BIOLOGY OF EGGS AND IMPLANTATION 



835 



plasm, that which was still in the pehvitel- 

 line space lashed about vigorously. These 

 observations are similar to those described 

 by Shettles (1960) in the human. As men- 

 tioned earlier, the technical problems in ob- 

 serving in vitro fertilization will no doubt be 

 solved when the molecular species of the 

 fluids forming the normal egg environment 

 is known. 



There is no specific information with re- 

 spect to the nature of the vitelline membrane 

 of the mammalian egg or to the changes it 

 may undergo on sperm entry. It would be de- 

 sirable to know whether the vitelline mem- 

 brane undergoes modification after penetra- 

 tion by the fertilizing spermatozoon. An 

 interesting procedure for measuring the so- 

 lidification of the egg membranes of salmo- 

 nid eggs has been described recently by Zotin 

 (1958). Even though there is no clear evi- 

 dence of a comparable phenomenon in mam- 

 malian eggs, some factor appears to control 

 the number of spermatozoa which enter the 

 vitellus. Cortical granules have been de- 

 scribed in the unfertilized hamster egg which 

 disappear on fertilization, but apparently 

 they are not associated with the block of 

 l)olyspermy (Austin, 1956a). Quantitative 

 data are necessary to clarify the relationship 

 between the number of spermatozoa which 

 may enter the periovarial space, the rate of 

 the "tanning" reaction of the zona, if such a 

 ])henomenon exists, and the reaction of the 

 perivitelline membrane which blocks the en- 

 try of further spermatozoa. 



Shrinkage of the vitellus after sperm pen- 

 etration has been described in the rabbit and 

 rat (Gilchrist and Pincus, 1932; Pincus and 

 Enzmann, 1932), but a comparable shrink- 

 age can be noted in unfertilized ova recov- 

 ered from the oviduct several hours after 

 ovulation, and thus shrinkage per se cannot 

 be used as a criterion for sperm penetration. 

 The shrinkage of the vitellus is related in 

 some way to changes in the vitelline mem- 

 brane because the numerous microvilli pres- 

 ent in the young ovarian egg have disap- 

 peared and the total surface of the egg has 

 been greatly reduced. 



E. FERTILIZ.\TIOX IN VITRO 



During the past century one of the most 

 challenging and frustrating problems was 



the attempt to fertilize mammalian ova in 

 vitro and to follow their cleavage. Even 

 though several successes were recorded, it 

 could not be maintained unequivocally until 

 the recent work of Chang ( 1959a) that sperm 

 penetration has been accomplished and that 

 the divisions of the eggs noted were the re- 

 sult of fertilization rather than of an "ac- 

 tivation" of the egg instituted by some other 

 factor in the environment, or just plain frag- 

 mentation. 



Relatively little has been added to our un- 

 derstanding of the mechanism of sperm pen- 

 etration into the ooplasm since the extensive 

 experiments of Long (1912) in which he at- 

 tempted to fertilize rat and mice eggs in 

 vitro. He described penetration of the fol- 

 licle cells and observed the sinuous move- 

 ments of the sperm as they advanced within 

 the cunmlus. The role of the spermatozoa in 

 the dispersal of the granulosa cells was noted 

 and this was interpreted as being due to the 

 lashing activities of the sperm fiagellum. 

 Long also described the formation of the 

 second polar body in eggs which had been 

 placed in sperm suspensions. Polar body for- 

 mation began within 2 hours and abstric- 

 tion was completed within 4 hours of the 

 time of immersion. Unfortunately, his de- 

 scription leaves one uncertain as to whether 

 penetration by the sperm was actually ob- 

 served or merely confirmed by sectioned ma- 

 terial. 



Some success with fertilization in vitro 

 was also achieved by Pincus (1930, 1939), 

 Pincus and Enzmann (1934, 1935), Venge 

 (1953), and Thibault and Dauzier (1960) in 

 their extensive experiments with both ovar- 

 ian and tubal eggs of rabbits. These in- 

 vestigators described the abstriction of the 

 second polar body, the shrinkage of the 

 vitellus, the penetration of the zona by 

 spermatozoa partially embedded within it, 

 and the presence of spermatozoa in the peri- 

 vitelline space in fixed and stained prepara- 

 tions. Transplantation of living eggs into the 

 oviducts of pseudopregnant rabbits, follow- 

 ing the addition of sperm to the eggs, re- 

 sulted in the birth of live young possessing 

 the genetic characteristics of coat color 

 which had been used as markers. It is sug- 

 gested in a later report (Chang and Pincus, 

 1951) that the results "may have been due 



