Ultrastructure of intrauterine eggs: Evidence of early ovoviviparity in the caryophyllidean cestode <Emphasis Type="Italic">Wenyonia virilis</Emphasis> Woodland, 1923

Ultrastructural evidence for early intraurerine embryonic development of Wenyonia virilis is presented. At the initial stage of egg formation, the fertilized oocyte or ovum is surrounded by numerous vitellocytes and newly formed eggshell. Individual vitellocytes undergo progressive fusion into a vitelline syncytium. During cleavage divisions, three types of blastomeres are formed: macromeres, mesomeres and micromeres. Two large macromeres contain a large nucleus with spherical nucleolus and numerous small heterochromatin islands dispersed in moderately electron-dense nucleoplasm. The granular cytoplasm shows a few large mitochondria. Medium-sized mesomeres contain a spherical nucleus with numerous heterochromatin islands, adjacent to the nuclear envelope, and a prominent electron-dense nucleolus. Their nuclei are embedded in granular cytoplasm with a few large and numerous small mitochondria and Golgi complexes. The small micromeres are characterized by presence of spherical nucleoli with large areas of highly condensed heterochromatin and a few islands of granular electron-lucent nucleoplasm. Their granular cytoplasm shows a few small lipid droplets and several spherical mitochondria. Majority of micromeres give rise to the hexacanth but many of them also undergo degeneration or apoptosis. Both mesomeres and macromeres are engaged in the formation of the oncospheral envelopes. The outer envelope is formed by a fusion of two macromeres whereas the inner envelope originates from a fusion of mesomeres. The intrauterine eggs of W. virilis usually contain an embryo at the early preoncopheral phase of development and possesses three primary envelopes: (1) thick eggshell; (2) thin cytoplasmic layer of the outer envelope and (3) inner envelope. Based on embryonic development, egg type and life-cycle characteristics, caryophyllideans tend to show closer affinities to spathebothriideans than to the former pseudophyllideans.     

Embryonic development of three Cestoda from the genus Acanthobothrium (Tetraphyllidea, Onchobothriidae) (author's transl)]

The embryonic development from the egg to the oncosphere is examined in three Cestoda: Acanthobothrium coronatum (Rud., 1819), Acanthobothrium filicolle, Zschokke, 1888 and Acanthobothrium zschokkei Baer, 1948 (Tetraphyllidea, Onchobothriidae). The three ontogeneses have in common the following data: -- Two vitelline cells pass with the zygote into the ootype where a thin shell is formed out of a material which comes from the vitelline cells. -- At first the cleavage is equal, then it becomes unequal resulting in the formation of four types of blastomeres: macromere, secondary macromere, mesomere and micromere. -- The preoncospheral phase is characterized first by the blastomere multiplication and later by their decreasing number and differentiation. -- The embryonic envelopes are formed within the shell. The vitelline layer includes the cytoplasm, a vitelline nucleus and possibly the secondary macromere, the nucleus of which always lies against the outer membrane of this envelope. The syncytial embryophore develops from mesomeres coming from the embryo. -- The oncosphere is limited by its owm membrane whose posterior region seems to double in order to form a kind of cap bending over the six hook tips. The final number of embryonic hexacantha cells is relatively low.     

Ultrastructure of the intrauterine eggs of <Emphasis Type="Italic">Didymobothrium rudolphii</Emphasis> (Monticelli, 1890) (Cestoda,Spathebothriidea, Acrobothriidae) and its phylogenetic implications

The intrauterine polylecithal eggs of the spathebothriidean cestode Didymobothrium rudolphii (Monticelli, 1890) were examined by means of transmission electron microscopy (TEM). Each unembryonated egg is composed of a fertilised oocyte or ovum and several vitelline cells, all surrounded by a newly formed shell. The lumen of the proximal uterus is packed with unutilised vitelline material and eggs at different stages of shell formation. In the proximal region of the uterus, the fertilised oocytes, initially surrounded by dense, discontinuous islands of eggshell material and containing long axonemes of spermatozoa in their cytoplasm, were frequently observed. Sperm axonemes also remain in the oocyte cytoplasm of eggs surrounded by a thick electron-dense shell until the sperm nucleus is transformed into a male pronucleus. Despite the fact that the two-pronuclei stage and cell divisions within the eggs of D. rudolphii were never observed, individual eggs containing several blastomeres of different sizes were seen in the middle and distal regions of the uterus. This provides indirect evidence that at least a few initial cleavage divisions must take place in the intrauterine eggs and direct evidence that the early embryonic development of D. rudolphii starts in utero. The several vitellocytes present in each egg contain nutritive reserves for the developing embryos; these are composed mainly of numerous lipid droplets and a moderate amount of glycogen. In the eggs containing early embryos composed of several blastomeres, the cytoplasm of the degenerating vitellocytes exhibits the presence of so-called ‘foci of cytoplasmic degradation’, which appear to be involved in the autolytic process of the vitellocyte cell components and inclusions, such as a high accumulation of lipids and glycogen. This progressive degeneration of the vitellocytes, considered as an example of programmed cell death or apoptosis, likely contributes towards the resorption of nutritive reserves by the developing embryo. Some of the results of this study are commented upon in relation to the affiliation of the spathebothriideans with other lower cestode groups.     

Early intrauterine embryonic development in <Emphasis Type="Italic">Khawia sinensis</Emphasis> Hsü, 1935 (Cestoda,Caryophyllidea, Lytocestidae), an invasive tapeworm of carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>): an ultrastructural study

Intrauterine embryonic development in the caryophyllidean tapeworm Khawia sinensis has been investigated using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. Contrary to previous light microscopy findings that reported the release of non-embryonated eggs of K. sinenesis to the external environment, the present study documents various stages of embryonation (ovoviviparity) within the intrauterine eggs of this cestode. At the initial stage of embryonic development, each fertilised oocyte is accompanied by several vitellocytes that become enclosed within the operculate, electrondense shell. Cleavage divisions result in formation of blastomeres (up to about 24 cells) of various sizes. Mitotic divisions and apparent rosette arrangment of the blastomeres, the latter atypical within the Eucestoda, are observed for the first time in the intrauterine eggs of K. sinenesis. The early embryo enclosed within the electrondense shell is surrounded by a thin membraneous layer which in some enlarged regions shows presence of nuclei. Simultaneously to multiplication and differentiation, some of the blastomeres undergo deterioration. A progressive degeneration of the vitellocytes within eggs provides nutritive reserves, including lipids, for the developing embryo. The possible significance of this atypical timing of the intrauterine embryonic development to (1) the ecology of K. sinensis and that of a recent introduction of another invasive tapeworm, the caryophyllidean Atractolytocestus huronensis Anthony, 1958 to Europe; and (2) the affiliation of caryophyllideans with other lower cestodes, are discussed.     

Egg ultrastructure of the amabiliid cestode Tatria biremis Kowalewski, 1904 (Cyclophyllidea, Amabiliidae), with an emphasis on the oncospheral envelopes

This is the first report on the ultrastructure of eggs in the cestode family Amabiliidae Braun, 1900. The gravid proglottides of Tatria biremis easily detach from the strobila. Their thick-walled saccate uterus contains numerous rounded or oval eggs measuring about 30-32 μm in diameter. In the early preoncospheral phase, three primary embryonic envelopes are formed around the developing and differentiating embryos, namely: (1) vitelline capsule originating from vitellocyte material; (2) outer envelope formed by two macromeres, and (3) inner envelope originating from a fusion of three mesomeres. Thus, both the outer and inner envelopes of T. biremis eggs are cellular in origin and syncytial in nature. During egg maturation, the three primary embryonic envelopes undergo differentiation into fully formed oncospheral or egg envelopes. Most significant changes were observed in the inner envelope which becomes progressively subdivided into 3 sub-layers: the extra-embryophoral sub-layer, the embryophore, and the intra-embryophoral sub-layer, containing mesomere nuclei. The mature hexacanth is covered by a thin layer of the oncospheral tegument. Within the infective hexacanth larva, five cell types were distinguished: (1) a binucleated subtegumental cell; (2) U-shaped penetration gland; (3) nerve cells; (4) somatic cells representing the myocytons of both somatic and hook musculature, and (5) large germinative cells. Ultrastructural characteristics of T. biremis eggs are compared with those described in representatives of other cestode taxa. Since the functional ultrastructure of cestode egg envelopes is defined by multiple factors such as the type of life cycles, habitats and behaviour of the intermediate hosts, mode of the intermediate host infection, etc., ultrastructural studies of the greater diversity of cestodes are needed to obtain comparative data for fruitful analysis of cyclophyllidean cestode adaptations to their diverse life cycles.     

Light and electron microscopy observations of embryogenesis and egg development in the human liver fluke, Opisthorchis viverrini (Platyhelminthes, Digenea)

Eggs of most species digenean flukes hatch in the external environment to liberate larvae that seek and penetrate a snail intermediate host. Those of the human liver flukes, Opisthorchis viverrini, hatch within the gastrointestinal canal of their snail hosts. While adult parasites are primarily responsible for the pathology in cases of human opisthorchiasis, their eggs also contribute by inducing granulomata and in serving as nidi for gallstone formation. In view of the peculiar biology of O. viverrini eggs and their contribution to pathology, we investigated embryogenesis in this species by light and transmission electron microscopy. Egg development was traced from earliest stages of coalescence in the ootype until full embryonation in the distal region of the uterus. Fully mature eggs were generally impermeable to resin and could not be examined by conventional electron microscopy methods. However, the use of high-pressure freezing and freeze-substitution fixation of previously fixed eggs enabled the internal structure of mature eggs, particularly the subshell envelopes, to be elucidated. Fertilization occurs in the ootype, and the large zygote is seen therein with a single spermatozoon wrapped around its plasma membrane. As the zygote begins to divide, the spent vitellocytes are pushed to the periphery of the eggs, where they progressively degrade. The early eggshell is formed in the ootype by coalescing eggshell precursor material released by approximately six vitelline cells. The early eggs have a thinner eggshell and are larger than, but lack the characteristic shape of, mature eggs. Characteristic shell ornamentation, the "muskmelon" appearance of eggs, appears after eggshell polymerization in the ootype. Pores are not present in the shell of O. viverrini eggs. The inner and outer envelopes are poorly formed in this species, with the outer envelope evident beneath the eggshell at the opercular pole of the mature egg. The miracidium has a conical anterior end that lacks the distinctive lamellar appearance of the terebratorium of other digeneans, such as the schistosomes. The miracidium is richly glandular, containing an apical gland in the anterior end, large cephalic gland, and posterior secretory glands. Each gland contains a secretory product with different structure. The paucity of vitelline cells associating with eggs, the reduced size of eggs, and reduced complexity of the extraembryonic envelopes are interpreted as adaptations to the peculiar hatching biology of the miracidia.     

Advanced stages of embryonic development and cotylocidial morphogenesis in the intrauterine eggs of Aspidogaster limacoides Diesing, 1835 (Aspidogastrea), with comments on their phylogenetic implications

Ultrastructural aspects of the advanced embryonic development and cotylocidial morphogenesis of the aspidogastrean Aspidogaster limacoides are described. The posterior or distal regions of the uterus are filled with eggs containing larvae at advanced stages of morphogenesis and fully-formed cotylocidia. Various stages and organs of this larva are described in detail, including the aspects of the developing and fully-differentiated cotylocidium, the body wall (tegument and musculature), glandular regions and the protonephridial excretory system. Blastomere multiplication by means of mitotic divisions takes place simultaneously with the degeneration or apoptosis of some micromeres; this frequently observed characteristic is compared and discussed in relation to corresponding reports for other neodermatans. During the advanced stages of the embryonic development of A. limacoides, the vitelline syncytium disappears and the size of the embryo increases rapidly. Evident polarization of the differentiating larva was observed; towards one pole of the egg, cytodifferentiation of the mouth, surrounded by the oral sucker and cephalic glands, takes place, whereas, towards the opposite pole, differentiation of the posterior sucker (incipient ventral disc) occurs. The oral and posterior suckers are formed from numerous embryonic cells which have differentiated into myocytes. The central part of the oral sucker undergoes invagination and forms the future pharynx and intestine. Fully-developed cotylocidia of A. limacoides have a neodermatan type of tegument, flame cells and two types of glandular structures. These results suggest a sister relationship between the Aspidogastrea and the Digenea, although the systematic position of aspidogastreans in relation to other platyhelminth taxa remains somewhat equivocal.     

An ultrastructural study of embryonic envelope formation in the anoplocephalid cestode Mosgovoyia ctenoides (Railliet, 1890) Beveridge, 1978

In this study the ultrastructural aspects of egg envelope formation in the anoplocephalid cestode Mosgovoyia ctenoides are described. In the early stage of oncospheral morphogenesis, formation of three following primary embryonic envelopes takes place: (1) the capsule, (2) the outer envelope, and (3) the inner envelope. The capsule is formed from the vitellocyte material. Two macromeres contribute to the formation of the outer envelope and three mesomeres take part in the formation of the inner envelope. The three primary envelopes undergo further differentiation and transformation into the secondary envelopes, the so-called oncospheral or egg envelopes. In the advanced preoncospheral phase, the inner envelope undergoes differentiation into three sublayers: (1) a thick extra-embryophoral cytoplasmic layer; (2) an electron-dense embryophore, as a stiff pyriform apparatus; and (3) a thin intra-embryophoral cytoplasmic layer containing mesomere nuclei. The oncosphere is located in the extended cupule-like part of the pyriform apparatus. The two embryophoral horns elongate and fuse, thus forming a rigid cone. Four egg envelopes surround the mature infective oncosphere of M. ctenoides: (1) a thick capsule; (2) the outer envelope; (3) the inner envelope with a characteristic embryophore, in the form of the pyriform apparatus; and (4) the oncospheral membrane. The differentiation and ultrastructure of the egg envelopes of M. ctenoides are compared, in particular to those described in other anoplocephalids, and in general to the oncospheres of other cestode species.An erratum to this article can be found at     

Ultrastructural study of vitellogenesis in Maritrema feliui (Digenea, Microphallidae)

During vitellogenesis in the microphallid trematode Maritrema feliui, we distinguished four stages: (I) a stem cell stage of the gonial type; (II) an early differentiation stage with the main cell activity concentrated on the initiation of protein synthetic activity and the beginning of shell globule formation; (III) an advanced differentiation stage concentrated on a rapid intensification of protein synthetic activity, the progressive fusion of individual shell globules into large shell globule clusters and the formation of saturated lipid droplets and a small amount of β-glycogen particles in the peripheral cytoplasm, considered as a store of nutritive reserves for the developing embryos; and (IV) the mature vitellocyte. Early vitellocyte maturation is characterised by: (1) an increase in cell volume; (2) extensive development of large, labyrinth-like cisternae of GER that produce proteinaceous granules; (3) the development of Golgi complexes engaged in packaging this material; and (4) a continuous enlargement of proteinaceous granules within vacuoles and their transformation into shell globule clusters composed of the heterogeneous material observed during vitellocyte cytodifferentiation. Mature vitelline cells are very rich in two types of cell inclusions accumulated in large amounts in their cytoplasm: (1) shell globule clusters, which play an important role in eggshell formation; and (2) a few osmiophobic lipid droplets of a saturated nature that undoubtedly represent nutritive reserves for the developing embryos. In addition, there are small numbers of β-glycogen particles in the peripheral cytoplasm of mature vitellocytes of this species. The general pattern and ultrastructure of vitellogenesis in M. feliui greatly resembles those observed in another microphallid trematode, Maritrema linguilla, in other digeneans and in some lower cestodes. Quantitative and qualitative variations in lipids (saturated and unsaturated) and glycogen (α-glycogen rosettes and β-glycogen particles) during platyhelminth vitellogenesis between the different species of trematodes and some lower cestodes are identified and discussed.     

Structure and origin of the eggshell ofQuinqueserialis quinqueserialis (Trematoda: Notocotylidae)

Ultrastructural studies were conducted on the eggshell and portions of the female reproductive system of the notocotylid trematodeQuinqueserialis quinqueserialis (Barker and Laughlin 1911). The eggshell consists of long bipolar filaments extending from an ovoid egg capsule. The egg capsule, formed within the ootype, is composed of an inner electron-dense layer of vitelline origin and an outer membranous layer containing many shallow pits derived from membranous secretions of the Mehlis' gland. Filaments are formed at the level of the fourth uterine loop from electron-dense secretions produced within the uterine epithelium. Filament elongation continues as eggs pass through the remaining uterine loops.     

Role of vitelline envelope during fertilization in the black tiger shrimp, Penaeus monodon

Animal eggs possess investments through which sperm must penetrate. The aim of the present study was to investigate the role of the egg coating, the vitelline envelope, during sperm-egg interactions in the black tiger shrimp, Penaeus monodon. The site(s) of primary binding between sperm and egg and the possible binding molecule(s) for sperm were identified. In vitro adsorption of the vitelline envelope protein onto the sperm surface showed that primary binding occurred between the sperm anterior spike of acrosome intact sperm and the vitelline envelope. Results from streptavidin blotting revealed that the component of the vitelline envelope that interacts with the sperm integral membrane protein is a 370 kDa protein. In addition, it was shown that the vitelline envelope protein had no ability to induce acrosome reaction. These results suggest that the function of the vitelline envelope is as a primary binding site for sperm in shrimp, but not a sole trigger for the acrosome reaction.     

Ultrastructure of eggshell and embryological development of Salvator merianae (Squamata: Teiidae)

The objective of this study was to characterize the external morphology of Salvator meriane embryos in different stages of embryonic development and establish a relationship with the ultrastructure of the shell from oviductal transit to hatching. A total of 120 embryos were analyzed to describe their external morphology, and 78 eggs were used for the analysis of the shell. For embryonic development, the series was established according to the total length of the body. We established 40 embryonic stages from the primitive streak. In the early stages, the external morphological features are the C-shaped body, the maxillary, and mandibular fusion processes with the frontal process and the fusion of the forelimb with the digital plate. In the middle stages, the eyelid appears, and there are claws on the toes, cornification of fingers, and the onset of pigmentation. The last stage of embryonic development is characterized by the beginning of the formation of the scales, appear the toenails, and finalize the entire pigmentation. Regarding the relationship that exists with the ultrastructure of the egg during development, it was possible to observe a marked change in the composition of the shell and well-marked compaction during embryonic development, which may be related to the transport of calcium during embryonic ossification. Our results allowed us to show the complete sequence of embryonic development, determining the laying stage for this species. It was possible to establish a relationship with the ultrastructure of the eggshell from the oviductal transit to the moment of hatching.     

New advances in human embryology: morphofunctional relationship between the embryo and the yolk sac

Organogenesis occurs during the first 8 weeks of human embryonic development; in consequence, early human growth and development take place before and in the absence of fully developed internal organs. During this period, normal development depends on several factors, but two are imperative: nutrition and a functional transport system for the distribution of nutrients and for waste disposal. The yolk sac (YS), a highly differentiated adnexal organ, is known to accomplish this fundamental task during early pregnancy. In this review, we summarize our contribution to the understanding of early human embryology, focusing interest on analysis of the morphofunctional link that is established between the human embryo and the YS during the embryonic period. Embryos were collected from the gestational sac after salpingectomies performed on patients with singleton pregnancies occurring in the fallopian tube. Samples of YS were taken from 20 human embryos at Carnegie stages ranging from 12 to 20. The age of the embryo was estimated from data of the patient's last menstrual history and confirmed from crown-rump length measurements and morphological characteristics of the specimen. The samples were fixed in 3% glutaraldehyde and then postfixed in 2% osmium tetroxide and prepared for light, transmission, and scanning electron microscopy according to conventional techniques. The samples were examined with a Philips 301 EM and an S-4000 Hitachi field emission SEM. The yolk stalk, and the YS wall with its corresponding endodermal, mesenchymal, and mesothelial layers, were analyzed. In accordance with their morphological features, the endodermal cells are equipped with organelles to fulfill several functions that are expressed in absorption from the vitelline cavity via microvilli present into the outer cell surface, in secretion to the extracellular space, and in the synthesis of numerous proteins which are transported by the bloodstream to the embryo. The mesothelial surface is provided with cell-surface differentiation that promotes a protective coat to prevent damage from compression or friction of the YS wall against the amnios, umbilical cord, and chorionic cavity wall during growth. The mesenchyme is the main site for blood vessel formation and gives rise to a network that provides the embryo with nutrients and a means of waste disposal. A critical analysis of the role of the endodermal vesicle in the production of fluid that is accumulated in the YS, and of the role that the vitelline duct play in the exchange function between the YS and intestinal tract, is presented. We have demonstrated that the vitelline duct is not functional after week 5 because of the closure of its lumen. This finding is discussed with reference to the biological meaning of the vitelline duct and its functional period of activity, and its possible role in the physiology of exchange during the embryonic period is assessed.     

Transmission electron micrograph studies of developing oncospheral envelopes ofFimbriaria fasciolaris (Hymenolepididae)

Studies of the ultrastructure of oncospheral envelopes ofFimbriaria fasciolaris in utero using transmission electron microscopy revealed three primary layers: uterine, outer and inner envelopes. A capsule was not found. The uterine envelope surrounds up to 12 oncospheres enclosed by the outer and inner oncospheral envelopes. The secondary envelopes arise during the embryos' development and, in part, are formed in a way different from that described for other Hymenolepididae. A thin, folded shell is a derivative of the embryo and the mother organism. The embryophore and oncospheral membrane originate intracellularly from the inner oncospheral envelope. The mode of formation and final shape of the definitive oncospheral envelopes inF. fasciolaris and other Hymenolepididae species as well as their terminology are discussed.     

Computer-controlled, multilevel, morphometric analysis of blastomere size as biomarker of fragmentation and multinuclearity in human embryos

BACKGROUND: Little is known about blastomere size at different cleavage stages and its correlation with embryo quality in human embryos. Using a computer system for multilevel embryo morphology analysis we have analysed blastomeres of human embryos and correlated mean blastomere size with embryonic fragmentation and multinuclearity. METHODS: A consecutive cohort of 232 human 2-, 3- and 4-cell embryos from patients referred for ICSI treatment were included. Sequences of digital images were taken by focusing at 5- micro m intervals through the embryo. Blastomere sizes and number of nuclear structures were evaluated based on these sequences. The degree of embryonic fragmentation was evaluated by normal morphological assessment prior to transfer and correlated to the blastomere sizes. RESULTS: As a result of normal cell cleavage, mean blastomere size decreased significantly from a volume of 0.28 x 10(6) microm(3) at the 2-cell stage to 0.15 x 10(6) microm(3) at the 4-cell stage (P < 0.001). Mean blastomere size decreased significantly (P < 0.001) with increasing degree of embryonic fragmentation, where highly fragmented embryos showed a 43-67% reduction in blastomere volume compared with embryos with no fragmentation. Multinucleated blastomeres were significantly larger than non-multinucleated blastomeres (P < 0.001). On average, multinucleated blastomeres were 51.5, 67.8 and 73.1% larger than their non-multinucleated sibling blastomeres at the 2-, 3- and 4-cell stage, respectively. Furthermore, the average volume of non-multinucleated blastomeres originating from multinucleated embryos was significantly smaller than the average volume of the blastomeres from mononucleated embryos (P < 0.001). CONCLUSIONS: The results of this study show that the average blastomere size is significantly affected by degree of fragmentation and multinuclearity, and that computer-assisted, multilevel analysis of blastomere size may function as a biomarker for embryo quality.     

Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence

It has been suggested that mitochondrial DNA defects that effect metabolic capacity may be a proximal cause of failures in oocyte maturation, fertilization, or early embryonic development. Here, the distribution of mitochondria was examined by scanning laser confocal microscopy in living human pronuclear oocytes and cleavage stage embryos, followed either by measurements of the net ATP content of individual blastomeres or anti-tubulin immunofluorescence to determine the relationship between mitochondrial distribution and microtubular organization. The results indicate that specific patterns of perinuclear mitochondrial aggregation and microtubular organization are related, and that asymmetrical mitochondrial distributions at the pronuclear stage can result in some proportion of blastomeres with reduced mitochondrial inheritance and diminished ATP generating capacity. While the inability to divide appears to be a development consequence for an affected blastomere, for the embryo, reduced competence may occur during cleavage if several blastomeres inherit a mitochondrial complement inadequate to support normal cellular functions. The findings provide a possible epigenetic explanation for the variable developmental ability expressed within cohorts of morphologically normal early cleavage stage human embryos obtained by in-vitro fertilization.     

Adenosinetriphosphatase (Mg-ATPase) activity in the plasma membrane of preimplantation mouse embryo as revealed by electron microscopy

The ultrastructural localization of Mg2+-ATPase activity was investigated in unfertilized eggs and in preimplantation mouse embryos. Enzyme activity was detected on the entire surface of unfertilized eggs and in one-cell embryos. From the two-cell stage up to the 12-cell embryo the reaction product was localized both at the exposed surface of the embryo and between blastomeres. At the late morula stage, activity was only present on the free surface of the peripheral cells outside the apical tight junctions. In the early blastocyst the membranar reaction was observed both on the outside membrane of the trophoblastic cells and on the inner surface facing the blastocoele, whereas in the late blastocyst the reaction remained unchanged on the outer surface of the embryo but was absent from the membranes bordering the blastocoele. These results are discussed with reference to the modification of the cell surface membrane during preimplantation development.     

Chorion formation and ultrastructure of the egg of the cat flea (Siphonaptera: Pulicidae)

Oocyte development in adult female cat fleas, Ctenocephalides felis (Bouché), was studied by light and electron microscopy to determine the formation and ultrastructural morphology of the eggshell. As oocytes develop, somatic follicle cells from the lining of the ovariole migrate around the oocytes. The follicle cells produce electron-dense granules that form the vitelline membrane around the developing oocyte. Subsequently, electron-lucent granules containing an electron-dense core (precursors of the chorion) are produced from the rough endoplasmic reticulum that appear as dilated and clear linear clefts in the cytoplasm of the follicle cells. Exocytosis and coalescence of the granules around the oocyte as the follicle cells disintegrate give rise to the chorion. The chorion was found to consist of 4 distinct layers. The external surface of the egg shell consists of a particulate layer approximately 760 nm thick, composed of an electron-lucent layer of widely dispersed granules. Embedded in this layer are electron-dense spheres that project above the surface of this granular layer. Beneath this outermost layer is a band of electron-dense material, consisting of densely packed granules and is half as thick as the outer particulate layer. The 3rd layer consists of relatively thick, weakly laminated chorion, with a felt-like appearance due to a meshwork of microfibrils. Projections of this network of microfibrils form pillars that attach this layer to a thin relatively compact 4th or basal layer. The pillars and the air-filled cavities lying between the 3rd and 4th chorionic layers constitute the chorionic meshwork known as the palisades or trabecular layer that form the major respiratory organ of the eggshell. The trabecular layer is connected to the external environment by means of the lateral and anterior aeroplyes. The vitelline membrane lies between the chorion and oocyte and is a granular, uniform, moderately electron-dense layer measuring approximately 260 nm thick. The micropyle at the posterior of the flea egg consists of a rosette of 50-80 apertures and possesses an internal electron-dense plug between the chorion and the vitelline membrane. An aeropyle at the anterior end of the egg consists of a rosette of 40-50 apertures. An inconspicuous aeropyle appears as a cluster of hexagonal or polygonal-shaped plaques on the lateral surface of the chorion. Each plaque contains 3-8 pores.     

Influence of E-Cadherin-Mediated Cell Adhesion on Mouse Embryonic Stem Cells Derivation from Isolated Blastomeres

Efforts to efficiently derive embryonic stem cells (ESC) from isolated blastomeres have been done to minimize ethical concerns about human embryo destruction. Previous studies in our laboratory indicated a poor derivation efficiency of mouse ESC lines from isolated blastomeres at the 8-cell stage (1/8 blastomeres) due, in part, to a low division rate of the single blastomeres in comparison to their counterparts with a higher number of blastomeres (2/8, 3/8 and 4/8 blastomeres). Communication and adhesion between blastomeres from which the derivation process begins could be important aspects to efficiently derive ESC lines. In the present study, an approach consisting in the adhesion of a chimeric E-cadherin (E-cad-Fc) to the blastomere surface was devised to recreate the signaling produced by native E-cadherin between neighboring blastomeres inside the embryo. By this approach, the division rate of 1/8 blastomeres increased from 44.6% to 88.8% and a short exposure of 24 h to the E-cad-Fc produced an ESC derivation efficiency of 33.6%, significantly higher than the 2.2% obtained from the control group without E-cad-Fc. By contrast, a longer exposure to the same chimeric protein resulted in higher proportions of trophoblastic vesicles. Thus, we establish an important role of E-cadherin-mediated adherens junctions in promoting both the division of single 1/8 blastomeres and the efficiency of the ESC derivation process.     

Preimplantation development of mouse and human embryos biopsied at cleavage stages using a modified displacement technique

A modified embryo biopsy method was tested on four- and eight-cell stage mouse embryos and used on human embryos to obtain blastomeres for preimplantation genetic diagnosis. The biopsy method tested combines zona drilling and fluid displacement to force one or two cells through an opening in the zona pellucida of the cleavage-stage embryo. Rates of cell division and the percentage of mouse embryos forming blastocysts following biopsy at the eight-cell stage were not significantly different from those observed in unoperated control embryos. The percentage blastocyst formation was not significantly different in embryos biopsied at the four-cell stage and in control embryos, although cell division was significantly retarded following biopsy. 96% of the mouse blastomeres isolated at the eight-cell stage were recovered intact and 96% of those placed in culture underwent cell division. Survival and division of cells isolated at the four-cell stage were 92 and 84% respectively. Most of the cultured blastomeres cleaved several times and formed small trophoblast vesicles. Chromosomes were observed in 59% of blastomeres incubated in the presence of colcemid. In the initial use of this biopsy technique for human preimplantation genetic diagnosis, blastocyst formation was observed in 9 of 13 human embryos biopsied at the 7- to 10-cell stage. These findings support the use of this biopsy method as an alternative to aspiration techniques.