Skip to main content Accessibility help

Expression and cellular distribution of zona pellucida glycoproteins in canine oocytes before and after in vitro maturation

  • Bartosz Kempisty (a1) (a2) (a3), Hanna Piotrowska (a4), Dorota Bukowska (a5), Magdalena Woźna (a5), Sylwia Ciesiółka (a2), Katarzyna Wojtanowicz-Markiewicz (a5), Renata Włodarczyk (a5), Karol Jopek (a2), Michal Jeseta (a6), Małgorzata Bruska (a3), Michał Nowicki (a2), Jędrzej M. Jaśkowski (a5), Klaus-Peter Brüssow (a7) and Maciej Zabel (a8)...


This study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus–oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization.


Corresponding author

All correspondence to: Bartosz Kempisty. Department of Histology and Embryology, Department of Anatomy, Poznan University of Medical Sciences, 6 Święcickiego St., 60–781 Poznań, Poland. Tel: +48 61 8546419. Fax: +48 61 8546455. e-mail:


Hide All
Abe, H. & Hoshi, H. (2003). Evaluation of bovine embryos produced in high performance serum-free media. J. Reprod. Dev. 49, 193202.
Antosik, P., Kempisty, B., Jackowska, M., Bukowska, D., Lianeri, M., Brüssow, K.P., Woźna, M. & Jaśkowski, J.M. (2010). The morphology of porcine oocytes is associated with zona pellucida glycoprotein 3 and integrin beta 2 protein levels. Vet. Med. (Praha) 55, 154–62.
Blackmore, D.G., Baillie, L.R., Holt, J.E., Dierkx, L., Aitken, R.J. & McLaughlin, E.A. (2004). Biosynthesis of the canine zona pellucida requires the integrated participation of both oocytes and granulosa cells. Biol. Reprod. 71, 661–8.
Chiu, P.C., Wong, B.S., Chung, M.K., Lam, K.K., Pang, R.T., Lee, K.F., Sumitro, S.B., Gupta, S.K. & Yeung, W.S. (2008). Effects of native human zona pellucida glycoproteins 3 and 4 on acrosome reaction and zona pellucida binding of human spermatozoa. Biol. Reprod. 799, 869–77.
Chiu, P.C., Wong, B.S., Lee, C.L., Lam, K.K., Chung, M.K., Lee, K.F., Koistinen, R., Koistinen, H., Gupta, S.K., Seppälä, M. & Yeung, W.S. (2010). Zona pellucida-induced acrosome reaction in human spermatozoa is potentiated by glycodelin-A via down-regulation of extracellular signal-regulated kinases and up-regulation of zona pellucida-induced calcium influx. Hum. Reprod. 25, 2721–33.
Concannon, P.W. (2011). Reproductive cycles of the domestic bitch. Anim. Reprod. Sci. 124, 200–10.
de Avila Rodrigues, B. & Rodrigues, J.L. (2003). Influence of reproductive status on in vitro oocyte maturation in dogs. Theriogenology 60, 5966.
Diederich, M., Hansmann, T., Heinzmann, J., Barg-Kues, B., Herrmann, D., Aldag, P., Baulain, U., Reinhard, R., Kues, W., Weissgerber, C., Haaf, T. & Niemann, H. (2012). DNA methylation and mRNA expression profiles in bovine oocytes derived from prepubertal and adult donors. Reproduction 144, 319–30.
Duranthon, V. & Renard, J.P. (2001). The developmental competence of mammalian oocytes: a convenient but biologically fuzzy concept. Theriogenology 55, 1277–89.
El-Mestrah, M., Castle, P.E., Borossa, G. & Kan, F.W. (2002). Subcellular distribution of ZP1, ZP2, and ZP3 glycoproteins during folliculogenesis and demonstration of their topographical disposition within the zona matrix of mouse ovarian oocytes. Biol. Reprod. 66, 866–76.
Gupta, S.K., Bhandari, B., Shrestha, A., Biswal, B.K., Palaniappan, C., Malhotra, S.S. & Gupta, N. (2012). Mammalian zona pellucida glycoproteins: structure and function during fertilization. Cell Tissue Res. 349, 665–78.
Hewitt, D.A. & England, G.C. (1997). Effect of preovulatory endocrine events upon maturation of oocytes of domestic bitches. J. Reprod. Fertil. 51, 8391.
Hidalgo, C.O., Gómez, E., Prieto, L., Duque, P., Goyache, F., Fernández, L., Fernández, I., Facal, N. & Díez, C. (2004). Pregnancy rates and metabolic profiles in cattle treated with propylene glycol prior to embryo transfer. Theriogenology 62, 664–76.
Hirao, Y. (2011). Conditions affecting growth and developmental competence of mammalian oocytes in vitro . Anim. Sci. J. 82, 187–97.
Ikawa, M., Inoue, N., Benham, A.M. & Okabe, M. (2010). Fertilization: a sperm's journey to and interaction with the oocyte. J. Clin. Invest. 120, 984–94.
Jackowska, M., Kempisty, B., Antosik, P., Bukowska, D., Budna, J., Lianeri, M., Rosińska, E., Woźna, M., Jagodziński, P.P. & Jaśkowski, J.M. (2009). The morphology of porcine oocytes is associated with zona pellucida glycoprotein transcript contents. Reprod. Biol. 9, 7985.
Kempisty, B., Jackowska, M., Piotrowska, H., Antosik, P., Woźna, M., Bukowska, D., Brüssow, K.P. & Jaśkowski, J.M. (2011). Zona pellucida glycoprotein 3 (pZP3) and integrin β2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test. Theriogenology 75, 1525–35.
Kempisty, B., Woźna, M., Piotrowska, H., Bukowska, D., Jackowska, M., Antosik, P., Jaśkowski, J.M. & Brüssow, K.P. (2012). The expression of genes encoding zona pellucida glycoproteins in canine cumulus–oocyte complexes cultured in vitro in media supplemented with progesterone and estradiol. Theriogenology 77, 684–93.
Kölle, S., Dubois, C.S., Caillaud, M., Lahuec, C., Sinowatz, F. & Goudet, G. (2007). Equine zona protein synthesis and ZP structure during folliculogenesis, oocyte maturation, and embryogenesis. Mol. Reprod. Dev. 74, 851–9.
Krisher, R.L. (2004). The effect of oocyte quality on development. J. Anim. Sci. 82 E-Suppl., E1423.
Luvoni, G.C., Luciano, A.M., Modina, S. & Gandolfi, F. (2001). Influence of different stages of the oestrous cycle on cumulus-oocyte communications in canine oocytes: effects on the efficiency of in vitro maturation. J. Reprod. Fertil. Suppl. 57, 141–6.
Martinova, Y., Petrov, M., Mollova, M., Rashev, P. & Ivanova, M. (2008). Ultrastructural study of cat zona pellucida during oocyte maturation and fertilization. Anim. Reprod. Sci. 108, 425–34.
Michelmann, H.W., Rath, D., Töpfer-Petersen, E. & Schwartz, P. (2007). Structural and functional events on the porcine zona pellucida during maturation, fertilization and embryonic development: a scanning electron microscopy analysis. Reprod. Domest. Anim. 42, 594602.
Nickson, D.A., Boyd, J.S., Eckersall, P.D., Ferguson, J.M., Harvey, M.J. & Renton, J.P. (1993). Molecular biological methods for monitoring oocyte maturation and in vitro fertilization in bitches. J. Reprod. Fertil. Suppl. 47, 231–40.
Otoi, T., Murakami, M., Fujii, M., Tanaka, M., Ooka, A., Une, S. & Suzuki, T. (2000). Development of canine oocytes matured and fertilized in vitro . Vet. Rec. 146, 52–3.
Otoi, T., Willingham, L., Shin, T., Kraemer, D.C. & Westhusin, M. (2002). Effects of oocyte culture density on meiotic competence of canine oocytes. Reproduction 124, 775–81.
Otoi, T., Shin, T., Kraemer, D. & Westhusin, M.E. (2004). Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization. Reprod. Nutr. Dev. 44, 631–7.
Parillo, F. & Verini Supplizi, A. (1999). Glycohistochemical investigation of canine and feline zonae pellucidae of preantral and antral oocytes. Acta Histochem. 101, 127–46.
Parillo, F., Zelli, R., Verini Supplizi, A., Fagioli, O. & Gargiulo, A.M. (2005). Topographical localisation of glucidic residues and their variations in the canine zona pellucida during folliculogenesis. J. Mol. Histol. 36, 131–7.
Rankin, T.L., O’Brien, M., Lee, E., Wigglesworth, K., Eppig, J. & Dean, J. (2001). Defective zonae pellucidae in ZP2-null mice disrupt folliculogenesis, fertility and development. Development 128, 1119–26.
Rath, D., Töpfer-Petersen, E., Michelmann, H.W., Schwartz, P., von Witzendorff, D., Ebeling, S., Ekhlasi-Hundrieser, M., Piehler, E., Petrunkina, A. & Romar, R. (2006). Structural, biochemical and functional aspects of sperm-oocyte interactions in pigs. Soc. Reprod. Fertil. Suppl. 62, 317–30.
Reynaud, K., Fontbonne, A., Marseloo, N., Viaris de Lesegno, C., Saint-Dizier, M. & Chastant-Maillard, S. (2006). In vivo canine oocyte maturation, fertilization and early embryogenesis: a review. Theriogenology 66,1685–93.
Rodrigues, B.A. & Rodrigues, J.L. (2010). In vitro maturation of canine oocytes: a unique conundrum. Anim. Reprod. 7, 315.
Rodrigues Bde, A., dos Santos, L.C. & Rodrigues, J.L. (2004). Embryonic development of in vitro matured and in vitro fertilized dog oocytes. Mol. Reprod. Dev. 67, 215–23.
Schwartz, P., Magerkurth, C. & Michelmann, H.W. (1996). Scanning electron microscopy of the zona pellucida of human oocytes during intracytoplasmic sperm injection (ICSI). Hum. Reprod. 11, 2693–6.
Sirard, M.A., Richard, F., Blondin, P. & Robert, C. (2006). Contribution of the oocyte to embryo quality. Theriogenology 65, 126–36.
Songsasen, N. & Wildt, D.E. (2007). Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog. Anim. Reprod. Sci. 98, 222.
St John, J.C. (2002). The transmission of mitochondrial DNA following assisted reproductive techniques. Theriogenology 57, 109–23.
Van Soom, A., Vandaele, L., Goossens, K., de Kruif, A. & Peelman, L. (2007). Gamete origin in relation to early embryo development. Theriogenology 68, S1317.
von Witzendorff, D., Ekhlasi-Hundrieser, M., Dostalova, Z., Resch, M., Rath, D., Michelmann, H.W. & Töpfer-Petersen, E. (2005). Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure. Glycobiology 15, 475–88.
Willingham-Rocky, L.A., Hinrichs, K., Westhusin, M.E. & Kraemer, D.C. (2003). Effects of stage of oestrous cycle and progesterone supplementation during culture on maturation of canine oocytes in vitro . Reproduction 126, 501–8.
Yuan, Y. & Krisher, R.L. (2012). In vitro maturation (IVM) of porcine oocytes. Methods Mol. Biol. 825, 183–98.
Yuan, Y.Q., Van Soom, A., Leroy, J.L., Dewulf, J., Van Zeveren, A., de Kruif, A. & Peelman, L.J. (2005). Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence. Theriogenology 63, 2147–63.


Related content

Powered by UNSILO

Expression and cellular distribution of zona pellucida glycoproteins in canine oocytes before and after in vitro maturation

  • Bartosz Kempisty (a1) (a2) (a3), Hanna Piotrowska (a4), Dorota Bukowska (a5), Magdalena Woźna (a5), Sylwia Ciesiółka (a2), Katarzyna Wojtanowicz-Markiewicz (a5), Renata Włodarczyk (a5), Karol Jopek (a2), Michal Jeseta (a6), Małgorzata Bruska (a3), Michał Nowicki (a2), Jędrzej M. Jaśkowski (a5), Klaus-Peter Brüssow (a7) and Maciej Zabel (a8)...


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.