MEIOTIC ORIGIN OF TRIPLOIDY IN THE FROG DETECTED BY GENETIC ANALYSIS OF ENZYME POLYMORPHISMS1

DAVID A. WRIGHT, CHUN-PING HUANG AND BARBARA D. CHUOKE

The University of Texas Cancer Center M . D. Anderson Hospital and Tumor Institute and The University of Texas Health Science Center Graduate School of Biomedical Sciences,

Houston, Texas 77030

Manuscript received January 9, 1976 Revised copy received June 1,1976

ABSTRACT

A female frog heterozygous at two unlinked loci, specifying electrophoretic forms of mannosephosphate isomerase (MPI) and malate dehydrogenase (MDH) was crossed to male frogs homozygous for different alleles at each locus. In the offspring approximately ten percent proved to be triploid accord- ing to nucleolar and chromosome counts of tail tip cells. Most of these triploids had both maternal alleles at the MDH and MPI loci suggesting that the fiist meiotic division was repressed. Others seemed to represent a repressed second meiotic division and one animal, a pentaploid, could only have resulted from inhibition of both meiotic divisions of the egg. Densitometer tracings of starch gels stained for 6 phosphogluconate and isocitrate dehydrogenases, expected to be heterozygous in a particular cross, demonstrated that the triploids had twice as much maternal as paternal gene product for each locus, similar to patterns found in triploids produced by nuclear transplantation.

ELECTROPHORETIC enzyme polymorphisms in frogs of the Rana pipiens complex have been of interest to us as gene products to study in early devel-

opment. Allelic differences between parents have allowed an assessment of nuclear and cytoplasmic contributions to the enzymatic phenotype of the hybrid embryo (WRIGHT and MOYER 1966,1968; WRIGHT and SUBTELNY 1971,1973).

Although some enzyme systems are easily understood by analogy to the same enzyme in other species, some can only be understood through a formal genetic analysis. To this end twelve enzyme loci were studied in the backcross progeny of a laboratory-reared F, hybrid (WRIGHT 1973,1975).

Occasionally in the course of routine genotyping of frogs used to produce hybrids between species or subspecies, naturally occurring heterozygotes for one or more loci will be discovered (WRIGHT 1975). This paper reports results from one such series of matings in which the inheritance and possible linkage relation- ships between enzyme loci were being analyzed. To our surprise, some of the enzyme patterns in the offspring resembled patterns seen previously in triploids produced by nuclear transplantation (SUBTELNY and WRIGHT 1969). Nucleolar and chromosome counts confirmed triploidy had occurred. Most significantly we

Supported by a research grant from the National Institutes of Health, HD 07021

Genetics 84: 319-332 October, 1976.

320 D. A. WRIGHT, C-P. HUANG A N D B. D. CHUOKE

were able to determine through segregation of maternal alleles for electrophoretic enzyme variants that either the first or second meiotic division of the ova could be affected.

MATERIALS AND METHODS

Crosses in various combinations were made between frogs of the Rana pipiens complex derived from different North American localities. Frogs from Tennessee, Rana sphenocephala, were supplied by MIKE TOLLEY, Nashville, Tennessee. Sinaloa, Mexico frogs, Rana berlandieri forreri, were supplied by Southwestern Scientific, Tucson, Arizona. Frogs from Vermont, Rana pipiens pipiens, were supplied by Hazen, Alburg, Vermont. Wisconsin frogs, R. pipiens pipiens, were supplied by Nasco, Oshkosh, Wisconsin. As used in crosses, each animal was assigned a number.

Artificial ovulation was accomplished by a modification of methods described by RUGH (1934, 1962) and WRIGHT and FLATHERS (1961). For ovulation in February, three female pituitary glands from R. p . pipiens plus one milligram progesterone (Sigma Chemicals) were injected and animals kept at 18" for 40-48 hours before eggs were fertilized.

In making crosses, eggs from each female producing eggs that day were fertilized with males from the three different geographic locations thereby producing F, hybrids of two types as well as controls.

Embryos were reared in a dilute growth medium containing 350 mg/L Na C1,13 mg/L Ca C1, 2H,O, 12 mg/L Mg C1, 6H,O, 7 mg/L KHCO, and a final concentration of 0.001 M Tris H C1 pH 7.6.

Preparation of extmcts for electrophoresis Brain, heart and muscle of adult frogs were removed, frozen and homogenized in a volume

of homogenization media in a ratio of 1:2 (w/v). Liver homogenate was prepared in a ratio of 1:3 (w/v). Homogenization media contains 0.01M Tris H C1 pH 7.5,O.OOlM p mercaptoethanol, 0.001M EDTA. Whole embryos, tadpoles with the gut removed or tadpole tails were collected singly and frozen. These samples were homogenized with a twice-concentrated homogenizing media in a ratio of 1 : l w/v) for whole tadpole and 1:2 (w/v) for tadpole tail.

All the homogenized samples were centrifuged at 12000 x g for 30 minutes before applying on starch gels for electrophoresis.

Gel electrophoresis Vertical starch gel electrophoresis was carried out as described in detail by SICILIANO and

SHAW (1976). Caunaught starch (Fisher Scientific) was used. Electrophoresis of gels was for four to six hours at 4" a t approximately l2v/cm. Three buffer systems used included the follow- ing: (1) tris-citrate pH 7.0, 0.135M tris, 0.043M citrate electrode buffer pH 7.0, 40 ml of this diluted to 600 ml for making the starch gel; (2) tris-versene borate pH 8.0, 0.5M tris, 0.016M EDTA, 0.6M borate pH 8.0 electrode buffer diluted 60 to 600 for the gel buffer; (3) a hybrid system used the tris citrate pH 7.0 electrode buffer and pH 7.5 gel buffer made by combining 35 ml of the tris-citrate p H 7.0 electrode buffer 5 ml of the tris versene borate pH 8.0 electrode buffer in a total of 600 ml.

Adult tissue extracts were analyzed by starch gel electrophoresis and specific staining for 20 different enzymes. Those in which variants were found for these studies included manose phos- phate isomerase (MPI) E.C.5.3.1.8, malate dehydrogenase (MDH) E.C.1.1.1.37, 6-phosphogl~- conate dehydrogenase (6PGD) E.C.1.1.1.44, isocitrate dehydrogenase (IDH) E.C.1.1.1.42, and lactate dehydrogenase (LDH) E.C.1.1.1.27. Stain mixtures were made from recipes given in SICILIANO and SHAW 1976, except for MPI which was modified from the method of NICHOLS, CHAPMAN and RUDDLE (1973) and included: 15 mg nicotinamide adenine dinucleotide phos- phate; 15 mg nitroblue tetrozolium; 1 mg phenazene methosulfate; 100 mg Mg Cl,; 50 mg Mannose-6-phosphate (disodium salt); 10 ml of 0.2M tris-HCL buffer pH 8.0; 40 ml distilled water; 200 units of glucose-6-phosphate isomerase; 160 units of glucose-6-phosphate dehydro- genase (all available from Sigma Chemical).

ENZYME GENETICS IN THE APPEARANCE O F TRIPLOIDS 32 1 Densitometry

Individual sample columns of stained starch gel after electrophoresis were run through a Photovolt densicord Model 542. Chart speed was set at two inches per minute by using 44-Tooth motor gear. The geometric figures determined by the densitometric tracings were cut out and their relative area estimated by a comparison of their weights.

Nucleoli and chromosome preparations One-fourth inch of tadpole tail was clipped and placed in a depression slide. The tail tip was

chopped up into small pieces in two drops of 0.2M tris-borate buffer containing 0.008M EDTA pH 8.0 which was removed one minute later. Two or three drops of Steinberg’s media pH 7.4 were added in order to transfer tail tip to slide and it was pressed gently with coverslip. Sixty or more cells were examined for nucleolar number under phase-contrast microscopy. Staining with methylene blue before applying the coverslip facilitated photography of nucleoli using a Baush and Lomb Balplan microscope.

The best chromosome preparatioiis result when one waits one week after first cutting a tail tip for the new tip to regenerate. The tadpole was injected with colchicine solution in a dosage of 50 pl/g body weight. The colchicine solution was prepared with 3 mg colchicine, 75 ml of 0.9 percent saline and 25 ml Amphibian Ringer’s. The regenerated tail tip was cut off 24 hours later and kept in .2M tris borate .008 M EDTA pH 8.0 buffer for two minutes, rinsed with a Ca++ M g f f free Steinberg’s Medium, then incubated two and three minutes in distilled water. Finally, it was stained with aceto-orcein and squashed.

RESULTS

Enzyme patterns of frogs used in crosses Tissue extracts from each frog were subjected to starch gel electrophoresis and

enzymes visualized using specific enzyme stains. Four of the enzymes showed differences in these animals useful for genetic analysis, manose phosphate iso- merase (MPI) , malate dehydrogenase (MDH) , 6 phosphogluconate dehydro- genase (6PGD) and isocitrate dehydrogenase (IDH) . The parental patterns for each of these are shown in Figures 1-5. Female 171 and male 173 are R. spheno- cephala from Tennessee, male 174 is an R. berlandieri forreri from Sinaloa, and male 176 is R. pipiens from Vermont. Analysis of the offspring of 171 female with each male should allow us to describe the inheritance of MPI and help understand the complex MDH patterns in this animal. If 171 proves to be het- erozygous at these two enzyme loci, possible linkage can be tested. The inheri- tance of 6PGD and IDH has been studied previously in haploid, diploid and polyploid animals and shall provide information for comparative purposes.

Manose phosphate isomerase Based on previous studies (NICHOLS, CHAPMAN and RUDDLE 1973; WRIGHT

1975) it seemed that female 171 was heterozygous ( a / b ) at the MPI locus result- ing in two bands of activity (Figure 1 ) characteristic of enzymes with a monomer structure (SHAW 1964). We predicted that in crosses with each of the three dif- ferent males (b /b , b/b, and c/c) two phenotypes should be found among the offspring, the results of segregation of the maternal alleles.

In the case of the offspring of the 171 (R. sphenocephala) female x 173 (R. spenocephala) male or 174 ( R . berlandieri) male crosses, the parental pheno- types were generated (Figure 6) . A satellite band slightly anodal to the main

322 D. A. \VRIGHT, C-P. HUANG A N D l3. D. CHUOKE

. - C

171 173 174 176 I , I, < ,'

L. C C I

II, P:, - A A 0

c

O b ' < { n h <

FIGURE 1.-Photograph of a portion of a Tris citrate pH 7.0 starch gel aftrr electrophoresis and staining for mannose phosphate isomerasr. Samples are heart extracts of (11) male 173, (a) female 171, (c) male 174, ((1) male 176. Note positions of two bands A and D in frmale 171 pattern and position of band C in male 176.

FIGURE 2.-Malate dehydrogenase patterns aftrr electrophoresis (in TC pH 7.0) and staining, liver samples from (a) female 171, (b) male 173. (c) male 174, (d) male 176.

FIGURE 3.--Malate drhydrogenase pattern9 of frog heart extracts run on Tris-citrate-TVD hybrid buffer system pH 7.5, (a), (b), (c) and (d) the same as in Figures 1 and 2. Note the pattern in frmale 171 (a) showing three differrnt kinds of supernatant MDH BD, DC and CC in addition to the mitochondrial AnlAlll hand. Note the position of supernatant band AA in 171(c) and 1 7F(d) and the mitochondrial BnlBm band in 174(c).

FIGURE 4.-Photograph of a portion of a starch gel stninrd for 6 phosphogluconate clrhydro- genase aftrr electrophoresis of liver extracts (a) female 171, (11) male 173, (c) male 174, ( (I) male 176.

FIGURE 5.-Pattrrns of Isocitrate dehydrogenase found in frogs (a) female 171, (b) male 173, (c) male 174, (d) male 176. The fastest migrating bond is the supernatant isozyme while the slower form is localized in the mitochondria.

zone of activity is seen in homozygotes c, d and e in Figure G . Seven of the 108 analyzed had both A and D but seemed to have more activity in the I3 band than the A; these are designated as nhb. In the cross of 171 female x 176 (R. pipiens) male, as expected, none of the phenotypes generated corresponded to the parental phenotypes. The two expected types are ac and bc, corresponding to the segre- gants of the maternal a and b alleles combined with the paternal c allelc (Figure G ) . Five of 32 analyzed had three-band abc phenotypes corresponding to the mobilities of the paternal plus both types of maternal allele products.

The data on the inheritance of MPI patterns is summarized in Table 1. If thc ahberant patterns thought to represent nonscgregation of maternal alleles are

ENZYME GENETICS IN TI11 .APPI'.?\RANCE OF TRIPI.0ID.S 32 3

I

F r r x n E 6.-Vannosr phosphate isomrrase pattrms in offspring of frnialr 171. a-e wholr lama evtracts from individuals 13, I $ . 17 and 11 from thr 171 frmale x 174 male cross. f-i whole larva extracts of inrlividuals 33, 31, 35, 36 rrsrilting from the 171 frmale x 176 male cross. N o t e the three band pattern in number 3.C in this cross ( g ) indicated hy the triangle.

FmunE i.--Malatc dehydroqrnasc pattrrns of diploid offspring of the 171 x 174 cross run on Tris-citratr pH 7.0 grl. Notc two kinds of pnttrms in the suprrnatant locus a, 11 and e rcprc- srnting the nh phmotypr while c and d rrprrzmt thc nc phcnotypr. Each has the mitochondrial 11DIl nh phrnotypr.

FicunE 8.-Malate clehydrogcnase pattrrns of individuals from the 171 x 171 cross. S o t e the presrrire of two iidivicliiiils c ( * 3 ) nritl j ( C t 0 ) tliilt h a w sunimiitiori of the oh and nc phenotypes.

FIGURE 9.-6 phosphogluconatr clchydrogrnnsc pnttrrns of the same individual used on the stained gel pictured in Iiigurc 8. Yotr that c ( f t 3 ) and j ( f10) have skewed pattrrns having more activity in the RR niaternal band than the CC patrrnal hand. Densitomctrr tracings indi- cate a relative activity for thc CC:CR:RR bands is npproximatclv I :+4 in c and i whilr for the other pattrrns a, h, (I, r, f , g. h, i, k, and 1, a I :? : 1 ratio of activity was found.

324 D. A. WRIGHT, C-P. H U A N G A N D B. D. CHUOKE

TABLE 1

Inheritance of mannose phosphate isomerase patterns

Cross Parental type Offspring

ab bb abb 171 x 173 24 22 6 ab bb abb 171 x 174 30 25 1 ac bc Q ~ C 171 x 176 12 15 5

Totals of maternal alleles a b ab 66 62 12

- - - ab X bb

ab x bb

ab X cc

- - -

- - -

- - -

ignored, the remainder of the data yields numbers of 66 individuals inheriting the maternal a allele and 62 the maternal b allele which is not significantly dif- ferent from the expected 1 : 1 ratio.

Mulate dehydrogenase MDH patterns in frogs consist of supernatant and mitochondrial isozymes as

shown by cell fractionation experiments (WRIGHT and SUBTELNY 1971). The patterns for the males used in these crosses illustrate the nature of the patterns usually found (Figures 2 and 3). The faster group of bands is supernatant and the slower band is mitochrondrial. The supernatant variants are the aa phenotype in 174 and 176 and the faster migrating bb phenotype in 173. Male frog 174 also shows the variant bb phenotype for the mitochrondrial MDH locus characteristic for R. berlundieri. Female frog 171 is different. Before genetic analysis, it was not clear if she was heterozygous at the mitochondrial or supernatant locus. Slightly different patterns are seen in a pH 7 tris citrate buffer system and a hybrid system in which the pH is raised to 7.5 (Figure 3 ) .

Our interpretation of electrophoretic patterns of the parents is as follows: The 171 female is heterozygous at the supernatant MDH locus. The B polypep- tide produced by the S-MDH b allele is the same as that found in the homozygous 173 male. The C polypeptide is produced by the other allele. Since the enzyme is a dimer (WRIGHT and SUBTELNY 1971) BB, BC and CC isozymes are formed. In the pH 7.0 tris citrate buffer system the CC-SMDH has the same mobility as the Am Am product of the mitochondrial (M)-MDH a allele. In pH 7.5 TC-TVB hybrid buffer system the CC and Am Am bands are resolved.

Analysis of MDH in the offspring of female 171 x male 173 shows that most individuals have either the maternal pattern (bc) or the paternal pattern (bb ) as shown in Figures 2 and 3. Six individuals had more activity in the BB band than the CC band (bcb) and one (ccb) had more activity in the CC than BB band (Table 2 ) . Four of the six individuals with bcb MDH pattern also had the ubb MPI pattern.

Each of the offspring of the 171 female x 174 male was heterozygous at the mitochrondrial MDH locus. AULAm, Am Bm and Bm Bm bands are present (Figure

ENZYME GENETICS I N T H E APPEARANCE OF TRIPLOIDS

TABLE 2

Inheritance of supernatant malate dehydrogenase patterns

325

Cross Parental type Offspring

171 X 173

171 X 174

171 x 176

bc x bb

bc x aa

bc x aa

bc bb bcb ccb 36 29 6 I ac ab bca 23 30 4 ac ab bca

- - - -

- - -

- - - 17 18 5

Totals of maternal alleles c b bc 76 77 15 - - .-

7) . In addition, all have the paternal AA supernatant MDH. Thirty of 57 had the BB band plus an AB band intermediate between AA and BB, the ab pheno- type. An approximately equal number (23 of 57) of offspring in this cross had the AA, the CC and AC bands (the ac phenotype) (Figures 7 and 8). Four indi- viduals had patterns which resembled summations of the two expected pheno- types, a bca type pattern (Figure 8, c and j). Due to overlapping mobilities of the CC S-MDH with one or the other of the M-MDH bands, we could not deter- mine the relative activities of the M-MDH bands in these animals. Two of the four animals with the bca MDH pattern also had an abb MPI pattern.

In the 171 x 176 cross a similar pattern of inheritance is seen for the super- natant MDH as in the 171 x 174 cross. There were 18 of the ab phenotype, 17 of the ac phenotype and 5 showing a bca phenotype. Each of the 5 individuals showing a bca MDH phenotype also had an abc MPI phenotype.

Summarizing the MDH data from the three crosses involving 171 female and ignoring the individuals thought to represent non-segregation of maternal alleles, 76 icherited the S-MDH c allele and 77 the S-MDH b allele (Table 2 ) .

The independent assortment of the MPI and MDH loci is indicated by the data in Table 3. In terms of maternal alleles inherited, 62 of the MPI a, MDH b plus MPI b, MDH c class compared to 63 of the MPI a, MDH c plus M I b, MDH b class (the parental and recombinant classes or vice versa).

6 phosphogluconate and isocitrate dehydrogenases The three-band pattern of GPGD of the 173 male indicated that he was het-

erozygous (6PGD a/GPGDb) at this locus so that in the offspring of the 171 female x 173 male cross two types of patterns were expected; those having the maternal phenotype bb and those having the paternal ab phenotype. The results were 33 ab and 32 bb out of 70 analyzed. There were 5 of the 70 that had pat- terns in which there was greater activity by visual examination and densitometer tracings in the B band than thc A, this was called a bba phenotype. If one expects an equal number of bbb as bba individuals and subtracts 5 from the 32 bb, the ratio of 27 to 33 is still not significantly different from the expected 1: 1. The data suggest that among animals indicated as being 3N from other enzymes or

326 D. A. WRIGHT, C-P. HUANG A N D B. D. CHUOKE

TABLE 3

Independent assortment of genes for MPI and MDH

Male 173 Male 174 Male 176 Totals for gametes gametes gametes maternal alleles

MPIb No. each MPIb No. each MPIc No. each Exp. MDHb genotype MDHa genotype MDHa genotype Obs. (1: l : l : l )

MPIa MPIa/b 10 MPIa/b 17 MPIa/c 6 33 31.25 MDHb

Female MPIb

Gametes 171 MDHc

MPIa MDHc

MPIb MDHb

MDHb/b MDHb/a MDHb/a

MPIb/b 12 MPIb/b 10 MPIb/c 7 29 31.25 MDHc/b MDHcja MDHc/a

MPIa/b 18 MPIa/b 11 MPIa/c 5 34 31.25 MDHc/b MDHc/a MDHc/a

MPIb/b 11 MPIb/b 12 MPIb/c 6 29 31.25 MDHb/b MDHb/a MDHb/a

x2=0.664 p>.80

TABLE 4

Enzyme genotypes in polyploid offspring of female frog #I71

171 X 173 nu chr ab X bb bc X bb

MPI MDH bb X ab

GPGD Probable meiotic division repressed

4 5 9

47

51 54 56 58 65 71 73

171 X 174 3

10 25

62

171 X 176 6

12 13 26 34

3nu - 3nu - 3nu - 5nu - 3nu 39 3nu 39 3nu 39

nu chr - _

3nu 33

nu chr - _

- _ - _ 3nu -

- ccb - bbb? - bcb abb bbb?

aab ccb abb bcb abb bcb aabbb? bbccb? abb bcb

abb bcb - -

ab X bb bc X bb MPI MDH bbb? bca

abb bca bbb? bca

abb bca

abc bca abc bca abc bca abc bca abc bca

ab X cc bc X aa MPI MDH

bba bba bba bba

bbb? bbb? bbb? bbbbb? bbb

bba

bbc

-

bh x cc GPGD

bbc bbc

bbc bb X aa

GPGD bba bba bba bba bba

2nd M? 2nd M ? 1st M? 1st M x-over MDH or 2nd M x-over MPI 2nd M 1st M 1st M 1st M & 2nd M ! 1st M ? 1st M

1 s t M x-over MPI or 2nd M x-over MDH 1st M 1st M x-over MPI or 2nd M x-over MDH 1st M

aab 1st M aab 1st M acrb 1st M nab 1st M aab 1st M

Probable meiotic division repressed

aa X bb Probable meiotic IDH division repressed

ENZYME GENETICS IN THE APPEARANCE O F TRIPLOIDS 327

nucleolar number, 5 were bba and 4 were bbb (Table 4). In the other crosses, 171 x 174 and 171 X 176 heterozygous 6PGD patterns with the two different paternal alleles were seen, bc and ba respectively. More activity was seen in the band representing pure maternal allelic products in those cases where both maternal alleles at the MPI or MDH loci were present (Table 4). In the 171 X 174 cross, 4 of 57 were bbc (Figure 9). In the 171 X 176 cross, 5 of 40 were bba.

Only in the 171 x 176 cross were there differences in the parental, superna- tant(s) IDH (Figure 4). At the S-IDH locus, 171 is aa and 176 bb.

The same 5 individuals which showed bba 6PGD patterns had patterns for S-IDH indicating the presence of more maternal allelic product i.e. an aab pat- tern. The remaining 35 individuals were ab for S-IDH.

Nucleolar and chromosome numbers After finding some of the MPI and MDH patterns suggesting nonsegregation

of maternal alleles in the early part of the study, we began a routine analysis of nucleolar number before sacrifice of the tadpoles for enzyme analysis. Nucleoli were counted in sixty or more cells and a tally sheet kept recording the number of cells with 1,2,3,4 or 5 nucleoli (Figures 10-14).

Some cells had low counts probably because of nucleolar fusion or close prox- imity of nucleoli. Even in diploids, one-half might appear to have one nucleolus. An occasional non-nucleolar body might be expected to be scored as a nucleolus also. Our criteria for judging ihe organism’s nucleolar number is to use the high- est nucleolar number of cells that account for 20% or more of the cells counted.

Two of the offspring of 171 x 174 and 8 of the 171 X 173 crosses were checked for chromosome number (Figures 15-1 7). In each case the chromosome number was consistent with the nucleolar counts.

Results of other crosses The offspring of two other females stimulated to ovulate in the same way as

171 female were analyzed for enzyme patterns. One was from the same ship- ment from Tennessee (R. sphenocephala) #185. The other was from Wisconsin (R . pipiens) #184. The analysis of enzyme patterns in the offspring of these two females showed normal inheritance of MDH and lactate dehydrogenase ( L D H ) when crossed to the same R. sphenocephala male #186 heterozygous at these unlinked loci.

The enzyme phenotypes of the parents were 184 female LDHaa, MDHaa; 185 female LDHbb, MDHbb; 186 male LDHbc, MDHab. In the 185 x 186 cross 62 offspring were analyzed; 30 had an LDHbb phenotype and 32 the LDHbc phenotype; 27 had the supernatant MDHbb phenotype and 35 had the ab pheno- type. There were 23 parental and 39 recombinants between these two loci indi- cating independent assortment for LDH and MDH. In the 184 X 186 cross 71 offspring were examined for enzyme patterns; 34 were LDHac and 37 were LDHab, 35 were MDHab and 36 were MDHaa. Forty of the animals of this cross were checked for nucleolar number prior to electrophoretic analysis of their tissue

328 D. A. WRIGHT, C-P, H U A N G A N D l3. D. CHUOKE

*' 4

' I

I .

. . ' , * \ ; ;'" .

. * I , .. - '

le. FIGURES 10. AND 11.-Tail tip cells photographed with phase contrast microscopy sho

differences in nuclrolar number between groups of cells in 2 nu (Figure 10) and 3 nu (Figure 11) individuals, 4281.

FIGURES 12., 13. AND 14.-Closc ups of nuclei from the 171 x 173 cross stained with mcth- ylcne blue showing 2 nucleoli #50 (Figure i n ) , 3 nucleoli # 5 (Figure 13). and 5 nuclroli $58 (Figure 14), 1725,.

FrcunEs 15, 16. AND 17.-Aceto-orcein stained preparations of chromosomrs of regrnerating tail tip cells from larvae or embryos treated with colchicine overnight. Approximately 39 chromosomes are seen in spread from 171 x 173 $65 (Figure 15) and i n 171 x 173 $73 (Figure 16). A haploid chromosome spread from an abnormal stage 23 rmbryo, the result of removal O f the female nuclear material just after fertilization (PORTER 1939) has 13 Chromo- somes (Figure 17), 7881.

ENZYME GENETICS IN THE APPEARANCE O F TRIPLOIDS 329

and all were 2 nu. None of the enzyme patterns displayed by the offspring of these crosses indicated anything but that expected fo,r diploid individuals.

DISCUSSION

Data on all of the offspring suspected of being polyploid on the basis of rela- tive activity of allelic enzyme products, cases of nonsegregation of maternal alleles, nucleolar number or chromosome number are summarized in Table 4. Although the data is incomplete on some individuals, it is clear that if the parents at a particular enzyme locus have different alleles, the relative activity of the allelic products in the offspring can be used to indicate multiple allelic dosage at that locus. If several unlinked genetic loci for enzymes or nucleolus organizer show similar phenomena, the case for polyploidy is strengthened. Chromosome counts on a few of these animals verify this.

The patterns obtained for enzymes where one allele was represented once and the other allele twice are identical to enzyme patterns obtained in triploid frog larvae produced by nuclear transplantation ( SUBTELNY and WRIGHT 1969). These patterns are similar to those seen in certain triploid species of fish (VRIJENHOEK 1975) and urodeles (UZZELL and GOLDBLATT 1967) suspected of originating through a hybridization step between species. This information together with studies of enzymes in diploid and triploid Drosophila (LUCCHESI and RAWLS 1973) suggest that all alleles function equally in a triploid cell. The data presented here on the expression olf three different MPI and MDH alleles would seem to verify this argument.

We were fortunate that for two apparently unlinked enzyme loci (MDH and MPI) the female frog used in these crosses was heterozygous. These circum- stances allow us to consider the meiotic division where triploidy of the offspring originated.

Triploidy could have occurred in the offspring of female 171 by three dif- ferent mechanisms. The first possibility is that the first meiotic division was repressed keeping the homologous chromosomes from separating. This was fol- lowed by a normal second meiotic division where the sister chromatids were separated.

In the second possible mechanism the first meiotic division was normal, segre- gating the homologous chromosomes. This was €ollowed by a repressed second meiotic division preventing the separation of sister chromatids. A high rate of crossing over between each of the MPI and MDH loci and their respective cen- tromeres would have to occur to yield the high number of triploids having both maternal alleles for these enzymes.

A third possible mechanism is that an endomitotic event occurred (CIMINO 1972) before meiosis. Normal meiosis involving these oocytes would lead to diploid ova and triploid offspring. In this case, one would expect for MPI a ratio of one triploid having aa maternal alleles to two having ab maternal alleles to one having bb maternal alleles. Likewise for MDH, a ratio of one triploid having the bb maternal allele to two with bc alleles to one with cc alleles is expected. Considering both loci togeether, one would expect a ratio of one triploid off-

330 D. A. WRIGHT, C-P. HUANG AND B. D. CHUOKE

TABLE 5 ~ ~

Maternal alleles ~

obs. exp. (1:Z:i)

MPI QQ

ab bb

MDH bb bc cc

Same at both loci Different at one locus Different at both loci

1 1 2 2

obs.

2 14 2

obs.

1 3

11

3.75 x2=5.54 7.5 3.75

exp. (1:Z:l)

4.5 ~ 2 ~ 5 . 5 6 9.0 P < .07 4.5

exp. (1 :Z: I )

3.75 7.5 3.75

spring with identical maternal alleles at both loci to two that have identical maternal alleles at one locus and different maternal alleles at the other locus to one that has different material alleles at both loci.

These same 1 : 2: 1 ratios are expected if crossing over between the enzyme loci and their respective centromeres was so frequent as to randomize the alleles on the homologous chromosomes in the first two possible mechanisms of triploid origin.

The data in Table 5 indicate more triploids showing both maternal alleles than expected for a 1:2: 1 ratio. There are significantly more of the triploids showing different maternal alleles at both loci than expected for a 1:2: 1 ratio. This sug- gests that triploidy observed here is not the result of an endomitotic event in the oogonia. It indicates that most of the tiploids arose by repression of the first mei- otic division followed by a normal second meiotic division. Both loci are appar- ently located close enough to their respective centromeres so that they are not in- dependent of the centromere.

The occurrence of normal first meiotic division but a repressed second meiotic division in some cases is likely. This is especially so since one animal, #58 in the 171 x 173 cross, was found to be a pentaploid, the result of repressions of both meiotic divisions of the egg.

The facility with which the second meiotic division of amphibian eggs can be interfered with experimentally by temperature shock ( FANKHAUSER 1945; RUGH 1962) or pressure (DASGUPTA 1962) suggests that triploidy in natural populations may be due to environmental factors occurring at the time of fertili- zation. Reports of triploids in species hybrids of Rana (KAWAMURA 1952) and Bufo (BOGART 1972) in which two sets of maternal chromosomes and one set of paternal chromosomes were identified suggests that suppression of a meiotic di- vision usually occurs in the female gamete, not the male, in the origin of triploid animals. The report of pentaploid Bufo hybrids (BOGART 1972) having four sets of maternal chromosomes is a further indication that suppression of both meiotic divisions can occur in anurans. The use of electrophoretic variants of enzymes

E N Z Y M E GENETICS IN T H E APPEARANCE OF TRIPLOIDS 331

in this study has demonstrated that triploids OCCUT by suppression of the first meiotic division as well as the second meiotic division.

The advice of DR. CHARLEEN MOORE on the cytology, DR. MICHAEL J. SICILIANO and MR. DONALD MOHIZOT on the genetic studies are greatly appreciated.

LITERATURE CITED

BOGART, J. P., 1972 Karyotypes pp. 171-195. In Evolution in the Genus Bufo. Edited by W. F.

CIMINO, M. C., 1972 Meiosis in triploid all-female fish (Poeciliopsis, Poeciliidae). Science 175:

DASGUPTA, S., 1962 Induction of triploidy by hydrostatic pressure. J. Exp. Zool. 151: 105-116. FANKHAUSER, G., 1945 The effects of change in the chromosome number on amphibian develop-

ment. Quart. Rev. Biol. 20: 20-78. KAWAMURA, T., 1952 Triploid hybrids of Rana japonica Gunther female x R m a temporaria

ornativentria Werner male. J. Sci. Hiroshima Univ. Ser. B., Div. 1, 12: 3946. LUCCHESI, J. D. and J. M. RAWLS, 1973 Regulation of gene function: A comparison of enzyme

activity levels in relation to gene dosage in diploids and triploids of Drosophila melanogaster. Biochem. Genet. 9: 41-51.

Polymorphism and linkage for man-

Androgenetic development of the egg of Rana pipiens. Biol. Bull. 77:

BLAIR, University of Texas Press, Austin.

1482-1486.

NICHOLS, E. A., V. M. CHAPMAN and F. H. RUDDLE, 1973

PORTER, K. R., 1939 nose phosphate isomerase in Mus musculus. Biochem. Genet. 8: 47-53.

233-257. RUGH, R., 1934 Induced ovulation and artificial fertilization in the frog. Biol. Bull. 66: 22-29. - , 1962 Experimental Embryology, Techniques and Procedures. Burgess Publishing, Minneapolis.

SHAW, C. R., 1964 The use of genetic variants in the analysis of isozyme structure. Brookhaven Symp. Biol. 17: 117-129.

Separation and localization of enzymes on gels. In: Chromatogruphic and Electrophoretic Techniques. Edited by IVOR SMITH, Vol. 2, fourth edition, ch. 8, pp. 185-209. The Yearbook Medical Publ. Chicago, Ill.

Biochemical polymorphisms in animals as models for man. pp. 17-53. In: Progress in Medical Genctics Vol. X. Edited by STEIN- BERG and BEARN. Grune & Stratton, Inc.

Genic expression in nuclear transplant hybrid embryos of the Ranu pipiens complex. J. Cell Biol. 43: (2) pt. 2: 141a (abstract).

Serum proteins of salamanders of the Ambystoma jeffer- sonianum complex and the origin of the triploid species of this group. Evolution 21: 345- 354,

Gene dosage in diploid and triploid unisexual fishes (Poeciliopsis, Poeciliidae). pp. 463475. In: Isozymes Vol. 4, Genetics and Evolution. Edited by C. MARKERT. Acad. Press, N. Y.

Inheritance of twelve enzyme loci in hybrids of Rana pipiens pipiens and R. p . berlundieri. Amer. Zool. 13: 1320 (abstract). -- , 1975 Expression of enzyme phenotypes in hybrid embryos. pp. 649-664. In: Isozymes, IV Genetics and Evolution. Edited by C. MARKERT. Academic Press, New York.

SICILIANO, M. J. and C. R. SHAW, 1976

SICILIANO, M. J., D. A. WRIGHT and C. R. SHAW, 1974

SUBTELNY, S. and D. A. WRIGHT, 1969

UZZELL, T. and M. GOLDBUTT, 1967

VRIJENHOEK, R. C., 1975

WRIGHT, D. A., 1973

3 32 D. A. WRIGHT, C-P. H U A N G A N D B. D. C H U O K E

WRIGHT, D. A. and F. H. MOYER, 1966 Parental influences on lactate dehydrogenase in the -,

Inheritance of frog lactate dehydrogenase patterns and the persistence of maternal

Nuclear and cytoplasmic contributions to dehydro-

fects of haploidy and hybridization on the activities of four dehydrogenases in frog embryos. Develop. Biol. 32: 297-308.

Facilitation of pituitary induced frog ovulation by progesterone in early fall. Proc. Soc. Exptl. Biol. Med. 106: 346-347.

Corresponding editor: F. H. RUDDLE

early development of hybrid frogs in the genus Ram. J. Exp. Zool. 163: 215-230. 1968 isozymes during development. J. Exp. Zool. 167: 197-206.

WRIGHT, D. A. and S. SUBTELNY, 1971 genase phenotype in hybrid frog embryos. Develop. Biol. 24: 119-140. __ , 1973 Ef-

WRIGHT, P. A. and A. R. FLATHERS, 1961