Skip to main content Accessibility help

Ultrastructural changes in goat interspecies and intraspecies reconstructed early embryos

  • Yong Tao (a1), Lizi Cheng (a1), Meiling Zhang (a1), Bin Li (a1), Jianping Ding (a1), Yunhai Zhang (a1) (a2), Fugui Fang (a1), Xiaorong Zhang (a1) and Poul Maddox-Hyttel (a2)...


The low efficiency of somatic cell nuclear transfer may be related to the ultrastructural deviations of reconstructed embryos. The present study investigated ultrastructural differences between in vivo-produced and cloned goat embryos, including intra- and interspecies embryos. Goat ear fibroblast cells were used as donors, while the enucleated bovine and goat oocytes matured in vitro as recipients. Goat–goat (GG), goat–cattle (GC) and goat in vivo-produced embryos at the 2-cell, 4-cell, 8-cell and 16-cell stages were compared using transmission electron microscopy. These results showed that the three types of embryos had a similar tendency for mitochondrial change. Nevertheless, changes in GG embryos were more similar to changes in in vivo-produced embryos than were GC embryos, which had more extreme mitochondrial deviation. The results indicate the effects of the cytoplast on mitochondria development. The zona pellucida (ZP) in all three types of embryos became thinner and ZP pores in both GC and GG embryos showed an increased rate of development, especially for GC embryos, while in vivo-produced embryos had smooth ZP. The Golgi apparatus (Gi) and rough endoplasmic reticulum (RER) of the two reconstructed embryos became apparent at the 8-cell stage, as was found for in vivo embryos. The results showed that the excretion of reconstructed embryos was activated on time. Lipid droplets (LD) of GC and GG embryos became bigger, and congregated. In in vivo-produced embryos LD changed little in volume and dispersed gradually from the 4-cell period. The nucleolus of GC and GG embryos changed from electron dense to a fibrillo-granular meshwork at the 16-cell stage, showing that nucleus function in the reconstructed embryos was activated. The broken nuclear envelope and multiple nucleoli in one blastomere illuminated that the nucleus function of reconstructed embryos was partly changed. In addition, at a later stage in GC embryos the nuclear envelope displayed infoldings and the chromatin was concentrated, implying that the blastomeres had an obvious trend towards apoptosis. The gap junctions of the three types of embryos changed differently and GG and GC embryos had bigger perivitelline and intercellular spaces than did in vivo-produced embryos. These results are indicative of normal intercellular communication at an early stage, but this became weaker in later stages in reconstructed embryos. In conclusion, inter- and intraspecies reconstructed embryos have a similar pattern of developmental change to that of in vivo-produced embryos for ZP, rough ER, Gi and nucleolus, but differ for mitochondria, LD, vesicles, nucleus and gap junction development. In particular, the interspecies cloned embryos showed more severe destruction. These ultrastructural deviations might contribute to the compromised developmental potential of reconstructed embryos.


Corresponding author

*All correspondence to: Zhang Xiaorong. Faculty of Animal Genetics, Breeding and Reproduction, College of Animal Science and Technology, Anhui Agricultural University, Changjiang West Rd 130, Hefei, 230036, China. Tel: +86 551 5782 488. Fax: +86 551 5785 543. e-mail:


Hide All
Abe, H., Otoi, T., Tachikawa, S., Yamashita, S., Satoh, T. & Hoshi, H. (1999). Fine structure of bovine morulae and blastocysts in vivo and in vitro. Anat. Embryol. 199, 519–27.
Abe, H., Matsuzaki, S. & Hoshi, H. (2002a). Ultrastructural differences in bovine morulae classified as high and low qualities by morphological evaluation. Theriogenology 57, 1273–83.
Abe, H., Yamashita, S., Satoh, T. & Hoshi, H. (2002b). Accumulation of cytoplasmic droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum free or serum-containing media. Mol. Reprod. 61, 5766.
Au, H.K., Yeh, T.S., Kao, S.H., Tzeng, C.R. & Hsieh, R.H. (2005). Abnormal mitochondrial structure in human unfertilized oocytes and arrested embryos. Ann. New York Acad. Sci. 1042, 177–85.
Baran, V., Vignon, X., LeBourhis, D., Renard, J.P. & Flechon, J.E. (2002). Nucleolar changes in bovine nucleotransferred embryos. Biol. Reprod. 66, 534–43.
Betteridge, K.J. (1988). The anatomy and physiology of pre-attachment bovine embryos. Theriogenology 29, 155–87.
Brogliatti, G.M., Palasz, A.T., Rodriguez-Martinez, H., Mapletoft, R.J. & Adams, G.P. (2000). Transvaginal collection and ultrastructure of llama (Lama glama) oocytes. Theriogenology 54, 1269–79.
Campos, Y., Huertas, R. & Bautista, J. (1993). Muscle carnitine deficiency and lipid storage myopathy in patients with mitochondrial myopathy. Muscle Nerve 16, 778–81.
Cohen, J., Malter, M., Fehilly, C., Wright, G., Elsner, C., Kort, H. & Massey, J. (1988). Implantation of embryos after partial opening of oocyte zona pellucida to facilitate sperm penetration. Lancet 2, 162.
Crosier, A.E., Farin, Dykstra, M.J., Alexander, J.E. & Farin, C.E. (2000). Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro. Biol. Reprod. 62, 1459–65.
Crosier, A.E., Farin, P.W., Dykstra, M.J., Alexander, J.E. & Farin, C.E. (2001). Ultrastructural morphometry of bovine blastocysts produced in vivo and in vitro. Biol. Reprod. 64, 1375–85.
Dalakas, M.C., Leon-Monzon, M.E. & Bernardini, I. (1994). Zidovudine induced mitochondrial myopathy is associated with muscle calmitine deficiency and lipid storage. Ann Neurol. 35, 482–7.
de Loos, F., van Vliet, C., van Maurik, P. & Kruip, T.A. (1989). Morphology of immature bovine oocytes. Gamete Res. 24, 197204.
Desai, N.N., Goldstein, J., Rowland, D.Y. & Goldfarb, J.M. (2000). Morphological evaluation of human embryos and derivation of an embryo quality, scoring system specific for day 3 embryos. A preliminary study. Hum. Reprod. 15, 2190–6.
Devreker, F. & Englert, Y. (2000). In vitro development and metabolism of the human embryo up to the blastocyst stage. Eur. J. Obstet. Gynecol. Reprod. Biol. 92, 51–6.
Dirnfeld, M., Shiloh, H., Bider, D., Harari, E., Koifman, M., Lahav-Baratz, S. & Abramovici, H. (2003). A prospective randomized controlled study of the effect of short coincubation of gametes during insemination on zona pellucida thickness. Gynecol. Endocrinol. 17, 397403.
Enders, A.C., Hendricks, A.G. & Binkerd, P.E. (1982). Abnormal development of blastocysts and blastomeres in the rhesus monkey. Biol. Reprod. 26, 353–66.
Evans, M.J., Gurer, C., Loike, J.D., Wilmut, I., Schnieke, A.E. & Schon, E.A. (1999). Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep. Nat. Genet. 23, 90–3.
Funahashi, H., Ekwall, H. & Rodriguez, M.H. (2000). Zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy. Biol. Reprod. 63, 1437–2.
Gabrielsen, A., Bhatnager, P.R. & Petersen, K. (2000). Influence of zona pellucida thickness of human embryos on clinical pregnancy outcome following in vitro fertilization treatment. J. Assist. Reprod. Genet. 17, 323–8.
Hamilton, H.M., Peura, T., Laurincik, J., Walker, S.K., Maddocks, S. & Maddox-Hyttel, P. (2004). Ovine cytoplasm directs initial nucleolar assembly in embryos cloned from ovine, bovine, and porcine cells. Mol. Reprod. Dev. 69, 117–25.
Hyttel, P., Laurincik, J., Zakhartchenko, V., Stojkovic, M., Wolf, E., Müller, M., Ochs, R.L. & Brem, G. (2001). Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from embryonic cells. Cloning 3, 6981.
Kerr, J.F., Winterford, C.M. & Harmon, B.V. (1994). Apoptosis, its significance in cancer and cancer therapy. Cancer 73, 2013–26.
Kress, A. & Selwood, L. (2004). Precedence of cell–zona adhesion over cell–cell adhesion during marsupial blastocyst formation prohibits morula formation and ensures that both the pluriblast and trophoblast are superficial. Cells Tissues Organs 177, 87103.
Laurincik, J., Zakhartchenko, V., Avery, B., Stojkovic, M., Brem, G., Wolf, W., Miiller, M. & Hyttel, P. (2000). Activation of ribosomal RNA in pre-implantation in vitro-produced and nuclear transfer bovine embryos. Reprod. Domest. Anim. 35, 255–9.
Laurincik, J., Zakhartchenko, V., Stojkovic, M., Brem, G., Wolf, E., Müller, M., Ochs, R.L., & Maddox-Hyttel, P. (2002). Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from granulosa cells. Mol. Reprod. Dev. 61, 477–87.
Liptau, H. & Viebahn, C. (1999). Expression patterns of gap junctional proteins connexin 32 and 43 suggest new communication compartments in the gastrulating rabbit embryo. Differentiation 65, 209–19.
Liu, Y., Zhang, X.R., Chen, D.Y., Zhang, Y.H., Zhang, Z.G., Jin, R.T., Wang, C.L., Zhang, M.L., Li, D.W., Li, B., Zhao, H. & Cheng, L.Z. (2004). Study on development of cloned embryo using bovine somatic cell and rabbit oocyte in vitro. Sci. Agric. Sinica. 37, 441–5.
Makarevich, A.V., Chrenek, P., Zilka, N., Pivko, J. & Bulla, J. (2005). Preimplantation development and viability of in vitro cultured rabbit embryos derived from in vivo fertilized gene-microinjected eggs: apoptosis and ultrastructure analyses. Zygote 13, 125–37.
Monika, O., Schernthaner, W., Sinowatz, F. & Wolf, E. (2002). Effects of bovine serum albumin and estrous cow serum on development and ultrastructure of in vitro-produced porcine embryos. Anat. Histol. Embryol. 31, 151–7.
Nottola, S.A., Makabe, S., Stallone, T., Familiari, G., Correr, S. & Macchiarelli, G. (2005). Surface morphology of the zona pellucida surrounding human blastocysts obtained after in vitro fertilization. Arch. Histol. Cytol. 68, 133–41.
Pelletier, C., Keefe, D.L. & Trimarchi, J.R. (2004). Noninvasive polarized light microscopy quantitatively distinguishes the multilaminar structure of the zona pellucida of living human eggs and embryos. Fertil. Steril. 81, 850–6.
Pereda, J., Cheviakoff, S. C. & Roxatto, H.B. (1989). Ultrastructure of a 4-cell human embryo developed in vivo. Hum. Reprod. 4, 680–8.
Pereda, J. & Coppo, M. (1987). Ultrastructure of a two-cell human embryo. Anat. Embryol. 177, 91–6.
Pereda, J. & Croxatto, H.B. (1978). Ultrastructure of a seven-cell human embryo. Biol. Reprod. 18, 481–9.
Pereira, D.C., Dode, M.A. & Rumpf, R. (2005). Evaluation of different culture systems on the in vitro production of bovine embryos. Theriogenology 63, 1131–41.
Plante, L. & King, W.A. (1994). Light and electron microscopic analysis of bovine embryos derived by in vitro and in vivo fertilization. J. Assist. Reprod. Genet. 11, 515–29.
Rivera, R.M., Kelley, K.L., Erdos, G.W. & Hansen, P.J. (2003). Alterations in ultrastructural morphology of two-cell bovine embryos produced in vitro and in vivo following a physiologically relevant heat shock. Biol. Reprod. 69, 2068–77.
Sabine, K., Miodrag, S., Sven, R., Horst-Dieter, R., Eckhard, W. & Fred, S. (2004). Effects of growth hormone on the ultrastructure of bovine preimplantation embryos. Cell Tissue Res. 317, 101–8.
Smith, S.D., Soloy, E. & Kanka, J. (1996). Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos. Mol. Reprod. Dev. 45, 444–50.
Steinborn, R., Schinogl, P., Zakhartchenko, V., Achmann, R., Schernthaner, W., Stojkovic, M., Wolf, E., Muller, M. & Brem, G. (2000). Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning. Nat. Genet. 25, 255–7.
Sun, Q.Y., Tan, J.H., Qin, P.C. & Yang, Q.Z. (1994). An ultrastructural study on 8–16 cell sheep embryos. J. Northeast. Agric. Uni. (in Chinese) 25, 380–4.
Thompson, J.G. (2000). In vitro culture and embryo metabolism of cattle and sheep embryos—a decade of achievement. Anim. Reprod. Sci. 61, 263–75.
Van Soom, A., Mateusen, B., Leroy, J. & de Kruif, A. (2003). Assessment of mammalian embryo quality: what can we learn from embryo morphology? Reprod. Biomed. Online 7, 664–70.
Vanroose, G., Nauwynck, H., Soom, A.V., Ysebaert, M.T., Charlier, G., Oostveldt, P.V. & de Kruif, A. (2000). Structural aspects of the zona pellucida of in vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study. Biol. Reprod. 62, 463–9.
Wen, D., Yang, C., Cheng, Y., Li, J., Liu, Z., Sun, Q., Zhang, J., Lei, L., Wu, Y., Kou, Z. & Chen, D. (2003). Comparison of developmental capacity for intra- and interspecies cloned cat (Felis catus) embryos. Mol. Reprod. Dev. 66, 3845.
Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. & Campbell, K.H. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–3.
Yang, D.S., Liu, D.J., Qi, M.G. & Shorgan, B. (2003). Ultrastructure of in vitro produced bovine embryos cultured in two conventional media. Acta. Veterinaria. Et. Zootechnica. Sinica. (in Chinese) 34, 54–8.
Zhang, X.R. & Liu, Y. (2004). Effect of some factors on the fusion rate of bovine–rabbit interspecies reconstructed eggs. Chin. J. Agric. Biotechnol 1, 135–8.
Zhang, Z.G., Zhang, X.R., Liu, Y., Jin, R.T., Wang, C.L., Zhao, H., Li, B., Cao, C.C., Li, D.W. & Cheng, L.Z. (2005). Serial nuclear transfer of goat–rabbit interspecies reconstructed embryos. Agric. Sci. Chin. 4, 629–33.


Ultrastructural changes in goat interspecies and intraspecies reconstructed early embryos

  • Yong Tao (a1), Lizi Cheng (a1), Meiling Zhang (a1), Bin Li (a1), Jianping Ding (a1), Yunhai Zhang (a1) (a2), Fugui Fang (a1), Xiaorong Zhang (a1) and Poul Maddox-Hyttel (a2)...


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed