Amphibians, whose embryos are commonly used for the bioassays of inductive interactions, are listed in Table 1. They can be commercially purchased, are easily maintained, and can be coaxed to breed in the laboratory. Urodela, such as Cynops and Ambystoma, lay relatively large eggs (approx 2-mm diameter), and their embryos develop relatively slowly, which are advantageous features for performing surgical manipulation. Consequently, urodele embryos have proved to be the most useful material for traditional (i.e., classical) experimental embryology (4). Urodele populations are, however, currently decreasing throughout the world. The amphibian most widely used today is the African clawed frog, Xenopus laevis. It is commercially available, produces large numbers (1000-2000) of eggs at one spawning in response to hormone stimulation, and its embryos develop relatively rapidly (hatch in 37 h at room temperature). Those features of Xenopus provide significant advantages for biochemical and molecular biological studies (5) when compared with urodeles.
Eggs of Ambystoma mexicanum (axolotl) are inseminated as they pass through the cloaca of the female. (After a ritual courtship, she picks up sperm packets [spermato-phores] fastened to the substrate [e.g., rocks] by a male.) Fertilized eggs are usually obtained by natural spawnings in the laboratory. Artificial insemination is also possible (6), but rather difficult when compared with the Xenopus artificial insemination procedure. To obtain natural axolotl spawnings, a male is placed together with a female in the mating container (e.g., plastic dishpan lined with pebbles) and left in the dark. Enhanced spawning frequencies can be obtained by injecting a female intramuscularly with 250 IU of human chorionic gonadotropin (6). The male is removed the next day after spermatophores have been deposited on the pebbles. The container with the female is then returned to the dark. The female usually starts to shed fertilized eggs about 24 h after she has picked up spermatophores. Staging series published by Schreckenberg and Jacobson (7) and by Bordzilovskaya and Dettlaff (8,9) are available for the staging of A. mexicanum embryos.
Amphibians Commonly Used in Experimental Embryology
Number Egg size Natural
Amphibia Popular name of eggs (mm) breeding period Locality
Ambystoma mexicanum Axolotl 200-1000 2.0 October to April Central Mexico
Cynops pyrrhogaster Japanese fire-salamander 50-250 2.0 March to June Japan (except Hokkaido) Anura
Xenopus laevis African clawed-toad 1500-2000 1.2 April to September South Africa
Although adult Cynops females can be commercially purchased, Japanese laboratories usually collect them from the ditches around rice fields in early spring and late autumn. The majority of females collected in those seasons already carry spermato-phores. The females are stored in water at 4-6°C and need not be fed. They retain the spermatophores for many months. To obtain fertilized eggs, females are injected with 50-100 IU of human chorionic gonadotropin (HCG; e.g., GESTRON®, Denka Seiyaku, Kawasaki, Japan) on alternate days for two or three injections (10), then placed in water at 20°C together with a polyethylene tape on which they can lay eggs. A robust female will lay as many as 250 eggs over 1 wk (Fig. 1A). A staging series for Cynops pyrrhogaster has been published by Okada and Ichikawa (11), based on external morphology and on features visible in dissected embryos.
Xenopus can be easily induced to spawn by HCG injection every 3 mo. To obtain fertilized eggs, the dorsal lymph sacs of both male and female are injected with 600800 IU of HCG. The animals are then placed in a container filled with water to approx 5 cm in depth and kept at 20°C overnight (Fig. 1B). Fertilized eggs laid on the bottom of the container are scraped off and collected with a wide-bore pipet (5 mm in diameter). For artificial insemination, HCG-injected females are kept at 20°C overnight to induce ovulation (Fig. 1C). Approximately 100 eggs are squeezed into a 60-mm plastic Petri dish and fertilized with 0.2-0.4-mL sperm suspension, which is prepared by suspending finely macerated testis in 2 mL modified De Boer's saline (MDB; 110.00 mM NaCl, 1.30 mM KCl, 0.44 mM CaCl2, 3.00 mM HEPES, pH 7.3) (12). Artificial insemination is advantageous, especially in the microinjection study, to simultaneously bring embryos to exactly the same developmental stage. A normal table published by Nieuwkoop and Faber (13) is available for staging Xenopus embryos.
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