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Reprod Biol Endocrinol
2004 Aug 05;2:60. doi: 10.1186/1477-7827-2-60.
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In vitro fertilization and artificial activation of eggs of the direct-developing anuran Eleutherodactylus coqui.
Toro E
,
Michael SF
.
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Although much is known about the reproductive biology of pond-breeding frogs, there is comparatively little information about terrestrial-breeding anurans, a highly successful and diverse group. This study investigates the activation and in vitro fertilization of eggs of the Puerto Rican coqui frog obtained by hormonally induced ovulation. We report that spontaneous activation occurs in 34% of eggs, probably in response to mechanical stress during oviposition. Artificial activation, as evidenced by the slow block to polyspermy and the onset of zygote division, was elicited both by mechanical stimulation and calcium ionophore exposure in 64% and 83% of the cases, respectively. Finally, one in vitro fertilization protocol showed a 27% success rate, despite the fact that about one third of all unfertilized eggs obtained by hormone injection auto-activate. We expect these findings to aid in the conservation effort of Eleutherodactylus frogs, the largest vertebrate genus.
Figure 1. Early egg development. A) Untouched, unfertilized egg. The surface of the egg under the jelly coat is featureless. B) Sperm-activated egg at six hours post-fertilization at the four-cell stage. Note the straight, ordered cleavage pattern (arrow). C) Artificially activated egg pseudocleaving. Note the jagged and disorganized cleavage pattern (arrow). D) An egg pseudocleaving at 16 hours. This egg was not handled and did not cleave within the first ten hours after deposition. At ten hours it was poked, and started pseudocleavage shortly thereafter (arrow). Scale bar = 1 mm.
Figure 2. Artificial activation of E. coqui eggs in relation to time after deposition. Unfertilized eggs were treated with calcium ionophore to induce activation at 0 (n = 12), 1 (n = 12), 2 (n = 10), 3 (n = 12), 4 (n = 12), 5 (n = 12), 10 (n = 18), and 24 (n = 18) hours. Eggs were scored for activation by the presence of cleavage furrows at 6 hours post treatment. Note that for the 10 and 24 hour time points, six eggs at each time point had already auto-activated by 6 hours post deposition. At the 10 hour time point, nine additional eggs activated later in response to ionophore treatment, while at 24 hours, no additional eggs were observed to activate after ionophore treatment.
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