Studies on zona hardening in rat oocytes that are matured in vitro in a serum‐free medium

The present study confirms that zonae pellucidae of rat oocytes became resistant to chymotrypsin digestion (zona hardening) after undergoing in vitro maturation (IVM) in a serum‐free medium. However, zona hardening and did not occur when empty zonae without oocytes were cultured in the same IVM conditions, suggesting that oocyte‐derived factor(s) is responsible for zona hardening in oocytes matured in the serum‐free medium. Zona hardening occurred primarily after dictyate oocytes were cultured for 6–8 hours in the IVM medium without serum. Zona hardening could be prevented or alleviated if oocytes were cultured in the IVM medium containing bovine foetal calf serum, a soybean trypsin inhibitor, or β‐mercaptoethanol, and in vitro fertilization rates for such oocytes were normal. Possible mechanisms of the phenomenon of zona hardening in oocytes matured in serum‐free conditions are discussed in relation to its possible relevance to the cortical reaction and the physiological block to polyspermy.

[1]  T. Iwamatsu,et al.  Sperm penetration in vitro of mouse oocytes at various times during maturation. , 1972, Journal of reproduction and fertility.

[2]  D. R. Kirby,et al.  DEVELOPMENT OF THE MOUSE BLASTOCYST TRANSPLANTED TO THE SPLEEN , 1963 .

[3]  R. Gwatkin,et al.  The zona reaction of hamster and mouse eggs: production in vitro by a trypsin-like protease from cortical granules. , 1973, Journal of reproduction and fertility.

[4]  M. C. Chang,et al.  Fertilization of rat eggs in vitro by epididymal spermatozoa and the development of eggs following transfer. , 1974, Journal of reproduction and fertility.

[5]  F. N. David,et al.  Principles and procedures of statistics. , 1961 .

[6]  B. Gulyas,et al.  Ovoperoxidase activity in ionophore treated mouse eggs. II. Evidence for the enzyme's role in hardening the zona pellucida , 1980 .

[7]  X. Zhang,et al.  Effects of follicle-stimulating hormone and ovarian steroids during in vitro meiotic maturation on fertilization of rat oocytes. , 1989, Gamete research.

[8]  B. Vanderhyden,et al.  Zona drilling increases the penetrability of rat oocytes matured in vitro. , 1989, Biology of reproduction.

[9]  R. Gwatkin.  Fertilization Mechanisms in Man and Mammals , 1977, Springer US.

[10]  C. Barros,et al.  Induction of Zona Reaction in Golden Hamster Eggs by Cortical Granule Material , 1971, Nature.

[11]  S. Pickering,et al.  The influence of cooling on the properties of the zona pellucida of the mouse oocyte. , 1988, Human reproduction.

[12]  R. Schultz,et al.  Precocious loss of cortical granules during mouse oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida. , 1990, Developmental biology.

[13]  D. Wolf,et al.  Induction of zonal and egg plasma membrane blocks to sperm penetration in mouse eggs with cortical granule exudate. , 1977, Biology of reproduction.

[14]  A. D. Fleming,et al.  Developmental capability of rat oocytes matured in vitro in defined medium , 1985 .

[15]  C. Thibault.  HAMMOND MEMORIAL LECTURE , 1977 .

[16]  S. Willadsen,et al.  Effect of follicular steroids on the maturation and fertilization of mammalian oocytes. , 1980, Journal of embryology and experimental morphology.

[17]  D. Armstrong,et al.  Reduction in fertilization rate in vitro of oocytes from immature rats induced to superovulate. , 1984, Journal of reproduction and fertility.

[18]  B. Vanderhyden,et al.  Role of cumulus cells and serum on the in vitro maturation, fertilization, and subsequent development of rat oocytes. , 1989, Biology of reproduction.

[19]  M. Felici,et al.  «Spontaneous» hardening of the zona pellucida of mouse oocytes during in vitro culture. II: The effect of follicular fluid and glycosaminoglycans , 1985 .

[20]  S. Downs,et al.  Serum maintains the fertilizability of mouse oocytes matured in vitro by preventing hardening of the zona pellucida , 1986 .

[21]  P. Wassarman,et al.  Characterization of a proteinase that cleaves zona pellucida glycoprotein ZP2 following activation of mouse eggs. , 1989, Developmental biology.

[22]  B. Shapiro,et al.  Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[23]  D. Loskutoff,et al.  Cultured granulosa cells produce two plasminogen activators and an antiactivator, each regulated differently by gonadotropins. , 1985, Endocrinology.

[24]  A. Trounson,et al.  Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. , 1977, Journal of reproduction and fertility.