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Ultrastructural and Autoradiographic study of Preimplantation rabbit embryos grown in conventional or uterine flushing-supplemented culture media

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Abstract

Rabbit morulae and blastocysts were cultured in conventional culture media [Ham’s F10 or BSM II supplemented with bovine serum albumin (BSA) or serum] or in Ham’s medium supplemented with synchronous or asynchronous uterine flushings, mostly for 2 days, and afterwards investigated by light and electron microscopy and by autoradiography. Ultrastructure and cell proliferation differed considerably between cultured embryos and noncultured controls. Cultured embryos displayed more dead cells. They were developmentally retarded (predominance of smooth endoplasmic reticulum rather than the age-specific rough endoplasmic reticulum, mitochondria still round to ovoid shaped) and showed nonspecific signs of cells damage (swollen mitochondria and Golgi complex vesicles, increased number of lysosomes). All these features were also present in embryos grown in uterine flushing-supplemented media, but were less pronounced. Cell damage and impaired cell proliferation had affected trophoblast cells more than embryoblast cells. Endoderm could be differentiated only if culture had been started with blastocysts—not with morulae—and seems to require uterine secretions. No significant ultrastructural differences were observed between embryos cultured in synchronous or in asynchronous uterine flushings. Present results indicate that cultured preimplantation rabbit embryos deviate clearly from those grown in vivo and maintain, for some time, a better cellular structure—and probably function —in the presence of uterine flushings than in conventional culture media. Specific abnormal morphologic features related to a particular medium could not be identified.

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References

  • Adams, C. E. The development of rabbit eggs after culture in vitro for 1–4 days. J. Embryol. Exp. Morphol. 23:21–34; 1970.

    PubMed  CAS  Google Scholar 

  • Adams, C. E. Asynchronous egg transfer in the rabbit. J. Reprod. Fertil. 35:613–614; 1973.

    PubMed  CAS  Google Scholar 

  • Adams, C. E.; Hay, M. F.; Lutwak-Mann, C. The action of various agents upon the rabbit embryo. J. Embryol. Exp. Morphol. 9:468–491; 1961.

    PubMed  CAS  Google Scholar 

  • Baltz, J. M.; Biggers, J. D. Oxygen transport to embryos in microdrop cultures. Mol. Reprod. Dev. 28:351–355; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Bavister, B. D. Studies on the developmental blocks in cultured hamster embryos. In: Bavister, B. D., ed. The mammalian preimplantation embryo. New York: Plenum Press; 1987:219–249.

    Google Scholar 

  • Beier, H. M. Oviducal and uterine fluids. J. Reprod. Fertil. 37:221–237; 1974.

    PubMed  CAS  Google Scholar 

  • Beier, H. M.; Mootz, U.; Fischer, B., et al. Growth and differentiation of rabbit blastocysts in defined culture media. In: Beier, H. M.; Lindner, H. R., eds. Fertilization of the human egg in vitro. Berlin: Springer-Verlag; 1983:371–386.

    Google Scholar 

  • Benos, D. F.; Balaban, R. S. Energy requirements of the developing mammalian blastocyst for active ion transport. Biol. Reprod. 23:941–947; 1980.

    Article  PubMed  CAS  Google Scholar 

  • Brachet, A. Récherches sur le déterminism héréditaire de l’oeuf des maramifères. Development in vitro de jeunes vesicules blastodermiques du lapin. Arch. Biol. (Paris) 28:447–504; 1913.

    Google Scholar 

  • Byatt-Smith, J. G.; Leese, H. J.; Gosden, R. G. An investigation by mathematical modelling of whether mouse and human preimplantation embryos in static culture can satisfy their demands for oxygen by diffusion. Hum. Reprod. 6:52–57; 1991.

    PubMed  CAS  Google Scholar 

  • Carnegie, J. A.; McCully, M. E.; Robertson, H. A. The early development of the sheep trophoblast and the involvement of cell death. Am. J. Anat. 174:471–488; 1985.

    Article  PubMed  CAS  Google Scholar 

  • Carney, E. W.; Foote, R. H. Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium. J. Reprod. Fertil. 91:113–123; 1991.

    PubMed  CAS  Google Scholar 

  • Chang, M. C. Development and fate of transferred rabbit ova or blastocyst in relation to the ovulation time of recipients. J. Exp. Zool. 114:197–216; 1950.

    Article  Google Scholar 

  • Chatot, C. L.; Ziomek, C. A.; Bavister, B. D., et al. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J. Reprod. Fertil. 86:679–688; 1989.

    PubMed  CAS  Google Scholar 

  • Cole, R. D.; Edwards, R. G.; Paul, J. Cytodifferentiation and embryogenesis in cell colonies and tissue culture derived from ova and blastocysts of the rabbit. Dev. Biol. 13:385–407; 1966.

    Article  PubMed  CAS  Google Scholar 

  • Copp, A. J. Interaction between inner cell mass and trophectoderm of the mouse blastocyst. I. A study of cellular proliferation. J. Embryol. Exp. Morphol. 48:109–125; 1978.

    PubMed  CAS  Google Scholar 

  • Daniel, J. C., Jr.; Kennedy, J. R. Crystalline inclusion bodies in rabbit embryos. J. Embryol. Exp. Morphol. 44:31–43; 1978.

    PubMed  Google Scholar 

  • El-Banna, A. A.; Daniel, J. C., Jr. The effect of protein fractions from rabbit uterine fluids on embryo growth and uptake of nucleic acid and protein precursors. Fertil. Steril. 23:105–114; 1972.

    PubMed  CAS  Google Scholar 

  • El-Shershaby, A. M.; Hinchliffe, J. R. Cell redundancy in the zona-intact preimplantation mouse blastocyst: a light and electron microscope study of dead cells and their fate. J. Embryol. Exp. Morphol. 31:643–654; 1974.

    PubMed  CAS  Google Scholar 

  • Enders, A. C.; Hendrickx, A. G.; Pinkerd, P. E. Abnormal development of blastocysts and blastomeres in the rhesus monkey. Biol. Reprod. 26:353–366; 1982.

    Article  PubMed  CAS  Google Scholar 

  • Fischer, B. Development retardation in cultured preimplantation rabbit embryos. J. Reprod. Fertil. 79:115–123; 1987.

    PubMed  CAS  Google Scholar 

  • Fischer, B. Embryonalentwicklung in vitro und in vivo. Charakterisierung entwicklungsspezifischer Strukturen und Lebensäuβerungen von Präimplantationsembryonen in vitro und in vivo. Stuttgart: Enke Verlag; 1988.

    Google Scholar 

  • Fischer, B. Effects of asynchrony on blastocyst development. J. Reprod. Fertil. 86:479–491; 1989.

    PubMed  CAS  Google Scholar 

  • Fischer, B.; Jung, T.; Hegele-Hartung, C., et al. Criteria to survey development of preimplantation embryos. In: Holstein, A. F.; Vogt, K. D.; Gräβlin, D., eds. Reproductive biology and medicine. Berlin: Diesbach; 1989:239–252.

    Google Scholar 

  • Fischer, B.; Jung, T.; Hegele-Hartung, C., et al. Development of preimplantation rabbit embryos in uterine flushing-supplemented culture media. Mol. Reprod. Dev. 27:216–223; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Fischer, B.; Mootz, U.; Denker, H.-W., et al. The dynamic structure of rabbit blastocyst coverings. III. Transformation of coverings under non-physiological developmental conditions. Anat. Embryol. 183:17–27; 1991.

    PubMed  CAS  Google Scholar 

  • Gregory, P. W. The early embryology of the rabbit. Contrib. Embryology 21:141–168; 1930.

    Google Scholar 

  • Guilbert-Blanchette, L.; Lambert, R. D. Analysis of the rabbit uterine fluid collected by a continuous collecting technique: evidence for an embryotoxic component. Biol. Reprod. 13:1125–1134; 1978.

    Article  Google Scholar 

  • Hadek, R.; Swift, H. A crystalloid inclusion in the rabbit blastocyst. J. Biophys. Biochem. Cytol. 8:836–841; 1960.

    Article  PubMed  CAS  Google Scholar 

  • Hafez, E. S. E. Effect of progestational stage of the endometrium on implantation, fetal survival and fetal size in the rabbit, Oryctolagus cuniculus. J. Exp. Zool. 151:217–226; 1963.

    Article  Google Scholar 

  • Ham, R. G. An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp. Cell Res. 29:515–526; 1963.

    Article  PubMed  CAS  Google Scholar 

  • Handyside, A. H.; Hunter, S. Cell division and death in the mouse blastocyst before implantation. Roux’s Arch. Dev. Biol. 195:519–526; 1986.

    Article  Google Scholar 

  • Hegele-Hartung, C.; Fischer, B.; Beier, H. M. Development of preimplantation rabbit embryos after in-vitro culture and embryo transfer: an electron microscopic study. Anat. Rec. 220:31–42; 1988a.

    Article  PubMed  CAS  Google Scholar 

  • Hegele-Hartung, C.; Schumacher, A.; Fischer, B. Ultrastructure of preimplantation rabbit embryos exposed to visible light and room temperature. Anat. Embryol. 178:229–241; 1988b.

    Article  PubMed  CAS  Google Scholar 

  • Hegele-Hartung, C.; Piegsa, K.; Fischer, B. Effect of osmolarity of the fixative on the ultrastructure of preimplantation rabbit embryos. Acta Anat. 136:79–88; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Hegele-Hartung, C.; Schumacher, A.; Fischer, B. Effects of visible light and room temperature on the ultrastructure of preimplantation rabbit embryos: a time course study. Anat. Embryol. 183:559–571; 1991a.

    PubMed  CAS  Google Scholar 

  • Hegele-Hartung, C.; Dreiner, U.; Beier, H. M. Effect of in vitro culture on the dynamics of uteroglobin distribution in rabbit blastocysts. Anat. Embryol. 183:119–128; 1991b.

    PubMed  CAS  Google Scholar 

  • Hesseldahl, H. Ultrastructure of early cleavage stages and preimplantation in the rabbit. Z. Anat. Entwicklungsgesch 135:139–155; 1971.

    Article  PubMed  CAS  Google Scholar 

  • Hoffman, L. H.; Olson, G. E. Crystalline inclusions in the rabbit blastocyst. Exp. Cell Res. 127:1–14; 1980.

    Article  PubMed  CAS  Google Scholar 

  • Jung, T. Protein synthesis and degradation in non-cultured and in-vitro cultured rabbit blastocysts. J. Reprod. Fertil. 86:507–512; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Jung, T.; Fischer, B. Correlation between blastocyst diameter and DNA or protein activity in rabbit blastocysts. Biol. Reprod. 39:1111–1116; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Jung, T.; Fischer, B.; Beier, H. M. Quantitative aspects of protein synthesis in non-cultured and cultured rabbit blastocysts. Hum. Reprod. 2:23–27; 1987.

    PubMed  CAS  Google Scholar 

  • Kane, M. T. Culture media and culture of early embryos. Theriogenology 27:49–57; 1987.

    Article  Google Scholar 

  • Kane, M. T.; Bavister, B. D. Protein-free culture medium containing polyvinylalcohol, vitamins, and amino acids supports development of eight-cell hamster embryos to hatching blastocysts. J. Exp. Zool. 247:183–187; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Kerr, F. E. R.; Bishop, C. F.; Searle, I. Apoptosis. Recent Adv. Histopathol. 12:1–15; 1984.

    Google Scholar 

  • Legge, M.; Sellens, M. H. Free radical scavengers ameliorate the 2-cell block in mouse embryo culture. Hum. Reprod. 6:867–871; 1991.

    PubMed  CAS  Google Scholar 

  • Lopata, A.; Kohlman, D.; Kellow, G. N. The fine structure of human blastocysts developed in culture. In: Burger, M. M., ed. Embryonic development, part B: Cellular aspects. New York: Alan R. Liss Inc.; 1982:62–85.

    Google Scholar 

  • Lutwak-Mann, C. Some physiological and biochemical properties of the mammalian blastocyst. Bull. Schweiz. Akad. Med. Wiss. 22:101–122; 1966.

    PubMed  CAS  Google Scholar 

  • Maurer, R. R. Advances in rabbit embryo culture. In: Daniel, J. C., Jr., ed. Methods in mammalian reproduction. New York: Academic Press; 1978:259–272.

    Google Scholar 

  • Maurer, R. R.; Beier, H. M. Uterine proteins and development in vitro of rabbit preimplantation embryos. J. Reprod. Fertil. 48:33–41; 1976.

    PubMed  CAS  Google Scholar 

  • McKiernan, S. H.; Bavister, B. D.; Tasca, R. J. Energy substrate requirements for in-vitro development of hamster 1- and 2-cell embryos to the blastocyst stage. Hum. Reprod. 6:64–75; 1991.

    PubMed  CAS  Google Scholar 

  • McReynolds, H. D.; Hadek, R. A comparison of the fine structure of late mouse blastocysts developed in vivo and in vitro. J. Exp. Zool. 182:95–118; 1972.

    Article  PubMed  CAS  Google Scholar 

  • Monis, H.; Bavister, B. D. Analysis of the inhibitory effect of inorganic phosphate on development of four-cell hamster embryos in vitro. J. Exp. Zool. 256:75–83; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Mootz, U. Die Regulationsbefahigung der Keimscheibe von Oryctolagus cuniculus nach totaler Rontgenbestrahlung. Zool. Jb. Anat. 88:587–682; 1971.

    Google Scholar 

  • Pedersen, R. A.; Spindle, A. I. Interference with in vitro development of mouse inner cell mass by 24-h treatment with 5-bromodeoxyuri-dine. Differentiation 9:43–48; 1977.

    Article  PubMed  CAS  Google Scholar 

  • Pincus, G.; Werthessen, N. T. The comparative behavior of mammalian eggs in vivo and in vitro. J. Exp. Zool. 78:1–18; 1938.

    Article  CAS  Google Scholar 

  • Schini, S. A.; Bavister, B. D. Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose. Biol. Reprod. 39:1183–1192; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Seshagiri, P. B.; Bavister, B. D. Assessment of hamster blastocysts derived from eight-cell embryos cultured in hamster embryo culture medium-2 (HECM-2): cell numbers and viability following embryo transfer. J. In vitro Fertil. Embryo Transfer 7:229–235; 1990.

    Article  CAS  Google Scholar 

  • Snow, M. H. L. Abnormal development of preimplantation mouse embryos grown in vitro with3H-thymidine. J. Embryol. Exp. Morphol. 29:601–615; 1973.

    PubMed  CAS  Google Scholar 

  • Spielmann, H. Analysis of embryotoxic effects in preimplantation embryos. In: Bavister, B. D., ed. The mammalian preimplantation embryo. New York: Plenum Press; 1987:389–431.

    Google Scholar 

  • Spindle, A.; Wu, K.; Pedersen, R. A. Sensitivity of early mouse embryos to (3H)thymidine. Exp. Cell Res. 142:397–405; 1982.

    Article  PubMed  CAS  Google Scholar 

  • Trounson, A.; Sathananthan, A. H. The application of electron microscopy in the evaluation of two- to four-cell human embryos cultured in vitro for embryo transfer. J. In Vitro Fertil. Embryo Transfer 1:153–166; 1984.

    Article  CAS  Google Scholar 

  • Trump, B. F.; Mergner, W. J.; Kahng, M. W., et al. Studies on the subcellular pathophysiology of ischaemia. Circulation 53:(Suppl)1:17–26; 1976.

    Google Scholar 

  • Van Blerkom, J.; Manes, C.; Daniel, J. C., Jr. Development of preimplantation rabbit embryos in vivo and in vitro. I. An ultrastructural comparison. Dev. Biol. 35:262–282; 1973.

    Article  PubMed  CAS  Google Scholar 

  • Wales, R. G.; Edirisinghe, W. R. Volume of fluid and concentration of cations and energy substrates in the uteri of mice during early pseudopregnancy. Reprod. Fertil. Dev. 1:171–178; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Wyllie, A. H. Cell death: a new classification separating apoptosis from necrosis. In: Bowen, I. D.; Lockshin, R. A., eds. Cell death in biology and pathology. London: Chapman and Hall; 1981:9–34.

    Google Scholar 

  • Wyllie, A. H. Apoptosis: cell death under homeostatic control. Mechanisms and models in toxicology. Arch. Toxicol. Suppl. II:3–10; 1987.

    Google Scholar 

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  1. Department of Anatomy & Reproductive Biology, Medical Faculty, RWTH Aachen, D-5100, Aachen, Germany

    Bernd Fischer, Manfred Lambertz & Christa Hegele-Hartung

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  1. Bernd Fischer
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  2. Manfred Lambertz
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  3. Christa Hegele-Hartung
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Fischer, B., Lambertz, M. & Hegele-Hartung, C. Ultrastructural and Autoradiographic study of Preimplantation rabbit embryos grown in conventional or uterine flushing-supplemented culture media. In Vitro Cell Dev Biol - Animal 28, 337–347 (1992). https://doi.org/10.1007/BF02877057

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