Chromosoma (Berl.) 77, 373 378 (1980) CHROMOSOMA �9 by Springer-Verlag 1980

Insect Sex Chromosomes

V. 3H-Uridine Induced Aberrations in the X Chromosomes of Tetraploid Spermatogonia from Gryllotalpa fossor

Paramjit Arora and S.R.V. Rao 1 Department of Zoology, University of Delhi, Delhi-110007, India (J addressee for reprint requests)

Abstract. The two X chromosomes in tetraploid spermatogonial cells from Gryllotalpa fossor respond differentially to the production of chromatid aberrations by 3H-uridine (3H-U). AS in diploid female somatic cells, only the euchromatic arm of one X shows such aberrations. The equivalent arm of the other X and the constitutive arms of both Xs are not affected. This differential response of the homologous arms of the two Xs appears to be due to a facultative heterochromatinization of one of them. It is suggested that an imprinting process, which has been assumed to occur during fertiliza- tion in other cases of X-inactivation, may not be necessary for the differential regulation of two X chromosomes in this case.

Introduction

In a previous paper (Rao and Arora, 1979), we have presented evidence for the occurrence of a presumptive X inactivation mechanism in cells from the gastric caeca of thc female mole-cricket, Gryllotalpa fossor (Gryllotalpidae, Or- thoptera). This was based on the induction of 3H-U-induced aberrations as a means of examining transcription at the whole chromosome level. Implicit in this approach is the assumption that 3H-U produces aberrations when it is momentarily in proximity with D N A during transcription. Aberrations then result from the localized ionization of the/?-particles of 3H-tritium when D N A acts either as a transcriptive or a replicative template. In female mole-crickets, 3H-U induces aberrations only in one arm of one of the two X chromosomes thus paralleling the behaviour in the single X chromosome of the male. That is, in both sexes, only one arm of one X shows aberrations. This suggests that in female Gryllotalpa only one arm of one X chromosome is transcriptional- ly active while the other arm and the whole of the other X, which are resistant to 3H-U, are transcriptionally inactive. Such behaviour can be explained on the assumption that one arm of one X chromosome is facultatively heterochro- matized while the second arm of both Xs is composed of constitutive heterochro-

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374 P. Arora and S.R.V. Rao

mat in . The s i tua t ion in female Gryllotalpa is thus reminiscent of tha t which occurs in female m a m m a l s (Rao and Aro ra , 1979). We have now ex tended our s tudy to spon taneous ly occurr ing t e t r ap lo id spe rma togon ia l cells, w h i c h also possess two X ch romosomes , to see whether bo th Xs are affected by 3H-U in the same way under these c i rcumstances . T h o u g h such t e t r ap lo id cells occur in the testes o f mos t indiv iduals o f Gryllotalpa, they are found in large number s in only a few of them. W e also present evidence tha t ~H-U- induced abe r r a t i ons are indeed due to t r ansc r ip t iona l activity.

Material and Methods

Four males belonging to a size range of 150-152 mg were each injected with 20 gc of 3H-U (44 C/mM, New England Nuclear). 1 h later, 50 gg of cold uridine per animal was again administered as a chase. The animals were sacrificed 13 14 h after injection of the label since samples at this time gave the maximum number of X chromosome aberrations. Colchicine (0.05 ml of a 0.1% solution) was injected 2 h prior to sacrifice. Chromosome preparations and the analysis of the induced aberrations were both carried out according to the procedure already described (Rao and Arora, 1979). For the sake of convenience we have pooled the data from both treatment times since, for any given time interval, the frequency of X chromosome aberrations remains the same.

In order to confirm that the 3H-U-induced aberrations are indeed due to transcriptional activity, experiments were performed on two females, weighing about 150-152 rag, using actinomycin-D (AD) before and after 3H-U administration. In the first protocol, AD (0.1 ~tg/animal) was injected first, followed by 3H-U (20 gc/animal, Sp. act. same as above) after 10 min and then cold uridine (50 gg/animal) after 1 h. In the second treatment, 3H-U (same dose) was administered first, followed after 1 h by AD (same dose) plus 50 gg cold uridine. Chromosome preparations and the analysis of aberrations were carried out according to the procedure already described (Rao and Arora 1979).

Observations and General Remarks

The d ip lo id n u m b e r in G.fossor is 23, X0, male and 24, XX, female. Acco rd ing ly the t e t r ap lo id spe rma togon ia l cells show 46 c h r o m o s o m e s with two large meta - centr ic X c h r o m o s o m e s (Fig. 1). Ne i the r in d ip lo id nor in t e t r ap lo id spe rma to - gonia l cells have we ever found the X c h r o m o s o m e to show the negat ive he tero- pycnos is so character is t ic o f g rasshoppers and crickets (Whi te 1973). One a r m of the X in Gryllotalpa is c o m p o s e d of const i tu t ive he te rochromat in . As expected, this a r m decondenses on cold t rea tment , br ight ly f luoresces with Q M and Hoechs t " 3 3 2 5 8 " ( A r o r a and Rao , 1979), is res is tant to 3H-U- induced abe r ra - t ions (Rao and Aro ra , 1979) and repl icates D N A asynchronous ly dur ing the S-phase (Aro ra and Rao , in Press).

As is charac ter i s t ic o f 3H-U t r ea tmen t ( N a t a r a j a n and Sharma , 1971 ; D o n a l d and Coope r , 1977; R a o and Aro ra , 1979), only c h r o m a t i d type ( ch roma t id and i s o c h r o m a t i d b reaks and gaps) abe r r a t i ons were found. W e believe these abe r r a t i ons are due to t r ansc r ip t iona l activity. This is borne out by the fact (Table 1) tha t when cells are exposed to A D before 3H-U (Pro toco l 1) the to ta l number o f abe r ra t ions (au tosomes and X c h r o m o s o m e ) are s ignif icant ly

Insect Sex Chromosomes. V 375

Table 1. Actinomycin D effects on the incidence of 3H-U-induced aberrations (14 h sample) in the hepatic caeca cells of female Gryllotalpa

Protocol Total cells Abnormal cells Aberrations (%)

X chromosome Autosomes

1. AD-3H-U 295 3.38 3 (3) 16 (8) 2. 3H-U-AD 385 18.96 43 (43) 90 (57)

Figures in parenthesis denote the number of cells

Table 2. Comparison of X chromosome aberrations induced by 3H-U between diploid somatic (Rao and Arora, 1979) and tetraploid spermatogonial cells for (13 14) h samples

Ploidy Total cells Frequency (%)

Aberrant cells X chromosome aberrations"

Diploid Female hepatic caeca 440 56.55 27.5 Male hepatic caeca 201 53.61 26.16

Tetraploid Spermatogonia 250 49.6 (124) 32.8 (82)

a Only one arm of one x chromosome shows chromatid aberrations in both diploid somatic and tetraploid spermatogonial cells. Figures in parenthesis denote number of cells

r educed as c o m p a r e d to those ob t a ined fo l lowing exposure to A D after 3H-U t r ea tmen t (P ro toco l 2). We appl ied the large sample test for equal i ty o f p r o p o r - tions. The test s tat ist ics for a u t o s o m a l and X c h r o m o s o m e abe r ra t ions were found to be --6.3962163 and --5.224208, respectively. The hypothes is tha t the behav iou r is al ike in the two p ro toco l s is re jected at the 0.00001% (for au to- somes) and 0.0001% (for X c h r o m o s o m e ) levels o f significance. Thus, the evidence tha t 3H-U- induced abe r ra t ions are indeed due to t r ansc r ip t iona l act ivi ty is p ro- ved b e y o n d reasonab le doubt .

Out o f a to ta l of 250 t e t r ap lo id cells 82 o f them (32.8%) showed one abe r ra - t ion per X ch romosome . This percentage is in good agreement wi th tha t o f d ip lo id cells t aken f rom a somat ic series. On average there was one a be r r a t i on per X c h r o m o s o m e in such a d ip lo id series (Rao and Aro ra , 1979 and see Table 2). W e failed to f ind a single case where bo th a rms o f an X were s imul tan- eously affected. Since one a rm of the X is const i tu t ive ly he t e roch roma t in i zed this is no t unexpected . Add i t iona l ly , we found only one t e t r ap lo id cell giving any ind ica t ion tha t bo th Xs m a y be affected. Even here the a be r r a t i on was clear in only one a rm of one X; in the o ther it was o f a doub t fu l nature .

376 P. Arora and S.R.V. Rao

Fig. 1. Tetraploid spermatogonial cell showing the two metacentric X chromosomes (X0, male diploid). Note the characteristic chromatid apposition in tl~e facultative arm (f) of one X and the constitutive arms (c) of both Xs. The chromatids of the euchromatic arm (e) are clearly separate. Bar represents 10 ~tm

Although the frequency of aberrations is low, it seems certain that this is not due to sample size. To confirm this we investigated the Poisson expectation of:

(1) An aberration in both arms of the X, and (2) Aberrations in both Xs. These calculations show that: (1) In 500 X chromosomes taken f rom 250

tetraploid cells we can expect 5.7 of these to have two aberrations and half of this, i.e., 2.85, to have a break in each arm of a given X chromosome, and (2) in 250 tetraploid cells we can expect 4.83 breaks to occur in both Xs.

We believe that the aberrations are restricted to only one arm, the euchroma- tic arm, of one X. As in diploid somatic cells, such aberrations can be found at proximal, interstitial and distal regions of this arm. That is, the sites of aberrat ion induction are similar to those in diploid hepatic caeca cells of both sexes. Thus, even in tetraploid spermatogonia, only one arm of one X shows an aberration. The euchromatic arm of the second X present in the tetraploid spermatogonia is resistant to 3H-U and is considered inactive as a result of facultative heterochromatinization. Thus, the response of the two X chromosomes to 3H-U is identical in both diploid female somatic cells and tetraploid male spermatogonial cells. Equally informative is the fact that, in some tetraploid spermatogonial cells, as in some of the female diploid somatic cells, the faculta- tive arm of the one X and the constitutive arms of both Xs, show a characteristic chromatid apposition. In the single euchromatic arm by contrast, sister chroma- tids are never closely apposed (Fig. 1).

Insect Sex Chromosomes. V 377

This situation in Gryllotalpa offers an interesting parallel to that found m tetraploid spermatogonial cells of a morabine grasshopper (White, 1970). In short-horned grasshoppers and crickets, unlike the situation in Gryllotalpa, the single X chromosome is negatively heteropycnotic in diploid spermatogonial divisions. In tetraploid male cells of the morabine grasshopper "P52a ", however, only one of the two Xs showed heteropycnosis ("Asymmetrical heteropycno- sis ").

In this respect, White (1970, pg. 59) comments that "The mechanism which exists in mammals has been considered to be a dosage compensating one, i.e. an adaptive mechanism with a long evolutionary history. Clearly, the phenomenon of asymmetrical heteropycnosis in tetraploid grass- hopper spermatogonia cannot be regarded as adaptive, since it is only manifested in abnormal cells which are not destined to give rise to functional gametes ... the possibility exists that the phenomenon we have described in the tetraploid spermatogonia is an indication of some more widespread mechanism of genetic regulation".

Our work on Gryllotalpa confirms this suggestion. It is true that the tetraploid spermatogonial cells in these insects have no

reproductive potential. Nevertheless, the evidence is of importance in relation to a possible mechanism for the evolutionary origin of facultative heterochroma- tinization.

In both tetraploid spermatogonial cells and female diploid cells (Rao and Arora, 1979) only one X chromosome is active. It seems reasonable to suggest, therefore, that there exists a molecular mechanism which regulates the activity of the X chromosome in both female somatic and male spermatogonial cells. Our unpublished studies on the female germ cells indicate again that only one arm of each of the X-chromosomes is affected by 3H-U and hence is transcrip- tionally active, a situation similar in principle to that which obtains in mammals (Gartler and Andina, 1976). Brown and Chandra (1977) suggest that, because of stringent limitations on the quantity of regulatory entities in the cells, at least one X chromosome escapes facultative heterochromatization in mammals. This appears to be the case too in Gryllotalpa.

It is well known that the X chromosome in heterogametic males is always maternally derived. It follows then, that the two Xs in tetraploid spermatogonial cells are of similar origin. Since only one of these is facultatively heterochromati- zed, it is possible this could be a random phenomenon.

In diploid spermatogonia, the euchromatic arm of the single X of Gryllotalpa is active (Rao and Arora, 1978). In other cases too it is only later in spermatogen- esis that the active X becomes inactive (Monesi, 1965; Odartchenko and Pavil- lard, 1970; Lifschytz and Lindsley, 1972). This change in activity status (active to inactive state) appears to reflect a more generalised phenomenon possibly related to the X chromosome inactivation found in female somatic cells. In Gryllotalpa, as we have seen, one of the two Xs shows differential behaviour (inactivation) even in tetraploid spermatogonia. In an attempt to explain such differential X chromosome behaviour in mammals, Chandra and Brown (1975) suggested that an imprinting process (inactivation of one of the two Xs) occurs in the ovum upon entry of the sperm nucleus. The present study demonstrates that it is possible to initiate the process of differential regulation without the intervention of an imprinting process which requires a fertilization event. A

378 P. Arora and S.R.V. Rao

similar conclusion was reached by Kaufman et al. (1978) who were able to demonstrate X chromosome inactivation in mouse eggs parthenogenetically acti- vated by the suppression of polar body formation (diploid parthenogenesis). Thus, it may be that the molecular mechanism which underlines these several forms of regulation will turn out to be the same in both mammals and Gryllotal- pa.

Acknowledgements. We thank Professor B. John, Department of Population Biology, Research School of Biological Sciences, Australian National University, Canberra City, Australia, for com- ments and suggestions and Dr. Asok Lahiri, Delhi School of Economics, University of Delhi, for statistical advice. This work was partly supported by a Senior Research Fellowship from the University Grants Commission, Delhi, to one of us (Paramjit Arora).

References

Arora, Paramjit., Rao, S.R.V.: Insect Sex Chromosomes II. Mitomycin induced aberrations in the X chromosome of the mole cricket, Gryllotalpa fossor (Scudder). The Nucleus (Calcutta) (in press, 1979)

Arora, Paramjit., Rao, S.R.V. : Insect Sex Chromosomes IV. DNA replication in the chromosomes of Gryllotalpa fossor. Cytobios (in press, 1980)

Brown, S.W., Chandra, H.S. : Chromosome imprinting and the differential regulation of homologous chromosomes. In: Cell Biol. 1, 11e~189 (L. Goldstein and D.M. Prescott, eds.). New York, London Academic Press Inc., 1977

Chandra, H.S., Brown, S.W. : Chromosome imprinting and the mammalian X chromosome. Nature (Lond.) 253, 165 168 (1975)

Donald, J.A., Cooper, D.W.: Studies on metatherian sex chromosomes III. The use of tritiated uridine-induced chromosome aberration to distinguish active and inactive X chromosome. Aust. J. biol. Sci. 30, 103-114 (1977)

Gartler, S.M., Andina, R.J.: Mammalian X-chromosome inactivation. In Advanc. Hum. Genet. 7, 99 140 (1976)

Kaufman, M.H., Guc-Cubrilo, M., Lyon, M.F. : X chromosome inactivation in diploid parthenoge- netic mouse embryos. Nature (Loud.) 271, 547 549 (1978)

Lifschytz, E., Lindsley, D.L.: The role of X-chromosome inactivation during spermatogenesis. Proc. nat. Acad. Sci. (Wash.) 69, 182-186 (1972)

Monesi, V. : Differential rate of ribonucleic acid synthesis in autosomes and sex chromosomes during male meiosis in the mouse. Chromosoma (Berl.) 17, 11-21 (1965)

Natarajan, A.T., Sharma, R.P.: Tritiated uridine induced chromosome aberration in relation to heterochromatin and nucleolar organisation in Microtus agrestis L. Chromosoma (Berl.) 34, 168-182 (1971)

Odartchenko, N., Pavillard, M.: Late DNA replication in male mouse meiotic chromosomes. Science 167, 1133-1134 (1970)

Rao, S.R.V., Arora, Paramjit. : Insect Sex Chromosomes I. Differential response to 5-Bromodeoxy- uridine of the two X chromosomes in females of the mole cricket. Gryllotalpa fossor (Scudder). Ind. J. exp. Biol. 16, 870 872 (1978)

Rao, S.R.V., Arora, Paramjit.: Insect Sex Chromosomes III. Differential susceptibility of homolo- gous X chromosomes of Gryllotalpa fossor to 3HUrd-induced aberrations. Chromosoma (Berl.) 74, 241-252 (1979)

White, M.J.D.: Asymmetry of heteropycnosis in tetraploid cells of a grasshopper. Chromosoma (Berl.) 30, 51-61 (1970)

White, M.J.D. : Animal Cytology and Evolution, 3rd ed. London, New York: Cambridge University Press 1973

Received November 6 - December i1, 1979 / Accepted December 11, 1979 by B. John Ready for press January 22, 1980