118 THE MAMMALIAN EGG 



tions, however, penetrated or fertilized eggs have been placed in 

 culture so as to permit further development under artificial condi- 

 tions (Appendix No. 2). Some authors combined storage or culture 

 with subsequent transfer to suitable recipient animals in order to 

 demonstrate that the treatment in vitro had no permanent ill-effect 

 upon the embryo (Chang, 1948a, b, c, 1950b; Adams, 1956; Biggers 

 and McLaren, 1958; McLaren and Biggers, 1958). 



Most success in culture has been had with rabbit eggs, which 

 undergo apparently normal cleavage from the i-ccll to the early 

 blastocyst stage, provided the medium contains about 50 per cent 

 or more of serum. Blastocyst expansion fails, however, and the 

 embryos collapse and become disorganized. The eggs of other 

 mammals have been found even more refractory to culture ; so far, 

 they have not been found to undergo more than one or two divisions 

 when placed in culture at the i-cell stage, but 4- to 8-cell mouse 

 eggs have often been shown capable of developing to blastocysts. 

 Here again, proteins, such as egg-white or serum, are evidently 

 essential components of the medium. 



Fertilization in vitro. It is evident that the ease with which the 

 fertilization of many non-mammalian eggs can be obtained under 

 artificial conditions fostered the belief that mammalian eggs should 

 readily undergo fertilization in vitro. As a result, the consequences 

 of placing eggs and spermatozoa together /'// vitro were often inter- 

 preted on the assumption that fertilization must inevitably be 

 occurring or have taken place and that the provision of proof would 

 be an act of supererogation. The need for a more critical evaluation 

 of observations became apparent as the appreciation grew that eggs 

 could be activated to a degree of parthenogenetic development by 

 conditions they encountered under experiment, that ejaculated 

 spermatozoa were accompanied by substances detrimental to eggs, 

 that the sperm concentrations that seemed appropriate in tests were 

 in fact vastly greater than those normally occurring in vivo, and that 

 spermatozoa require to undergo capacitation before they become 

 capable of fertilization. In addition, the pitfalls inherent in some of 

 the experimental procedures have not always been clearly recog- 

 nized. Undoubtedly, the best criterion of the occurrence of fertiliza- 

 tion in vitro is the development of foetuses or the birth of young 

 from eggs transferred to recipient animals after treatment with 

 spermatozoa. Preferably, the progeny should in addition be of both 

 sexes and genetically distinguishable as deriving from the transferred 



