Triploid and gynogenetic diploid Xenopus laevis

Abstract

Triploid and gynogenetic diploid Xenopus laevis frogs were produced using pressure to suppress the formation of the second polar body. Five to ten minutes after in vitro insemination, eggs were subjected to six minutes of hydrostatic pressure a t 4,800 pounds per square inch. All of the surviving embryos tested were triploid. Application of the same pressure technique to eggs fertilized with ultraviolet irradiated sperm resulted in gynogenetic diploid embryos in every case where the sperm nucleus was inactivated. Triploid and gynogenetic diploid embryos developed normally through the larval period and metamorphosis.Triploid cells are useful cell markers in grafting experiments since amphibian cells and nuclei vary in size in proportion to the number of haploid sets of chromosomes in the nuclei (Fankhauser, '55). Triploid cells have been distinguished from diploid cells in chimerae by cell size, DNA content (Turpen and Volpe, '741, number of nucleoli (Namenwirth, '74), as well as chromosome counts. In these ways, the fates of cells of surgically defined origin in chimaerae can be followed, for triploid cells are as viable as diploid cells.The formation of the second polar body of amphibian eggs can be suppressed in a variety of ways including heat shock, cold shock, and pressure (Smith, '58; Briggs, '47; Fankhauser, '55; Dasgupta, '62). If normal fertilized eggs are so treated a t an appropriate time, triploid embryos are produced. Triploidy has not been used extensively in studies of Xenopus laeuis probably because the method available (Smith, '58) produces triploidy in only slightly more than half the treated embryos.Gynogenetic diploid amphibians have been produced by suppressing second polar body formation in eggs fertilized with sperm whose nuclei are incapable of participating in subsequent development. (Volpe and Dasgupta, '62). Gynogenetic diploids are of interest in studying sex determination, in screening wild populations of animals for mutant genes and mapping mutant genes relative to the centromeres of the chromosomes which bear them (Volpe and Dasgupta, '62; Lindsley et al., '56).In this report methods for producing triploid and gynogenetic diploidXenopus laeuis are de- scribed which are reliable as judged by chromosome counts and genetic analysis. MATERIALS AND METHODSNormal Xenopus laevis were obtained from Nasco, Fort Atkins, Wisconsin. Periodic albino (aP/aP) Xenopus laevis (Hoperskaya, '75; Tompkins, '77) were generously provided by Doctor A. J. Brothers. Ovulation was induced by the method of Gurdon ('67). Artificial insemination by the method of Wolf and Hedrick ('72) yielded poor results, usually less than 10% fertilization. Therefore, the following method was devised. Males were sacrificed and their testes removed and washed in 10% Ringer's solution a t 0°C. One testis was macerated in 10 mi of ice cold 100% Ringer's solution and this suspension was kept on ice until needed. Good fertilization was obtained up to 12 hours after maceration. Eggs were stripped from ovulating females into...

A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis

“…Gynogenesis utilizing high pressure has been used to great success in X. laevis (Müller et al, 1978;Tompkins, 1978;Reinschmidt et al, 1979;Columbelli et al, 1984;Krotoski et al, 1985;Tompkins and Reinschmidt, 1991). Given that X. tropicalis embryos develop at a slightly Fig.…”

“…While three generations are required to isolate homozygous mutants in the traditional manner, the approach we used, based on the methods developed for making mutants in wild-caught animals in X. laevis (Tompkins, 1978) reduces this requirement to two generations (Fig. 1A).…”

Fixation‐Associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs

“…Hazen and Co., Alburg, VT) and Xenopus laeuis (South African Snake Farm, Capetown, South Africa), were used. In addition, a few non-chimeric 3N adult Xenopus laeuis (9,24) were also used.…”

Xenopus, the next generation: X. Tropicalis genetics and genomics

“…Haploid embryos develop abnormally, potentially obscuring detection of recessive phenotypes. However, if haploid embryos are subjected to several thousand pounds of hydrostatic pressure within a few minutes of fertilization, the extrusion of the second meiotic polar body is suppressed, thereby generating diploid embryos with only a maternal gene complement (Tompkins, 1978). One can then score these embryos for developmental phenotypes.…”