830 



SPERM, OVA, AND PREGNANCY 



Many further attempts to demonstrate 

 the therapeutic value of hyaluronidase in 

 mammalian infertility have met with fail- 

 ure (see Siegler, 1947; Tafel, Titus and 

 Wightman, 1948; Johnston and Mixner, 

 1950). The generally poor results obtained 

 by the addition of hyaluronidase to semen 

 introduced into the vagina or uterus by 

 artificial insemination may be explained by 

 the later experiments of Leonard, Perlman 

 and Kurzrok (1947), which conclusively 

 demonstrated that hyaluronidase inserted 

 into the lower reproductive tract is not 

 transported to the oviducts. The systematic 

 studies of Austin (1949b) and Chang (1947, 

 1951a) revealed that in the rabbit only 100 

 to 1000 spermatozoa reach the site of fer- 

 tilization. Even though in one experiment 

 600,000,000 spermatozoa were artificially 

 introduced into the female reproductive 

 tract, only approximately 2000 of them were 

 found in the tubes. An even smaller number 

 (10 to 50) have been shown to reach the 

 ampulla of the rat oviduct at the time of 

 sperm penetration (Blandau and Odor, 

 1949; Moricard and Bossu, 1951). 



It is probably correct to assume that any 

 hyaluronidase which reaches the cumulus at 

 the time of semination is transported by 

 relatively few spermatozoa. Although the 

 enzyme has not been localized in the sperm 

 itself, it is assumed that it is an integral 

 part of the cell and is liberated in a rela- 

 tively localized region as the spermatozoon 

 makes its way through the cement sub- 

 stance. The spermatozoon is remarkably 

 permeable in that such large molecules as 

 cytochrome c or hyaluronidase can detach 

 themselves from the sperm cell and pass 

 into the extracellular en^'ironment by the 

 so-called "leakage" phenomenon (Mann, 

 1954). 



In vitro tests have shown that the enzyme 

 hyaluronidase diffuses into the suspending 

 fluid at a definite rate depending on the type 

 of medium and the temperature. New for- 

 mation of the enzyme by spermatozoa does 

 not seem to occur (Meyer and Rapport, 

 1952). The possibility exists that the en- 

 zyme may be able to exert its action while 

 still bound to the sperm cell. 



A recent development in the study of 

 hyaluronidase action and its possible role 



in fertilization has been the attempt to 

 utilize certain inhibitors of the enzyme as 

 systemic contraceptives. Among the natu- 

 rally occurring and extraneous inhibitors 

 may be listed heavy metals, heparin, qui- 

 nones, "rehibin" or trigentisic acid, and 

 antihyaluronidase antibodies, as well as a 

 nonspecific, electrophoretically identifiable 

 serum factor (Leonard and Kurzrok, 1945; 

 Beiler and Martin, 1947; Glick and Moore, 

 1948; Meyer and Rapport, 1952; Hahn and 

 Frank, 1953; Parkes, 1953). Many of these 

 substances are highly active inhibitors of 

 hyaluronidase and may reduce or prevent 

 fertilization when added to semen in vitro 

 before artificial insemination. Attempts to 

 inhibit fertilization by giving these sub- 

 stances orally or by injection have not been 

 repeatedly successful, but several deriva- 

 tives of hyaluronic acid obtained by acety- 

 lation or nitration and added to rabbit 

 semen in vitro seemed to have inhibited dis- 

 persion of follicle cells and to have im- 

 paired fertility (Pincus, Pirie and Chang, 

 1948). 



It has now been demonstrated repeatedly 

 that ova in the ampulla of the oviduct may 

 have been penetrated by spermatozoa with- 

 out evident dispersal of the granulosa cells 

 (Lewis and Wright, 1935; Leonard, Perl- 

 man and Kurzrok, 1947; Austin, 1948b; 

 Bowman, 1951; Odor and Blandau, 1951, in 

 the rat; Chang, 1950b, in the rabbit; Amo- 

 roso, personal communication, in the cat). 

 Again, dog spermatozoa do not contain 

 hyaluronidase yet they are capable of pene- 

 trating the many layers of granulosa cells 

 comprising the cumulus. Inasmuch as a gen- 

 eralized dispersal of the cells of the cumu- 

 lus does not occur at the time of sperm 

 penetration, the pendulum has swung to the 

 present view that the individual spermato- 

 zoon carries sufficient enzyme to make a 

 path for itself through the cumulus layer 

 and the gel matrix. If rat spermatozoa are 

 added to slides containing cumulus masses 

 from freshly ovulated eggs and their move- 

 ment through the cumulus matrix observed 

 with phase objectives, one is led to conclude 

 that an intact cumulus is essential if sperm 

 penetration is to be successful, i.e., the 

 cumulus may act as a base against which 

 the sperm flagellum can push as it moves 



