Changing localizations of site-specific sperm surface antigens during sea urchin fertilization

Indirect immunofluorescence studies show that monoclonal antibody (mAb) J18/2 binds site-specifically to surface antigens localized over the acrosome and tail regions of mature Strongylocentrotus purpuratus spermatozoa. Within 5 min after induction of the acrosome reaction by exposure to egg jelly or ionophore A23187, these surface antigens become detectable over the lateral region of the head so that the entire surface of the spermatozoon is labeled. Polyspermically fertilized S. purpuratus eggs fixed at varying times after insemination and exposed to mAb J18/2 reveal that these surface antigens are quickly incorporated into the egg plasma membrane and begin to disperse as early as 1.5 min after insemination. At subsequent times, they undergo further dispersal so that by 45 min they are distributed over the entire surface of the egg. These results suggest that the sperm surface components recognized by mAb J18/2 gain the ability to disperse laterally during the acrosome reaction and proceed to do so in the egg plasma membrane after fertilization.     

Changing localizations of site-specific surface antigens during sea urchin spermiogenesis

Whole mount preparations of dissociated testicular cells from the sea urchin, Strongylocentrotus purpuratus, were exposed to monoclonal antibodies (mAbs) directed against sperm surface proteins. Indirect immunofluorescence microscopy and Western immunoblot analysis show that mAb J18/29 binds to the entire surface of the mature spermatozoon and membrane proteins ranging in relative molecular masses from 25 to 340 kDa. MAb J18/2 binds to the acrosomal and tail regions of the mature spermatozoon and mainly to a 210-kDa membrane protein. MAb J17/30 binds to the midpiece and tail regions and monospecifically to a 60-kDa membrane protein. MAb J16/33 binds specifically to the sperm midpiece but does not bind to Western immunoblots of sperm membrane proteins. With the exception of J16/33, which shows a punctate binding pattern, all of these mAbs show uniform binding over the entire surface of the early spermatid. This uniform and complete surface binding is observed through all stages of spermiogenesis for mAb J18/29. By the midspermatid stage, when tail formation first begins, but before the nucleus condenses and the cytoplasm decreases in volume, localized binding patterns of mAbs J17/30 and J16/33 become evident. Localized binding of mAb J18/2 is not observed until the late spermatid stage. These results show that the sea urchin sperm surface is composed of at least four different domains and provide the first insight into differentiation of the cell surface during sea urchin spermatogenesis.     

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.     

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.     

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.     

Characterization of a monoclonal antibody that induces the acrosome reaction of sea urchin sperm

A monoclonal antibody, J18/29, induces the acrosome reaction (AR) in spermatozoa of the sea urchin Strongylocentrotus purpuratus. J18/29 induces increases in both intracellular Ca2+ and intracellular pH similar to those occurring upon induction of the AR by the natural inducer, the fucose sulfate-rich glycoconjugate of egg jelly. Lowering the Ca2+ concentration or the pH of the seawater inhibits the J18/29-induced AR, as does treatment with Co2+, an inhibitor of Ca2+ channels. The J18/29-induced AR is also inhibited by verapamil, tetraethylammonium chloride, and elevated K+. All these treatments cause similar inhibition of the egg jelly-induced AR. J18/29 reacts with a group of membrane proteins ranging in molecular mass from 340 to 25 kD, as shown by immunoprecipitation of lysates of 125I-labeled sperm and Western blots. The most prominent reacting proteins are of molecular masses of 320, 240, 170, and 58 kD. The basis of the multiple reactivity appears to reside in the polypeptide chains of these proteins, as J18/29 binding is sensitive to protease digestion but resistant to periodate oxidation. There are approximately 570,000 sites per cell for J18/29 binding. J18/29 is the only reagent of known binding specificity that induces the AR; it identifies a subset of sperm membrane proteins whose individual characterization may lead to the isolation of the receptors involved in the triggering of the AR at fertilization.     

Surface activity at the egg plasma membrane during sperm incorporation and its cytochalasin B sensitivity: Scanning electron microscopy and time-lapse video microscopy during fertilization of the sea urchin Lytechinus variegatus

Sperm incorporation and the formation of the fertilization cone with its associated microvilli were investigated by scanning electron microscopy of eggs denuded of their vitelline layers with dithiothreitol or stripped of their elevating fertilization coats by physical methods. The activity of the elongating microvilli which appear to engulf the entering spermatozoon was recorded in living untreated eggs with time-lapse video microscopy. Following the acrosome reaction, the elongated acrosomal process connects the sperm head to the egg surface. About 15 microvilli adjacent to the attached sperm elongate at a rate of 2.6 μm/min and appear to engulf the sperm head, midpiece, and sperm tail. These elongate microvilli swell to form the fertilization cone (average height, 6.7 ± 2.0 μm) and are resorbed as the sperm tail enters the egg cytoplasm 10 min after insemination. Cytochalasin B, an inhibitor of microfilament motility, completely inhibits the observed egg plasma membrane surface activity in both control and denuded eggs. These results argue for a role of the microfilaments found in the egg cortex and microvilli as necessary for the engulfment of the sperm during incorporation and indicate that cytochalasin interferes with the fertilization process at this site.     

Mouse sperm antigens that participate in fertilization. I. Inhibition of sperm fusion with the egg plasma membrane using monoclonal antibodies

Monoclonal antibodies (mAbs) have been generated to determine the sperm components responsible for interaction with an egg that results in fertilization. Here, we report upon a group of six different mAbs, all of which localize to a restricted region of the sperm head, the equatorial segment. Several of these mAbs demonstrated cross-reactivity with sperm from the other species tested (human, hamster, rabbit); when cross-reaction occurred, the mAb distribution was restricted to the equatorial segment despite the various configurations that this homologous region assumes in different species. When tested for an effect upon the fertilization process in vitro, ascites fluids containing two of the six mAbs, M29 and M37, displayed significant inhibition. The concentration dependency of this inhibition was observed using purified M29 immunoglobulin M, over a range of 0 to 0.2 mg/ml. The mAb inhibition of fertilization was independent of the presence of either the cellular (the cumulus) or acellular (the zona pellucida) layers surrounding the egg, indicating that the specific locus of inhibition for both of these antisperm mAbs was the egg plasma membrane. Immunologic detection of sperm components separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels followed by transfer to nitrocellulose sheets was used to identify the sperm components recognized by two of the mAbs in this group: M29, which inhibited fertilization, and M2, which did not inhibit fertilization. Using M29 mAb, a single sperm component with an apparent subunit molecular weight of approximately 40,000 was detected, whereas in the nitrocellulose strips incubated with M2 mAb two components displayed reactivity, a very prominent band at approximately 44,000 and a tight cluster of bands at approximately 36,000. Parallel nitrocellulose strips of mouse liver did not display these reactivities, consistent with indirect immunofluorescence data in which only testis and sperm, and not liver, kidney, ovary, and epididymal epithelium, demonstrated positive reactivity. These results indicate that the use of mAbs permits identification of sperm components that participate, putatively, in individual events of the fertilization process. Furthermore, using this strategy, we have identified a specific sperm component that appears to be a candidate for a role in sperm fusion with the egg plasma membrane.     

Ultrastructure of the spermatozoa and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine fish

Ultrastructure of sperm and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine teleost, was examined by scanning and transmission electron microscopy. The results showed that the sperm do not have an acrosome but have a very long mid-piece (one to two times the sperm head length) containing numerous well-developed elongated mitochondria. The sperm also have two tails (is biflagellate) each consisting of nine peripheral and one central pair (9 ± 2) of microtubules. This long mid-piece and the biflagellate nature of the sperm appear to be associated with the long life-span of the sperm and with sperm dispersal in the ovary to fertilize the eggs internally. The ocean pout eggs are enveloped by a porous chorionic membrane similar to that found in other teleosts but have two micropyles, a condition likely related to a mechanism of egg fertilization which increases the egg fertlity in the presence of low sperm numbers. Following insemination, some biochemically undefined excretions appeared on the surface of fertilized eggs and led to the acquisition of adherent capability of the eggs which formed a tightly associated egg mass in sea water. © 1995 wiley-Liss, Inc.     

Analysis of a sperm surface molecule that binds to a vitelline envelope component of Xenopus laevis eggs

To analyze sperm surface molecules involved in sperm–egg envelope binding in Xenopus laevis, heat‐solubilized vitelline envelope (VE) dot blotted onto a polyvinylidene difluoride (PVDF) sheet was incubated with a detergent extract of sperm plasma membrane (SP‐ML). The membrane components bound to the VE were detected using an antibody library against sperm plasma membrane components, and a hybridoma clone producing a monoclonal antibody (mAb) 16A2A7 was identified. This mAb was used in a Far Western blotting experiment in which VE was separated by electrophoresis, and then transferred to a PVDF strip that was incubated with SP‐ML. It was found that SP‐ML binds to the VE component gp37 (Xenopus homolog of mammalian ZP1). The antigens reactive to mAb 16A2A7 showed apparent molecular weights of 65–130 and 20–30 kDa, and were distributed relatively evenly over the entire sperm surface. Periodate oxidation revealed that both the pertinent epitope on the sperm surface and the ligands of VE gp37 were sugar moieties. VE gp37 was exposed on the VE surface, and the mAb 16A2A7 dose‐dependently inhibited sperm binding to VE. The sperm membrane molecules reactive with mAb 16A2A7 also reacted with mAb 2A3D9, which is known to recognize the glycoprotein SGP in the sperm plasma membrane and is involved in interactions with the egg plasma membrane, indicating that the sperm membrane glycoprotein has a bifunctional role in Xenopus fertilization. Mol. Reprod. Dev. 77: 728–735, 2010. © 2010 Wiley‐Liss, Inc.     

Sperm binding and fertilization envelope formation in a cell surface complex isolated from sea urchin eggs

An isolated surface complex consisting of the vitelline layer, plasma membrane, and attached secretory vesicles has been examined for its ability to bind sperm and to form the fertilization envelope. Isolated surface complexes (or intact eggs) fixed in glutaraldehyde and then washed in artificial sea water are capable of binding sperm in a species-specific manner. Sperm which bind to the isolated surface complex exhibit the acrosomal process only when they are associated with the exterior surface (vitelline layer) of the complex. Upon resuspension of the unfixed surface complex in artificial sea water, a limiting envelope is formed which, based on examination of thin sections and negatively stained surface preparations, is structurally similar to the fertilization envelope formed by the fertilized egg. These results suggest that the isolated egg surface complex retains the sperm receptor, as well as integrated functions for the secretion of components involved in assembly of the fertilization envelope.     

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.     

A Fine Structural Study of the Fertilization Process of the Jellyfish Cladonema uchidai

This study described the fertilization process of the jellyfish Cladonema uchidai by means of transmission electron microscopy. Female pronucleus was situated in close vicinity to the animal pole of the spawned egg, where the surface of the egg was flat or slightly depressed. Microvilli were observed except on the surface at the animal pole. The egg was entirely covered with a coat composed of fibrous materials. The spermatozoon was of the primitive type, and the proacrosomal vesicles were found immediately beneath the plasma membrane of the antero-lateral region of the sperm head. Within 15 sec after insemination, spermatozoa were incorporated in the egg cytoplasm only at the microvilli-free surface at the animal pole. Neither opening of the proacosomal vesicles nor formation of the acrosomal process was observed. No appreciable changes of cortical cytoplasm could be detected, although the egg became sticky after fertilization. Decondensation of the incorporated sperm nucleus occurred without breakdown of the original nuclear envelope. Within 10 min after insemination, the sperm nucleus still under the process of its decondensation fused with the female pronucleus. These findings were discussed in comparison with the fertilization process of higher metazoans as well as of other cnidarians.     

MAGNESIUM ION-REQUIRING STEP IN FERTILIZATION OF SEA URCHINS*

The magnesium ion-requiring step in fertilization of sea urchins was investigated. When eggs were inseminated in Mg-free sea water, several spermatozoa were found to bind to each egg surface with their reacted acrosomes without elevation of fertilization membrane. The number of binding jelly-treated spermatozoa to an egg did not differ regardless of the presence or virtual absence of magnesium ions. Although fertilization did not occur in Ca, Mg-deficient sea water (CM-deficient SW) even when jelly-treated spermatozoa were employed, some eggs could be fertilized by the addition of magnesium to the CM-deficient SW 60 sec after insemination, when jelly-treated spermatozoa had completely lost their fertilizing capacity in the CM-deficient SW. The acrosomal process of jelly-treated spermatozoa appeared to penetrate the vitelline layer in the CM-deficient SW. DTT- or pancreatin-treated eggs could not be fertilized in the virtual absence of magnesium. Re-fertilization using the fertilized eggs deprived of fertilization membrane did not occur under conditions of magnesium deficiency. These results suggest that external magnesium ions are indispensable at least for the fertilization process following penetration of the vitelline layer by the spermatozoa, such as fusion of the plasma membrane between an egg and a reacted spermatozoon, or the subsequent step(s) such as sperm penetration into egg interior and egg activation which precedes the cortical reaction.     

Time course and pattern of cortical granule breakdown in hamster eggs after sperm fusion.

We have determined the temporal relationship between sperm fusion and cortical granule breakdown in the hamster egg. Sperm fusion was determined by the Hoechst-transfer method (Stewart-Savage and Bavister: Dev Biol 128:150-157, 1988), and cortical granules were visualized with fluorescein isothiocynate-conjugated Lens culinaris agglutinin (Cherr et al. J Exp Zool 246:81-93, 1988). By 55 min after insemination, there was an 85% reduction in the density of cortical granules (fewer than four granules/100 microns2). Taking this value as the completion of the cortical reaction, analysis of the data indicate that the cortical reaction was completed 9 min after sperm fusion and 3 min after the formation of the zona and cell surface blocks to polyspermy. There was no obvious spatial pattern of granule loss in eggs that had a Hoechst-positive sperm but had not completed the cortical reaction.     

Voltage Noise Changes during Monospermic and Polyspermic Fertilization of Mature Eggs of the Anuran, Rana temporaria

The electrical response of mature anuran eggs to the fertilizing sperm consists of a rapid depolarization and a decrease in resistance of the plasma membrane (fertilization potential) and serves as a fast block to polyspermy. We report here that the fertilization potential, previously thought to be the earliest electrical response of the egg, is preceded in Rana temporaria by changes in voltage noise. Voltage noise was recorded after insemination and compared in monospermic and NaI-induced polyspermic eggs. Fertilization potential in monospermic eggs arised at 1 min 45 sec to 2 min 15 sec after insemination, and that in NaI-induced polyspermic eggs did at 3 min to 3 min 30 sec after insemination. However, the increase in voltage noise was detected at the similar time (1–2 min 30 sec) after insemination in both the eggs. The duration of voltage noise increase before the fertilization potential was larger in polyspermic eggs (50–105 sec) than in monospermic eggs (10–40 sec). Polyspermic fertilization in Rana temporaria induced by NaI was checked by visualizing multiple sperm entry sites with the scanning microscope. The process of sperm entry and the development of the fertilization body are similar to those occurring with monospermic fertilization; furthermore all supernumerary sperm fuse only with the animal hemisphere of the egg. Although the physiological basis of the changes in voltage noise is unclear, these alterations appear to be the earliest electrical response to sperm yet reported.     

The production of cloned fish in the medaka (Oryzias latipes)

The measurement of cellular DNA content by DNA microfluorometry revealed that medaka embryos that were fertilized with normal sperm and exposed to heat shock (41 degrees C for 3 min) or hydrostatic pressure (700 kg/cm2 for 10 min) at 85-95 min after insemination were tetraploid. Embryos fertilized with normal sperm and exposed to heat shock (41 degrees C for 2 min at 2-3 min after insemination) were triploid. These results suggest that heat shock or hydrostatic pressure at 85-95 min after insemination arrests the first cleavage, while heat shock at 2-3 min after insemination arrests the second meiotic division. Medaka clones have been produced by the following method: Eggs from orange-red or variegated variety were activated by UV-irradiated, genetically impotent sperm of wild-type fish (UV sperm). The haploid eggs obtained were diploidized by preventing the first cleavage with heat shock or hydrostatic pressure to produce homozygous females. Each of the two homozygous females was mated with vasectomized male in isotonic balanced salt solution to collect unfertilized eggs. The collected eggs were activated with UV sperm and converted from haploid to diploid by arrest of the second meiotic division with heat shock. Hatched fry of each homozygous diploid (all females) were fed with a methyltestosterone-containing diet (40 micrograms/gm diet) to produce sex-reversed males, which were mated with brood females, and thus two cloned lines were obtained.     

Surface Architecture of Sperm Tail Entry into the Hamster Oocyte

At various times after artificial insemination in vivo , fertilized eggs were flushed from the ampulla of the oviduct of the hamster. The processes of sperm tail entry into the oocyte were studied with phase-contrast and electron microscopes. At 6–7 hr after insemination, the sperm head was incorporated completely into the ooplasm, but the entire length of the sperm tail still projected freely over the oocyte surface. The region on the oocyte surface where the second polar body was extruded was different from where the first polar body emerged. At 8–9 hr after insemination, the sperm tail was attached in a wave-like fashion to the oocyte surface. Where some portions of the tail were attached, they were trapped by the microvilli of the oocyte and had begun to sink into the ooplasm. Thus, the entire length of the sperm tail was incorporated into the ooplasm successively but almost synchronously. From the present observations, we have proposed a model for the mechanism of sperm tail entry into the vitellus in vivo .     

Membrane potential measurements of unfertilized and fertilized Xenopus laevis eggs are affected by damage caused by the electrode

Upon penetration in an unfertilized Xenopus egg bathed in 1/10 Ringer, the voltage recorded by a microelectrode shows an abrupt jump to a negative voltage (Ep) followed by a rapid depolarization to a steady value (Er) (Ep = -39.4 +/- 1.9 mV and Er = -11.5 +/- 0.5 SE, 54 eggs from 9 females). The same is true for fertilized eggs impaled 16-35 min after insemination (Ep = -29.5 +/- 2.1 mV, Er = -11.5 +/- 0.9 mV, SE, 18 eggs from 3 females). The voltage recorded by a second microelectrode inserted into the same egg does not show the transient initial negativity. The stationary level of the membrane potential is close to the diffusion potential calculated from the Goldman equation with equal permeabilities for all the relevant ions. It is concluded that the low resting potentials measured in Xenopus eggs before and after fertilization are largely due to damage caused by the electrode. Using an upper limit of -39 mV for the true membrane potential and correlating the input resistance with the stationary membrane potential, a lower limit of 22 M omega (about 1 M omega cm2) for the membrane resistance can be obtained. Insertion of a microelectrode during the first 3 min after insemination shows a steady positive potential while, at later times (3-16 min post-insemination), a positive peak followed by a repolarization can be observed. This indicates that the measurement of the peak of the fertilization potential is not seriously affected by the electrode penetration while its time course after the first 3 min may be deformed by the presence of a large leakage conductance.     

Fine structural investigation of the insemination response in Urechis caupo.

Electron microscopy of Urechis eggs revealed no changes in the egg cortex or investing layers until 4 min after insemination at 172C. From 4 min to about 30 min after insemination the surface coat gradually elevates, widening the perivitelline space. During this period, microvilli separate from the tightly woven layer of the surface coat, fibrogranular aggregates resembling surface coat material appear in the perivitelline space, and some cortical granules are extruded from the egg cortex into cytoplasmic processes. There is no statistically significant decrease in the number of cortical granules remaining in the egg surface during the first 95 min after insemination; many cortical granules persist in postgastrulae. Most of the cortical granules remain in fertilized eggs after removal of the surface coat with glucose-EGTA. We found no morphological correlates of the polyspermy block which is established within 1 min of insemination (Paul, 1975).