BIOLOGY OF EGGS AND IMPLANTATION 



833 



or disintegrated in rat and rabbit eggs by 

 buffers with pH values from 3 to 5 (Hall, 

 1935; Harter, 1948; Braden, 1952). Various 

 reducing agents such as glutathione and 

 cysteine in Tyrode's solution cause rapid 

 dissolution of the zona. Oxidizing agents 

 such as the hydrogen peroxide which is pro- 

 duced by sperm (Tosic and Walton, 1946) 

 are particularly efficacious in removing this 

 membrane. Several investigators favor the 

 possibility that a specific mucolytic en- 

 zyme, "zona lysin" (Austin and Bishop, 

 1958) may be secreted by the sperm as it 

 makes contact with the zona pellucida (Le- 

 blond, 1950; Austin, 1951b). It seems likely 

 that the passage of the spermatozoon 

 through the zona pellucida may occur in a 

 variety of ways in different animals. Too 

 few observations have been made to sig- 

 nificantly implicate any of the physical, 

 chemical, or mechanical mechanisms sug- 

 gested for sperm penetration of the zona 

 l)ellucida in the mammalian egg. 



It has been suggested that the physical 

 jiroperties of the zona pellucida in the dog, 

 hamster, and sheep are altered after the 

 first sperm passes through it and enters the 

 vitellus. It is postulated that a substance is 

 secreted by the vitellus which "tans" the 

 zona so that additional sperm cannot pene- 

 trate it (Braden, Austin and David, 1954). 

 Smithberg (1953) reported that the zonae 

 l)ellucidae of the unfertilized mouse eggs 

 are more readily removed by proteolytic 

 enzymes than those of fertilized eggs. 



Chang and Hunt (1956) tested the effects 

 of a variety of proteolytic enzymes on the 

 zonae pellucidae of fertilized and unferti- 

 lized eggs of rabbits, rats, and hamsters. 

 Even though none of the fertilized hamster 

 eggs contained more than one sperm, there 

 was no evidence that the zonae pellucidae 

 of the fertilized eggs were more resistant to 

 digestion than those of unfertilized eggs. In 

 contrast Austin (1956c) reported that the 

 zonae pellucidae of fertilized hamster eggs 

 were dissolved more quickly by trypsin 

 than those of unfertilized eggs. Blockage of 

 the zona pellucida in the rat and rabbit 

 egg is not as definite, yet there are indica- 

 tions that fertilized and unfertilized eggs 

 react differently to proteolytic enzymes. 



In many animals the sequence of the re- 



productive processes are arranged in such 

 a manner that spermatozoa must wait at 

 the site of fertilization for several hours be- 

 fore ovulation occurs and the eggs have ar- 

 rived in the ampullae. If freshly ejaculated 

 spermatozoa of rats or rabbits are trans- 

 ferred directly to oviducts containing newly 

 ovulated eggs, relatively few if any of the 

 eggs will be fertilized. If, however, sperma- 

 tozoa are introduced into the genital tract 

 several hours l)efore the expected time of 

 ovulation, they undergo some kind of change 

 by which they gain the capacity to fertilize 

 eggs on contact. Chang ( 1951 ) was the first 

 to report this j^henomenon in the rabbit and 

 termed it "development." In the same year, 

 Austin (1951) working in Australia inde- 

 pendently described the phenomenon and 

 called it "capacitation." Chang (1959a) fur- 

 ther api)i-oached this question by artificially 

 inseminating rabbits that acted as "incuba- 

 tor" hosts. He subsequently withdrew sperm 

 samples at stated intervals and injected 

 them into the oviducts of rabbits that had 

 just ovulated. Chang concluded that 6 

 hours of such "host incubation" was neces- 

 sary before rabbit sperm could fertilize the 

 majority of ova ovulated. Similar observa- 

 tions by Austin (1951), Noyes (1953), and 

 Noyes, Walton and Adams (1958) on rats 

 indicated that approximately 3 hours is the 

 time required for capacitation in this ani- 

 mal. There has been some success in the 

 intrajieritoneal insemination of the rabbit 

 doe 8 hours before ovulation with sperm 

 which had been washed several times in a 

 sodium citrate buffer solution (Hadek, 

 1958). Attempts to induce capacitation in 

 vitro by exposing rabbit spermatozoa for 

 varying lengths of time to a variety of 

 physiologic solutions and solutions contain- 

 ing endometrial tissue have been largely 

 unsuccessful (Chang, 1955b). Partial ca- 

 pacitation has been reported when rabbit 

 spermatozoa are incubated in diverticula 

 of the bladder and colon which had been 

 created surgically (Noyes, Walton and 

 Adams, 1958). Capacitation was also ef- 

 fected when spermatozoa were stored in the 

 seminal vesicles and anterior chamber of the 

 eye. There is no evidence as yet which favors 

 the need for capacitation in the mouse and 

 guinea pig during normal mating. According 



