Ralph B. L. Gwatkin

Immunization of Mice with Heat-Solubilized Hamster Zonae: Production of Anti-Zona Antibody and Inhibition of Fertility

Immunization of female mice with heat-solubilized hamster zonae pellucidae resulted in the production of antibody specific for the zona pellucida of several species, including that of primates. Complete, although temporary, infertility was produced in the mice. The zonae of ovulated eggs from the immune mice were found to contain antibody and complement. Many also appeared abnormal, but no apparent effect was observed on either vitelli or any of the other tissues that were examined. These results suggest the feasibility of developing a zona vaccine for contraception.

The Prevention of Polyspermy

Fusion of the cortical granules with the oolemma and a discharge of their contents into the perivitelline space occurs when the sperm reaches the vitellus (Szollosi, 1967; Piko, 1969). Studies with hamster gametes in vitro have shown that the trigger for this so-called cortical reaction is fusion between the gamete membranes and that simple contact is not sufficient (Gwatkin et al., 1976b). Penetration of the sperm into the vitellus is not needed, since it was found that capacitated spermatozoa would induce the reaction even after they were frozen and thawed so as to prevent penetration. The cortical reaction is propagated around the egg from the point of association of the fertilizing sperm with the vitelline membrane (Braden et al., 1954).

Fertilization In Vitro

As already indicated, recent progress in our knowledge of fertilization mechanisms is due primarily to the development of in vitro fertilization techniques. Despite many early claims (e.g., by Shenk in 1878; see review by Thibault in 1969), the first successful method was not devised until 1954, when Dauzier et al. obtained cleavage of rabbit eggs after exposing them to sperm recovered from the uterus. Since then methods have been developed for at least 11 different mammalian species, including man (Table 3).

The Acrosome Reaction

No morphological change has been observed in mouse (Bryan, 1974) or rabbit (Bedford, 1969) spermatozoa lying free in the ampulla of the oviduct. However, as the spermatozoa pass through the cumulus oophorus the outer acrosomal membrane may invaginate to form membrane-bound vesicles within the acrosome followed by loss of the plasma membrane (Jones, 1973; Roomans and Afzelius, 1975), or multiple fusions may develop between the plasma and outer acrosomal membranes (Barros et al., 1967; Franklin et al., 1970; Yanagimachi and Noda, 1970b). These vesiculation processes mark the start of the acrosome reaction and probably permit the release of hyaluronidase (Figure 18b). Discharge of hyaluronidase by the sperm presumably facilitates their passage through the cumulus. Hyaluronidase release has been used as a criterion for the completion of the in vitro capacitation of rabbit (Lewis and Ketchell, 1972) and hamster (Rogers and Morton, 1973b) sperm and for the onset of the acrosome reaction in guinea pig sperm (Rogers and Yanagimachi, 1975a).

Fusion of the Sperm with the Vitellus

Studies with hamster gametes in vitro have shown that the microvilli of the vitellus wrap around the head of the sperm and fuse with its plasma membrane in the postacrosomal region (Yanagimachi and Noda, 1972). The role of the postacrosomal plasma membrane in initiating fusion between mammalian gametes is in striking contrast to gamete fusion in marine invertebrates in which initial contact with the egg membranes is made by an apical process extending from the inner acrosomal membrane (Colwin and Colwin, 1967; Epel, 1975; Summers et al., 1975).

Attachment and Binding of the Sperm to the Zona Pellucida

Sperm appear to bind to the surface of the zona pellucida by the plasma membrane overlying their acrosomes (Franklin et al., 1970; Gwatkin et al., 1976a). However, participation of the post-acrosomal region has not been excluded. Observations made by Hartmann et al. (1972) with a low-power light microscope have shown that hamster gametes in vitro associate by a complex series of interactions. During this time the sperm acrosome reaction which might explain some of their observations is probably taking place, but unfortunately, this event was not recorded by Hartmann et al. It will be important to repeat their experiments using the scanning electron microscope to reveal the full story of gamete association.

The value of semen analysis and sperm function assays in predicting pregnancy among infertile couples**Supported by grant 84-45 from The Physician Services Incorporated Foundation, Toronto, Canada.

Over a 2-year period, 227 couples were evaluated by an extended assessment of the male partners ejaculate. This extended assessment comprised sperm penetration of denuded hamster oocytes, ability of sperm to penetrate synthetic mucus, and the adenosine triphosphate (ATP) content of whole semen. In proportional hazards analysis adjusting for the contribution of clinical and other seminal variables, the sperm penetration assay (SPA) test was a predictor of pregnancy in the subgroup with normal conventional seminal variables (>40 million motile sperm per ejaculate). When the SPA result was 20% or more the probability of pregnancy was 3.7 times higher; the performance of the SPA as a diagnostic test was better in the couples with treated tubal disease than in other diagnostic categories.

Fate of Nonfertilizing Spermatozoa and Interaction of Spermatozoa with Somatic Cells

Of the millions of mammalian sperm inseminated into the female genital tract, only a very few ever reach the site of fertilization. The major mechanism by which excess spermatozoa are eliminated is by leukocytes that invade the uterus (Austin, 1960; Yanagimachi and Chang, 1963b; Bedford, 1965; Mahajan and Menge, 1966; Moyer et al., 1967) and uterine cervix (Moyer et al., 1970) after copulation. Phagocytosis of spermatozoa by epithelial cells of the endometrium (Moyer et al., 1967) and oviducal mucosa (Austin, 1960; Zamboni, 1971a) has also been reported. Nonfertilizing sperm persisting in the female tract may also occasionally be phagocytized by embryos at the two-cell (Thompson and Zamboni, 1974) and blastocyst (Tachi and Kraicer, 1967; McReynolds and Hadek, 1971) stages. In rodents and lagomorphs clearance is rapid, occurring within 24–36 hr after copulation (Howe, 1967; Bishop, 1969). However, little is known concerning the clearance from the female tract of spermatozoa in species such as the dog, horse, and ferret in which sperm survive much longer, a property possibly correlated with the longer estrus period in these mammals.

Penetration of the Zona Pellucida by the Sperm

Evidence that acrosin is responsible for digesting a pathway for the sperm through the zona pellucida comes first from the fact that acrosin isolated from rabbit sperm will dissolve the zona pellucida of rabbit eggs (Stambaugh and Buckley, 1969). The second line of evidence is that trypsin-acrosin inhibitors inhibit fertilization both in vitro and in vivo. Stambaugh et al. (1969) found that ovomucoid and soybean trypsin inhibitors, each at 1 mg/ml, inhibited the in vitro fertilization of rabbit eggs by 36 and 90%, respectively. This corresponded to their relative effectiveness in blocking the action of rabbit acrosin on synthetic substrates. Pretreatment of capacitated rabbit sperm with the synthetic trypsin inhibitor, tosyl-L-lysyl chloroketone (TLCK), but not the chymotrypsin inhibitor, tosyl-L-phenylalanyl chloroketone (TPCK), inhibits the ability of the sperm to fertilize eggs on subsequent insemination (Zaneveld et al., 1970). The in vivo fertilization of rabbit eggs is also inhibited by incubating capacitated sperm prior to insemination with pancreatic trypsin inhibitor or partially purified rabbit seminal plasma trypsin inhibitor (Zaneveld et al., 1971). Similarly the fertilization of frog eggs is blocked when sperm are incubated in naturally occurring or synthetic trypsin inhibitors prior to insemination (Greenslade et al., 1973).

Metabolic Changes Associated with Fertilization

Because of the limited number of eggs available for experimentation, little is known of the metabolic changes which follow fertilization in mammals. However, considerable knowledge has been obtained with echinoderm eggs (Monroy, 1965, 1973; Epel, 1975).