha6 and beta1 subunits before and after fertilization by confocal laser microscopy. At an early stage of sperm-egg fusion, the integrin alpha6 and beta1 subunits were accumulated at the sperm binding site. The frequency of cluster formation was closely related to that of sperm-egg fusion, indicating that integrin receptors are accumulated by sperm destined for fusion. Taken together, these results strongly suggest that the integrin alpha6beta1 is involved in sperm-egg binding leading to fusion via direct association of the integrin alpha6 with sperm.     

Selective Identification of Sperm Fused with the Surface of Echinoderm Eggs by DNA-Specific Bisbenzimide (Hoechst) Fluorochromes

When unfertilized echinoderm eggs are treated with the DNA-specific bisbenzimide fluorochrome Hoechst 33342 and then fertilized with unlabeled sperm, a single spermatozoan bound to the egg surface becomes fluorescent. Several lines of evidence, including correlative scanning electron microscopic studies, indicate that the fluorescent sperm is, in fact, the fertilizing sperm which acquires fluorescence as a consequence of membrane fusion between the sperm and egg. Comparative studies show that several fluorochromes structurally related to H33342 can be used to selectively identify the fertilizing sperm at the egg surface and that H33258 possesses a distinct advantage when used to visualize the male and female pronuclei in eggs fixed prior to fluorochrome exposure. Finally, none of the fluorochromes tested in these studies have any discernible effect on development from the first cell division through the pluteus larva stage. These observations suggest that the fluorochrome-transfer technique for identifying the fertilizing sperm may be useful in a wide variety of studies of gamete interaction as a simple and rapid cytological indicator for sperm-egg fusion.     

Morphological changes in the oocyte and its surrounding vestments during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata

This light and transmission electron microscopical study shows that the first polar body is given off before ovulation and that part of its cell membrane and that of the surrounding oocyte have long microvilli at the time of its ejection. Several layers of cumulus cells initially surround the secondary oocyte and first polar body, but the ovulated oocytes in the oviducts in the process of being fertilized do not have cumulus cells around them. Partly expelled second polar bodies occur in the oviduct; they are elongated structures that lack organelles and have electron-dense nuclei. A small fertilization cone appears to form around the sperm tail at the time of sperm entry into the egg and an incorporation cone develops around the sperm head in the egg cytoplasm. In three fertilized eggs a small hole was seen in the zona, which was presumably formed by the spermatozoon during penetration. Cortical granules, present in ovarian oocytes, are not seen in fertilized tubal or uterine eggs; release of their contents probably reduces the chances of polyspermy, although at least one polyspermic fertilized egg was seen and several other fertilized eggs had spermatozoa within the zona pellucida. In the zygote the pronuclei come to lie close together, but there was no evidence of fusion. A "yolk mass," which becomes eccentric before ovulation, is extruded by the time the two-cell embryos are formed, but many vacuoles remain in the non-yolky pole of the egg. A shell membrane of variable thickness is present around all uterine eggs but its origin remains undetermined.     

The movements and fusion of the pronuclei at fertilization of the sea urchin Lytechinus variegatus: Time-lapse video microscopy

The penetration of the sperm into the egg, and the movements of the male and female pronuclei were followed from sperm attachment through pronuclear fusion, using time-lapse video microscopy of gametes and zygotes of the sea urchin Lytechinus variegatus (23° C). The pronuclei move in four stages: I. Sperm Entry Phase, following sperm-egg fusion and a rapid radiating surface contraction (5.9 ± 1.3 μm/second) when egg microvilli engulf the sperm head, midpiece, and tail to form the fertilization cone and the sperm tail beats in the egg cytoplasm; II. Formation of the Sperm Aster, which pushes the male pronucleus centripetally at a rate of 4.9 ± 1.7 μm/minute starting 4.4 ± 0.5 minutes after sperm-egg fusion, as the male pronucleus undergoes chromatin decondensation; III. Movement of the Female Pronucleus, the greatest and fastest of the pronuclear motions at a rate of 14.6 ± 3.5 μm/minute at 6.8 ± 1.2 minute after sperm-egg fusion, which establishes the contact between the pronuclei; and IV. Centration of the Pronuclei to the egg center at a rate of 2.6 ± 0.9 μm/minute by 14.1 ± 2.6 minutes after sperm-egg fusion. Pronuclear fusion typically occurs after stage IV and proceeds rapidly starting 14.7 ± 3.6 minutes after sperm-egg fusion with the male pronucleus coalescing into the female pronucleus at a rate of 14.2 ± 2.6 μm/minute.     

Integration of sperm and egg plasma membrane components at fertilization

Studies examining the integration of the sperm and egg plasma membranes, subsequent to gamete fusion in the surf clam, Spisula solidissima, were carried out employing the concanavalin A-horseradish peroxidase-diaminobenzidine procedure (Con A-HRP-DAB). When unfertilized Spisula eggs were incubated in Con A, either prior to or after aldehyde fixation and reacted with HRP-DAB, enzymatic precipitate was found associated with the vitelline layer and plasmalemma. The plasma membranes of sperm treated in a similar manner failed to stain. The plasma membranes of fertilized eggs reacted with Con A-HRP-DAB and examined by 1 min postinsemination were associated uniformly with enzymatic precipitate except at sites of sperm incorporation. These portions of unstained plasma membrane were derived from the spermatozoon and delimited the contents of the fertilization cone. From 2 to 4 min postinsemination, HRP-DAB reaction product became associated with the plasma membrane delimiting the fertilization cone. By 4 min postinsemination no difference in staining of the plasma membranes derived from the egg or the sperm (plasmalemma delimiting the fertilization cone) was detected. Evidence is presented suggesting that the acquisition of HRP-DAB reaction product by the former sperm plasmalemma is due to the movement of Con A binding sites from the egg plasma membrane.     

Studies on the participation of epididymal sperm protein DE/CRISP-1 in egg activation.

Protein DE (32 kDa) associates with sperm during epididymal maturation and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. In the present work we investigated the participation of DE in two mechanisms probably involved in egg activation: the ability of DE to trigger activation by its interaction with the binding sites on the egg surface (receptor model) and its ability to regulate intracellular calcium channels (sperm factor model). The incubation of eggs with DE did not promote activation parameters such as calcium oscillations or meiosis resumption. Secondly, microinjection of DE into eggs was ineffective in either eliciting calcium release or modifying oscillations induced by an activating sperm extract. Together, these results argue against the participation of DE in egg activation, restricting the activity of this protein and its egg binding sites to the sperm-egg fusion process.     

Sperm surface proteins are incorporated into the egg membrane and cytoplasm after fertilization

Using an antibody to sperm surface proteins, we have investigated the fate of the sperm membrane after fertilization. By immunofluorescence, two distinct loci of sperm surface proteins were found in the embryo: a large, restricted domain on the embryo surface and a smaller locus that apparently had moved from the point of sperm entry into the egg cytoplasm. The surface domain was initially over the fertilization cone and slowly disappeared, so that by 2 hr after fertilization it was no longer seen. The small internal locus of staining remained intact throughout the one-cell stage. When fluorescein isothiocyanate (FITC)-labeled sperm were used to fertilize eggs, the FITC-patch in the eggs was at a site distinct from either locus of sperm surface proteins. Thus, while most sperm surface proteins are incorporated into the egg surface at the site of fertilization and then slowly disappear, other sperm surface components are internalized to be retained for longer times. The differential handling of these sperm cytoplasmic components by the embryo raises the possibility that some of the sperm components may play a role in later embryonic events.     

Observations of hamster sperm-egg fusion in freeze-fracture replicas including the use of filipin as a sterol marker

We have extended the observations of previous transmission electron microscopy studies of sperm-egg fusion to include those of freeze-fracture replicas showing sperm-egg interactions before, during, and following sperm head fusion with the egg membrane. Hamster eggs were incubated with hamster sperm under polyspermic conditions and were observed after a period of 5-30 minutes. After fixation, the eggs and sperm were exposed to filipin, which binds beta-OH-sterols to form visible complexes in freeze-fracture replicas. Filipin can act as a marker for egg plasma membrane wherein it is abundant, while filipin is relatively scarce in the acrosome-reacted hamster sperm membrane, found only in the plasma membrane of the equatorial segment. The earliest sperm-egg interactions are observed between the egg microvilli and the perforatorium and the equatorial segment of the sperm, and the initial fusion between egg and sperm occurs in the vicinity of the equatorial segment. At later stages of fusion involving the postacrosomal segment, a clear line of demarcation is observed between the filipin-rich egg membrane and the filipin-poor sperm postacrosomal segment, suggesting that filipin binding lipids from the egg intercalate into the sperm membrane following membrane fusion. The anterior segment of the sperm does not fuse with the egg but is instead incorporated into a cytoplasmic vesicle derived from both sperm and egg membranes. In this latter step, filipin-sterol complexes are not found in sperm-derived membranes suggesting that there may be barriers to the movement of filipin binding lipids from the egg into these sperm membranes.