BIOLOGY OF EGGS AND IMPLANTATION 



829 



the follicle ruptures. Although only limited 

 observations have been made, some reports 

 indicate that the cumulus oophorus in the 

 preovulatory follicles cannot be dispersed as 

 readily by the methods that are effective in 

 ovulated eggs (Farris, 1947; Shettles, 1953). 

 It is important to determine what chemical 

 or physical alterations occur in the inter- 

 cellular cement substances of the cumulus 

 during the time the follicle is ripening and 

 to learn why this should differ in the cells 

 surrounding the egg from other similar 

 cells lining the walls of the follicle. 



The existence of a "cumulus-dispersing" 

 factor in mammals was brought to light by 

 the experiments of Gilchrist and Pincus 

 (1932), Yamane (1935), Pincus (1936), 

 and Pincus and Enzmann (1936). These in- 

 vestigators demonstrated that either living 

 sperm suspensions or sperm extracts of the 

 rabbit, rat, and mouse rapidly disperse the 

 cells of the cumulus oophorus of tubal ova. 

 Yamane (1930) inferred that the presence 

 of a proteolytic enzyme in the spermatozoa 

 was responsible for both follicle-cell dis- 

 persion and "activation" of the egg to pro- 

 duce the second polar body. 



In a series of carefully controlled experi- 

 ments Pincus (1936) showed that a heat- 

 labile substance was present in sperm ex- 

 tracts which caused follicle-cell dispersion, 

 but that this substance would not effect 

 second polar body formation. Pincus dem- 

 onstrated further that the rate of cell dis- 

 persion in vitro was roughly proportional 

 to the number of spermatozoa in the sus- 

 pension. It was discovered later that the 

 "cumulus-cell-dispersing substance" was 

 the enzyme hyaluronidase (Duran-Reyn- 

 olds, 1929). The enzyme depolymerizes and 

 liydrolyzes the hyaluronic acid cement sub- 

 stance binding the granulosa cells together. 

 This discovery at first seemed to provide a 

 happy solution to the problem of how sper- 

 matozoa penetrate the cumulus oophorus 

 (McClean and Rowlands, 1942; Fekete and 

 Duran-Reynolds, 1943; Leonard and Kurz- 

 rok, 1945). Numerous observations cpickly 

 demonstrated that the testes and spermato- 

 zoa of mammals are the richest sources of 

 animal hyaluronidase. The enzyme first ap- 

 pears in the testes when spermatogenesis 

 begins in the pubertal animal and before 



fully developed spermatozoa are present in 

 the tubules (Riisfeldt, 1949). 



It became clear that there is a propor- 

 tional relationship in vitro between sperm 

 count and the hyaluronidase concentration ; 

 further, that the enzyme is associated with 

 the spermatozoa and not with the seminal 

 plasma (Werthessen, Berman, Greenberg 

 and Gargill, 1945; Kurzrok, Leonard and 

 Conrad, 1946; Swyer, 1947a; Michelson, 

 Haman and Koets, 1949). Hyaluronidase 

 concentration per sperm is highest in the 

 bull and rabbit, somewhat less in the boar 

 and man, still lower in the dog, and very 

 low in birds and reptiles (Swyer, 1947a, b; 

 Mann, 1954). Observations on the in vitro 

 dispersal of granulosa cells by hyaluroni- 

 dase suggested that large numbers of sper- 

 matozoa are necessary in the semen in order 

 to provide a sufficient concentration of the 

 enzyme. 



The in vitro observations of Pincus and 

 Enzmann (1936) strengthened this assump- 

 tion when they demonstrated that a mini- 

 mum number of 20,000 spermatozoa per 

 cubic millimeter of rabbit semen is neces- 

 sary if the cumulus cells surrounding one 

 ovulated egg are to be dispersed. Such ob- 

 servations seemed to explain the necessity 

 of the "sperm swarms" described in the 

 oviducts of mated rabbits. The swarms 

 created and maintained a sufficiently high 

 concentration of the enzyme to permit the 

 denudation of the eggs so that certain of 

 the spermatozoa could approach and pene- 

 trate the zona pellucida. 



Attempts were then made to increase the 

 fertilizing capacity of a subnormal number 

 of spermatozoa by adding hyaluronidase 

 extracts to semen suspensions used for arti- 

 ficial inseminations. In 1944, Rowlands pro- 

 posed that such a procedure had increased 

 the fertilizing capacity of rabbit spermato- 

 zoa. This could not be confirmed by Chang 

 (1950b) ; indeed, it was observed that semi- 

 nal plasma in which the hyaluronidase had 

 been inactivated by heat was as effective as 

 untreated plasma. Kurzrok, Leonard and 

 Conrad (1946) outlined a method for adding 

 bull hyalurodinase to oligospermic speci- 

 mens of human semen which was to be used 

 for artificial insemination. This method was 

 employed in the treatment of sterility and 

 reported to have been notably successful. 



