Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

Tveden‐Nyborg, Pernille; Peura, Teija; Hartwich, K.M.; Walker, Shawn; Maddox-Hyttel, Poul

doi:10.1530/rep.1.00850

Cited by 17 publications

(11 citation statements)

References 42 publications

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“…Some of these features correspond to those previously reported in studies examining only one of the two developmental processes, either elongation or gastrulation [17], [18], [19], [20], [22], [39], [40], [41]. Here, however, we describe for the first time a high incidence of E/EE uncoupling, a result which emerged from our concomitant analysis of the two processes.…”

Section: Discussionsupporting

confidence: 90%

Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

et al. 2012

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Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way.

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Section: Discussionsupporting

confidence: 90%

Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

et al. 2012

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“…As this phenomenon was equally common in spherical/ovoid embryos and the more advanced filamentous embryos, it seems as if embryonic growth, at least for a period, is not disturbed by the lack of an epiblast. Similar findings have been reported for ovine SCNT embryos (Tveden-Nyborg et al 2005). It may be speculated that hypoblast formation is a dominant process, which, if the inner cell mass is low in cell number, consumes all cells of the inner cell mass.…”

Section: Discussionsupporting

confidence: 87%

“…In sheep, it has been documented that serum-enriched embryo culture and SCNT procedures cause a retardation of embryo development and cell differentiation on D7-19 of gestation (Tveden-Nyborg et al 2005). In addition, Bertolini et al (2002) found that on D16 of gestation, the length of bovine IVP embryos was shorter than embryos produced in vivo and, moreover, they displayed ED deviations.…”

Section: Introductionmentioning

confidence: 99%

Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss

Alexopoulos¹,

Maddox-Hyttel²,

Tveden‐Nyborg³

et al. 2008

Reproduction

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In ruminants, the greatest period of embryonic loss coincides with the period of elongation when the embryonic disc is formed and gastrulation occurs prior to implantation. The impact of early embryonic mortality is not only a major obstacle to the cattle breeding industry but also impedes the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy, immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect to ultrastructural features and differentiation markers. Reproduction (2008) 136 433-445

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“…In addition to cattle, vimentin protein expression has been detected in the epiblast of in vivo embryos in the case of other ungulates, including pigs (day 9) (Prelle et al. 2001) and sheep (day 11) (Tveden‐Nyborg et al. 2005).…”

Section: Discussionmentioning

confidence: 99%

Dynamic Expression of Vimentin in Bovine Blastocysts in ExtendedIn VitroCulture

Ikeda

Sugimoto

Kume

2010

Reproduction in Domestic Animals

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In mammalian blastocysts, the inner cell mass (ICM) differentiates into the epiblast and the hypoblast. In vitro culture of bovine pre-implantation embryos is generally carried out for a limited duration at most up to the blastocyst formation, therefore, it is unclear whether and when the differentiation of ICM occurs in vitro. In bovine species, epiblast formation is characterized by the expression of the intermediate filament protein vimentin. In the present study, the spatial and temporal expression of vimentin in bovine blastocysts maintained under extended in vitro culture (EIVC) was investigated. Bovine blastocysts produced by in vitro maturation, fertilization, and culture were further cultured in vitro in modified synthetic oviduct fluid up to day 12 (fertilization = day 0). Early, expanded, or hatched blastocysts on day 8 and hatched blastocysts on days 9 and 12 were individually subjected to indirect immunofluorescence analysis with an anti-vimentin monoclonal antibody and to semi-quantitative reverse transcription-polymerase chain reaction analysis to examine the vimentin expression. Most of the blastocysts (25/26) did not exhibit any significant immunostaining for the vimentin protein on day 8. On the other hand, the vimentin protein was observed in 59.1% (13/22) and 86.4% (19/22) of the hatched blastocysts on days 9 and 12, respectively. Vimentin was detected as filaments localized within a portion of the ICM, and its expression levels varied among the embryos. Vimentin transcript could not be detected in 50% (3/6) of the blastocysts on day 8, whereas it was detected in all the blastocysts on days 9 and 12. Examination of the day 8 blastocysts revealed that compared with the slow developers, the fast developers (blastocysts which had already expanded by 168 h post-insemination) had significantly higher levels of vimentin transcripts. These results indicate that the ICM differentiates dynamically in bovine blastocysts maintained under EIVC, as reflected by the expression of vimentin and that the higher vimentin expression may reflect the higher developmental competence of embryos.

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Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

Cited by 17 publications

References 42 publications

Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss

Dynamic Expression of Vimentin in Bovine Blastocysts in ExtendedIn VitroCulture

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