The Properties of the Oviducal Pars Recta Protease which Mediates Gamete Interaction by Affecting the Vitelline Coat of a Toad Egg

SDS-PAGE analyses of the vitelline coats (VCs) of coelomic eggs (CEVC) and uterine eggs (UEVC) of Bufo japonicus revealed that the UEVC lacks the 40K–52K molecular weight components present in the CEVC; this is concomitant with the increased stainability of a 39K component and the appearance of a 36K component. These macromolecular alterations, accompanied by the acquisition of egg fertilizability, were induced when coelomic eggs were treated with the contents of secretory granules obtained from the oviducal pars recta (PRG). Gel-filtration of PRG in combination with hydrolytic assays employing either fluorescamine-labeled CEVC or a variety of synthetic substrates showed that the CEVC to UEVC alterations are ascribable to the action of a protease hydrolyzing specifically peptidyl-Arg-MCAs in a highly Ca²⁺-dependent way. This enzyme, which has an optimal pH of 8.0–8.2, is inhibited by soybean trypsin inhibitor and leupeptin, as well as by such serine protease inhibitors as DFP and p-APMSF. On the basis of a SDS-PAGE analysis, its molecular weight is estimated to be 66K. Treatment of coelomic eggs with the partially purified PR protease did not render the eggs fertilizable, although CEVC to UEVC macromolecular alterations were effected. We conclude that the action of this oviducal protease in partially hydrolyzing the VC is a prerequisite but insufficient in itself to render the coelomic eggs fully accessible to a fertilizing sperm.     

Classification,inhibition, and specificity studies of the vitelline coat lysin from toad sperm

The sonicated supernatant of the sperm of the toad, Bufo japonicus, can digest easily the vitelline coat (VC) of uterine eggs, and to a lesser extent the VC of coelomic eggs, but not that of activated eggs. The VC lysis and fertilization were competitively inhibited in the presence of t-butyloxycarbonyl-L-Gln-L-Arg-L-Arg-4-methylcoumaryl-7-amide (Boc-Gln-Arg-Arg-MCA), suggesting the involvement of proteases in the fertilization process. Starting from a sonicated supernatant, a potent VC lysin, possessing hydrolytic activity on Boc-Gln-Arg-Arg-MCA, was obtained by anion-exchange chromatography and gel filtration. The activity of the partially purified lysin was inhibited by diisopropyl fluorophosphate (DFP) and by such trypsin inhibitors as soybean trypsin inhibitor, leupeptin, and (p-amidinophenyl) methanesulfonyl fluoride hydrochloride, but not by chymostatin, E-64, and ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. The molecular weight of the lysin was estimated to be 32K, based on the fluorographic image of ³H-DFP binding to the lysin on sodium dodecyl sulfate gel electrophoresis. The VC lysin was most active at pH 7.0–7.6 and under low ionic strength equivalent to fresh water. The release of the VC lysin was induced upon incubation of sperm with the contents of oviducal pars recta granules (PRG), which are known to induce the acrosome reaction. We conclude that the protease studied here represents the VC lysin of toad sperm that is involved in fertilization by digesting the VC of uterine eggs, probably released as a result of the acrosome reaction induced by PRG.     

Analyses of Oviductal Pars Recta-Induced Fertilizability of Coelomic Eggs in Xenopus laevis

The acquisition of fertilizability in coelomic eggs of Xenopus laevis has been shown to be correlated with the physical, biochemical, and ultrastructural alterations of the egg envelope [coelomic envelope (CE)] induced during the passage of eggs through the pars recta portion of the oviduct. However, no direct evidence that the pars recta renders eggs fertilizable has yet been presented. In this study, we show that coelomic eggs are highly fertilizable when they are incubated with continuous shaking for 4 h at 15 degrees C in pars recta extract (PRE) derived from females prestimulated by pregnant mare serum gonadotropin. The PRE from pituitary-stimulated Bufo japonicus was as potent as homologous PRE in rendering Xenopus eggs fertilizable. Incubation of coelomic eggs in PRE for 30 min induced a dramatic increase in the rates of sperm binding to the envelope to a level equivalent to that exhibited by the envelope from uterine eggs (VEs). The CE-to-VE ultrastructural conversion and a 43k-to-41k hydrolysis of the envelope glycoprotein component started 5 min after, and were completed by 15 min after, the start of incubation in PRE and were accompanied by an exposure of a new N-terminal sequence typical to gp41. Thus, the biochemical and ultrastructural conversions and the sperm-binding activity of the envelope induced by PREs, although being prerequisite, were not sufficient to render coelomic eggs fully accessible to fertilizing sperm.     

Oviducal Contribution to Alteration of the Vitelline Coat in the Frog, Rana japonica. An Electron Microscopic Study

The vitelline coat (VC) surrounding coelomic eggs of the frog, Rana japonica , comprises bundles of filaments running both parallel and perpendicular to the egg surface. The coat gives little or no staining reaction with PA-CrA-Silver methenamine. In contrast, in the VC of uterine eggs the filament bundles are less conspicuous. and the interstices between the filament bundles stain strongly for carbohydrate. This alteration occurs during passage of the eggs down the first 1/20 th of the oviduct, the pars recta. The epithelium of the p. recta contains secretory cells, which contain electron-dense granules distinct from those in the jelly-secreting cells in more caudal portions of the oviduct. Treatment of coelomic eggs with an extract of p. recta followed by exposure to a sperm suspension resulted in marked swelling and softening of the VC. These results indicate that the contents of the granules secreted from the epithelial cells in the p. recta are deposited in the VC to increase its susceptibility to a fertilizing sperm.     

Involvement of carbohydrate moieties of the toad egg vitelline coat in binding with fertilizing sperm

Vitelline coats (VC) were isolated from the eggs of Bufo japonicus , and were added with sperm in reconstituted salt solution, which mimics the physiological role of jelly envelopes, to determine the rates of sperm binding per unit area (0.2 mm²) of VC. The rate of sperm binding to VC from uterine eggs was high, but was low to VC from coelomic eggs and eggs activated in 1/20 De Boer's solution (DB) and moderately low to VC from eggs activated in DB. The binding rate increased when VC from coelomic eggs were treated with extracts of the pars recta portion of the oviduct. The sperm that bound to VC were not acrosome-reacted and their binding to VC required both a low salinity, assuring motility of sperm, and sufficiently high levels of Ca²⁺ and Mg²⁺. The rate of sperm binding was reduced by either coexisting solubilized VC materials, periodate-oxidation of VC or the pretreatment of VC with Fab fragments of anti-VC antibodies, which reacted mostly to carbohydrate residues of VC glycoproteins. Sperm-VC binding assays in combination with gel-filtrated VC components revealed that the fractions containing 36–39 kDa components were most effective both in inhibiting the binding and in neutralizing the antibody induced inhibition of binding. These results indicate that carbohydrate moieties in 36–39 kDa glycoproteins of VC, exposed as a result of hydrolysis by the oviducal pars recta protease, are involved in binding with fertilizing sperm.     

Role of oviductal pars recta extract on interspecific fertilization between anurans

During their journey through the oviductal pars recta, the vitelline envelope (VE) of Bufo arenarum oocytes encounter structural alterations that make them sensitive to attack by sperm lysin and thus to penetration by sperm cells. The role of pars recta (PR) on the specificity of fertilization between amphibians was analyzed by conditioning Bufo arenarum oocytes with either homologous PR extract (PRE) or Leptodactylus chaquencis PRE. The oocytes were thereafter exposed to sperm lysin preparations from both species. Lysis of the VE only took place when the oocytes were exposed to the homologous PRE. The pattern of protein composition of PRE of these species was strikingly different as shown by Coomassie blue staining of SDS-PAGE. Moreover, antibodies against PR fluid (PRF) of Bufo arenarum produced seven bands of immunoprecipitation in electrophoresed homologous PRE and only one faint band in Leptodactylus chaquencis PRE. Here we show that: (i) the biological activity of PR from Bufo arenarum and Leptodactylus chaquencis over the VE of Bufo arenarum oocytes is species-specific; (ii) this specificity seems to be based in differences in protein structure, which was indicated by the fact that proteins from PRE of Leptodactylus chaquencis and Bufo arenarum were antigenically distinct; (iii) the specificity was solely related to PR activity and not to sperm lysin activity since sperm lysin preparations from both species showed comparable activity.     

Oviductal protease and trypsin treatment enhance sperm-envelope interaction in Bufo arenarum coelomic eggs

We describe the morphological and biochemical changes in Bufo arenarum coelomic egg envelopes (CE) following passage through the oviduct. In this species, the transformation of the CE into the vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein component. Electrophoretic patterns indicate that a pars recta oviductal protease selectively hydrolyzes in vitro the 84 and the 55 kDa glycoproteins of the CE. During the CE to VE transformation, the relative concentrations of gp48, 42 and 39 kDa also change. In in vitro tests, sperm binding to envelope glycoprotein occurs when they are exposed to VE but not when treated with CE, and VE labeled glycoproteins bind to the head and mid piece of the sperm. The gp39 VE component has 100% identity with internal domains of the sequence deduced from ovarian cDNA for the homologous zona pellucida glycoprotein type C (ZPC) protein precursor in B. arenarum. The effects of trypsin as a substitute for oviductal protease were also examined. Trypsin selectively attacks the 84 and the 55 kDa glycoproteins without hydrolyzing other components and renders coelomic eggs fertilizable in a jelly water preparation. Therefore, trypsin can mimic in vitro the biological action of the oviductal protease. However, it does not wholly mimic the biological action of the oviduct which, in B. arenarum at least, exceeds a mere proteolytic effect. This fact was verified by the lower fertility rates and the abnormal embryo development found when trypsin-treated coelomic eggs were fertilized in vitro.     

Acrosome reaction in sperm of the toad,bufo bufo japonicus

The acrosome in the sperm of the toad, Bufo bufo japonicus, consists of a membrane-limited acrosomal cap and a fibrous perforatorium. When sperm are incubated with the oviducal pars recta extract (PRE) for 30–60 min, the outer acrosomal membrane fuses with the overlying plasma membrane at several points with concomitant loss of the contents of the acrosomal cap. The inner acrosomal membrane thus exposed fuses with the plasma membrane at the caudal end of the acrosomal region. This PRE-induced acrosome reaction is completely inhibited by soybean trypsin inhibitor. Sperm found in the innermost jelly layer of inseminated eggs possess an intact acrosome, but those either passing through the vitelline coat or localizing in the perivitelline space are acrosome-reacted in the same manner as when treated with PRE. These observations, combined with recent evidence showing involvement of the pars recta substance in fertilization, indicate that the acrosome reaction occurring in a fertilizing sperm at or near the surface of the vitelline coat is a response to a substance that is derived from the pars recta and deposited in the vitelline coat.     

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.     

Egg exudate-induced reduction of sperm lysin sensitivity in the vitelline coat after fertilization of Bufo japonicus and its participation in polyspermy block

The jellyless eggs of Bufo japonicus or those from which the vitelline coats (VCs) had been removed (denuded eggs) were electrically activated. The exudate that accompanied egg activation (AEX) was collected to study its role in preventing polyspermy. When dejellied (but VC intact) eggs were treated with AEX, the eggs lost not only fertilizability but also the sensitivity of their VCs to the sperm lysin. By contrast, denuded eggs treated with AEX were fertilizable; even activated eggs were highly fertilizable, provided they were deprived of their VCs and inseminated 30 min after activation. The loss of sensitivity to sperm lysin occurred in VCs 3-5 min after activation either in De Boer's or 1/20 De Boer's solution. The activity of AEX to reduce the sensitivity of VCs to sperm lysin was heat-sensitive and dependent on Ca2+, but it was not affected at all by the variety of protease inhibitors used. The activity was lost by the preincubation of AEX with fragmented VCs in the presence of Ca2+, suggesting Ca(2+)-dependent binding of AEX molecules to the VC at fertilization. Immunocytochemical studies employing anti-AEX rabbit serum showed that the pertinent antigens were localized in the cortical granules of unfertilized eggs and in both the inner surface of VCs and the perivitelline space of fertilized eggs. We conclude that the AEX-induced loss of lysin sensitivity in VCs and the deposition of cortical granule materials on the inner wall of VCs constitute a slow and permanent block to polyspermy.     

Morphological Modifications of Oocyte Vitelline Envelope from Bufo arenarum during Different Functional States

The submicroscopic morphology of the vitelline envelope of Bufo arenarum's oocyte change significantly during the maturation and fertilization processes. The morphological changes are related to physiological activity in vivo and can be triggered in vitro by experimental procedures. It is our scope to present the ultrastructure differences of fascicular components of the vitelline envelope in coelomic, "pars recta" conditioned, oviductal, oviposited and fertilized oocytes. Our experimental results indicate that artificial "pars recta" treatment of coelomic oocytes arrange the fascicular components as those of oviposited oocyte, although differences still remain indicating that additional maturation processes take place while the egg pass througth the oviduct. Fertilized or activated oocytes which are refractary to sperm penetration, change the vitelline envelope fascicular components orientation giving a submicroscopical image quite different to those of none fertilized oocytes. These ultrastructural changes define in a clear cut manner the functional states of Bufo arenarum's oocyte.     

Potential involvement of C(3) complement factor in amphibian fertilization

We have assessed the potential involvement of C(3), the third complement factor, and its receptor in Bufo arenarum fertilization. We show that a polyclonal antibody against a B. arenarum C(3)-like factor (C(3)Ba) reacts specifically with components of the extracellular matrix (ECM) of coelomic eggs and the cell membrane of uterine eggs. Interestingly, we have identified a 163 kD protein immunoreactive with a monoclonal antibody against the CD11b alpha chain of the human C(3) receptor on the cell membrane of the animal pole of uterine eggs, the site of entry of the sperm, but not in coelomic eggs (CR3Ba). Treatment of coelomic eggs with a pars recta oviductal-like protease, trypsin, induced the translocation of C(3)Ba from the ECM to the cell membrane. Furthermore, inhibition of CR3Ba by trypan blue, as well as inhibition of C(3)Ba by anti-C(3)Ba on uterine eggs impaired fertilization, whereas identical treatment on sperm cells did not alter percentage fertilization. Our results suggest, (A) that changes in the localization of C(3)Ba from the ECM to the cell membrane may be triggered by trypsin-like proteases during passage of eggs through the oviduct; and (B) that C(3)Ba/CR3Ba may be involved in B. arenarum fertilization.     

Characterization of the acrosome reaction-inducing substance in Xenopus (ARISX) secreted from the oviductal pars recta onto the vitelline envelope

We previously demonstrated that Xenopus sperm undergo an acrosome reaction on the vitelline envelope (VE) in response to the materials secreted from the oviductal pars recta [Dev. Biol. 243 (2002), 55]. A monoclonal antibody against the acrosome reaction-inducing substance in Xenopus (ARISX) was obtained by immunizing mice with pars recta extract (PRE). The acrosome reaction by PRE or on the VE was effectively inhibited by the intact anti-ARISX antibody as well as its Fab fragment, indicating that the antibody recognizes the epitopes localized on the acrosome reaction-inducing substance. On Western blots, the anti-ARISX antibody recognized a molecule with an apparent molecular mass of 300 kDa in PRE and in the VE, but this molecule was not detected in the coelomic envelope. The amount of ARISX in PRE was increased by the treatment of females with pregnant mare serum gonadotropin. Periodate oxidation of PRE completely abolished the acrosome reaction-inducing activity, indicating the involvement of the carbohydrate moieties of ARISX in inducing the acrosome reaction. On immunofluorescence observation, ARISX was localized in the epithelial cells in the posterior region of the pars recta and on the VE as fibrous structures.     

Localization of cortical granule constituents before and after exocytosis in the hamster egg

Electrical activation of the hamster egg was used to study cortical granule constituents before and after exocytosis. The activated hamster eggs underwent cortical granule decondensation just prior to and at the time of exocytosis. Some of the cortical granules of aged, unactivated eggs underwent similar changes. FITC- and gold-conjugated Lens culinaris agglutinin (LCA) bound intensely to the surfaces of activated but not unactivated eggs. This labelling was associated with the microvilli. Permeabilized eggs exhibited discrete cortical labelling before activation, with a subsequent decrease following the cortical reaction. Gold-conjugated LCA specifically bound to cortical granules when incubated with thin sections. FITC-soybean trypsin inhibitor (SBTI) bound in discrete foci in the cortex of unactivated eggs. Following activation, cortical labelling by SBTI decreased. Aprotinin and benzamidine hydrochloride inhibited FITC-SBTI from binding to the egg cortex. Gold-avidin localization of biotin-SBTI in the electron microscope demonstrated that condensed cortical granules did not bind SBTI but decondensed or exocytosing granules did. This suggests that a cortical granule protease is exposed just prior to exocytosis. Activated eggs exhibited dramatic decreases in the number of hamster sperm penetrating the cytoplasm, suggesting that a plasma membrane block to polyspermy is temporally related to cortical granule exocytosis.     

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.     

Participation of the 39-kDa glycoprotein (gp39) of the vitelline envelope of Bufo arenarum eggs in sperm-egg interaction

The acquisition of egg fertilizability in Bufo arenarum takes place during the oviductal transit and during this process the extracellular coelomic envelope (CE) of the eggs is converted into the vitelline envelope (VE). It has been stated that one of the necessary events leading to a fertilizable state is the proteolytic cleavage of CE glycoproteins in the oviductal pars recta by oviductin, a serine protease. Consequently, there is a marked increase in the relative quantity of glycoproteins with 39 (gp39) and 42 kDa (gp42) in the VE. In the present study, sperm-VE binding assays using heat-solubilized biotin-conjugated VE glycoproteins revealed that both gp39 and gp42 have sperm binding capacity. According to this result, our study was focused on gp39, a glycoprotein that we have previously reported as a homologue of mammalian ZPC. For this purpose, rabbit polyclonal antibodies against gp39 were generated at our laboratory. The specificity of the antibodies was confirmed with western blot of VE glycoproteins separated on SDS-PAGE. Immunohistochemical and immunoelectron studies showed gp39 distributed throughout the width of the VE. In addition, immunofluorescence assays probed that gp39 bound to the sperm head. Finally, as an approach to elucidate the possible involvement of gp39 in fertilization, inhibition assays showed that pretreatment of eggs with antibodies against gp39 generated a significant decrease in the fertilization rate. Therefore, our findings suggest that gp39, which is modified by oviductal action, participates as a VE glycoprotein ligand for sperm in Bufo arenarum fertilization.     

Serum antigens detected in pars recta luminal fluid and coelomic envelope surrounding Bufo arenarum eggs

In anurans, protease activity from the pars recta portion of the oviduct (under regulation by 17β-estradiol), is known to cause ultrastructural alterations on the oocyte surface rendering fertilizability. In mammals, the presence of serum proteins in oviductal fluid via transudation is also well known. In the present study we determined the plasma proteins of the anuran Bufo arenarum that are present in pars recta fluid and oocyte extracellular matrix and characterized the 17β-estradiol-induced proteins synthesized de novo and secreted into the pars recta lumen. Rabbit polyclonal antibodies against the soluble proteins in pars recta fluid cross-reacted with anuran plasma proteins and with the extracellular matrix of coelomic eggs based on immunoelectrophoresis and immunohistochemistry, respectively. Using radiolabeled leucine in the absence and presence of 17β-estradiol, we show that a polypeptide of 66 kDa molecular mass is the principal protein synthesized and secreted into the pars recta lumen.     

Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10^?5 to 10^?3M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10^?3M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the egg's surface, which give rise to cortical projections in the presence of SBTI.     

A hydrogen peroxide block to polyspermy in the sea urchin Arbacia punctulata

Fertilization by more than one sperm in sea urchins inevitably leads to uneven division and death of the embryo. We provide evidence for a block against this polyspermy involving the hydrogen peroxide release by the egg during fertilization that is triggered by entry of the successful sperm. Polyspermy in 100% of fertilized eggs was demonstrated when catalase was added to destroy hydrogen peroxide immediately after fertilization. Soybean trypsin inhibitor, another polyspermic agent, is shown to prevent the formation of hydrogen peroxide in the fertilized egg. This suggests that the protease released from egg cortical granules during fertilization plays a role in the hydrogen peroxide generating system.     

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.