Developing a national food defense guideline based on a vulnerability assessment of intentional food contamination in Japanese food factories using the CARVER+Shock vulnerability assessment tool.

The awareness of food terrorism has increased following the September 11, 2001 terrorist attacks in New York City, United States, and many measures and policies dealing with this issue have been established worldwide. Suspected deliberate food-poisoning crimes have occurred in Japan, although they are not regarded as acts of food terrorism. One area of concern is that the small- to medium-sized companies that dominate Japan's food industry are extremely vulnerable to deliberate food poisoning. We conducted a literature research on food defense measures undertaken by the World Health Organization and in the United States and Europe. Using the Carver+Shock vulnerability assessment tool, eight food factories and related facilities in Japan were evaluated and we found the level of awareness of food defense to be low and the measures inappropriate. On the basis of this evaluation, we developed a set of guidelines that Japanese food companies can use to help develop their food defense strategies and to serve as a reference in considering specific measures.

Tentative food defense guidelines for food producers and processors in Japan.

OBJECTIVES: With increasing global interest in intentional food contamination, expert meetings have been held by the G8, while the U.S. government has proposed policies for preventing food terrorism and intentional contamination. However, Japan has no food defense policy, and some food companies are concerned about an impending terrorism and contamination crisis. METHODS: We developed a Food Defense Checklist for Food Producers and Processors and published the details on the website. We also developed tentative Food Defense Guidelines for Food Producers and Processors on the basis of the checklist. In this study, we tested the usability of the guidelines through a hearing survey regarding food plants. We also compared the checklist with the implementation manual for the approval system of Comprehensive Sanitation Management and Production Process (the Japanese equivalent of the HACCP). RESULTS: We organized the comments gleaned from the hearing survey and provided a detailed explanation of the guidelines. As the HACCP has been adopted by Japanese food companies, we included both precautionary measures and the HACCP perspective in the explanation regarding the rapid dissemination of information. CONCLUSION: The guidelines are useful for Japanese food companies, and it is important to disseminate knowledge on this topic and implement food defense measures.

Identification of the sperm motility-initiating substance in the newt, Cynops pyrrhogaster, and its possible relationship with the acrosome reaction during internal fertilization.

Motility initiation is a key event during internal fertilization of female-stored sperm, although the underlying mechanisms remain unclear. In internally fertilizing urodeles, quiescent sperm initiate motility on the surface of the egg-jelly, a thick extracellular matrix that accumulates around the egg in oviduct. By immunizing mice with egg-jelly extracts, we successfully generated an alpha34 monoclonal antibody (mAb) which neutralized sperm motility-initiating activity in the egg-jelly of the newt, Cynops pyrrhogaster, in a dose-dependent manner. The alpha34 mAb recognized an unglycosylated 34 kDa protein in the outermost of the six layers that comprise egg-jelly. Under nonreducing conditions, immunoblotting with alpha34 mAb produced many bands in addition to the 34 kDa protein, suggesting that the 34 kDa protein associates not only with the jelly matrix itself, but also with additional substances present in the matrix. Our current results are compatible with the supposed features of sperm motility-initiating substance (SMIS), indicating that the 34 kDa protein itself, or a complex consisting of the 34 kDa protein and some other molecules, is the SMIS in C. pyrrhogaster. Immunofluorescence staining further indicated that SMIS was distributed in a dot-like pattern in the outermost jelly layer and was fully covered with acrosome reaction-inducing substance (ARIS). Immunocytochemical and scanning electron microscopic examinations of the outermost jelly layer strongly suggests that the 34 kDa protein localized in granules (2 microm) and that ARIS was distributed covering the granules and in the sheet-like structure above the granules. These data suggest that the initiation of sperm motility is mediated by the acrosome reaction.

Identification of egg-jelly substances triggering sperm acrosome reaction in the newt, Cynops pyrrhogaster.

Our previous studies have shown that the acrosome reaction (AR) occurs in egg-jelly of the Japanese newt, Cynops pyrrhogaster. This is analogous to the substances of echinoderms but distinct from those of many other vertebrates derived from the egg envelope or its derivative, the zona pellucida. To identify the AR-inducing substances in newt egg jelly, a monoclonal antibody (mAb) was generated against the jelly by screening the culture supernatants to find the one that best neutralized the AR-inducing activity of the jelly substance. The mAb specifically reacted to protein bands in the jelly. These proteins, with apparent molecular weights of 122 and 90 kDa, exhibited AR-inducing activity, indicating that they are definitely AR-inducing substances. Western blotting using the mAb indicated that the 122 and 90 kDa proteins are present only in the egg jelly's outermost layer, where AR-inducing activity is known to occur. Both proteins were recognized with wheat germ agglutinin (WGA), a lectin that inhibits AR-induction in egg jelly extract. Taken together, these findings indicate that the 122 and 90 kDa proteins are the AR-inducing substances in the egg jelly of C. pyrrhogaster. The WGA recognition of the proteins was lost by N-glycosidase digestion, suggesting that N-linked carbohydrate moieties in these proteins may be responsible for the AR-inducing activity.

Two states of active spermatogenesis switch between reproductive and non-reproductive seasons in the testes of the medaka, Oryzias latipes.

Seasonal change in spermatogenesis was examined in the restricted spermatogonium-type testes of a teleost, Oryzias latipes. Histological observation revealed that the number of each stage of germ cells during most of the non-reproductive season, from October to January (O-J period) was nearly half of that during the reproductive season, from May to July (M-J period), except for type B spermatogonia (B-gonia), which was actually equal. As a result, the ratio of primary spermatocytes (P-cytes) to B-gonia was remarkably small in the O-J period. Despite the differences between both time periods, the proliferative activity of type A spermatogonia (A-gonia), B-gonia, or P-cytes was at a similar level in both periods. Moreover, in cultured testes treated with bromodeoxyuridine as a cell-lineage tracer, P-cytes differentiated to spermatids in 11-15 days in both M-J and O-J periods. These indicate that spermatogenesis is active in each period at a different state. In the spermatogenic testis, A-gonial proliferation was maintained by human follicle stimulating hormone/luteinizing hormone in culture. Whereas cell death of B-gonia and/or P-cytes gradually increased in the M-J period in spite of those cells being constant in population sizes. In transition to the O-J period, A-gonia and P-cytes first decreased, which was accompanied by a decrease in proliferative activity of A-gonia and relative increase of dead cells from B-gonia and/or P-cytes against live P-cytes. These suggest that A-gonial proliferation and cell death of B-gonia and/or P-cytes that is induced coordinately with B-gonial differentiation are critical for the spermatogenic control.

Egg jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope.

The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+). Fluorescein isothiocyanate-labeled DBA bound specifically to the acrosomal cap of the intact sperm in the presence of a Ca(2+)-chelating agent, EDTA, suggesting that binding of DBA to the native receptor for the egg jelly substance on the acrosomal region took the place of the egg jelly substance-induced acrosome reaction. In contrast, the sperm that had been acrosome reacted by DBA treatment did not bind to the vitelline envelope of the egg whose jelly layers were removed. Subsequent addition of jelly extract caused the sperm binding to vitelline envelope, indicating that the egg jelly of the newt contains substances that are involved in not only inducing the acrosome reaction but also binding to the vitelline envelope. This is the first demonstration of the involvement of egg jelly substance in the binding of acrosome-reacted sperm to the vitelline envelope.

The outermost layer of egg-jelly is crucial to successful fertilization in the newt, Cynops pyrrhogaster.

The significance of egg-jelly layers in internal fertilization was evaluated in the newt, Cynops pyrrhogaster. In this species, six egg-jelly layers, J1, J2, J3, J4, J5 and the outermost J6 layers, are accumulated on the surface of the fertilizable eggs in pars convoluta of the oviduct. When a large number of sperm (about 6 x 10(5)) were placed on eggs having different numbers of jelly layers, all the eggs were fully fertilized, although many of the eggs developed abnormally. Upon insemination using about 600 sperm, only eggs with the full set of jelly layers were fertilized at a high rate with normal development. Since around 300 (the range of 48-1,192) sperm were observed on and in the egg-jelly in naturally spawned eggs, we conclude that the J6 layer must be present on the outermost surface of the egg-jelly for successful internal fertilization of the newt. Previous studies have suggested that the J6 layer is a prerequisite for the initiation of sperm motility and the acrosome reaction. In the present study, the fertilization rate decreased in eggs with a full set of jelly layers when inseminated using acrosome-reacted and motile sperm. However, the fertilization rate was high when motile sperm with intact acrosome was used. These results suggest that induction of the sperm acrosome reaction in the J6 layer is an important step in the internal fertilization of the newt.

Ordered progress of spermiogenesis to the fertilizable sperm of the medaka fish, Oryzias latipes, in cell culture.

Spermiogenesis is significant for producing sperm with equipment for achieving fertilization. Although multiple events occur in a particular order during spermiogenesis, it is unclear how the timing of those events is controlled. In the present study, we found that primary spermatocytes obtained from the spermatogenic testes of Oryzias latipes synchronously differentiated into sperm without contact with somatic cells in culture. Because those sperm can fertilize with mature eggs (Saiki et al., 1997), any events of spermiogenesis that are essential for achieving fertilization are completed in the in vitro spermiogenesis. In the in vitro spermiogenesis, the protamine gene expression was observed in the early period and mitochondrion localization was established in the same period. Those results suggest that both nuclear remodeling and organelle replacement begin in the early period of spermiogenesis. The cytoplasmic lobe was formed after the mitochondrion localization had been established. In most spermatids differentiated in cell culture, a flagellum began to elongate during the early period and continued to elongate up to 3 days. These results revealed the timings of the spermiogenetic events under the intrinsic control of the cultured spermatids toward the formation of fertilizable sperm in O. latipes.

Characteristics of sperm motility induced on the egg-jelly in the internal fertilization of the newt, Cynops pyrrhogaster.

Most urodeles undergo internal fertilization and sperm are directly inseminated onto the surface of egg-jelly. Feature of sperm motility induced on the egg-jelly was examined in the newt, Cynops pyrrhogaster. When sperm were directly inseminated onto an egg-jelly, sperm motility was immediately induced on its surface. The egg-jelly of C. pyrrhogaster was composed of six sublayers that were added by turns in oviduct. When the eggs with various sets of the sublayers were obtained and sperm were inseminated onto the egg-jelly, the immediate activity for the initiation of sperm motility was observed only on the outermost sublayer. Similarly, the immediate initiation of sperm motility was induced in the sperm suspended in the extract of the egg-jelly (JE). The initiation of sperm motility was affected by the external pH, and the motility was activated in the moving sperm. A K(+)-channel antagonist, charybdotoxin (CTX), or a Ca(2+)-channel antagonist, gallopamil inhibited the initiation of sperm motility in a dose dependent manner. These results demonstrated the feature of the mechanism regulating sperm motility under stable surroundings in the internal fertilization of amphibians.

CpZPC, a newt ZPC molecule, localizes to the inner surface of the egg envelope.

Zona pellucida-3 is an essential molecule for the binding of sperm to the egg envelope and for induction of the acrosome reaction in mice. Its homologous molecules, ZPCs, have been widely identified in the eggs of many vertebrates, except for urodeles. In this study, to investigate the participation of ZPC in newt fertilization, we cloned the cDNA of newt ZPC from the ovary of Cynops pyrrhogaster by reverse transcription polymerase chain reaction (RT-PCR). The cDNA was constructed from 1,397 nucleotides and included one open reading frame corresponding to a sequence of 439 amino acids. The deduced amino acid sequence had identities at 52, 47 and 45% with Xenopus gp41, mouse ZP3 and medaka L-SF, respectively. It included four potential N-linked glycosylation sites and 12 highly conserved cysteine residues of mammalian ZP3/ZPC molecules. This result suggests that CynopsZPC (cpZPC) has molecular features similar to those of mammalian ZP3/ZPCs. Messenger RNA for cpZPC was detected in the ovary and faintly in the testis. Two bands corresponding to 84 kDa and 70 kDa in the egg envelopes were detected by immunoblotting with an antiserum raised against a 9 amino acid peptide in the C-terminus domain of cpZPC. The molecular size of 84 kDa fits with the size of a putative sperm-binding protein reported by Nakai et al. (1999), suggesting that cpZPC may contribute to sperm binding to the egg envelope in C. pyrrhogaster. The results of immunohistochemistry suggest that cpZPC was localized in the inner surface of the egg envelope. Similar localization is seen only in fish, suggesting that cpZPC is a unique molecule which may allow us to investigate the functional evolution of the egg envelope in vertebrates.

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.

The activity for the induction of the sperm acrosome reaction localises in the outer layers and exists in the high-molecular-weight components of the egg-jelly of the newt, Cynops pyrrhogaster.

Localisation of the acrosome reaction inducing activity in egg-jelly was examined in the newt, Cynops pyrrhogaster. The jelly has six layers: the J0, J1, J2, J3, J4 and st layers. Jelly was mechanically dissected and placed on a Millipore filter. When sperm were added from the outer surface side of the jelly, most of them exhibited the acrosome reaction after passing through the jelly. When egg-jelly was divided into four layers, strong activity for the induction of acrosome reaction was detected in the outer layers, J4+st. These findings suggest that the acrosome reaction is induced by a substance in the outer layers of the egg-jelly. Among jelly components separated by SDS-PAGE, a fraction of more than 500 kDa in molecular weight induced the acrosome reaction. Wheat germ agglutinin (WGA), Griffonia simplicifoliar agglutinin 1 (GS-1), Maclura pomifera agglutinin (MPA) and Arachis hypogaea agglutinin (PNA) inhibited the induction of the acrosome reaction by jelly extract, and WGA did so in a dose-dependent manner. Those lectins precipitated some molecules of over 500 kDa. These results suggest that the acrosome reaction is induced by the high molecular-weight components of egg-jelly in C. pyrrhogaster.

Egg-jelly structure promotes efficiency of internal fertilization in the newt, Cynops pyrrhogaster.

Egg-jelly is composed of a network of fibrous components and contains substances regulating the sperm-egg interaction. Many studies on the latter have been conducted, whereas the role of the egg-jelly structure in fertilization has not yet been fully assessed. In this study, we examined the fertilization efficiency in the presence and absence of the structure around the egg of the newt, Cynops pyrrhogaster, using a gelatin gel system. Although de-jellied eggs of C. pyrrhogaster can be fertilized with an adequate number of sperm, the fertilization rate was dramatically increased through the use of the gelatin gel. Sperm showed forward motility with straight morphology in the gel, whereas they swam in circles in solution. This result indicates that the gel structure is significant for sperm guidance to the egg surface, and its presence raises the fertilization efficiency in C. pyrrhogaster. When sperm were entangled in the gel structure, they were immediately folded and never showed any forward motility. Sperm with zigzag morphology were observed in the gelatin gel as well as in the egg-jelly, indicating the elimination of sperm by the gel structure. The effect of sperm elimination on successful fertilization was estimated using gelatin gels of different thickness. Though the variation did not affect the fertilization rate, the rate of normal development gradually increased in the thicker gels. This result indicates that sperm elimination in egg-jelly can function in the fertilization system. The roles of sperm guidance and sperm elimination under the physiological condition of internal fertilization of the newt are discussed.

The urodele egg-coat as the apparatus adapted for the internal fertilization.

Fertilization is a significant event for reproducing offspring. It is achieved under a species-specific environment, which influences the conditions to assure the successful fertilization in some cases. Several studies about the basic mechanism of fertilization suggest that the fertilization mechanism is modified among species to be suited for the fertilization environment. In amphibians, many anurans undergo external fertilization while most urodeles do internal fertilization. An amphibian egg is surrounded by egg-coats, which are composed of vitelline envelope and layered egg-jelly. They are significant as fields for the sperm-egg interaction at fertilization. The fertilization processes that take place in the egg-coats are supposed to be easily influenced by the fertilization environment, because they, especially egg-jelly, are exposed to the surroundings at fertilization. In the present article, we describe the fertilization system equipped in newt egg-coats. Newt sperm are stored in spermatheca that exists in cloaca of a female and directly inseminated on the surface of egg-jelly. Sperm motility and acrosome reaction are induced in the outermost portion of the egg-jelly. Motion of the moving sperm becomes vigorous in the egg-jelly and sperm are guided to vitelline envelope by the aid of egg-jelly structure. Most of the sperm passing through the egg-jelly, as the result, has been induced acrosome reaction and those sperm can bind to the vitelline envelope to contribute to the successful fertilization. This fertilization system has a distinct feature from the known system in species undergoing external fertilization. The feature of the system in the newt egg-jelly is discussed with the view to achieving the successful fertilization in the internal environment.

Activation of Xenopus Eggs by an Extract of Cynops Sperm: (fertilization/egg-activation/polyspermy block/sperm protease).

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca2+ ions, [Ca2+ ]0 , was reduced to 1.5 µM, but it was enhanced when [Ca2+ ]0 was increased to 340 µM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca2+ ]0 was 340 µM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca2+ ions that results in voltage-insensitive activation of the egg.

Cloning of Protamine cDNA of the Medaka (Oryzias latipes) and Its Expression during Spermatogenesis: (protamine/gene expression/spermatogenesis/in situ hybridization/medaka).

Protamines or sperm specific basic proteins are highly basic low molecular weight proteins that substitute histones in the chromatin of sperm during spermatogenesis. They condense sperm DNA into a highly compact, stable and inactive complex. In this study, cDNA of protamine of the medaka, Oryzias latipes, was cloned to elucidate the molecular mechanisms involved in spermatogenesis. A medaka testis cDNA library constructed in lambda gt11 showed 2.78X106 independent recombinants. Several positive clones were obtained by immunoscreening with polyclonal antiserum against medaka protamine. Sequencing showed that one of these positive clones, named MP-1, encoded arginine clusters characteristic of protamine. The putative amino acid sequence of MP-1 revealed a remarkable extent of homology with other fish protamines, such as 71% identity with thynnin Y, a sperm specific basic protein isolated from the bluefin tuna, Thunnus thynnus. Northern hybridization using a MP-1 cDNA probe showed that MP-1 mRNA is present exclusively in the testes and that it gave three detectable bands: a major band of 280 b, and two others of 400 b and 500 b. In situ hybridization of a complementary RNA probe (digoxigenine-UTP-labeled MP-1 RNA) revealed that MP-1 mRNA is localized in some secondary spermatocytes and spermatids, but not in primary spermatocytes or spermatogonia. These results differ from those obtained in studies on the rainbow trout by solution hybridization and in situ hybridization.

Time Sequence of Early Events in Fertilization in the Medaka Egg: (micropyle/exocytosis/sperm movement/calcium release/medaka egg).

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes. The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 108 /ml at 23°-25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 µm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5-16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 µm area, and became constant at about 12 µm/sec from 100 µm to 500 µm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 µm away from the sperm attachment point occurs simultaneously.

Fertilization of the eggs ofCynops pyrrhogaster (japanese newt) after immersion in water.

Cynops pyrrhogaster oviducal eggs with and without jelly envelopes (jelly egg and dejellied egg respectively) were immersed in water, and then inseminated artificially. After 1 h of immersion in water, more than half the dejellied eggs were fertilized and developed, but no jelly eggs developed. The rapid decrease in the ability of jelly eggs to be fertilized after immersion in water is not due to a deficiency in the egg. Our results make it clear that hydrated jelly envelopes prevent the eggs from fertilizing. The ability of the egg to be fertilized decreases for a long time in water and this decrease proceeds more slowly in De Boer's solution or Holtfreter's balanced salt solution than in water.

Fertilization of newt coelomic eggs in the absence of jelly envelope material.

Fertilization ofCynops pyrrhogaster (Japanese newt) coelomic eggs was studied in the absence of jelly envelope material or synthetic high polymer. An undiluted sperm fluid from the vas deferens fertilized coelomic eggs in the absence of the jelly envelope material or synthetic high polymer. The fertilized eggs developed beyond gastrulae and formed tail bud embryos. These results indicate that the fertilization process does not depend upon the presence of jelly envelope material or synthetic high polymer and that the sperms within the vas deferens are already capable of fertilizing the eggs inC. pyrrhogaster. The sperm suspension in Holtfreter's balanced salt solution (H-sperm) fertilized the coelomic eggs without the jelly envelope material or synthetic high polymer. These eggs had been suspended in Holtfreter's balanced salt solution (H) or in 1/20 strength H (1/20 H) prior to insemination (H-eggs or 1/20 H-eggs). In contrast, the sperm suspension in 1/20 H (1/20 H-sperm) did not fertilize 1/20 H-eggs, but dit H-eggs. In the latter case, H surrounding the eggs may affect sperms, allowing them to be fertilized. The 1/20 H-sperms regained their ability to fertilize 1/20 H-eggs on re-exposure to H. The 1/20 H-sperm also fertilized jelly eggs. The results of the dejellied egg experiment showed the same pattern. These results indicate that the sperms within the vas deferens lose their capacity to fertilize 1/20 H-eggs on exposure to low ionic strength solution (1/20 H); this capacity is restored by exposure to high ionic strength solution (H) or to jelly envelope.

Immunocytochemical location of vitellin in the egg of the silkworm,Bombyx mori, during early developmental stages.

Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.