HAPLOID CELLS IN RAT KANGAROO CORNEAL ENDOTHELIUM CULTURES AND X-RAY-INDUCED CHROMOSOME ABERRATIONS

KENNETH T. S. YAO

U.S. Department of Health, Education, and Welfare Bureau of Radiological Health, Division of Biological Effects, Rockville, Maryland 20852

Received July 30, 1970

N the reproduction of mammals? germ cells undergo two maturation divisions I to yield haploid gametes which, through union with gametes from the opposite sex, give rise to the next generation. Based on the existence of haploid status in gametes, investigators in the 1940’s and 50’s experimentally induced haploid cells in parthenogenetically developed blastocysts or in very early embryos of rabbits (AMOROSO and PARKES 1947), rats (AUSTIN and BRADEN 1954), and mice (BEATTY 1954; EDWARDS 1958). FREED (1962) and FREED and MEZGER-FREED ( 1969) successfully established haploid cell lines from androgenetic frog embryos. We have noted the presence of haploid cells in cell cultures derived from a somatic tissue. corneal endothelium of an adult rat kangaroo, and have used X rays to induce chromosome aberrations in the haploid cells. Owing to the advantages of using mammalian haploid cell lines in mutagenic studies, cloning and selective isolation experiments are underway in an attempt to establish haploid cell lines from these cultures.

MATERIALS A N D METHODS

The cell cultures used in this study were initiated in February 1967 from corneal endothelial tissue of a female rat kangaroo (Potorous tridactylis) by using the double chamber technique (ELLINGSON and YAO 1966). The cultures have been transferred through 42 passages of subcul- ture. For the first 20 passages the cells were grown in Eagle’s minimum essential medium sup- plemented with 20% calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, plus 100 units of penicillin and 100 microgram equivalents of streptomycin per ml of medium. After the 20th passage they were grown in medium 199 supplemented with 20% fetal bovine serum plus other ingredients as above. Cell morphology and growth pattern of the cultures will be described later.

The cell cultures were grown in T-60 flasks. For X irradiation, the cells were trypsinized and transferred into T-30 plastic flasks at a concentration of approximately IO5 cells per ml with 4 ml of celI suspension per flask. The transplanted cells were incubated at 37” C for 24 hr before they were washed with Hanks’ balanced salt solution and exposed to X rays. There was no fluid in the flasks during exposure. The cells were exposed to 0, 20, 50, 100, or 150 roentgens of X rays from a Westinghouse 250 kVcp X-ray machine with hvl 0.55 mm Cu, 15 mA, without ad- ditional filtration, at an air distance of 120 cm and at the rate of approximately 31.3 roentgens per minute.

Fresh medium was added to the flasks after exposure and the cells were reincubated for 6, 12,24, 36, or 48 hr before they were fixed with 1 :3 (acetic acid : ethanol) mixture to make in situ air-dry slides. Prior to fixation the cells were treated with 0.03 microgram of Colcemid per ml of medium for 3 hr, and immediately before fixation they were treated with hypotonic solution for 8 min. The slides were stained with Giemsa and scored for mitotic indices, number of

Genetics 67: 399-409 March, 1971.

400 K. T. S. YAO

mitotic haploid cells per 50 mitotic diploid cells, and chromosome aberrations of the haploid and diploid cells. Mitotic index was recorded as percentage of mitotic cells in 1,000 cells observed randomly across the slide. Chromosome aberrations were observed for chromatid breaks, isochro- matid breaks, dicentrics, rings, and chromatid exchanges. Chromosomal analyses were performed with a Carl Zeiss Photomicroscope I1 with an oil immersion lens using bright light as well as phase contrast fields to cross-verify the observations.

Analysis of variance was employed to evaluate the effect of culture age and X-ray effects on the increase in the number of haploid cells and mitotic indices in the culture. Numbers of haploid cells per 50 diploid cells were converted to percentages. The percentages were trans- formed by use of arc sine (2 s i n - l d x ) to stabilize the variance of the percentages. The method of fitting constants, as described by BANCROFT (1968), was used in the analysis of variance to adjust for missing plots. Variances between repetitions of the mitotic indices were used in a “t” test to measure the statistical significance of differences between control and X-irradiated cul- tures.

RESULTS

Corneal endothelium cultures and haploid cells: The endothelial cells grew fast in culture and morphologically were flat and epithelial-like during the first 6 passages. They formed a transparent monolayer in culture flasks. After the 6th passage fibroblast-like cells emerged in the culture. The number of fibroblast-like cells continuously increased until finally, at around the 16th passage, all the cells in the culture were fibroblast-like. In the meantime the cell culture became multi-layered; the spindle-shaped fibroblast-like cells crossed each other with two long cytoplasmic processes as if they were being woven into a sheet of fabric. The multiple layer of the heavily inoculated culture detached from the bottom of the flasks after only two or three days of growth. When a heavy culture was sub- cultured and trypsinized, the entire sheet of cells came off the glass in a few minutes; the sheet of cells was very difficult to break up by pipetting. In the light- er cultures, the cells were also easily detached from the glass floor after trypsini- zation but the cells formed small clumps of five or more cells that could not be separated even in Versene (disodium ethylenediamine tetraacetate) and/or Darvan (R. T. Vanderbilt Co., Inc.) were added in the trypsin solution. After the 20th subculture. the medium used for the cultures was changed from Eagle’s minimum essential medium to medium 199; the culture grew in a similar pattern with no noticeable changes. The culture, transplanted at a cell density of ap- proximately 9 x lo4 cells per ml had a cell growth doubling-time of 16.4 hours

The chromosome configuration of the cells was not examined until after the 20th passage of subculture. Of the mitotic cells in the culture, a majority were normal diploids. Many of them were found to have a haploid number of chromo- somes, and a fraction of the mitotic cells were polyploid. The percentage of hap- loid cells in the culture fluctuated from 0 to about 20%. The haploid cells had the chromosome complement of a single genome: five autosomes and one X chromo- some (Figure 1 ) . The six individual chromosomes could be distinguished readily. The first one was long and subacrocentric; the second, long and submetacentric; the third, median-sized and submetacentric with its short arm relatively shorter than the short arm of the second chromosome; the fourth, short and metacentric; the fifth, the shortest and metacentric. The X chromosome, similar in size to the

IRRADIATED TISSUE CULTURES 401

FIGURE l.-Chromosomes of a haploid cell.

third chromosome, had a secondary constriction on its long arm next to the centromere.

X-ray aberrations: The cell cultures grew well after being washed with Hanks’ salt solution. held without medium at room temperature for more than two hours, X-irradiated, and incubated with fresh medium. Mitotic indices of these cultures ranged from 8.4 to 12.7% at 6 hr after irradiation, decreased rapidly with time, and reached 0.1 to 0.6% in 48 hr. Average indices of four experiments are pre- sented in Table I. Overall, the indices appeared to decrease with increasing X-ray dose as well as with increasing culture age. The decrease in mitotic indices with culture age was highly significant (P < O.Ol), but was not significant with X-ray dose. However, when mitotic indices of the X-rayed cultures were compared with those of the controls at different time intervals separately, mitotic indices of the X-rayed cultures at 6 hr after exposure were significantly higher than that of the control; those at 24 hr were about the same as the control; and those at 48 hr were significantly lower than that of the control. These results can be explained by radiation mitotic delay. Some cells were at radiation-sensitive stages (G? and M) (Yu and SINCLAIR 1967) when they were exposed, then joined the cells that were not at sensitive stages, and entered mitosis to increase the population’s mitotic index about 6 hr after exposure. Radiation death or damage decreased mitotic indices of the X-rayed cultures 48 hr after exposure.

The average number of haploid cells per 50 diploids increased with increasing culture age and X-ray dose (Table 1). Statistical analysis of the data revealed that the increase in number of haploid cells with age of the cultures was highly significant (P < 0.01) but was not significant with X-ray dose. Nevertheless, the number of haploid cells increased with X-ray doses exceeding 100 roentgens as

402 K. T. S. YAO

IRRADIATED TISSUE CULTURES

- Mitotic Index - 20 - #-A--

/ E

18 - \ ' 2

16 - 14 - 1 - 11 -

-- - - 10 I I I 0

0 10 50 100 1 so

X-RAY WSE ( w n t w )

403

FIGURE 2.-X-ray-dose effect on number of haploid cells and mitotic indices.

shown in Figure 2. The average number of haploid cells and the mitotic indices of the cultures showed opposite trends in relation to culture age and X-ray dose as illustrated in Figures 2 and 3. The correlation coefficient between these two variables is estimated to be - 0.7013 with 74 df (P < 0.01). The larger the num- ber of the haploid cells in the culture, the smaller the mitotic index.

Among 950 diploid and 950 haploid cells scored for chromosomal aberrations, 156 and 52 cells, respectively, had chromosome aberrations and were considered to be abnormal cells. The percentages of abnormal cells at various culture ages and X-ray exposure levels are presented in Table 2. The X-irradiated haploid cell population consisted of 0 to 14% abnormal cells, while the diploid cells had 6 to 30 %. The percentages of abnormal diploid cells averaged 2-3 times greater than those of haploid cells at all time intervals and exposure levels observed. The per- centage of abnormal diploid cells decreased with culture age but increased with X-ray dose. This trend was not as pronounced in haploid cells as in the diploids illustrated in Figures 4 and 5. Nevertheless, X-irradiated cultures had greater

24 t .A

U 16 0 0

14

12

10

A / \

\ Mitotic Index \

A- I I 1 I --4

1 l 4

6 12 24 36 48

HOURS AFTER X-IRRADIATION

FIGURE 3.-Average number of haploid cells and mitotic indices of corneal endothelium cell culture at various intervals after exposure.

404 K. T. S. YAO

TABLE 2

Percentage of X-ray-induced abnormul cells in haploid ( h ) and diploid ( d ) cell populations

X-ray dose (roentgens) Culture age Or 20r 5 Or 1 OOr 150r Average

(hours) h d 11 d h d h d h d 11 d

6 h r 0 2 10 28 6 18 . . . . . . . . 8.0 Z3.0 12 hr 2 20 4 10 2 30 . . 2.7 20.0 24hr 0 0 6 12 2 8 14 18 8 28 7.6 16.6 36 hr 4 6 4 6 6 18 . . . . 4.7 10.0 48hr 0 0 12 12 0 12 2 14 4 10 4.7 12.0

Average 0 0.6 6.8 15.6 3.2 10.8 6.0 20.0 6.0 19.0 5.4 16.4

percentages of abnormal cells than nonirradiated cultures in both haploid and diploid cell populations.

Frequencies of chromosome aberrations observed in diploid cells are presented in Table 3. and those in haploid cells in Table 4. Dicentrics were by far the most prevalent in both diploid and haploid cells. Secondary constrictions occurred mostly on chromosome number 1 and were not counted as breaks. The secondary constriction of the X chromosome was not recorded. Average number of chromo- some breaks per cell ranged from 0.2320 to 0.3700 in X-irradiated diploid cells, and 0.0440--0.1400 in haploid cells. The average number of chromosome breaks per cell per roentgen ranged from 0.0025 to 0.0142 for diploid cells, and 0.0009- 0.0046 for haploid cells. The average numbers of chromosome breaks per cell in- dicate that larger X-ray doses induce higher chromosome aberration frequencies. The average numbers of chromosome breaks per cell per roentgen indicate that the larger doses are less efficient in inducing aberrattions. About three times more

v-

6 1 2 24 36 48

HOURS AFTER X-IRRADIATION

FIGURE 4.-Age of culture and percentage of X-ray-induced abnormal cells in haploid and diploid cell populations.

IRRADIATED TISSUE C U L T U R E S 405

24t Diploid Cells ye-*

- 20 - J J W

----A-- - - - --. -A/ - - Haploid Cells \

I I 0 20 50 100 150

X-RAY DOSE ( roentgen )

FIGURE 5.-X-ray-dose effect on percentage of abnormal cells.

TABLE 3

Number of chromosomal aberrations observed in X-irradiated diploid cells

Number of breaks X-ray Number Iso- Dicen- Secondary Total dose of cells Chromatid chromatid trics Rings constrictions number Per Per cell

(roentgens) observed breaks breaks ( 2 ) ( 2 ) (0) breaks' cell per roentgen

O r 150 0 0 1 0 3 2 0.0133 . . . . . 20r 250 3 2 30 3 2 71 0.284-0 0.0142 5 0 r 250 1 5 18 8 5 58 0.2320 0.0046

l O O r 200 9 9 25 1 0 70 0.3500 0.0035 150 r 100 4 5 14 0 0 37 0.3700 0.0025

Total 950 17 21 88 12 10 238 0.2950 . . . . .

*Each dicentric, ring, or chromatid exchange was caunted as two breaks as indicated in parentheses. Secondary constrictions were not caunted as breaks.

TABLE 4

Number of chromosome aberrations in X-irradiaied haploid cells

Number of breaks X-ray Number Iso- Dicen- Secondary Total dose of cells Chromatid chromatid trics Rings constrictions number Per Per cell

(roentgens) observed breaks breaks ( 2 ) ( 2 ) ( 0 ) breaks' cell per roentgen

O r 150 0 0 0 0 0 0 20r 250 1 0 9 2 5 23 00920 00046 5 0 r 250 1 0 4 2 1 13 0.0440 00009

100 r 200 2 3 5 2 3 19 00950 00009 150 r 100 0 2 5 1 0 14 01400 00009

Total 950 4 5 23 7 9 69 0 0863

*Each dicentric, ring, or chromatid exchange was counted as two breaks as indicated in parentheses. Secondary constrictions were not counted as breaks.

406 K. T. S. YAO

chromosome aberrations were found in diploid cells than in haploid cells at all X-ray doses examined.

DISCUSSION

Two meiotic divisions occur in mature mammalian germ cells. Reduction di- vision separates homologous chromosomes and halves the chromosome number of the nucleus. Chromosomes in a germ cell undergo a series of changes to accom- plish pairing and segregation of each pair of homologues so that in resulting gametes, each has a single chromosome complement (set). Parthenogenetic de- velopment of the haploid gametes can lead to the production of haploid cells o r ani- mals (BEATTY 1957). That is actually the case in the establishment of haploid frog cell lines (FREED and MEZGER-FREED 1969) and in some insects such as bees or wasps (Hymenoptera) and aphids (Homoptera) .

Our endothelial cell culture has been subcultured 42 times. Karyotype of these cells was not observed until at the 20th subculturing passage. I t is unlikely that the haploid cells were carried over from the original tissue, since no haploid cell could exist in normal mammalian corneal endothelia.

SCHWARZACHER and PERA (1969) followed through the mitosis of the binu- cleated cells in culture derived from kidney tissue of Microtus agrestis and de- termined by DNA measurements the ploidy of the daughter nuclei. They found that two diploid nuclei of a binucleated cell fused to form a common metaphase plate with a tetraploid number of chromosomes; it yielded three daughter nuclei from a tripolar mitosis. Of the daughter nuclei, one was triploid. one haploid, and one tetraploid. The haploid nucleus was assumed to be produced by a selective alignment of the chromosomal sets on the multipolar metaphase plate. Since they were working with a primary culture, haploidy of the nucleus could have been inherited from the original tissue. However, the authors did not show the chromo- somal configuration of the haploid nucleus.

In this study haploid cells clearly exhibited a single set of the chromosomal complement. Also, a large number of haploid cells appeared in the culture, al- though diploid cells were still predominant. Mammalian cells in culture usually undergo endomitosis and become polyploid cells which. through nondisjunction or other mechanisms, lose a certain number of single chromosomes and stabilize around a certain chromosome number (stem cells). The culture thus becomes a heteroploid cell population (Hsu, BILLEN and LEVAN 1961). The reduction in chromosome number is irregular and perhaps random. The haploid cells in this study must be formed in a strictly regulated way since each cell receives precisely one set of chromosomes of one genome.

BERGER (1938) and GRELL (1946) studied chromosomes in cells of iliac epi- thelium of the mosquito larva (Culex pipiens) and found that repeated duplica- tion of chromonemata in the cells throughout larval life resulted in the formation of multiple chromosome complexes which can reach as high a ploidy as 128 n. During metamorphosis chromosome reduction divisions took place, and the cells resumed a lower level of ploidy around 4 n to form adult tissue. The process of each reduction involved two cell divisions. In the first division there appeared

IRRADIATED TISSUE CULTURES 407

FIGURE 6.-Paired chromosomes.

chromosome pairing or somatic synizesis at anaphase and there was no duplica- tion or longitudinal division of chromonemata in the interphase. In the second division the paired chromosomes reappeared at prophase, separated. and moved toward opposite poles at anaphase to complete the reduction division.

In the present study somatic chromosome pairing at the prophase stage must occur (see Figure 6). The ensuing anaphase separates the homologues, completes the somatic reduction, and produces haploid cells. Chromosome pairing and the absence of chromonemata1 duplication in mosquito larval cells were probably monitored by a metabolic mechanism of the larva. In the present tissue culture, the presence of haploid cells probably can be attributed to certain nutritional de- ficiencies that reduce DNA synthesis. At the same time there might be certain other elements or factors which promote cell division. The significant increase in number of haploid cells with age of the culture probably is such an indication (Table 1).

The cultures were at mid-log phase of growth when they were exposed to X rays. The p w t h rate of the cells was increased by the addition of fresh medium to the culture after X irradiation. Due to overcrowding. mitotic indices of the cultures decreased rapidly from 11.1 to 0.3% in 48 hr (Table 1). A negative cor- relation coefficient of -0.70 between number of haploid cells and mitotic index suggests that overcrowding was another possible factor in producing haploid cells.

A haploid cell has six chromosomes only, while a diploid has 12. The size of the nucleus of a haploid cell is about half of that of a diploid. Hence, during the

408 K. T. S. YAO

X-ray exposure haploid cells represent smaller targets for direct bombardment by X rays; this could result in a smaller percentage of damaged haploid cells than diploids. On the other hand, the haploid cells may grow slower than diploid cells. Slow metabolic activity and long generation time of the haploid cells might also have contributed to the haploid cells’ exhibiting three times fewer aberrations than diploid cells at the same radiation dose. However, the possibly high radiation lethality and the comparatively less favorable growth conditions of the haploid cells in the culture have not been taken into account in this study. Further study with an established haploid cell line is needed to clarify the X-ray sensitivity of the haploid cells.

The author wishes to thank Dr. GORDON L. JESSUP for assistance in statistical analysis, Mr. WAH LEE and Miss LYNDA D. K ~ A M E R for their X-irradiation service, and Mrs. DINNIEMAUD J. ELLINGSON for her valuable assistance in initiating the rat kangaroo endothelium cell culture with double Rose-chamber technique. The assistance of Mr. DONALD HVDGE in preparing the manuscript is acknowledged.

SUMMAliY

Many haploid cells were observed among the mitotic figures of rat kangaroo corneal endothelial cell cultures. The cultures were exposed to 250 kVcp X rays. The percentages of haploid cells in cultures varied from 18 to 32, and increased with culture age but not with X-ray dose until the X-ray dose exceeded 100 roent- gens. The number of haploid cells was inversely related to mitotic index with a negative correlation coeflicient of -0.70. The percentages of X-ray-damaged hap- loid cells (0-14%) and diploid cells (6-30%) decreased with the culture age but increased with X-ray dose. The results also showed that larger X-ray doses pro- duced a greater average number of chromosome breaks per cell, but larger doses were less efficient in inducing chromosome aberrations. In all cases, X-ray damage to chromosomes in haploid cells appeared to be three times less than that in diploid cells.

LITERATURE CITED

AMVROSO, E. C. and A. S. PARKES, 1947 Effects on embryonic development of X-irradiation of rabbit spermatozoa in vitro. Proc. Roy. Soc. London B 134: 57-78.

AUSTIN, C. R. and A. W. H. BRADEN, 1954 tilized rat eggs. Nature 173: 999-1000.

Nucleus formation and cleavage induced in unfer-

BANCROFT, T. A., 1968 Topics in Intermediate Statistical Methods, Vol. 1, pp. 16-24. Iowa

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BEATTY, R. A., 1954 Haploid rodent eggs. Proc. 9th Intern. Congr. Genet. 2: 784. __ ,

BERGER, C. A., 1938 Multiplication and reduction of somatic chromosome groups as a regular developmental process in the mosquito, Culex pipiem. Carnegie Inst. Washington Publ. 476: 209-232.

EDWARDS, R. G., 1958 The number of cells and cleavages in haploid, diploid, polyploid, and other heteroploid mouse embryos at 3% days gestation. J. Exptl. Zaol. 138: 189-207.

IRRADIATED T I S S U E C U L T U R E S 409 ELLINGSON, D. J. and K. T. S. YAO, 1966 A double-chamber technique for the separation

and in vitro growth of mammalian corneal cells. In Vitro 2 : 127. FREED, J. J., 1962 Continuous cultivation of cells derived from haploid Rana pipiens embryos.

Exptl. Cell Res. 26: 327-333.

FREED, J. J. and L. MEZGER-FREED, 1969 Stable haploid cultured cell lines from frog embryos. Proc. Natl. Acad. Sci. U.S. 65: 337-344.

GRELL, M., 1946 Cytological studies in Culex. I: Somatic reduction divisions; 11: Diploid and meiotic divisions. Genetics 31 : 60-76; 77-94.

Hsu, T. C., D. BILLEN and A. LEVAN, 1961 Mammalian chromosomes in vitro. XV: Patterns of transformation. J. Natl. Cancer Inst. 27: 515-541.

SCHWARZACHER, H. G. and F. PERA, 1969 Multipolar mitosis and somatic segregation in cell cultures of Microtus agrestis. pp. 186191. In: Comparative Mammalian Cytogenetics. Edited by K. BENIRSCHKE. Springer Verlag, New York.

Mitotic delay and chromosomal aberrations induced by Yu, C. K. and W. K. SINCLAIR, 1967 X rays in synchronized Chinese hamster cells in vitro. J. Natl. Cancer Inst. 39: 619-631.