DIPLOID GYNOGENESIS IN THE MEXICAN AXOLOTL

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1 DIPLOID GYNOGENESIS IN THE MEXICAN AXOLOTL THOMAS M. TROTTIER AND JOHN B. ARMSTRONG Department of Biology, University of Ottawa, Ottawa, Canada KIN 6N5 Manuscript received October 3, 1975 Revised copy received March 21, 1976 ABSTRACT Gynogenetic diploid axolotls were produced by activating eggs with ultraviolet-inactivated sperm, and then subjecting the activated eggs to heat shock. Optimal conditions for ultraviolet inactivation of the sperm, and for suppression of the second meiotic division by heat shock, were established. Gynogenetic diploids produced by these procedures included progeny homozygous for recessive alleles carried by a heterozygous mother. Gynogenesis could, therefore, be used to uncover new mutations more rapidly than by conventional inbreeding techniques. However, some difficulty was encountered in recognizing mutant phenotypes because of the high incidence of abnormalities and deaths. Defective embryos probably resulted from a combination of heat-shock-induced damage to the eggs and the expression of deleterious recessive alleles carried by the mother. IN recent years, interest in vertebrate genetics has increased as many biologists turn from relatively simple organisms to the study of such complex problems as development. Attempts to adapt the powerful techniques of bacterial and fungal genetics have met with some success for the study of somatic cells in culture (DAVIDSON 1974), but as yet have been of little use to whole animal genetics. The amphibian embryo presents many advantages to the experimental embryologist (MALACINSKI ar?d BROTHERS 1974), and of all amphibian species, the best studied genetically is the Mexican axolotl, Ambystoma mexicanum (HUMPHREY 1975). However, mutant isolation has generally resulted from chance observation or inbreeding programs. As shown in Table 1, if one starts with a single sperm carrying a recessive mutation a, two generations are required, even with full-sib inbreeding, before a small percentage of mutant individuals would be homozygous for the mutation. One possible alternative to such prolonged inbreeding is parthenogenesis (TYLER 1941). If homozygous diploids can be induced from unfertilized eggs, then mutants should be obtained in much shorter time (Table 1 ). At least two routes are open for the induction of diploid parthenogens (BEATTY 1957). One is suppression of first cleavage, where the resultant animals would be homozygous for all loci. The other is suppression of meiosis I1 (retention of the second polar body). In this case, progeny may be heterozygous if crossing over has occurred, and in fact this method has been used to determine gene- Genetics 83 : August, 1976.

2 784 T. M. TROTTIER AND J. B. ARMSTRONG Mutant isolation-a Inbreeding TABLE 1 comparison of inbreeding and parthenogenesis Diploid Parthenogenesis mutant sperm a e99 A mutant sperm a, egg A adult Aa adult Aa eggs 112 A 112 a sperm A + J, -1 adults 1/2 AA 112 Aa eggs 1/2 A 112 a 11 parthenoqenesi s progeny 112 AA 112 aa inbreeding 259 of matings are Aa x Aa of the progeny from these matings are aa Requires 1 generation, 1/2 progeny are mutants 2 generations required, and a maximum of 1/16 of the progeny will be mutants kinetochore linkage (VOLPE 1970; NACE, RICHARDS and ASHER 1970). The utility of the second method is based on the fact that, for most vertebrates, mature eggs have not extruded the second polar body. In the axolotl, FANKHAUSER and HUMPHREY (1942) showed that cold treatment of eggs after fertilization led to triploid larvae, presumably by suppression of meiosis 11. Heat shock has also been used (SLADECEK and LANZOVA 1959; ROTT and BETINA 1964). Cold shock has been combined with radiation-induced gynogenesis to produce diploid trout, plaice and flounder (PURDOM 1969). Mortality of the gynogenetic diploids was greater than for the controls, and the incidence of abnormalities was high. PURDOM (1969) supposed that this was due to increased homozygosity and consequent inbreeding depression, but no genetic markers were used to demonstrate this. In some strains of turkeys, parthenogenetic development occurs without cold shock or similar treatments (OLSEN 1969). OLSEN (1966) demonstrated that females heterozygous for plumage color produced parthenogens of both types of homozygote, plus one individual out of 33 that was heterozygous and could have resulted from crossing over. OLSEN (1969) concluded that diploidy was restored through retention of the second polar body. In the frog, TOKUNAGA (1949) produced albinos from a wild-type female by parthenogenesis. Though these were presumably diploid, the chromosomes were Rot counted, and there was no treatment to increase the incidence of diploids.

3 GYNOGENETIC AXOLOTLS 785 VOLPE and DASGUPTA (1962) produced gynogenetic dipleids by activating eggs with sperm of another species, followed by heat shock to induce retention of the second polar body. Of 811 treated eggs, only 70 were diploid, and all but 17 died before metamorphosis. The authors postulated that most had died as a result of being homozygous for lethal mutations. NACE, RICHARDS and ASHER (1970) report carrying out a large number of similar experiments for the purpose of developing inbred lines. They reported zero to 34% viable embryos in 65 experiments. As part of these studies. RICHARDS, TARTOF and NACE (1969) isolated a melanoid variant. In this paper we will describe our attempts to develop a reliable method for obtaining gynogenetic axolotls for the purpose of uncovering recessive mutations. MATERIALS AND METHODS Materials: Animals were gifts from R. R. HUMPHREY (Indiana University), H. C. DALTON (Pennsylvania State University), and L. DELANNEY (Ithaca College), or had been raised in our laboratory. The following genetic markers were involved in the experiments: d, white (HAECKER 1907), which reduces the number of both melanophores and xanthophores; a, albino (HUMPHREY 1967), which suppresses melanin synthesis; ax, axanthic (LYERLA and DALTON 1971), which eliminates iridophores and xanthophores; m, melanoid (HUMPHREY and BAGNARA 1967), which eliminates iridophores, reduces xanthophores and increases melanophores. The melanoid phenotype was assessed in newly hatched larvae primarily on the basis of the presence or absence of iridophores in the iris. One of the gynogenesis experiments involved an animal heterozygous for the gene p, premature death. Animals homozygous p/p die about stage 36 (HUMPHREY 1975) of SCHRECKENBERG and JACOBSON S (1975) series. Chorionic gonadotropin was obtained from the Sigma Chemical Co. (St. Louis, MO.). Folliclestimulating hormone was from Ormond Veterinary Supply (Hamilton, Ontario). Luteinizing hormone was purchased from Schwarz/Mann (Orangeburg, N.Y.). Amphibian culture medium (Wolf and Quimby) was from Grand Island Biological Co. (Grand Island, N.Y.). Our modified Holtfreter s solution contained 3.46 g NaCl, 200 mg MgSO,, 200 mg NaHCO,, 100 mg CaCl,, and 50 mg KCl per liter distilled water. Procedures: The artificial insemination procedure was based on that previously described (TROTTIER and ARMSTRONG 1975), but with a number of modifications. Instead of one large dose of follicle-stimulating hormone (FSH), the female was given a combined dose of 12.5 mg of FSH and 50pg of luteinizing hormone (LH) the morning prior to the experiment, and a second dose of 12.5 mg of FSH in the afternoon. The double injection seemed to make the interval between injection and laying more reproducible. The female usually began to lay eggs 24 hr after the first injection. At the time of the second injection, the males were injected with 500 IU of chorionic gonadotropin, and the dummy females were given 4 mg of FSH and 50,pg LH. Though we previously reported (TROTTIER and ARMSTRONG 1975) that the sperm remained viable up to 8 hr after collection, in some recent experiments sperm viability appeared to decrease with time. For this reason the male was not placed with the dummy female until the experimental female began to lay eggs. If more than one male was used, the second was not placed with the dummy female until the sperm from the first had all been used. Spermatophores were collected as previously described, except that only 2 or 3 spermatophores were used with 1.0 ml culture medium, and they \?-ere not homogenized until immediately before use. As many eggs as were produced in a 20-min period were placed directly in the tube with the sperm suspension. The stoppered tube was gently agitated at room temperature (18 to ZOO), generally for one hour, though min appeared to be sufficient. For ultraviolet inactivation, sperm were collected as described above, placed in a hemicylindrical plastic trough on ice, and irradiated at a distance of 4 cm from a General Electric G4Sll 4-watt germicidal lamp. Ultraviolet intensity was measured with an Ultra-Violet Products, Inc.

4 786 T. M. TROTTIER AND J. B. ARMSTRONG (San Gabriel, Calif.) model J-225 short wave meter. Immediately after the irradiation, artificial insemination was carried out as described above. After the I-hr insemination period, the eggs were transferred to 10% modified Holtfreter s solution, and either heat shocked, or allowed to continue development at room temperature. Eggs were scored as fertile if there was evidence of cleavage. A daily record was kept of developing animals, but no attempt was made to determine ploidy until hatching. The tailtips were removed from hatched larvae, fixed in 5% formaldehyde, and nucleoli counted in squashes examined under phase contrast. In some cases part of the tailtip was placed in amphibian culture medium with 45 mg per 100 ml colchicine for 3 hours. Following incubation the tailtips were washed in water, fixed in Carnoy s fixative or 30 min, and squashed in 50% acetic acid. The squashes were stained for 10 min in 2% aceto-orcein, and the chromosomes counted in mitotic figures. In all cases chromosome counts confirmed determinations of ploidy based on number of nucleoli. RESULTS Our method of diploid gynogenesis in the axolotl is similar to that used by PURDOM ( 1969), VOLPE and DASGUPTA ( 1962), and NACE, RICHARDS and ASHER (1970) for other species. It involved inactivation of the sperm by radiation, followed by heat shock to suppress meiosis I1 afid restore diploidy. In order to establish optimal conditions for gynogenesis, we separately examined conditions for optimal ultraviolet inactivation of the sperm and for suppression of meiosis 11. Ultraviolet inactivation of sperm: Sperm from several males were irradiated with doses ranging from 660 to 7920 ergs/mmz, and then used to artificially inseminate a total 656 eggs from 4 females. As controls, an additional 205 eggs were inseminated with unirradiated sperm. For the control, 60 % were judged fertile when examined after 24 hr. For the experimental groups, fertility was significantly lower at the intermediate ( ergs/mmz) doses, and also at the highest dose, but not at either 660 or 5280 ergs/mmz (Table 1). Survival, calculated as the percentage of fertile eggs which hatched, was not significantly lower than the control at any dose. Ploidy was determined for all survivors at hatching, and the percentage of larvae which were haploid is shown in Figure 1. In these experiments, three of the four females were melanoid, and the males were wildtype (non-melanoid). All the haploid larvae from such inseminations were melanoid, confirming sperm inactivation. TABLE 2 EfJect of ultraviolet irradiation of sperm on fertilization and deuelopment No of eggs Pei cent Dose (ergs/mmz) inseminated fertile control (53-67)t (58-81) (22-41) (30-4f3) (33-50) (47-65) (10-34) Perrent survival * Percentage of fertile eggs which hatched. None of the differences were significant at the 5% level. + 95% confidence interval.

5 GYNOGENETIC AXOLOTLS 78 7 ULTRAVDLET DOSE,ERGS/MM* x IO-^ FIGURE 1.-Production of haploids by fertilization of eggs with ultraviolet-irradiated sperm. Ploidy was determined only for embryos which hatched, and was assessed primarily on the basis of nucleolar count. The effect of irradiation on the ability of sperm to activate eggs, and on subsequent development, is shown in Table 2. Suppression of meiosis ZZ: Optimal conditions were established on the basis of the efficiency of triploid induction after heat shock to normally inseminated eggs. Eggs were collected at 20-min ietervals, kept at room temperature for various periods of time, then exposed to 36 for 5 min, and returned to room temperature. At hatching, ploidy was determined primarily on the basis on nucleolar count. In addition to diploids and triploids, haploids, mosaics, and a single pentaploid were obtained (Table 3). TABLE 3 Effect of heat shock at different times after fertilization Time of Duration No. of eggs Percent Chromowme no. Percent shock (min) (min) shocked survival N 2N 3N N/ZN 2N/3N triploid + control ~1~ (0-.71)$ a (3-18) a (6-28) a (1)s 8 (3-18) b (16-33) b (16-38) b (20-56) c c (2-37) + 3N plus N/2N mosaics. $95% confidence interval. pentaploid; see text. * chi-square test for significance; values not followed by the same letter differ significantly at the 5% level.

6 788 T. M. TROTTIER AND J. B. ARMSTRONG A haplo-diploid mosaic would arise if, after fertilization, one of the haploid egg nuclei remained separate (HUMPHRIES 1956, Figure 3). Pentaploids would arise from suppression of meiosis I (HUMPHRIES 1956), and a diplo-triploid mosaic would likely arise if the egg nuclei remained separate after suppression of meiosis I. BRIGGS (1947) and SLADECEK and LANZOVA (1959) have proposed that the haploids are androgenetic, resulting from complete destruction of the egg nucleus by the heat shock. On the basis of these reports, we have grouped the haplodiploid mosaics with triploids (Table 3) to obtain a more accurate assessment of our success in suppressing meiosis 11. This grouping resulted in nearly identical triploid yields for 45 and 60 min; but fewer mosaics were obtained at 60 min, and it was, therefore, chosen as the optimal time. When the duration of the shock was increased to 7.5 min, the percentage of triploids increased slightly. A further increase to 10 min yielded an even higher percentage, but reduced survival significantly. Diploid gynogenesis: The optimal conditions established above were combined in a group of experiments designed to determine whether diploidy could be restored in eggs activated with ultraviolet-treated sperm. Sperm were irradiated with an ultraviolet dose of 5280 ergs/mm2. The eggs were heat shocked at hr after they had been mixed with the irradiated sperm: the shock lasted 7.5 min. Control eggs were inseminated with unirradiated sperm, and allowed to develop without heat shock. Four experiments were performed, in which the female was homozygous melanoid (mjm), albino (a/u) or axanthic (axlax), while the male was wild-type. The success of the artificial inseminations (60.3% fertile) compared favorably with results obtained previously (TROTTIER and ARMSTRONG 1975), but mortality was extremely high. Of 292 fertile eggs, 101 (34.6%) survived to hatching, and 71 (24%) were diploid. However, only 58 (20%) appeared normal, and many of these died early in post-embryonic life. In contrast, 47 of 59 control embryos survived to hatching, and all were normal. Of the 101 experimental larvae which survived to hatching, 4 were wild-type in color, but otherwise abnormal. Since the females were all homozygous for recessive color markers, these likely arose from incomplete sperm inactivation. All the other progeny were of the same phenotype as their mother, indicating a gynogenetic origin. The oldest surviving animal from this group, a female, produced a normal spawning at 14 months, and otherwise appeared completely normal. Uncovering recessive mutations: The objective of these experiments was to demonstrate that progeny homozygous for a recessive mutation could be obtained irom a heterozygous mother by means of the techniques developed above. Since different genetic markers were involved, the experiments will be described separately. In experiment one, the female was m/m, +/ax. The male, as in all the following experiments, was homozygous wild-type for these genes. Of a total of 34 diploid larvae which survived to hatching, color could be determined for only 29, 8 of which were axanthic. Three of the larvae were grossly abnormal, and 14 others showed a particular mutant phenotype characterized by a pear-shaped

7 GYNOGENETIC AXOLOTLS 789 form resulting from the accumulation of fluid. No heartbeat or circulation of red blood cells was apparent. The larvae did not feed, and died several days after hatching. The mutation thus seemed to be similar, if not identical, to the cardiac lethal (c) described by HUMPHREY (1972). The female was sent to HUMPHREY and mated with a male known to be +/c. The spawning yielded 121 normal and 44 c/c larvae, a fair approximation of the expected 3: 1 ratio (x' = 0.293). In experiment 2, the female was m/m, +/ax, +/p. Because the embryos showed a range of abnormalities, p/p homozygotes could not be identified with complete certainty. However, of 13 embryos which developed past stage 30,4 to 6 appeared to show the characteristic phenotype of p/p animals, and died before hatching. Five larvae did hatch, one of which appeared to be pentaploid, and one a wild-type diploid, presumably not of gynogenetic origin. Of the remaining three, two were axanthic. In experiment 3, the female was +/d, +/ax, +/a. Mortality was high and only three embryos hatched. Two were albino, with apparently normal xanthophores, while the third was wild-type. All three displayed a mutant phenotype similar to that observed in experimeni one, and none survived to feed. The female was sent to HUMPHREY and confirmed to be+/c when mated with a known +/c male. The spawning yielded 28 normal to 12 c/c larvae (x' = 0.53). In the last two experiments, the female was +/d. Unfortunately only 6 diploid progeny were obtained from 93 fertile eggs. One was white. In the entire group of experiments, the success of the artificial inseminations (63.3%) was similar to the first group, but mortality was even higher. Of 299 fertile eggs, 80 (26.8%) survived to hatching, and 43 (14.4%) were diploid. Only 22 (7.4%) appeared normal, but 8 were still surviving after 7 months. In comparison, 77 of 110 control embryos survived to hatching. All were normal. DISCUSSION The results of the gynogenesis experiments indicate that the techniques used for Rana pipiens (VOLPE and DASGUPTA 1962; NACE, RICHARDS and ASHER 1970) can be adapted to the axolotl. Our primary objective was to show that gynogenesis can be used to uncover recessive mutations, and that it is, therefore, a practical and efficient alternative to inbreeding. Though no new mutations were discovered, the experiments employing females heterozygous for known recessive mutations indicated that the approach is valid. These results are summarized in Table 4. In spite of our success, and the success of the labs of NACE and VOLPE with color and pattern mutants of Rana pipiens, some difficulty may be encountered in detecting new developmental mutations. The color variants were easy to distinguish, but even in this case a few of the gynogenetic embryos were too grossly abnormal for their phenotype to be scored. The cardiac lethal mutants, with their distinctive phenotype, were also relatively easy to distinguish. These experiments, more than any of the others, confirmed the usefulness of the method, since neither female was known to carry the mutation prior to the experiments. However, the premature death mutation, which acts earlier in development and has a

8 790 T. M. TROTTIER AND J. B. ARMSTRONG TABLE 4 Uncovering recessive mutations by gynogenesis Female Proaenv +/ax 10/35 ax/ax +/d 1/9 d/d +/a 2/3 a/a +/P P/P + /c 17/37 c/c less distinctive phenotype. was difficult to recognize, even though the female was known to carry the mutation. The reason for this was that morality was high in all the gynogenesis experiments, and death could not usually be ascribed to a single obvious cause. Several reasons for the high mortality have been suggested. Physiological damage to the egg by the heat shock may affect subsequent development. Incomplete inactivation of the sperm may lead to aneuploidy. Normally fertilized eggs (arising through failure of both sperm inactivation and heat shock) may carry dominant lethals from the irradiation of the sperm. Finally, the female may carry several recessive lethals, a large proportion of which would be expressed by the gynogenetic progeny. The effect of heat shock on subsequent development has not been fully established, though several workers have studied its effects in triploid induction experiments. In the axolotl, SLADECEK and LANZOVA (1959) observed 21 % cytolysis of heat-shocked eggs. They also observed 33% haploids developing shortly after heat shock, and suggested that the high mortality might be due to the death of haploids. BRIGGS (1947), in studies on Rana pipiens, found a high proportion of both haploids and mosaics, neither of which developed normally. SLADECEK and LANZOVA (1959) and BRIGGS (1947) attributed haploidy to inactivation of the egg nucleus. If this occurred in the gynogenesis experiments, there would, of course, be no functional nucleus, though limited cleavage might still occur. However, mosaics would not likely be found, since they depend on a functional sperm nucleus ( HUMPHRIES 1956). BRIGGS (1947) also suggested that heatshock-induced damage might include aneuploidy, though no aneuploids were observed. POGANY (1971) suggested that partial sperm inactivation should also lead to aneuploidy, and consequent impairment of development. However, his experiments with irradiated Rana pipiens sperm showed a sharp transition from diploid to haploid embryos with increasing exposure, and no evidence of aneuploidy. In our own experiments, aneuploids were not observed, and at the usual ultraviolet dose (5280 ergs/mm2), survival of embryos judged 'haploid' solely on the basis of nucleolar count was not significantly lower than that of control diploids. Low doses did impair the ability of the sperm to induce cleavage, but subsequent survival was not significantly lower than that of the unirradiated control.

9 GYNOGENETIC AXOLOTLS 79 1 While we do not regard aneuploidy as a major contributor to the observed mortality, ultraviolet induction of dominant lethals may be a factor. About 4% of the sperm survived irradiation. In the sperm inactivation experiments, 4% diploids were still obtained at 5280 ergs/"?, while in the first set of gynogenesis experiments, 4 of 101 embryos which survived to hatching were wild-type for the color marker. Aside from color, these 4 were all abnormal, and died shortly after hatching.* Others, more seriously abnormal, might have died earlier in development and not been counted. In the second set of gynogenesis experiments, one normal wild-type embryo was obtained among 38 embryos from two m/m females. If the expression of recessive lethals carried by the mother accounts for a portion of the mortality, then it is likely that which occurs during the late stages. In the axolotl, all known early developmental lethals show a maternal pattern of inheritance (MALACINSKI and BROTHERS 1974; HUMPHREY 1975), and would not likely be expressed among the gynogenetic progeny of a heterozygous female. We therefore believe that the early deaths must be accounted for by a factor such as heat-shock-induced damage, though late deaths could well be accounted for by the expression of recessive lethals. Aside from the uncovering of new mutations, there are at least three other uses for gynogenesis: the identification of known recessives carried by a female, the establishment of 'inbred' lines (OLSEN 1969; PURDOM 1969; NACE, RICHARDS and ASHER 1970), and gene-kinetochore mapping (VOLPE 1970; NACE, RICHARDS and ASHER 1970). Our data (Table 4) could be used for gene-kinetochore mapping, but the small number of progeny would not yield very accurate results. We thank R. R. HUMPHREY for his advice, for supplying some of the animals, and for his assistance in identifying the +/c females. We also thank L. HRONOWSKI for his assistance with some of the experiments. This work was supported by a grant from the National Research Council of Canada. LITERATURE CITED BEATTY, R. A., 1957 Parthenogenesis and Polyploidy in Mammalian Deuelopment. Cambridge University Press, Cambridge, England. BRIGGS, R., 1947 The experimental production and development of triploid frog embryos. J. Exptl. Zool. 106: DAVIDSON, R. L., 1974 FANKHAUSER, G. and R. R. HUMPHREY, 1942 Induction af triploidy and haploidy in axolotl eggs by cold treatment. Biol. Bull. 83: HAECKER, V., 1907 Gene expression in somatic cell hybrids. Ann. Rev. Genetics 8: Uber Mendelsche Verebung Bei Axolotin. Zool. Anz. 39: HUMPHREY, R. R., 1967 Albino axolotls from an albino tiger salamander through hybridization. J. Heredity 58: , 1972 Genetic and experimental studies on a mutant gene (c) determining absence of heart action in embryos of the Mexican axolotl (Ambystoma mexicanum). Devel. Bid. 27: , 1975 The axolotl, Ambystoma mexicanum. In: Handbook of Genetics, Vol. 4. Edited by R. C. KING. Plenum Press, New York. Only one was triploid; heat shock must have failed to suppress meiosis I1 in the other three cases.

10 792 T. M. TROTTIER AND J. B. ARMSTRONG HUMPHREY, R. R., and J. T. BAGNARA, : A color variant in the Mexican axolotl. J. Heredity HUMPIXRIES, A. A., 1956 A study of meiosis in coelomic and oviducal oocytes of Triturus uiridescens, with particular emphasis on the origin of spontaneous polyploidy and the effects of heat shock on the first meiotic division. J. Morphology 99: LYERLA, T. A. and H. C. DALTON, 1971 Genetic and developmental characteristics of a new color variant, axanthic, in the Mexican axolotl, Ambystoma mericanum, Shaw. Devel. Biol. 24: MALACINSKI, G. M. and A. J. BROTHERS: : Mutant genes in the Mexican axolotl. Science NACE, G. W., C. M. RICHARDS and J. H. ASHER, JR., 1970 Parthenogenesis and genetic variability. I. Linkage and inbreeding estimations in the frog, Rana pipiens. Genetics 66: OLSEN, M. W., 1966 Segregation and replication of chromosomes in turkey parthenogenesis. Nature 212: , 1969 Potential uses of parthenogenetic development in turkeys. J. Heredity 60: POGANY, G. C., 1971 Effects of sperm ultraviolet irradiation on the embryonic development of Rana pipiens. Devel. Biol. 26: PURDOM, C. E., 1969 Radiation-induced gynogenesis and androgenesis in fish. Heredity 24: RICHARDS, C. M., D. T. TARTOF and G. W. NACE, 1969 A melanoid variant in Rana pipiens. Copeia No. 4, pp ROTT, N. N. and M. I. BETINA, 1964 Obtaining of triploid larvae of the axolotl by a thermal action. (in Russian) Tsitologiia 6: SCHRECRENBERG, G. M. and A. G. JACOBSON, 1975 Normal stages of development of the axolotl, Ambystoma mezicanum. Devel. Biol. 42 : SLADECEK, F. and J. LANZOVA, 1959 Cytological mechanism of the production of heteroploids by cold or heat shock in axolotl. Folia Biol. 5: TOKUNAGA, C., 1949 Albino frogs produced by artificial parthenogenesis. J. Heredity 40: TROTTIER, T. M. and J. B. ARMSTRONG, 1975 Hormonal stimulation as an aid to artificial insemination in Ambystoma mezicanum. Can. J. Zool. 53 : TYLER, A., 1941 Artificial parthenogenesis. Biol. Rev. Camb. Phil. Soc. 14: VOLPE, E. P., 1970 Chromosome mapping in the leopard frog. Genetics 64: VOLPE, E. P. and S. DASGUPTA, 1962 Gynogenetic diploids of mutant leopard frogs. J. Exptl. Zool. 151: Corresponding editor: D. BENNETT