836 



SPERM, OVA, AND PREGNANCY 



to adherent sperm effecting fertilization in 

 the fallopian tubes." 



The mammalian egg may be "activated" 

 to various degrees according to the balance 

 of thermal, osmotic, and chemical factors in 

 its environment. Thus eggs "activated" by 

 being placed in a cold environment may form 

 double nuclei which closely resemble normal 

 pronuclei (Thibault, 1947a, b, 1948). The 

 eggs of the opossum, rat, mouse, hamster, 

 mink, and ferret also will show varying de- 

 grees of "activation" and may be difficult to 

 differentiate from normally cleaving ova 

 (Smith, 1925; Chang, 1950a; Austin, 1951a, 

 1956c; Blandau, 1952). Attempts to fertilize 

 the timed human ovarian ova recovered by 

 Corner, Farris and Corner (1950), were un- 

 successful. Rock and Menkin (1944) and 

 Menkin and Rock (1948) also attempted to 

 achieve fertilization of human ovarian eggs 

 in vitro and reported several successes. The 

 first egg recovered from a large follicle was 

 cultured in the patient's serum for 27 hours. 

 It was then placed in a washed suspension of 

 sperm for 1 hour and observed continuously. 

 Penetration of the ovum by sperm was not 

 observed. When the same egg was inspected 

 40.5 hours later, it consisted of two blasto- 

 meres each measuring 86 /a in diameter. A 

 second egg treated in much the same manner 

 also was found to contain two blastomeres 

 45 hours after exposure to spermatozoa. The 

 stage of maturation of these ovarian eggs 

 could not be determined and it is assumed 

 that the meiotic divisions occurred in vitro. 

 Since the fertilizable life of the human ovum 

 is unknown, and there is no specific informa- 

 tion on sperm penetration, the role of the 

 flagellum in semination, pronuclei formation, 

 karyogamy, and the rate of cleavage, it is 

 clear that the true identification of a fer- 

 tilized human ovum has not been achieved. 

 In the rat, for example, one can find unfer- 

 tilized cleaved ova which on first inspection 

 closely resemble fertilized eggs even contain- 

 ing modified nuclei or nuclear fragments. 

 When examined in detail the fragmenting 

 eggs do not contain the flagellae of spermato- 

 zoa, a positive indication that penetration 

 has not been accomplished (compare 2 and 3 

 in Figure 14.15). 



Various criteria have been accepted as an 

 indication of fertilization in vitro such as 

 polar body formation, shrinkage of the vitel- 



lus, presence of one or more pronuclei, and 

 cleavage of the ooplasm. As emphasized ear- 

 lier, all of these phenomena have been ob- 

 served many times in eggs which have not 

 been penetrated by a spermatozoon and 

 which are in varying stages of degeneration 

 and fragmentation. Too little is known con- 

 cerning the processes of semination and fer- 

 tilization in mammals, with the possible ex- 

 ceptions of the rat and rabbit, to judge 

 uneciuivocally whether normal sperm pene- 

 tration and fertilization have been accom- 

 plished in vitro. 



The freshly ovulated eggs of most mam- 

 mals are notoriously sensitive to changes in 

 environment and one is concerned lest the 

 eggs cultured in vitro may simulate the 

 events occurring in vivo without activation 

 by a spermatozoon. If sperm penetration and 

 the various fertilization i)henomena cannot 

 be followed continuously by direct visuali- 

 zation, it is generally agreed that, unless 

 viable embryos are obtained by transplant- 

 ing the supposedly fertilized eggs to recipient 

 animals, the success of fertilization is not 

 sufficiently proven. Recently Chang (1959a) 

 was successful in fertilizing the rabbit egg 

 in vitro and obtaining living young by trans- 

 planting them to host animals. Thus for the 

 first time a repcatable procedure for fertiliz- 

 ing mammalian ova in vitro has been per- 

 fected. Chang obtained unfertilized rabbit 

 eggs by intravenous injection of sheep pi- 

 tuitary extract into estrous rabbits. Sperm 

 were obtained 12 hours after mating females 

 with fertile bucks by washing the uterus with 

 a Krebs-Ringer bicarbonate solution. Un- 

 fertilized ova were obtained by flushing the 

 oviducts of the animals which had received 

 the gonadotrophins. Both sperm and eggs 

 were placed in a small Carrel flask and kept 

 at 38°C. Three to 4 hours later the ova were 

 transferred to a second Carrel flask contain- 

 ing rabbit serum and cultured for another 

 18 hours. At this time the eggs were recov- 

 ered and examined, and those that appeared 

 to be cleaving were transferred to recipient 

 rabbits. Approximately 42 per cent of the 

 transferred ova that appeared to be ferti- 

 lized were delivered at term as viable young. 



F. FATE OF THE UNFERTILIZED EGG 



Evidence that ovulation without fertiliza- 

 tion is followed by rapid degeneration and 



