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RESEARCH |
Department of Animal and Veterinary Basic Sciences, Royal Veterinary and Agricultural University, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark, 1 Sydney IVF Ltd, 4 O’Connell Street, Sydney, NSW 2000, Australia and 2 South Australian Research and Development Institute, Turretfield Research Centre, Rosedale, SA 5350, Australia
Correspondence should be addressed to Pernille Tveden-Nyborg; Email: ptn{at}kvl.dk
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Abstract |
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Introduction |
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Proper placental formation requires several developmental processes. Sheep placentation is chorioallantoic and is initiated with the budding of the allantois from the endoderm of the hind gut. Subsequently, placental vascularization is formed by the visceral mesoderm surrounding the allantois. Hence, a proper formation of the germ layers (endoderm, mesoderm and ectoderm) during the process of gastrulation is a key requirement for normal placental development.
In this investigation, we included three experimental groups: in vivo embryos as a control, in vitro cultured (IVC) embryos cultured in a medium supplemented with 20% human serum in order to provoke embryonic stress leading to LOS changes (Thompson et al. 1995, Walker et al. 1996a), and SCNT embryos.
The present study aimed to examine IVC and SCNT embryos in order to determine whether placental abnormalities previously associated with these procedures may have their origins during differentiation of the germ layers and formation of the allantoic bud. Pre- (days 7, 9, 11 and 13) and peri-implantation (days 17 and 19) ovine embryos produced by in vitro culture or SCNT were compared with in vivo derived embryos. All embryos were submitted to stereo-microscopy, and embryos collected on days 7, 9, 11 and 13 were processed for either transmission electron microscopy (TEM) or immunohistochemical examination of germ layer formation with the markers alpha-1-fetoprotein and vimentin (Maddox-Hyttel et al. 2003).
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Materials and Methods |
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Preparation of ewes
Donor ewes
Oestrus was synchronized by the administration of an intravaginal progestagen pessary (flugestone acetate; 45 mg; Laboratorie Pharmaceutique Porges, Paris, France) for 12 days. Superovulation was induced with follicle-stimulating hormone (FSH) (10 ml, i.m.; Folltropin, Vetrepharm, Ontario, Canada) administered in six decreasing does commencing 48 h before pessary removal and thereafter at 12-h intervals. Pregnant mare serum gonadotrophin (PMSG, 500 IU i.m. Pregnecol; Horizon Technology, North Ryde, NSW, Australia) was administered concurrently with the initial FSH injection. At 12 h after the final FSH injection, gonadotropin-releasing hormone (GnRH, 0.3 ml i.m.; Fertagyl, Intervet International, Boxmeer, Netherlands) was administered. Donors were artificially inseminated directly into the uterine lumen with a laparoscope, with approximately 1 x 107 motile sperm 14 h after GnRH treatment. Semen was collected from rams of proven fertility and diluted with PBS containing 5% heat inactivated sheep serum (HISS). The day of ovulation was designated as day 0.
Recipient ewes
Oestrus was synchronized as for donor ewes with a progestagen pessary. PMSG (0.4 ml i.m.) was administered at the time of pessary removal. All recipients were synchronized within ±12 h of donor ewes.
Generation of embryos
All reagents were obtained from Sigma and were of cell culture grade unless otherwise specified.
In vivo embryos
Twelve ewes were used to generate in vivo embryos. Putative zygotes were recovered from the oviducts of superovulated donors and transferred (10–20 per ewe) directly to the oviducts of synchronized intermediate recipients (n = 2). Embryos were subsequently recovered on day 6, and resultant blastocysts transferred (10–15 per ewe) to final recipients (n = 20) for recovery on days 7, 9, 11, 13, 17 and 19. For recovery of embryos, ewes were killed by a lethal dose of phenobarbitone (Virbac Australia Pty Ltd, Peakhurst, New South Wales, Australia), and the intact reproductive tract was excised. Once each tract had been trimmed of excess tissue, PBS with 5% HISS was flushed through the uterine horns in a retrograde direction from the uterotubal junction. Fluid passing out through the body of the uterus was collected into 90 mm Petri dishes. Embryos were identified under a stereo-microscope and fixed as detailed below.
In vitro cultured (IVC) embryos
Putative zygotes were recovered from superovulated donor ewes and placed directly into pre-equilibrated modified synthetic oviduct fluid (mSOF) medium (Tervit et al. 1972) supplemented with 20% heat-inactivated human serum (HIHS). Subsequent culture was conducted at 38.5 °C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. On day 6, the majority of resultant blastocysts were transferred to synchronized recipients (n = 8), as described above (10–15 blastocysts per ewe), and subsequently recovered from the reproductive tract on days 9, 11, 13, 17 and 19. The remaining blastocysts were cultured to day 7 and then fixed directly from the culture medium.
Somatic cell nuclear transfer (SCNT) embryos
SCNT embryos were constructed according to previously described protocols, using oocytes from adult ovaries for cytoplast production and adult granulosa cells as karyoplasts (Peura 2003).
Karyoplast derivation
Granulosa cells were obtained by removing cumulus cells from immature in vivo sourced oocytes and culturing them to passage 3 in Glasgow’s medium (Gibco, Life Technology, Auckland, New Zealand) supplemented with sodium pyruvate, L-glutamine, DMEM, penicillin, streptomycin and 5% heat-inactivated fetal calf serum (FCS) (CSL, Parkville, Australia). Once cells were 90% confluent, they were frozen in a 10% DMSO and 90% FCS solution in Nunc cryotubes (Nunc, Roskilde, Denmark) at a concentration of approximately 200 000 cells/ml and stored in liquid nitrogen. On the day of use, a cryotube of frozen cells was thawed in a 45 °C water bath, washed with fresh Glasgow’s medium and centrifuged (2500 r.p.m., 20 min) to remove cryoprotectants. The cell pellet was then resuspended with modified Glasgow’s medium and held at 38.5 °C until used for cell injection (within 1–4 h).
Cytoplast production
Briefly, ovaries were collected from a local slaughterhouse. Follicles of >2 mm were aspirated, and the cumulus–oocyte-complexes (COCs) were placed in HEPES-buffered TCM-199 (Sigma) supplemented with 2% HISS and 100 i.u./ml heparin. COCs were washed in and then transferred to four-well plates (Nunc; 25 per well) containing 0.6 ml maturation medium (TCM-199; Sigma) supplemented with 20% HISS; 5 µg/ml FSH, 5 µg/ml luteinizing hormone (LH) and 1 µg/ml oestradiol. The medium was overlaid with mineral oil (0.3 ml), and culture occurred at 38.5 °C in a humidified atmosphere of 5% CO2 in air. Cumulus cells were removed from COCs 18 h later with hyaluronidase (500 i.u./ml) and pipetting. Denuded oocytes were placed in mSOF supplemented with 4 mg/ml BSA (Gibco Life Technologies) and amino acids at ovine oviduct fluid concentrations (Walker et al. 1996b) until enucleated. As required, groups of oocytes (maximum 20) were incubated in hSOF (HEPES-buffered SOF with 4 mg/ml BSA) containing 7.5 µg/ml cyto-chalasin B and 5 µg/ml Hoechst (33342) for 5 min and then placed in enucleation medium (hSOF containing 7.5 µg/ml cytochalasin B). Enucleation involved the removal of a minimal amount of cytoplasm from the area directly underlying the polar body. All material removed from the oocyte was viewed under UV light to confirm that the metaphase II plate had been removed. After enucleation, the karyoplast was immediately injected into the perivitelline space through the enucleation hole in the zona pellucida. After reconstruction, cytoplast–karyoplast couplets were held in culture media until the time of fusion.
Fusion
Nuclear fusion was performed with a Genaust Fusion Machine (Genetics Australia, Bacchus Marsh, Australia) in a mannitol fusion medium (0.3 M mannitol, 0.05 mM CaCl2 and 0.1 mM MgSO4). The manipulation chamber comprised two parallel, 0.1 mm diameter platinum wires separated by 0.2 mm. An AC-alignment pulse of 400 kHz and 10 V was followed by two DC pulses of 1250 V/cm and 80 µs. Fusion was assessed after 30 min, and unfused couplets were re-pulsed. Fused couplets were placed in 10 µM Ca2+-ionophore in protein-free hSOF for 5 min, before incubation in 2 mM 6-DMAP for a minimum of 2 h. Finally, reconstructed embryos were placed in 0.6 ml mSOF overlaid with mineral oil and cultured in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2 at 38.5 °C.
Embryo development
Cellular cleaving was recorded on day 2 and blastocyst development on day 6. Day 7 blastocysts were recovered directly from the culture medium and fixed. Good-quality blastocysts (and a small number of compacted morulae) scheduled for collection on days 9–19 were vitrified on day 6 by the open pulled straw method (Vajta et al. 1996). These embryos were subsequently transferred to synchronized recipients (n = 20) after thawing. Only embryos without apparent cell lysis in the inner cell mass and presenting a reformed blastocoele cavity were transferred. Up to 24 blastocysts were transferred per ewe, except for ewes designated for recovery on days 17 and 19, where only up to nine embryos were transferred. Embryos were recovered from recipients on days 9, 11, 13, 17 and 19 and fixed.
Embryo fixation and processing
After recovery, embryos were fixed for either TEM or immunohistochemistry. For TEM, embryos were fixed in 3% glutaraldehyde in 0.1 M sodium phosphate buffer (PB, pH 7.2) for 1 h at 4 °C, and then transferred to 0.1 M PB and transported from Australia to Denmark in 0.1 M PB with 3 mg/ml polyvinyl alcohol (PVA). Post-fixation took place in 1% 0.1 M PB followed by dehydration and embedding in Epon (Hyttel & Madsen 1987). Embryos for immunohistochemistry were fixed in 4% paraformaldehyde (PFA) in 0.1 M PB (pH 7.2) for 1 h at 4 °C and then transferred to 1% PFA in 0.05 M PB. Embryos were transported in 1% PFA before dehydration and embedding.
After initial fixation, embryos were submitted to stereo-microscopy and digital image processing. A total of 52 in vivo, 38 IVC and 62 SCNT embryos were analysed in this way (Table 1
). Subsequently, the embryos or parts thereof, including the embryo proper, were embedded in agar blocks for further processing. When only the embryo proper was processed, the material was orientated in the blocks to produce sagittal sections through the embryo proper. Days 7, 9, 11 and 13 embryos were submitted to sectioning and further analysis.
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Immunohistochemistry
Serial sections (4 µm) of each embryo were cut. Selected sections were stained with haematoxylin-eosin and examined by LM for evaluation and orientation. A total of 51 paraffin-wax-embedded embryos were evaluated. Of these, 17 in vivo, 13 IVC and 20 SCNT embryos were selected for immunohistochemistry (Table 2). Remaining sections were kept at 4 °C until further use.
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Results |
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. In general, the embryos collected at days 7 and 9 were spherical blastocysts; day 11 embryos were larger and some clearly ovoid, whereas a combination of spherical, ovoid and tubular embryos was collected on day 13. In the ovoid and tubular embryos, an embryonic disc (ED) could, in most instances, be identified. On days 17 and 19, all analysed embryos displayed a clearly identifiable embryo proper.
The IVC embryos and, in particular, the SCNT embryos were slower in hatching than the in vivo embryos (Table 1). At day 7, the diameter of the SCNT embryos was significantly smaller than that of the in vivo and IVC embryos (Fig. 1), whereas at day 9, both the hatched SCNT and IVC embryos were smaller than the hatched in vivo embryos. At day 13, the SCNT embryos were shorter than the IVC and in vivo embryos, and there was an associated reduction in the size of their ED (Fig. 2). At day 17, no significant differences between the three groups of embryos were noted with respect to the length of the embryo proper. However, the width of the allantois as well as the number of somites was greater in the in vivo embryos than in the IVC and SCNT embryos. At day 19, the length of the embryo proper, the width of the allantois and the number of somites were greater in in vivo embryos than in IVC and SCNT embryos (Fig. 3).
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At day 7, five of six in vivo embryos presented a well-defined inner cell mass (ICM), and three had initiated the formation of the hypoblast (Fig. 4). Consequently, the remaining cells of the ICM are referred to as the epiblast. The hypoblast initially appeared directly beneath the epiblast and proceeded along the inside of the trophoblast, but none of the embryos had a complete hypoblast lining. At the ultrastructural level, the epiblast cells were uniform and rounded, presenting large nuclei and prominent nucleoli and a cytoplasm dominated by mitochondria, polyribosomes and lipid droplets. The hypoblast cells were squamous and linked by tight junctions. The trophectoderm cells were particularly rich in intermediate filaments, some of which engaged in the formation of desmosomes, along with which tight junctions were noted.
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). At day 9, four of six in vivo embryos presented a well-defined epiblast. All in vivo embryos had a complete hypoblast lining the inside of the trophoblast; in two embryos, the hypoblast directly beneath the epiblast had assumed a more cuboidal appearance. At the ultrastructural level, Rauber’s layer, while still intact, appeared as a thin layer of cytoplasmic trophectoderm processes.
In four of seven IVC embryos, a well-defined ICM or epiblast was found, while six embryos presented a complete hypoblast. At the ultrastructural level, IVC embryos displayed features comparable with day 7 in vivo embryos. Only one of the six SCNT-embryos presented a well-defined ICM or epiblast. This embryo also had an incomplete hypoblast. A complete and an incomplete hypoblast were, however, also noted in two embryos without an apparent epiblast.
At day 11, five of six in vivo embryos presented a well-defined ED consisting of an apparently pseudostratified or, in some regions, bilayered epiblast with its underlying hypoblast (Fig. 5). The hypoblast extended into a complete lining of the trophectoderm. Evidence of mitotic activity was observed primarily in the upper layer of the epiblast, and dome-shaped epiblast protrusions were seen at the apical epiblast surface. The hypoblast cells directly beneath the epiblast were cuboidal. At the ultrastructural level, the epiblast cells appeared to have formed a well-defined superficial layer of cells that displayed microvilli and were connected by putative tight junctions (Fig. 6). Intercellular junctions were not observed elsewhere in the epiblast. At the transition from superficial epiblast to trophectoderm, putative tight junctions were observed between these two cell types. In three embryos, evidence of initial gastrulation appeared as a single cluster or small clusters of cells in the space between the epiblast and hypoblast. These putative endoderm and mesoderm precursor cells were rounded or slightly cuboidal and presented a large nucleus, prominent nucleoli and mitochondria as dominant features. The precursor cells made contact to each other through short cytoplasmic processes.
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). Signs of a pseudostratified or bilayered organization of the epiblast were noted in only three of six IVC embryos. In seven of 13 SCNT embryos, an epiblast was found together with a complete hypoblast, but signs of a pseudostratified or bilayered organization of the epiblast were evident in only a single embryo. Among the six embryos without an ICM or epiblast, five presented a complete hypoblast. At the ultrastructural level, the epiblast of the SCNT embryos resembled the earlier (day 9) in vivo epiblasts. Remnants of Rauber’s layer were seen frequently along the surface of the apical epiblast. The hypoblast cells directly beneath the epiblast were not as cuboidal or as closely apposed as in the in vivo and IVC embryos, and initial gastrulation was not observed in any of the SCNT embryos.
At day 13, all nine in vivo embryos displayed a well-defined ED presenting a well-developed ectoderm and endoderm as well as mesoderm, of which the extra-embryonic parts were clearly divided into visceral and somatic portions (Fig. 7). The upper ectoderm cells presented marked dome-shaped protrusions and numerous mitoses. In three embryos, amniotic folds were present as uni- or bilaterally trophectoderm upfoldings in close proximity to the ectoderm. At the ultrastructural level, the ectoderm cells resembled their epiblast precursors on day 11 in vivo embryos. Again, mesoderm cells made contact with each other through cytoplasmic processes.
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Immunohistochemistry
Immunohistochemical staining was performed on paraffin sections for alpha-1-fetoprotein and vimentin (Table 2).
Alpha-1-fetoprotein
Staining was confined to the cytoplasm of hypoblast cells. Weak staining was observed in a single, day-11 in vivo embryo. Evident staining was seen in in vivo and IVC embryos on day 13, whereas none of the SCNT embryos exhibited staining.
Vimentin
All in vivo embryos displayed a positive cytoplasmic staining for vimentin in the ventral part of the epiblast/ectoderm and in all components of the mesoderm on day 13 (Fig. 8). A single embryo, without evidence of gastrulation, showed staining in the ventral portion of the epiblast on day 11. All IVC embryos displayed staining of the ventral epiblast/ectoderm on day 13 and, if present, in the mesoderm. On day 11, one of three embryos stained positive in the ventral portion of the epiblast. Among the SCNT embryos, three of six day-13 embryos showed positive staining of a few cells in the ventral parts of the epiblast (Fig. 8). Similar staining was noted in a single embryo on day 11.
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Discussion |
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The stereo-microscopic measurements clearly showed that in vivo embryos, with respect to the size of the embryo, the length and development of the embryo proper, and the size of the allantois, consistently developed at a faster rate than SCNT embryos and, to a lesser degree, IVC embryos. The magnitude of this effect is probably even more pronounced than what appears from the presented data, as embryos that were most impaired after the period of in vivo development in the recipient were potentially eliminated from the IVC and SCNT groups. Moreover, the variation in developmental measures was much more pronounced among IVC embryos and, in particular, SCNT embryos than among their in vivo counterparts, a phenomenon that would render only the most advanced IVC and SCNT individuals likely to continue development to term (Holm et al. 1996, Koo et al. 2002). Sheep embryos containing low cell numbers have been shown to display decreased survival rates and increased sensitivity to asynchrony in the uterine environment (Willadsen 1981). Furthermore, the degree of DNA fragmentation is increased in SCNT embryos, mainly in the ICM cells, indicating that SCNT embryos with low cell numbers have a higher rate of apoptosis (Fahrudin et al. 2002). Both the increased variation in embryo size and observed delay in development could thus be expected to have damaging effects on the continued embryonic growth of the IVC embryos and, particularly, the SCNT embryos.
During the pre- and peri-implantation development, embryos of the large domestic species undergo three sequential rounds of differentiation where cells are recruited from an initially totipotent but, later, pluripotent cell population. At the first round of differentiation, the terminally differentiated trophoblast and the pluripotent ICM are formed, while the second round of differentiation results in delamination of the hypoblast from the ICM, a process after which the latter cell population remains pluripotent and is referred to as the epiblast. Gastrulation represents the third round of differentiation where the basic germ layers are formed by a complex process including establishment of the primitive streak and involution of cells serving as precursors of endoderm and mesoderm. The endoderm cells are inserted into the upper portion of the hypoblast lining, whereas the mesoderm develops into embryonic and extra-embryonic portions, of which the latter was divided into somatic and visceral parts. Marked differences between the three different groups of embryos were noted with respect to the differentiation of the ICM into hypoblast and epiblast, particularly in the timing of this event. On day 7, hypoblast and epiblast were seen in all three groups of embryos, though at a higher frequency among the in vivo embryos. On day 9, all in vivo embryos and most of the IVC embryos had formed a complete hypoblast, which was, on the other hand, observed in only a single SCNT embryo. Interestingly, this embryo did not possess a recognizable epiblast. This was also noted in several of the IVC and SCNT embryos on days 9 and 11 respectively. As the hypoblast is formed by the delamination of ICM cells (Wintenberger-Torrès & Fléchon 1974, Maddox-Hyttel et al. 2003), it seems likely that, in some cases, the formation of the hypoblast consumes the complete cell population contained in the ICM. This observation renders hypoblast formation a predominant event in the developing blastocyst. A proportion of the IVC embryos and, in particular, the SCNT embryos also lacked an identifiable ICM before the differentiation into hypoblast and epiblast. Such embryos have probably been impaired even earlier and may have contained sufficient viable cells only to form the trophoblast during the first round of differentiation. The presence of a functional ICM would not have been easily recognized before transfer (van Soom et al. 1997, McEvoy et al. 2000), and hatching of blastocysts devoid of an evident ICM has been reported in cattle (Avery et al. 1995). Thus, trophoblast differentiation may be a developmental event similar in predominance to hypoblast formation. Such so-called trophoblastic vesicles have experimentally been shown to be capable of maintaining intrauterine growth and elongation in the sheep, thus enabling the persistence of such vesicles (Heyman et al. 1984, Fléchon et al. 1986).
Large differences were noted between the experimental groups with respect to the process of gastrulation. In half of the in vivo embryos, initial gastrulation was noted on day 11, whereas this event was only in its very early beginning in a single IVC embryo and was not seen at all in SCNT embryos. In agreement with earlier findings in the sheep (Guillomot et al. 2004), mesoderm and endoderm precursors were present before any morphological evidence of primitive streak formation. Gastrulation is preceded by the formation of a pseudostratified or bilayered organization of the epiblast, as has earlier been reported in farm animals (Betteridge & Fléchon 1988, Flechon et al. 2004), a phenomenon which was, in general, delayed by approximately 2 days in IVC embryos and, in particular, SCNT embryos. When the timing of gastrulation was assessed with immunohistochemical markers, a similar delay was observed, especially in SCNT embryos.
IVC and SCNT embryos also displayed delay with respect to the formation of the extra-embryonic membranes, including the amnion and the allantois, which are crucial for continued development. In the large domestic species, the amnion is formed by budding of the trophoblast into chorionic folds (Bryden et al. 1972, Maddox-Hyttel et al. 2003).This process was initiated in half of the in vivo embryos on day 13, whereas none of the IVC and SCNT embryos exhibited amniotic folds. Allantois formation marks an important stage of development when the embryonic portion of the future chorioallantoic placenta is prepared. On day 17, but particularly on day 19, the width of the allantoic bud was greater in in vivo embryos than among their IVC and SCNT counterparts. Placental formation is a process demanding a certain synchrony between embryonic (the allantois and associated vascularization) and maternal (the endometrium) components, and the impaired allantoic development may well form the basis of the placental deviations noted later during SCNT-based gestation (Hill et al. 1999, Heyman et al. 2002). Furthermore, a deviant placenta could cause perinatal deaths in SCNT offspring brought to term (Chavatte-Palmer et al. 2004).
In conclusion, SCNT and IVC embryos develop slower than in vivo embryos, a phenomenon that may have detrimental effects on placentation, thus partly explaining why high embryo and fetal loss is associated with these techniques.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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