Embryonic disc formation following post-hatching bovine embryo development in vitro

in Reproduction
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  • 1 Departamento de Reproducción Animal, INIA, Madrid, Spain

Correspondence should be addressed to P Ramos-Ibeas or P Bermejo-Álvarez; Email: priscilaramosibeas@gmail.com or borrillobermejo@hotmail.com
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Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

Supplementary Materials

    • Supplementary Table 1. Details of primers used for qPCR.
    • Supplementary Table 2
    • Supplementary Figure 1. Superovulation protocol employed to obtain in vivo derived embryos
    • Supplementary Figure 2. Details for specific methods used. A) Preparation of agarose tunnels using opposing combs prepared with glass capillaries. Once the gel is prepared it needs to be washed prior to its use for embryo culture. B) Picture of the system used to take tridimensional microscopy images. PBS drops retained by circles made by PAP pen on a coverslip are covered by an incubation chamber, preventing embryo crushing.
    • Supplementary Figure 3. Immunofluorescence detection of hypoblast cells underneath the trophoblast layer. A) Immunofluorescence staining for SOX17 and CDX2 of a D15 embryo cultured in agarose layer, z-section in confocal microscopy. B) Magnification of the region indicated in A. Scale bars = 300 µm for A and 50 µm for B.

 

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