Ultrastructural characterization of in vivo-produced ovine morulae and blastocysts (original) (raw)
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Oocyte ultrastructure in bovine primordial to early tertiary follicles
Anatomy and Embryology, 1997
The aim of the present study was to describe in detail the changes occurring in the cytoplasmic ultrastructure of the bovine oocyte from the onset of growth in the primordial follicle until the completion of growth in the tertiary follicle. Bovine oocytes from primordial, primary, secondary and early to mid-antral follicles were processed and analysed by light and transmission electron microscopy. The primordial follicular oocyte was characterized by numerous coated pits on the oolemma and the accumulation of free and organelle-related smooth (SER) and rough (RER) endoplasmic reticulum, round mitochondria and Golgi complexes around the nucleus, which was located slightly off centre. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, elongated mitochondria and Golgi complexes. During the secondary follicular stage, formation of the zona pellucida, development of gap junctions between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and reduction in the number of coated pits on the oolemma were seen. In the tertiary follicular oocyte up to 100 µm in diameter, the number of Golgi complexes and lipid droplets increased and the organelles were dislocated to the deep cortical region. During the final growth of the oocyte up to >120 µm, the organelles were dislocated further to the peripheral region, the extent of the free SER and RER compartments were reduced, the number of individual cortical granules increased, hooded mitochondria became abundant and the perivitelline space developed. In conclusion, the growth of the bovine oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte specific structures such as the zona pellucida and cortical granules.
A scanning electron microscope study of the hatching of bovine blastocysts in vitro
Reproduction, 1978
The blastocysts were recovered from cows 7-10 days after oestrus and cultured. The zona pellucida has a spongy fibrous structure. Hatching begins, about 24 h after culture, through a relatively small hole out of which the blastocyst appears to escape by its own activity. Later the zona becomes split and the two edges of the slit surround part of the blastocyst. The emerging cells were large or small blastomeres which were generally covered by a dense mass of microvilli.
Reproduction, 2003
The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphasized the need for basic knowledge about epiblast formation and gastrulation in bovine embryos. The aims of this study were to define stages of bovine post-hatching embryonic development and to analyse functional mechanisms of germ-layer formation. Embryos developed in vivo were collected after slaughter from superovulated cows on days 9, 11, 14 and 21 after insemination and processed for transmission electron microscopy (n = 26) or immunohistochemistry (n = 27) for potential germ-layer characterization (cytokeratin 8 for potential ectoderm; alpha-1-fetoprotein for potential endoderm; and vimentin for potential mesoderm). On day 9, the embryos were devoid of zona pellucida and presented a well-defined inner cell mass (ICM), which was covered by a thin layer of trophoblast cells (the Rauber's layer). Formation of the hypoblast from the inside of the ICM was ongoing. On day 11, the Rauber's layer was focally interrupted and adjacent underlying ICM cells formed tight junctions. The hypoblast, which formed a thin confluent cell layer, was separated from the ICM and the trophoblast by intercellular matrix. The embryos were ovoid to tubular and displayed a confluent hypoblast on day 14. The epiblast was inserted into the trophoblast epithelium and tight junctions and desmosomes were present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells. In some embryos, the epiblast was more or less covered by foldings of trophoblast in the process of forming the amniotic cavity. Cytokeratin 8 was localized to the trophoblast and the hypoblast underlying the epiblast; alpha-1-fetoprotein was localized to most hypoblast cells underlying the trophoblast; and vimentin was localized to most epiblast cells. On day 21, the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites and allantois development. These embryos depicted the gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate and lateral plate mesoderm. Cytokeratin 8 was localized to the trophoblast, the hypoblast and the surface and neural ectoderm; and alpha-1-fetoprotein was localized to the hypoblast, but not the definitive endoderm, the intensity increasing with development. Vimentin was initially localized to some, but not all, cells positioned particularly in the ventral region of the primitive streak, to presumptive definitive endoderm cells inserted into the hypoblast, and to mesoderm. In conclusion, within 2 weeks of hatching, bovine embryos complete formation of the hypoblast and the epiblast, establishment of the amniotic cavity, ingression of epiblast cells for primitive streak formation, involution of cells through the node and the streak for endoderm and mesoderm fomation, neurulation and differentiation of the mesoderm. The recruitment of cells from the epiblast to form the primitive streak as well as the endoderm and mesoderm is associated with expression of the intermediate filament vimentin.
Ultrastructural characterization of fresh and cryopreserved in vivo produced ovine embryos
Theriogenology, 2009
Controlled slow freezing and vitrification have been successfully used for ovine embryo cryopreservation. Selection of embryos for transfer is based on stereomicroscopical embryo scoring after thawing, but the subjectivity inherent to this selection step has been demonstrated by ultrastructural studies of controlled slow frozen, in vivo produced ovine morulae and blastocysts. These studies have shown that certain abnormalities remain undetected by stereomicroscopy only. In the present study, using ovine in vivo produced morulae and blastocysts, we have studied the ultrastructural alterations induced by open pulled straw vitrification (OPS) and controlled slow freezing, compared stereomicroscopical embryo scoring with light microscopy evaluation of embryo's semithin sections, and related the ultrastructural cellular damage with the embryo classification by stereomicroscopical embryo scoring of embryos' and semithin section evaluation by light microscopy. The ultrastructural lesions found for OPS-vitrified and controlled slow frozen embryos were similar, independently of embryo stage. A significant higher number of grade 3 embryos was found at stereomicroscopical scoring after controlled slow freezing (P = 0.02), and a significant higher number of grade 3 blastocysts was found at semithin sectioning after OPS vitrification (P = 0.037). The extension of ultrastructural damage, especially of mitochondria and cytoskeleton, was related to the semithin classification but not to stereomicroscopical scoring at thawing. This suggests that semithin scoring is a useful tool for predicting ultrastructural lesions and new improvements in cryopreservation and thawing methods of ovine embryos are still warranted, including in the case of blastocysts cryopreserved by OPS vitrification. #
Ultrastructural observations of preimplantation stages of the sheep
Journal of embryology and experimental morphology, 1976
Sheep embryos were examined with the electron microscope in order to characterize their organelles and the changes that occur during the preimplantation period. The first sign of differentiation of trophoblast cells was the appearance of junctions between external cells at the 16-cell stage. Nucleoli developed a granular component suggesting the synthesis of ribosomal RNA at the 16-cell stage also. Centrioles were seen as early as the 8-cell stage. Intracytoplasmic vesicles were present in large numbers in all cleavage stages but disappeared at blastulation. Mitochondria progressed from a very electron-dense hook- or U-shaped form with a few cristae to a cylindrical or spherical form of light density with many transverse cristae. Microvilli were not seen until the blastocyst stage and then only on the exterior surface of the trophoblast cells. Crystalloid or virus inclusions were not observed. It was concluded that the fine structure and developmental changes in the early sheep embr...
Prenatal Differentiation of Bovine Oviductal Epithelium: An Electron Microscopic Study
Anatomia, Histologia, Embryologia, 2008
ABSTRACT In this study, we investigated the ultrastructural changes during the prenatal differentiation of oviductal epithelium in 16 bovine embryos and fetuses from CRL of 18.0 cm to a CRL of 94.0 cm. Ciliated and secretory cells of bovine uterine tube, a derivative of the Müllerian duct, differentiate to distinct development stages in the prenatal period. The typical cellular pattern, which is generally characteristic for the adult bovine oviduct, is also obtained during fetal life. In the early stages (CRL 18.0/20.4 cm), the bovine oviductal epithelium appears mostly undifferentiated. The epithelial cells show only a few mitochondria, some cisternae of rough endoplasmic reticulum (rER) and a small Golgi-complex. Most of the cytoplasm is filled with a large amount of glycogen, which decreases during later development. Interspersed between the undifferentiated epithelial cells, a few cells undergoing ciliogenesis can be observed. Ciliogenesis increased significantly during the later prenatal developmental stages. At a CRL of 55.0 cm, ciliated cells appear fully differentiated with mature cells covering their luminal surface. Formation of cilia usually use the acentriolar pathway. Fibrous granules occurred initially in association with the Golgi-apparatus and r(ER) in the supranuclear cytoplasm. Fibrous granules later fuse with deuterosomes and give rise to procentrioles, which are translocated to the luminal plasma membrane. There they become arranged in a line just beneath the apical cell membrane and further differentiate to basal bodies from which the formation of cilia and striated rootlets take place. Clear signs of differentiation of secretory cells were first seen in our material in fetuses with a CRL of 51.0 cm and 64.0 cm. These cells contain a well developed rER and Golgi-apparatus with dilated cisterns. In the supranuclear cytoplasm, the number of secretory granules continuously increases during later development and the cells adapt to the morphology of mature secretory cells at the CRL 94.0 cm.
Comparative ultrastructure of hatched human, mouse and bovine blastocysts
Reproduction, 1982
A hatched human blastocyst obtained after in-vitro fertilization and culture was examined by transmission electron microscopy and the ultrastructural features compared with hatched mouse and bovine blastocysts. The human blastocyst contained a continuous layer of trophoblast cells with apical junctional complexes, an inner cell mass and the beginning of a primitive endoderm layer. Certain ultrastructural features were common to the blastocysts of all 3 species; these included characteristic junction regions between adjacent trophoblast cells, an abundance of microvilli on the external surfaces of the blastocysts and the presence of well developed mitochondria and numerous ribosomes in the trophoblast cells. The features that were dissimilar included the extent of development of the endoderm layer, the appearance of the inner cell mass and the nature and extent of vesicular inclusions in the trophoblast cells. Ultrastructurally, however, in-vitro cultured blastocysts show no development of endoderm cells 0022-4251/82/060499-11S02.
Molecular …, 2002
The objective of this study was to compare bovine and ovine oocytes in terms of (1) developmental rates following maturation, fertilization, and culture in vitro, (2) the quality of blastocysts produced in vitro, assessed in terms of their ability to undergo cryopreservation, and (3) the ultrastructural morphology of these blastocysts. In vitro blastocysts were produced following oocyte maturation/fertilization and culture of presumptive zygotes in synthetic oviduct fluid. In vivo blastocysts were used as a control from both species. In Experiment 1, the cleavage rate of bovine oocytes was significantly higher than that of ovine oocytes (78.3% vs. 58.0%, respectively, P < 0.001). The overall blastocyst yield was similar for both species (28.7% vs. 29.0%). However, when corrected for cleavage rate, significantly more ovine oocytes reached the blastocyst stage at all time-points (36.6% vs. 50.0% on day 8, for bovine and ovine, respectively, P < 0.001). Following vitrification, there was no difference in survival between in vivo produced bovine and ovine blastocysts (72 hr: 85.7% vs. 75.0%). However, IVP ovine blastocysts survived at significantly higher rates than IVP bovine blastocysts at all time points (72 hr: 47.4% vs. 18.1%, P < 0.001). At the ultrastructural level, compared with their in vivo counterparts, IVP blastocysts were characterized by a lack of desmosomal junctions, a reduction in the microvilli population, an increase in the average number of lipid droplets and increased debris in the perivitelline space and intercellular cavities. These differences were more marked in bovine IVP blastocysts, which also displayed electron-lucent mitochondria and large intercellular cavities. These observations may in part explain the species differences observed in terms of cryotolerance. In conclusion, the quality of ovine blastocysts was significantly higher than their bovine counterparts produced under identical in vitro conditions suggesting inherent species differences between these two groups affecting embryo quality. Mol. Reprod. Dev. 62: 320–327, 2002. © 2002 Wiley-Liss, Inc.
Journal of Veterinary Medicine Series A, 2000
The ultrastructure of day 7 bovine blastocysts developed in vitro in either of two culture systems was compared with that of morphologically normal blastocytes collected non-surgically from su erovulated cows on day 7 (Day 0 = day of insemination). The in vitro-embryos were medium (SFM, i.e. TCM 199 supplemented with BSA (10 mg/ml), insulin (5 pg/ml), transferrin (5 pg/ml) and selenium (5 ng/ml) or in a serum-supplemented medium (TCM 199 and 10 % (v/v) oestrous cow serum) together with bovine oviduct epithelial cells (BOEC). Five of the 8 blastocysts developed in SFM fulfilled the criteria set for normal morphology of the in vivodevelo ed blastocytes. In contrast, 6 out of 8 blastocysts developed in co-culture with BOEC normality. In vitro-developed blastocysts with deviated morphology showed a higher degree of cytoplasmic vacuolation, short, less developed cell-to-cell contacts between trophoblast as well as between inner cell mass (icm)-cells, less developed apical microvilli on the trophoblast and wide inter-cellular spaces. Additionally, numerous cytoplasmic vesicles, phagosomes, lipid droplets and hooded mitochondria were commonly present, both in trophoblast and in icmcells. The results indicate that a high proportion of blastocysts developed in co-culture with BOEC were morphologically deviated compared to those cultured in medium where serum and somatic cells were replaced by BSA, insulin, transferrin and selenium. obtaine s after culture of in vitro-matured and -fertilized oocytes either in a serum-free, cell-free were c r assified as morphologically deviated, and only 2 reached the criteria for morphological