Control of spermatozoa penetration and polyspermy by cumulus cells in porcine oocytes matured in culture

  • Published : 2003.12.01

Abstract

The functional role of the cumulus cells on sperm penetration and polyspermy during in vitro fertilization was examined. The penetration rate was significantly higher(p<0.01) in oocytes with(61%) than without(25%) cumulus cells. No significant differences, however, was observed in polyspermy. When the hyaluronidase was supplemented to the fertilization medium with different concentrations, penetration rates in oocytes with cumulus cells were higher than oocytes without cumulus cells at 0(61 vs 34% ; p<0.05), 0.01(56 vs 35% ; p<0.05), 0.1(66 vs 30% ; p<0.05) and 1.0 mg/$m\ell$(39 vs 27%). On the other hand, the polyspermy rates were lower oocytes without than with cumulus cells, and had a tendency to decrease with high concentrations of hyaluronidase. In another experiment, the penetration and polyspermy rates had a tendency to increase as time of sperm-oocytes culture was prolonged. At 16 and 20hrs after insemination, the penetration rates were significantly higher(p<0.05) in oocytes with(48 and 62% for 16 and 20hrs) than without(25 and 31% for 16 and 20hrs) cumulus cells in medium with hyaluronidase. However, the polyspermy rates were significantly(p<0.05) lower in oocytes without(3 and 16%) than with(37 and 48%) cumulus cells at 16 and 20hrs after insemination. In cumulus-free oocytes inseminated in medium with or without hyaluronidase at different concentrations of cumulus cells, the penetration rates were significantly(p<0.05) higher in medium with than without hyaluronidase at different concentrations of cumulus cells. The proportions of polyspermy were lower in medium without than with hyaluronidase at 0 (10 vs 0%), 10$^2$(25 vs 0%), 10$^4$(24 vs 14%) and 10$\^$6/(29 vs 10% ; p<0.05) cumulus cells/ml. These results suggest the advantage of culture in medium with cumulus cells and denuded oocytes to inhibit polyspermy with no decrease in the penetration rates during the fertilization in vitro in the porcine.

Keywords

References

  1. Moor RM, Smith MW, Dawson MC. 1980. Measurement of intercellular coupling between oocytes and cumulus cells using intracellular markers. Exp Cell Res 126:15-29 https://doi.org/10.1016/0014-4827(80)90466-8
  2. Sirard MA, Coenen K. 1993. The coculture of cumulus-enclosed bovine oocytes and hemisections of follicles: Effects on meiotic resumption. Theriogenology 40:933-942 https://doi.org/10.1016/0093-691X(93)90361-8
  3. Boatman DE, Robbins RR. 1991. Detection of a soluble acrosome reaction including factor, different from serum albumin, associated with the ovulated egg-cumulus complex. Mol Reprod Dev 30:396-400 https://doi.org/10.1002/mrd.1080300415
  4. Motlik J, Fulka J, Flechon JE. 1986. Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro. J Reprod Fertil 76:31-37
  5. Yanagimachi R. 1988. Mammalian fertilization. In Knobil E, Neill J Ed. The physiology of Reproduction. Vol. 1. Raven Press, New York: 135-185
  6. Corselli J, Talbot P. 1987. In vitro penetration of hamster oocyte-cumulus complexes using physiological numberss of sperm. Dev Biol 122:227-242 https://doi.org/10.1016/0012-1606(87)90348-4
  7. Cross PC, Brinster RL. 1970. In vitro development of mouse oocytes. Biol Reprod 3:298-307
  8. Ward CR, Storey BT. 1984. Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev Biol 104(2):287-296 https://doi.org/10.1016/0012-1606(84)90084-8
  9. Soupart P, Strong PA. 1974. Ultrastructural observations on human oocytes fertilized in vitro. Fertil Steril 25(1):11-44
  10. Bavister BD. 1982. Evidence for a role of post-ovulatory cumulus components in supporting fertilizing ability of hamster spermatozoa, J Androl 3:365-372
  11. Tesarik J. 1985. Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fertil 74:383-388
  12. Lorton SP, First NL. 1979. Hyaluronidase does not disperse the cumulus oophorus surrounding bovine ova. Biol Reprod 21(2):301-308 https://doi.org/10.1095/biolreprod21.2.301
  13. Fukui Y. 1990. Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro. Mol Reprod Dev 26(1):40-46 https://doi.org/10.1002/mrd.1080260107
  14. Rouillier P, Matton P, Sirard MA, Guilbault LA. 1996. Follicle-stimulating hormone-induced estradiol and progesterone production by bovine antral and mural granulosa cells cultured in vitro in a completely defined medium. J Anim Sci 74(12):3012-3019
  15. Robertson JE, Baker RD. 1969. Role of female sex steroids as possible regulators of oocyte maturation. 2nd Ann SSR, (abstr 57)
  16. Kennedy JF, Donahue RP. 1969. Human oocytes : maturation in chemically defined media. Science 164(885):1292-1293 https://doi.org/10.1126/science.164.3885.1292
  17. Bedford JM, Kim HH. 1993. Cumulus oophorus as a sperm sequestering device in vivo. J Exp Zool 265:321- 328 https://doi.org/10.1002/jez.1402650314
  18. Drobnis EZ, Yudin AI, Cheer GN, et al. 1988. Hamster sperm penetration of the zona pellucida : kinematic analysis and mechanical implications. Dev Biol 130:311-323 https://doi.org/10.1016/0012-1606(88)90437-X
  19. Ball GD, Leibfried ML, Lenz RW, et al. 1983. Factors affecting successful invitro fertilization of bovine follicular oocytes. Biol Reprod 28:717-725
  20. Madhadevan MM, Traunson AO. 1985. Removal of the cumulus oophorus from the oocyte for in vitro fertilization. Fertil Steril 43:263-267
  21. Itagaki Y, Toyoda Y. 1992. Effects of prolonged sperm preincubation and elevated calcium concentration on fertilization of cumulus-free mouse eggs in vitro. J Reprod Fertil 38:219-224
  22. Katska L, Kauffold P, Smorag Z, et a1. 1989. Influence of hardening of the zona pellucida on in vitro fertilization of bovine oocytes. Theriogenology 32:767-779 https://doi.org/10.1016/0093-691X(89)90465-2
  23. Gianfortoni JG, Gulyas BJ. 1985. The effects of short-term incubation(aging) of mouse oocytes on in vitro fertilization, zona solubility, and embryonic development. Gamete Res 11:59-68 https://doi.org/10.1002/mrd.1120110107
  24. Chian RC, Okuda K, Niwa K. 1995. Influence of cumulus cells on in vitro fertilization of bovine oocytes derived from in vitro maturation. Anim Reprod Sci 38:37-48 https://doi.org/10.1016/0378-4320(94)01357-R
  25. Bavister BD. 1986. Animal in vitro fertilization and embryo development. In Gwatkin RBL. Ed. Developmental Biology. Plenum Press, New York. 4:81-14
  26. Bedford JM, Cooper GW. 1978. Membrane fusion event in the fertilization of vertebrate eggs. In Poste G, Nicolson GL. Ed, Cell Surface. Reviews. NorthHolland, Amsterdam. 5:65-125
  27. Hunter RHF, Nichol R. 1988. Capacitation potential of the fallopian tubes: A study involving surgical insemination and the subsequent incidence of polyspermy. Gamete Res 21:255-266 https://doi.org/10.1002/mrd.1120210307
  28. Chian RC, Niwa K, Sirard MA. 1994. Effects of cumulus cells on male pronuclear formation and subsequent early development of bovine oocytes in vitro. Theriogenology 41:1499-1508 https://doi.org/10.1016/0093-691X(94)90201-S
  29. Downs SM, Schroeder AC, Eppig JJ. 1986. Serum maintains the fertilizability of mouse oocytes matured in vitro by preventing hardening of the zona pellucida. Gamete Res 15:115-122 https://doi.org/10.1002/mrd.1120150203