Association between HSP70 Genotypes and Oocytes Development on In vitro Maturation/Fertilization in Pig

  • Wee, M.S. (Animal Genetic Resources Station, National Institute of Animal Science) ;
  • Park, C.K. (College of Animal Resource Science, Kangwon National University) ;
  • Cho, S.R. (Animal Genetic Resources Station, National Institute of Animal Science) ;
  • Lee, S.S. (Animal Genetic Resources Station, National Institute of Animal Science) ;
  • Yeon, S.H. (Animal Genetic Resources Station, National Institute of Animal Science) ;
  • Kim, C.D. (Animal Genetic Resources Station, National Institute of Animal Science) ;
  • Cho, C.Y. (Animal Genetic Resources Station, National Institute of Animal Science) ;
  • Choi, S.H. (Animal Genetic Resources Station, National Institute of Animal Science) ;
  • Sang, B.D. (Animal Genetic Resources Station, National Institute of Animal Science) ;
  • Son, D.S. (Animal Genetic Resources Station, National Institute of Animal Science) ;
  • Li, Z.D. (College of Biological Science, China Agricultural University) ;
  • Jin, H.J. (Animal Genetic Resources Station, National Institute of Animal Science)
  • Received : 2007.10.24
  • Accepted : 2008.02.17
  • Published : 2008.10.01


This study was performed to clarify whether the variation of stress related heat shock protein 70 (HSP70) (GenBank X68213) gene was associated with the nuclear morphological change of in vitro maturation and in vitro capacitation in oocytes of pig ovaries obtained at the slaughterhouse. The nucleic acid substitution of C to G at the 483rd position was found out in HSP70 K1 (290-512) from X68213. The ovaries were categorized into CC, CG, and GG genotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (BsiHKA I). After the second in vitro maturation of immature fresh oocytes, the relation of nuclear morphological change in oocytes with the genotype of HSP70 K1 gene was such that the MII ratios of the genotype GG and CG (46.93% and 42.20%, respectively) were significantly higher than that of the CC genotype (10.71%) (p<0.05). With respect to in vitro maturation of frozen-thawed oocytes by an open pulled straw (OPS) method, the percentage of oocytes matured to MII stage of the CG genotype showed a higher trend than CC and GG genotypes. After the in vitro maturation of immature fresh oocytes and frozen-thawed oocytes by the OPS method, the relation of the pronuclei change in oocytes matured in vitro with HSP70 genotype was assessed, and the result showed that the enlarged sperm heads (ESH) of matured fresh oocytes and frozen-thawed oocytes were 80.0% and 60.0% in the CC genotype, respectively. The CC genotype group had a significantly higher rate of ESH than the CG and the GG genotype group (p<0.05). The ratios of polyspermic invasion were not different among HSP70 of the three genotypes. It was considered that the rate of in vitro maturation of fertilized oocytes was expected to differ according to genotype of the stress related gene.


Fertilization;Maturation;Porcine Oocytes;HSP70 Genotypes


Supported by : National Institute of Animal Science


  1. Allen, R. L., D. A. O'Brien, C. G. Jones, D. L. Rockett and E. M. Eddy. 1988. Expression of heat shock proteins by isolated mouse spermatogenic cells. Mol. Cell Biol. 8:3260-3266.
  2. Alliston, C. W., B. J. Howarth and L. C. Ulberg. 1965. Embryonic mortality following culture in vitro of one- and two-cell rabbit eggs at elevated temperatures. J. Reprod. Fertil. 9:337-341.
  3. Ananthan, J., A. L. Goldberg and R. Voellmy. 1986. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Sci. 232:252-254.
  4. Ball, G. D., M. L. Leibfried, R. W. Lenz, R. L. Ax, B. D. Bavister and N. L. First. 1983. Factors affecting successful in vitro fertilization of bovine follicular oocytes. Biol. Reprod. 28: 717-725.
  5. Baumgarner, A. P. and C. L. Chrisman. 1981. Cytogenetic analysis of ovulated mouse oocytes following hypothermic stress during meiotic maturation. Exp. Cell Res. 132: 359-366.
  6. Welch, W. J. 1992. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol. Rev. 72(4):1063-1081.
  7. Xu, K. P., R. Heier and T. Greve. 1987. Dynamic changes of estradiol concentration in two culture system for bovine oocytes maturation in vitro. Theriogenol. 27:297.
  8. Zakeri, Z. F., W. J. Welch and D. J. Wolgemuth. 1990. Characterization and inducibility of hsp 70 proteins in the male mouse germ line. J. Cell Biol. 111:1785-1792.
  9. Park, C. K., O. Ohgoda and K. Niwa. 1989. Penetration of bovine follicular oocytes by frozen-thawed spermatozoa in the presence of caffeine and heparin. J. Reprod. Fertil. 86:577-582.
  10. Petters, R. M. and K. D. Wells. 1993. Culture of pig embryos. J. Reprod. Fertil. Suppl. 48:61-73.
  11. Rothschild, M. F. and A. Ruvinsky. 1998. The genetics of the pig. Cab. International, 135-197.
  12. Vajta, G., P. J. Booth, P. Holm, T. Greve and H. Callesen. 1977. Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method. Cryo- Letter, 18:191-195.
  13. SAS. SAS system, Release 8.1, SAS Inst Inc, Cary, NC, USA 1999.
  14. Miller, S. A., D. D. Kykes and H. F. Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucl. Acids Reg. 16:1215.
  15. Monaghan, P., C. Carolan, P. Lonergan, H. Sharif, H. Wahid and I. Gordon. 1993. The effect of maturation time on the subsequent in vitro development of bovine oocytes. Theriogenol. 39:270.
  16. Nagai, T., A. Takenaka, T. Mori and M. Hirayama. 1994. Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture. Mol. Reprod. Dev. 37(4):452- 456.
  17. Neuer, A., S. D. Spandorfer and P. Giraldo. 1999. Heat shock protein expression during gametogenesis and embryogenesis. Infect. Dis. Obstet. Gynecol. 7:10-16.<10::AID-IDOG3>3.0.CO;2-7
  18. Kawarsky, S. J. and W. A. King. 2001. Expression and localisation of heat shock protein 70 in cultured bovine oocytes and embryos. Zygote, 9:39-50.
  19. Lu, K. H., I. Gordon, M. Gallegher and H. Ncgovern. 1987. Pregnancy established in cattle by transfer of embryos derived from in vitro fertilization of follicular oocytes matured in vitro. Vet. Rec. 121:159-260.
  20. Kikuchi, K., T. Nagai, J. Motlik, Y. Shioya and Y. Izaike. 1993. Effect of follicle cells on in vitro fertilization of pig follicular oocytes. Theriogenol. 39:593-599.
  21. Mattioli, M., M. L. Bacci, G. Galeati and E. Seren. 1991. Effect of LH and FSH on the maturation of pig oocytes in vitro. Theriogenol. 36:95-105.
  22. King, Y. T., W. C. Lee, M. S. Gao, J. L. Wang, C. F. Tu, S. C. Wu and Y. H. Kuo. 2000. Synthesis of 60 and 72 kDa heat shock proteins in early porcine embryogenesis. Anim. Reprod. Sci. 63:221-229.
  23. McGaughey, R. W. and C. Polge. 1971. Cytogenetic analysis of pig oocytes matured in vitro. J. Exp. Zool. 176:383-396.
  24. Lenz, R. W., G. D. Ball, M. L. Leibfried, R. L. Ax and N. L. First. 1983. In vitro maturation and fertilization of bovine oocytes are temperature dependent processes. Biol. Reprod. 29:173- 179.
  25. Lindquist, S. 1986. The heat shock response. Annu. Rev. Biochem. 55:1151-1191.
  26. Jin, H. J., B. Y. Park, J. C. Park, I. H. Hwang, S. S. Lee, S. H. Yeon, C. D. Kim, C. Y. Cho, Y. K. Kim, K. S. Min, S. T. Feng, Z. D. Li, C. K. Park and C. I. Kim. 2006. The effects of stress related genes on carcass traits and meat quality in pigs. Asian-Aust. J. Anim. Sci. 19(2):280-285.
  27. Ju, J. C. and J. K. Tseng. 2004. Nuclear and cytoskeletal alterations of in vitro matured porcine oocytes under hyperthermia. Mol. Reprod. Dev. 68:125-133.
  28. Hendrey, J. and I. Kola. 1991. Thermolability of mouse oocytes is due to the lack of expression and/or inducibility of Hsp 70. Mol. Reprod. Dev. 28:1-8.
  29. Huang, S. Y., Y. H. Kuo, Y. P. Lee, H. L. Tsou, E. C. Lin, C. C. Ju and W. C. Lee. 2000. Association of heat shock protein 70 with semen quality in boars. Anim. Reprod. Sci. 63:231-240.
  30. Huang, S. Y., M. Y. Chen, E. C. Lin, H. L. Tsou, Y. H. Kuo, C. C. Ju and W. C. Lee. 2002. Effect of single nucleotide polymorphisms in the 5'-flanking region of heat shock protein 70.2 gene on semen quality in boars. Anim. Reprod. 70:99-109.
  31. Jin, H. J., I. C. Kim, M. S. Wee, S. H. Yeon, C. D. Kim, C. Y. Cho, S. H. Choi, S. R. Cho, D. S. Son, Y. K. Kim, J. H. Jung, H. S. Choi, C. K. Park and C. I. Kim. 2005. Relationship between HSP70 gene polymorphism and IVF embryo development in pigs. Kor. J. Emb. Trans. 20(3):289-295.
  32. Edwards, J. L. and P. J. Hansen. 1996. Elevated temperature increases heat shock protein 70 synthesis in bovine two-cell embryos and compromises function of maturing oocytes. Biol. Reprod. 55:340-346.
  33. Edwards, J. L. and P. J. Hansen. 1997. Differential responses of bovine oocytes and preimplantation embryos to heat shock. Mol. Reprod. Dev. 46:138-145.<138::AID-MRD4>3.0.CO;2-R
  34. Fukui, Y., T. Sconoyama, H. Mochizuki and H. Ono. 1991. Effects of heparin dosage and sperm capacitation time on in vitro fertilization and cleavage of bovine oocytes matured in vitro. Theriogenol. 34:579-591.
  35. Hansen, P. J. 1999. Possible roles for heat shock protein 70 and glutathione in protection of the mammalian preimplantation embryo from heat shock. Ann. Rev. Biomed. Sci. 1:5-9.
  36. Cox, J. F. 1991. Effect of cumulus on in vitro fertilization of in vitro matured cow and sheep oocytes. Theriogenol. 35:191.
  37. Davis, D. L. 1985. Culture and storage of pig embryos. J. Reprod. Fertil. Suppl. 33:115-124.
  38. Dezeure, F., M. Vaiman and P. Chardon. 1993. Characterization of a polymorphic heat shock protein 70gen in swine outside the SLA major histocompatibility complex. Biochim. Biophys. Acta. 1174:17-26.
  39. Ealy, A. D., M. Drost and P. J. Hansen. 1993. Developmental changes in embryonic resistance to adverse effects of maternal heat stress in cows. J. Dairy Sci. 76:2899-2905.
  40. Abeydeera, L. R. and B. N. Day. 1997. Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified Tris-buffered medium with frozen-thawed ejaculated spermatozoa. Biol. Reprod. 57:729-734.