Placental Type Interactions and Evolutionary Trends of Development of Uterus in Cestodes

In Proteocephalus thymalli and P. torulosus, a contact of the placental type in uterus was shown to be formed at two different levels. At the first level an interaction occurs between outgrowths of uterine epithelium and thin capsule of embryos closely adjacent to uterine wall. The next level is formation of contact between neighboring egg capsules, which allows distributing nutrients among fetuses present in the uterine cavity. Placental interactions in Clestobothrium acheilognathi are limited in time and space. First, a relatively small number of eggs are involved in interaction of the placental type in the uterine sac, while uterine duct is filled with freely lying eggs. Second, the closest contact is observed in eggs with non-sclerosed egg shell. One of the main evolutionary tendencies in cestodes has been shown to be a modification of uterus for formation of close interrelations with embryonic membranes in the course of transition from the extrauterine to the intrauterine type of embryonic development. Uterus in parasites with a polylecital type of the egg is suggested to serve to the greater extent as a reservoir, whereas in cestodes with oligolecital eggs, uterus performs its direct function—supply of developing embryos with nutrients. As a result, modifications of uterine epithelium are formed: from the appearance of the placental type interactions formed repeatedly in phylogenetically distant groups of cestodes to formation of branched outgrowths separating the uterine space into units or disintegration to actively functioning uterine capsules.     

Oogenesis and egg-shell formation in Aspiculuris tetraptera Schulz (Nematoda: Oxyuroidea).

The ovary of Aspiculuris tetraptera has a prominent terminal cap cell. This is considered to be part of the ovarian epithelium. Oogonia detach from the short rachis and increase in size from 6 to 60 microns; accumulating hyaline granules, shell granules and glycogen. The hyaline granules persist in the eff cytoplasm after shell formation has been completed and are considered to be lipoprotein yolk. The shell granules contribute to the non-chitin fraction of the chitinous layer. A classification of the cytoplasmic inclusions of the nematode oocyte is proposed. Upon fertilization a vitelline membrane is formed which constitutes the vitelline layer of the egg-shell. The chitinous layer is secreted in the perivitelline space, between the vitelline layer and the egg oolemma. Upon completion of chitinous layer synthesis, the egg cytoplasm contracts away from its inner surface. The material of the lipid layer is secreted at the surface of the egg cytoplasm and adheres to the inner surface of the chitinous layer. During secretion of the chitinous and lipid layers by the egg cytoplasm, the uterine cells secrete the unit membrane-like external uterine layer and the crystalline internal uterine layer. A complex system of interconnecting spaces develops in the internal uterine layer. This system is open to the exterior via breaks in the external uterine layer. There is no direct involvement of the uterine cells in the formation of this structure.     

Morphology and ultrastructure of the uterus of Lineolepis scutigera (Dujardin, 1845) Karpenko, 1985 (Cestoda,Cyclophyllidea, Hymenolepididae) in formation of uterine capsules

The microanatomy and ultrastructure of the uterus in different stages of ontogenesis were studied in a cyclophyllidean cestode Lineolepis scutigera. In the early stages, developing embryos lie freely in the cavity of a single unbroken uterus, which is later fragmented into separate compartments. As a result, numerous spherical uterine capsules are formed; each of them contains one formed egg. Capsules represent a fragmented but actively functioning uterus. Muscular cells containing numerous lipid inclusions are located around them. In the final stages, placenta-like relationships are formed between eggs and the epithelium of capsules. A comparative morphofunctional analysis of uterine capsules in cestodes is presented. Attention is paid to the formation of close interactions between the uterine epithelium and developing eggs.     

Oogenesis and egg shell formation in Heterakis gallinarum (Nematoda)

The structure of the developing ova and egg shell formation of Heterakis gallinarum has been described. The oogonia are small, undifferentiated cells which are arranged around a central cytoplasmic rachis. The oogonia and young oocytes are in cytoplasmic continuity with the rachis and it is suggested that the rachis may influence synchronous development of the oocytes. The oocytes contain two types of granule; refringent, which give rise to the ascaroside layer of the egg shell, and another kind which appear to be a type of yolk for the developing egg. After fertilization the spermatozoon produces numerous ribosomes; a second unit membrane appears beneath the oolemma, and the chitinous layer of the shell forms between the oolemma and this inner membrane. The refringent granules later produce the ascaroside layer of the shell between the chitinous layer and the inner membrane. The outermost layer of the shell is produced from material secreted by the cells of the uterus.     

Structure and permeability of the egg capsule of the bonnethead shark, Sphyrna tiburo

In the viviparous bonnethead shark, Sphyrna tiburo, a fluid-filled, acellular egg capsule surrounds fertilized eggs and developing embryos throughout gestation. Like other placental shark species, the capsule remains intact even at the placental implantation site. Although its intervention between the uterine and embryonic tissues of the placenta has long been thought to mediate physiological exchange, little information is available concerning even its basic structure or permeability to solutes. The 1 mum thick capsule wall consists of an inner layer of gelatinous material and an outer layer consisting of at least three laminae of orthogonally arranged fibrous material. These fibers are irregular and often branched. Permeability experiments showed that solutes less than 1,355 Da diffuse across the egg capsule whereas those greater than 6,000 Da do not pass through the membrane. Solute movement across the capsule is a concentration-dependent phenomenon indicating diffusion rather than active transport. Experimental data also suggest that there is an increase in the permeability of the egg capsule to low molecular weight materials during mid- and late gestation. These observations are discussed in relation to the function of the egg capsule as a mediator of maternal-embryonic interactions in matrotrophic sharks.     

Ultrastructural studies on the nematode Xiphinema diversicaudatum: Egg shell formation

The ultrastructure of the formation of the egg shell in the longidorid nematode Xiphinema diversicaudatum is described. Upon fertilization a vitelline membrane, which constitutes the vitelline layer of the egg shell, is formed. The chitinous layer is secreted in the perivitelline space, between the vitelline layer and the egg cell membrane. On completion of the chitinous layer, the material of the lipid layer is extruded from the egg cytoplasm to the outer surface, through finger-like projections. Both chitinous and lipid layers are secreted by granules in the egg cytoplasm that disappear as the layers are completed. Chitinous and lipid layers are formed during the passage of the egg through the oviduct. The vitelline layer is enriched with secretions produced by the oviduct cells and then by phospholipids secreted by the cells of the pars dilatata oviductus. The inner uterine layer is also formed by deposition of secretory products apposed on the egg shell in the distal uterine region and Z-differentiation. In the proximal part of the uterus, the egg has a discontinuous electron-dense layer, the external uterine layer. Tangential sections between chitinous and uterine layers revealed the presence of holes, possibly egg pores, delimited by the two uterine layers.     

Quantification of the maternal-embryonal nutritional relationship of elasmobranchs: case study of wobbegong sharks (genus Orectolobus)

The present study used wobbegong sharks (genus Orectolobus) to assess the threshold value proposed by previous research to categorize strict lecithotrophic from incipient histotrophic species. Totals of 236 and 135 ornate wobbegong Orectolobus ornatus and spotted wobbegong Orectolobus maculatus, respectively, were collected from the New South Wales commercial fishery between June 2003 and May 2006. Eight pregnant gulf wobbegong Orectolobus halei were also recorded outside the sampling period for the first time. The three species were reproductively synchronous with a gestation of c. 10-11 months. Embryos started to be macroscopically visible during January and external yolk sacs were fully absorbed by June to July when embryos were c. 200 mm total length (L(T) ). Internal yolk sacs were first observed during April to May when embryos were c. 160 mm L(T) , reached a peak during June and persisted in embryos immediately prior to parturition. The total wet mass from uterine egg to full-term embryos increased by 44-89% and 45-62%, whereas the total organic mass decreased by 32-33% and 26%, for O. ornatus and O. maculatus, respectively, suggesting that these species are strict lecithotrophic yolk-sac viviparous sharks with no maternal nutrient input. A review of the literature identified various issues and suggested that the previously proposed organic mass loss threshold value separating strict lecithotrophic species from incipient histotrophic species might not be appropriate. Instead, it is recommended that a combination of methods (e.g. estimation of organic mass gain or loss between ovarian egg and developed embryo, histology and electron microscopy of the uterus, radio-tracer assay and uterine fluid analysis throughout gestation) is used to discern between strict lecithotrophic and incipient histotrophic species.     

Reproductive role of attaching filaments on the egg envelope in Xenopoecilus sarasinorum (Adrianichthidae, teleostei)

Eggs of Xenopoecilus sarasinorum possess two distinct types of filaments on the surface of the egg envelope (chorion), long, attaching filaments restricted to the vegetal pole and weak, nonattaching filaments around the animal pole (micropyle). Both types are formed during oogenesis. After mature eggs were spawned through the urogenital pore, they were fertilized and hung in an abdominal concavity of the female. Oviposition never took place in the presence of embryos in the concavity because of the retardation of oogenesis. The loosely tangled tips of the attaching filaments that are retained within the ovarian cavity plug the urogenital pore by forming a hard complex with the epithelial cells. Into this plug structure that fuses with the inner wall of the urogenital pore, capillaries are provided. Within 5 days after the initiation of hatching, this plug degenerates and is released from the urogenital pore. Thus, in female X. sarasinorum, the reproductive cycle seems to be regulated by the physiological function ofthe plug structure formed by the attaching filaments in response to the presence of developing embryos.     

Follicular epithelium, theca and egg envelope formation in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The follicular epithelium and theca of oocytes in Serrasalmus spilopleura differentiates during the initial primary growth phase. The follicular cells are squamous and the thecal cells are disposed in two layers. During the secondary growth phase, follicular cells become cuboidal, acquire characteristics typical of protein- or glycoprotein-producing cells, and show dilated intercellular spaces. Formation of the egg envelope in S. spilopleura begins in the previtellogenic oocytes as a layer of amorphous electron-dense material is laid down on the oolemma. During vitellogenesis, another layer of electron-dense material appears beneath the first layer. Also during this phase, a layer of amorphous, less electron-dense material is formed adjacent to the follicular epithelium. The secondary egg envelope appears at the postvitellogenic phase and is composed of a filamentous and undulant material. The morphology of the egg envelopes in S. spilopleura reflects not only its oviparous nature but also the fact that its eggs are adhesive.     

Laparoscopic transfer of ovine and cervine embryos using the transpic technique

A transpic technique was developed to transfer embryos to 352 sheep and 4 deer recipients using a laparoscope, a modified pair of Allis forceps and a modified Cassou aspic normally used for laparoscopic uterine insemination. The overall proportion of uncomplicated transfers in Experiment 1 in 216 recipient ewes was 90.7% (range between groups 80 to 100%), 3.7% of the transfers were presumed to be loss of embryos during expulsion from the transpic, and 5.6% were apparent transfers into the uterine wall. In Experiment 2,83% of transfers into 136 ewe recipients were uncomplicated, 5% were presumed to be loss of embryos during expulsion, 1% was apparent transfer into the uterine wall, and 11% involved 2 attempts at transfer. Only 34% of 116 recipients receiving low-quality frozen-thawed embryos were pregnant and 24% of the 226 embryos survived to term. In contrast, high pregnancy rates (>80%) and embryo survival rates (>70%) were achieved following uncomplicated and twice attempted transfers of fresh embryos. Pregnancy rates and embryo survival rates were low (<2%) following the presumed loss of embryos during expulsion and apparent transfers into the uterine wall. All 4 deer transfers were uncomplicated and 2 2 good-quality embryos survived to term compared with 0 2 low-medium quality embryos. The transpic technique is a moderately invasive technique which permits fast (15 to 20/h) and reliable transfer of embryos in small ruminants. With appropriate care, nearly all of the embryos can be correctly placed in the uterus, and high pregnancy rates and embryo survival rates can be achieved using this technique.     

Morphology and ultrastructure of reproductive organs of <Emphasis Type="Italic">Monocercus arionis</Emphasis> (Sibold, 1850) Villot, 1982 (Cestoda: Cyclophyllidea)

The morphology of the reproductive system at different stages of ontogenesis and ultrastructural peculiarities of the copulatory organs and uterine in Monocercus arionis (Sibold, 1850) Villot, 1982 (Cyclophyllidea) have been studied. The muscle of the outer wall of the cirrus bag has a higher organizational level than typical smooth-muscle cells in cestodes. The cirrus is armed with typical filamentous and bladelike microtriches. The uterine epithelium contacts the thin capsule of the developing embryos located closely to the uterus wall, and the embryos contact each other in the uterine cavity in what can be interpreted as placental interactions. The specificity of the structure and arming of the copulatory apparatus has been considered, and the ultrastructural peculiarities of the uterus in the members of different orders of cestodes have been compared and analyzed.     

Structural changes to the uterus of the dwarf ornate wobbegong shark (Orectolobus ornatus) during pregnancy

Embryos of the viviparous dwarf ornate wobbegong shark (Orectolobus ornatus) develop without a placenta, unattached to the uterine wall of their mother. Here, we present the first light microscopy study of the uterus of O. ornatus throughout pregnancy. At the beginning of pregnancy, the uterine luminal epithelium and underlying connective tissue become folded to form uterine ridges. By mid to late pregnancy, the luminal surface is extensively folded and long luminal uterine villi are abundant. Compared to the nonpregnant uterus, uterine vasculature is increased during pregnancy. Additionally, as pregnancy progresses the uterine epithelium is attenuated so that there is minimal uterine tissue separating large maternal blood vessels from the fluid that surrounds developing embryos. We conclude that the uterus of O. ornatus undergoes an extensive morphological transformation during pregnancy. These uterine modifications likely support developing embryos via embryonic respiratory gas exchange, waste removal, water balance, and mineral transfer.     

Capsule walls as barriers to oxygen availability: Implications for the development of brooded embryos by the estuarine gastropod Crepipatella dilatata (Calyptraeidae)

Encapsulation of developing embryos imposes potential restrictions, because the capsule wall must allow for adequate inward diffusion of oxygen and for increased diffusion of oxygen as metabolic demand increases with continued development. Samples of egg capsules from the gastropod Crepipatella dilatata were used to document surface characteristics, composition of the different capsule wall layers, and alterations in wall thickness during development. The diffusion coefficient and capsule wall permeability were determined experimentally for capsules containing embryos at different developmental stages. We also determined oxygen consumption rates for various embryonic stages and for nurse eggs, which provide food for embryos during development. The capsule wall of C. dilatata possesses 2 differentiated layers: the external capsular wall (ECW) and the internal capsular wall (ICW). The ECW is compact and fibrous, features that remain invariable during development, and lacks surface features that might make some portions of the capsule wall more permeable to oxygen than others. On the other hand, the ICW is initially spongy and thick, but significantly decreases in thickness over time, particularly before the embryos begin feeding on nurse eggs. Although the capsule wall is a serious barrier to diffusion, permeability to oxygen increases over time by 112% due to the dramatic thinning of the inner capsule wall layer. Nurse eggs consume oxygen but at very low rates, supporting the idea that they correspond to living embryonic cells that have stopped their development. Respiration measurements indicated that embryos are initially supplied with enough oxygen within the egg capsules to carry out the activities characteristic of embryogenesis, even though the capsular walls show their maximum thickness and lowest permeability at this time. However, as the embryo develops its velum and becomes more active, capsule wall thickness decreases and capsule permeability to oxygen increases. Correspondingly, the oxygen demands of metamorphosed but still encapsulated specimens are approximately 135% higher than those of pre-metamorphosed sibling embryos.     

三种胚胎移植技术方法比较

以近交系FVB、DBA／2小鼠作为供体胚小鼠,封闭群CD-1小鼠作为受体小鼠,对实验小鼠胚胎移植的三种方法进行比较,即：对小鼠受精卵1细胞期胚(或2细胞期胚)经由受体小鼠输卵管口移植、经由受体小鼠输卵管壁移植及对小鼠进行子宫移植(8细胞期胚)的方法进行了实验研究,并对各方法适用于使用的动物对象、实验要求、产仔率、优势与不足等做了进一步探讨,为今后的实验动物提供参考依据。结果表明,输卵管壁移植优于输卵管伞口移植和子宫移植。     

Co-culture of mouse embryos with oviduct and uterine cells prepared from mice at different days of pseudopregnancy

Oviduct and uterine cell cultures were prepared from mice at different days of pseudopregnancy and their effects on the development of 1- and 8-cell mouse embryos in co-culture were examined. One-cell mouse embryos in co-culture with oviduct cells from 20 h to 120 h after hCG had a mean (+/- s.e.) cell number of 70.1 +/- 3.6, significantly (P less than 0.001) higher compared with those cultured in Whittingham's T6 medium supplemented with 5% fetal calf serum (T6 + 5% FCS) (30.4 +/- 1.6). Transfer of embryos, at 96 h after hCG, to synchronous pseudopregnant recipients showed that more embryos in oviduct co-culture formed fetuses than those cultured in T6 + 5% FCS. Co-culture of 1-cell embryos with uterine cells did not confer an advantage in cell numbers over T6 + 5% FCS. However, more 8-cell embryos formed blastocyst outgrowths after 100 h in co-culture with uterine cells prepared from mice at Day 3 of pseudopregnancy than with uterine cultures prepared from mice at Day 1 of pseudopregnancy or oviduct cells. In addition, there was further improvement when the Day 3 uterine co-cultures were supplemented with 1 or 10 ng progesterone/ml. These results highlight the importance of the oviduct and uterine cells during the different stages of preimplantation embryo development.     

Fertilization in a crab: I. Early events in the ovary,and cytological aspects of the acrosome reaction and gamete contacts

Early events in fertilization were studied in Carcinus maenas by in vitro experiments and ultrastructural analysis; some were found to occur in the lumen of ripe ovaries. The acrosome reaction generally conformed to the usual Reptantia Decapoda pattern. However, a prominent membrane system continuous with the nuclear envelope and located close to the base of the acrosome tubule characterized the type of spermatozoon observed in Carcinus maenas. Such complex anatomical connections linking the three parts of the reacted spermatozoon (acrosome tubule, membrane system and nucleus envelope) may be significant in relation to the membrane system's contribution to the acrosome reaction. The outer layer of the everted acrosomal vesicle was found to comprise tubular elements ending in bell-shaped corpuscles, deeply interdigitated with the oolemma microvilli during the establishment of the initial contacts between the reacted spermatozoon and the egg plasma membrane. At the site of contact, the oolemma formed a minute fertilization cone, locally depressed by the acrosome tubule. During these early fertilization events, the nucleus, like the other spermatozoon components, was seen to penetrate the egg coatings first, and later to be located near the oolemma.     

Reproduction,placentation, and embryonic development of the Atlantic sharpnose shark,Rhizoprionodon terraenovae

The Atlantic sharpnose shark Rhizoprionodon terraenovae (Richardson) is a small carcharhinid that is a common year-round resident along the southeast coast of the United States. It is viviparous and its embryos develop an epithelio-vitelline placenta. Females enter shallow water to give birth in late May and early June. Mating occurs shortly after parturition, and four to seven eggs are ovulated. Fertilized eggs attain the blastoderm stage in early June to early July. Separate compartments for each egg are formed in the uterus when the embryos reach 3–30 mm. Embryos depend on yolk for the first 8 weeks of development. When embryos reach 72 mm their yolk supply is nearly depleted and they shift to matrotrophic nutrition. When the embryos reach 40–55 mm, placental development begins with the vascularization of the yolk sac where it contacts the uterine wall. Implantation occurs at an age of 8–10 weeks by which time the embryos reach 70–85 mm. The expanding yolk sac engulfs the maternal placental villi, and its surface interdigitates with the villi to form the placenta. The rest of the lumenal surface of the uterus is covered by non-placental villi that appear shortly after implantation. Histotrophe production by the non-placental villi begins just after their formation. The placenta grows continuously during gestation. The egg envelope is present throughout gestation, separating maternal and fetal tissues. Embryos develop numerous appendiculae on the umbilical cord. Young sharks are born at 290–320 mm after a gestation period of 11 to 12 months. © 1993 Wiley-Liss, Inc.     

MEGAGAMETOPHYTE ORGANIZATION IN A POLYEMBRYONIC LINE OF LINUM USITATISSIMUM

A haploid-diploid twin-producing polyembryonic line “RA91” of Linum usitatissimum L., flax, known to produce twin embryos with a frequency in excess of 32% was employed to determine the cytological factors relating to the production of haploid embryos using light and transmission electron microscopy. Potentially polyembryonic flax megagametophytes contain at maturity either a conventional 3-celled egg apparatus (36% of megagametophytes), or contain supernumerary eggs forming either a 4- (50%) or 5-celled (14%) egg apparatus. In megagametophytes with supernumerary eggs, one egg cell appears to occupy the conventional position, whereas additional supernumerary eggs surmount the synergids and are smaller cells presumably derived from division of the true egg. Although supernumerary eggs appear similar in ultrastructure, they differ in location, size, and unlike typical egg cells, appear to have a complete, contiguous cell wall adjacent to the synergid. Such supernumerary cells are postulated as the origin of such haploid embryos, with the diploid member formed by conventional fertilization events with the micropylar egg.     

Intracellular translocation of symbiotic bacteroids during late oogenesis and early embryogenesis of Bradysia tritici (syn. Sciara ocellaris) (Diptera: Sciaridae)

Abstract. The distribution of symbiotic prokaryotes (bacteroids) in ovarian follicles and young embryos of Bradysia ( Sciara ) was studied using light and electron microscopy. In mid-vitellogenic follicles (prior to oosome formation) isolated from 8-h-old midges, most symbionts were scattered in the ooplasm. Later, during oogenesis and concomitant with the formation of the oosome, symbionts aggregated at the posterior pole of the follicle. During early embryogenesis, large symbiont clusters were seen between the oolemma and oosome and lateral to the oosome. When the presumptive pole-cell nuclei moved into the oosome, the bacteroids became scattered in and around the prospective germ plasm, and many of them became incorporated into the pole cells. At the anterior pole, a nearly symbiontfree area could be recognized at first (anterior cone), but later on symbionts aggregated there (usually best seen in 1-h embryos). Thereafter, smaller symbiont clusters spread into more lateral parts of the thickened anterior cortex. Finally, the aggregates became dispersed when cleavage nuclei moved into the anterior egg cortex. The distribution of symbionts may reflect cytoplasmic streaming or other transport phenomena during development which could participate in oosome formation or in the histological differentiation of the anterior egg cortex.     

Aberrant expression of retinol-binding protein, osteopontin and fibroblast growth factor 7 in the porcine uterine endometrium of pregnant recipients carrying embryos produced by somatic cell nuclear transfer

The technique of somatic cell nuclear transfer (NT) is a useful tool to produce cloned animals for various purposes, but the efficiency to generate cloned animals using this technique is still very low. To improve the low efficiency in production of cloned pigs it is critical to understand the reprogramming process during development of cloned embryos, but it is also essential to understand the uterine function interacting with the transferred cloned embryos during implantation and placentation. Thus, to understand the uterine responsiveness to NT cloned embryos during pregnancy, we investigated expression of retinol-binding protein (RBP), osteopontin (OPN) and fibroblast growth factor 7 (FGF7), which play important roles in implantation and/or maintenance of pregnancy as a transport protein, an extracellular matrix protein and a growth factor, respectively, in the uterine endometrium in pigs. The uterine tissue samples were obtained by C-section from pigs with NT cloned normal (NT-normal) embryos and NT cloned abnormal (NT-abnormal) embryos and pigs with non-NT (Non-NT) embryos at term. Immunoblot analysis showed that expression of RBP and FGF7 decreased in the uterine endometrium of recipient gilts carrying NT embryos than in the endometrium of gilts carrying Non-NT embryos. Levels of OPN protein of 70 and 45kDa were not different in between the uterine endometrium of gilts carrying Non-NT and NT-normal embryos, but in the uterine endometrium of gilts carrying NT-abnormal embryos 70 and 45kDa OPN proteins increased compared to those in the endometrium of gilts carrying Non-NT embryos. Immunohistochemistry results showed that RBP expression was lower in the endometrial glandular epithelial cells, while OPN expression was higher in the endometrial luminal epithelial cells of the uterus of gilts carrying NT embryos than in the uterus of gilts carrying Non-NT embryos. Results of this study showed that maternal uterine genes were aberrantly expressed in the uterine endometrium of gilts carrying NT cloned embryos in varying degrees depending on the normality of the developing embryos. These results indicate that abnormal maternal-fetal interactions of the uterus carrying the developing NT cloned embryos may cause problems in development of cloned embryos.