In vitro fertilization
The average fertilization efficiency for natural matings conducted in our laboratory was 72% (see table 2). As roughly one third of all unfertilized eggs laid in response to hormone treatment auto-activate (table 1), and so only approximately 66% of the eggs in a given clutch will actually be receptive to sperm. If we assume these two factors to be independent -because we hypothesize that the eggs auto-activate during laying, a problem that doesn't arise in natural matings- then only 48 of every hundred eggs will be available for IVF (100*0.66*0.72). However, despite these complications, we were able to obtain an in vitro fertilization efficiency of 27% -or, rather, 56% of all receptive eggs (27/0.48) – by simply mincing the testes and adding them directly over the eggs (see table 2). Other IVF techniques were not as successful. Using sperm diluted in SDB resulted in only a 12% total fertilization efficiency (table 3).
Table 2  Natural mating fertilization percentages for E. coqui in captivity.
Table 3  In vitro fertilization of E. coqui eggs   Sperm concentration may play a role in fertilization efficiency as the use of diluted sperm resulted in decreased fertilization. In support of this possibility, polyspermy has been observed in this species and is apparently not deleterious to fertilization and development [40]. A second possibility is that fertilization efficiency is linked to sperm capacitation and acrosome reaction. We attempted to study this possibility by examining the acrosomes of fresh and treated sperm using Lysosensor green fluorescent dye (Molecular Probes, Eugene, OR). This dye concentrates in low pH vesicles of living cells through an unknown mechanism and was shown to accumulate and preferentially stain the acrosome in X. laevis sperm [42]. However, we were unable to observe acrosome-specific staining in E. coqui sperm using this dye (data not shown). We also examined the possibility that a component of the egg jelly coat may be important for sperm capacitance. To test this, we incubated sperm with SDB and jelly, or SDB alone, and injected this under the jelly coat of eggs. None of 12 eggs were fertilized by sperm incubated with SDB alone, while pre-incubation of sperm with an extract of the jelly coat in SDB resulted in one fertilization out of 10 eggs (10%). This is considerably less than the 27% efficiency following direct placement of minced testes over the eggs, but suggests that interactions between sperm and the jelly coat may play a role in sperm capacitance and subsequent fertilization.
To test the functional response of the eggs, we attempted to fertilize artificially activated eggs. As was explained above, we were able to achieve an IVF success rate of 27%. However, if we poked the eggs fifteen minutes prior to direct fertilization, none (0/36) developed (see table 3). If we assume our expected fertilization rate to be 25%, the possibility of this result being due to chance is (1-0.25)36 = 3.2 × 10-5, or less than one in ten thousand. This shows that fifteen minutes after being poked the eggs have established a block to polyspermy, one of the defining functional characteristics of activation. Hence, although the first visible indication of activation –the formation of the first cleavage furrow- will not be seen for six hours, we can conclude that the egg is undergoing the normal activation processes within minutes of being stimulated.