P. GAUR AND R. R. TEWARI. of Zoology, University of Allahabad. Received March 19, 1980

Transcription

1 JAPAN. J. GENETICS Vol. 55, No. 4: (1980) MALE MEIOSIS IN FLESH-FLIES OF THE GENUS PARASARCOPHAGA (SARCOPHAGIDAE: DIPTERA) D. KAUL, P. GAUR AND R. R. TEWARI Department of Zoology, University of Allahabad Allahabad , India Received March 19, 1980 Male meiosis in 6 species of the sarcophagid genus Parasarcophaga- P, miseya, P, albiceps, P, argyyostoma, P, orchidea, P, ruficornis and P, knabi-was investigated with a view to obtain information regarding the degree of homology betweeen the X and Y chromosomes. Both autosomal as well as the sex bivalents are achiasmate in all the six species. The X and Y chromosomes in all the species, except P, knabi, show a close side by side association right from prophase I to metaphase I. In P, knabi, the extremely long X and Y chromosomes are spatially separate on emergence from the resting nucleus at prophase I and do not show close association at metaphase I. The role of C-band positive heterochromatic material in the pairing of X and Y chromosomes during first meiotic prophase has been discussed. INTRODUCTION In a previous paper on the cytogenetics of various species of flesh-flies belonging to the genus Parasarcophaga, a role of extraordinarily high level of variation in morphology of sex chromosomes in the karyotypic divergence of species within the genus has been emphasized (Kaul et al. 1978). It is also interesting to ncte that an increase or decrease in size of the X chromosome is accompanied by a parallel increase/decrease in size of the Y chromosome. In order to obtain more information regarding the degree of homology between the X and Y chromosomes it was thought desirable to undertake a comparative study of the chromosome behaviour at meiosis in these species. In this paper, we present a comparative analysis of meiosis in six species of fleshflies of the genus Parasarcophaga, namely P, miseya, P, albiceps, P, argyyostoma, P. orchidea, P. ruficornis and P, knabi. MATERIALS AND METHODS Flesh-flies used in this study were collected from nature and inbred colonies are

2 268 D. KAUL, P. GAUR AND R. R. TEWARI being maintained in the laboratory. Meiosis was examined in 8-10 day old pupae and/or freshly emerged adults. Conventional were made from fresh material stained in lactic aceto-orcein mixture of 85% lactic acid and glacial acetic acid). the testicular cells of squash preparations (2% orcein in a 1: 1 RESULTS AND DISCUSSION The present results have demonstrated achiasmate male meiosis in all the examined members of the genus Parasarcophaga. These findings add another piece of evidence supporting the earlier findings that the super order Brachycera (with perhaps the only exception of Megaselia scalaris of Phoridae) is characterised by the presence of achiasmate meiosis in the heterogametic sex (White 1973). A critical observation of the nuclear contents of the early spermatocyte of all the Fig. 1. a-f. phase I; h. Male meiosis in P, misers. a. Prophase I; II; e. Metaphase II; f. Anaphase II. g-j. Metaphase I; i. Metaphase II; j. Anaphase b. Metaphase I; c. Anaphase Male meiosis in P. albiceps. g. Prophase II. Bar represents 10pm. I; d. Pro-

3 MALE MEIOSIS IN FLESH-FLIES 269 Fig. 2. a-f. Male meiosis in P, orchidea. a. Early prophase I; b. Late prophase I; I; d. Anaphase I; e. Prophase II; f. Metaphase II. g-m. Male meiosis in Early prophase I; h. Late prophase I; i. Metaphase I; j. Anaphase I; k. 1, Metaphase II; m. Anaphase II. Bar represents 10 rim. c. Metaphase P. knabi, g. Prophase II;

4 270 D. KAUL, P. GAUR AND R. R. TEWARI Fig. 3. a-f. Male meiosis in P. argyrostoma. a. Prophase I; b. Metaphase I; c. Anaphase I; d. Prophase II; e. Metaphase II; f. Anaphase II. g-k. Male meiosis in P, ruffcornis. g. Prophase I; h. Metaphase I; i. Prophase II; j. Metaphase II; k. Anaphase II. Bar represents 10 pm.

5 MALE MEIOSIS IN FLESH-FLIES 271 six species shows that the earliest spermatocyte nuclei of P. knabi, P, ruficornis, P. oychidea and P, argyrostoma show a faintly stained reticulum and a very prominent heteropycnotic body, while in P, misery and P, albiceps the heteropycnotic body is not visible. At first meiotic prophase chromosomes reappear from the resting nucleus as five autosomal bivalents, which are mostly in a completely paired condition, and a X-Y complex (sex bivalent) (Figs. la, g; 2a, g; 3a, g). The sex bivalent in P, misers and P. albiceps appears in the form of a dot in which the X and Y chromosomes cannot be differentiated. In case of P, argyrostoma, P, ruficornis and P. oychidea the heterochromosomes show an intimate side-by-side association. In many cells the heteromorphic nature of the sex bivalent (X-Y complex) could easily be discerned in striking contrast to the other five autosomal bivalents by the fact that there is a noticeable difference in size of the two elements forming the sex bivalent. The small distal segment of the longer element, the X chromosome, remains unpaired. The extremely large X and Y chromosomes of P, knabi are particularly interesting. In this species the sex chromosomes do not emerge paired from the resting spermatocyte nuclei but tend to remain independent of each other-the apparent lack of pairing becoming exaggerated during later stages (Figs. 2g, h). Nevertheless, the X and Y chromosomes usually lie near one another without actually touching each other upto metaphase I. In some of the metaphase I plates they do show a tendency to make contact. The possiblity of a weak heterochromatic attraction being involved in such a behaviour of the X and Y chromosomes cannot be excluded (see later). In transition from prophase I to metaphase I there is only a progressive contraction and separation of the paired homologues. As in most of the higher dipterans, showing achiasmate meiosis, typical diplotene figures are not encountered in the present study. By the time the bivalents come to lie on metaphase I plates they attain their most compact form and in majority of the cells the autosomal bivalents show a very clear reductional split; the homologues are almost seen lying parallel to each other without any contact (Figs, lb, h; 2c, i; 3b, h). Anaphasic separation of the chromosomes shows no abnormality and the homologues move to opposite poles synchronously (Figs. lc; 2d, j; 3c). After interkinesis, during the second meiotic cycle, the chromosomes emerge out at late prophase as fairly condensed threads (Figs, id; 2e, k; 3d, i) which gradually contract to the metaphase II. The completion of metaphase II is indicated by the division of chromosomes into sister chromatids except at the centromeres (Figs. le, i; 21, 1; 3e, j). The daughter chromosomes pass to the opposite poles at anaphase II synchronously (Figs, if, j; 2m; 3f, k). The nature of factors involved in the association of X and Y chromosomes during first meiotic prophase deserves some comment. Our earlier findings with the C-banding technique have shown that the telocentric X chromosomes in P, ruficornis and P. argyrostoma show a small lightly stained segment towards the distal end, but the telocentric Y chromosomes are entirely C-band positive. However, the extremely long metacentric X and Y chromosomes in P, knabi differ markedly with respect to the distribution of C-band positive heterochromatin. The X chromosome shows two lightly

6 272 D. KAUL, P. GAUR AND R. R. TEWARI stained regions, one each in both the arms, lying between three usual C-bands. The Y chromosome has only one lightly stained region in the middle of its long arm (Kaul et al. 1978). If the C-band positive regions of the X and Y chromosomes can be taken as a measure of homology then it is not without significance that, in the present study, the X and Y chromosomes in P, knabi do not show an intimate side-by-side association from prophase I upto metaphase I which is so characteristic of the behaviour of the x and Y chromosomes in the remaining species. In view of this it appears probable that attraction between the C-band positive heterochromatin may well lead to X and Y chromosomes establishing association during the first meiotic prophase. However, the question of interpretation of the role of C-band positive material in meiotic pairing of the sex chromosomes remains for the present largely a matter of conjecture. Extending the discussion to a more general level, several studies have shown that the C-band positive areas are rich in highly repetitive DNA (Pardue and Gall 1970; Gall et al. 1971; Yunis et al. 1971; Rae 1972; Jones 1973; Arrighi and Saunders 1973; Cordeiro et al. 1975; Brutlag et al. 1977, among others). Among the various functions that have been postulated for the highly repetitive DNA, one is that the repetitive DNA (satellite DNA) plays some important role in the meiotic recognition process (Bostock 1971; Comings 1972; Walker 1972; Swift 1973; Hsu 1975; Peacock et al. 1977). However, Yamamoto and Miklos (1977, 1978) have elegantly shown that satellite DNA is not involved in meiotic recognition process. They do not dismiss the agency of satellite DNA altogether, but suggest that "if satellite DNA per se were to be indispensable for meiotic or mitotic recognition processes, then the amount that is obligatory is extremely small." Their tenet seems to be amply supported by the fact that there are instances where C-band positive areas are found to be devoid of highly repetitive DNA (Gall et al. 1971; Hennig 1973; Arrighi et al. 1974; Samols and Swift 1979). All this is merely to say that if C-band positive material has been a factor in the meiotic pairing of the sex chromosomes, it is likely that some factor other than the highly repetitive DNA is at work. In this connection it is worthwhile to mention that Comings et al. (1973) have pointed out the importance of proteins in the production of C-bands. Hagele (1979) has also shown that the heteropycnotic X chromosome of Schistocerca gregaria reveals 2-3 large heavily stained segments after heat denaturation and differential reassociation in a Giemsa buffer or acridine orange buffer solution. These segments were presumed to represent DNA reassociation complexes which are formed only in the presence of histones. Thus, the possibility of the involvement of proteins in the meiotic pairing is worthy of serious consideration. One need mention only that Comings and Riggs (1971) have suggested that allosteric proteins (folding proteins) bound to specific DNA sites and to each other would control chromosome pairing. According to them a repeated DNA sequence could recognize a specific folding protein which would be capable of self association after binding DNA. To maintain the claim for the role of allosteric proteins, one would have to speculate that the X and Y chromosomes of P, knabi fail to pair properly during meiosis because of the absence of heterochromatin-specific-folding proteins in the lightly stained regions, however, they might show a weak tendency to come together due to presence of the same in darkly stained regions.

7 MALE MEIOSIS IN FLESH-FLIES 273 ACKNOWLEDGMENTS Thanks are due to Prof. U. S. Srivastava for providing necessary laboratory facilities and encouragement throughout the work. Authors are grateful to Dr. Rokuro Kano, Dean, Faculty of Medicine, Tokyo Medical and Dental University, for identification of the specimens. Assistance of Mr. Mohd. Hussain in photography is thankfully acknowledged. Financial assistance from C.S.I.R., India in the form of a S.R.F. to one of us (R.R.T.) is gratefully acknowledged. LITERATURE CITED Arrighi, F. E., and G. F. Saunders, 1973 The relationship between repetitious DNA and constitutive heterochromatin with special reference to man. Symp. Medica-Hoechst 6: Schattauer, Stuttgart, New York. Arrighi, F. E., T. C. Hsu, S. Pathak, and H. Sawada, 1974 The sex chromosomes of the Chinese hamster: Constitutive heterochromatin deficient in repetitive DNA sequences. Cytogenet. Cell Genet. 13: Bostock, C. J., 1971 Repetitious DNA. Advanc. Cell Biol. 2: Brutlag, D., R. Appels, E. S. Dennis, and W. J. Peacock, 1977 Highly repeated DNA in Drosophila melanogaster. J. Mol. Biol. 112: Comings, D. E., 1972 The structure and function of chromatin. Advanc. Hum. Genet. 3: Comings, D. E., and A. D. Riggs, 1971 Molecular mechanisms of chromosome pairing and function. Nature 233: Comings, D. E., E. Avelino, T. A. Okada, and H. E. Wyandt, 1973 The mechanism of C- and G-banding of chromosomes. Exp. Cell Res. 77: Cordeiro, M., L. Wheeler, C. S. Lee, C. D. Kastritsis, and R. H. Richardson, 1975 Heterochromatic chromosomes and satellite DNAs of Drosophila nasutoides. Chromosoma (Berl.) 51: Gall, J. G., E. H. Cohen, and M. L. Polan, 1971 Repetitive DNA sequences in Drosophila. Chromosoma (Berl.) 33: Hagele, K., 1979 Selective staining of X chromosome segments in Schistocerca gregaria after denaturation and reassociation procedures. Chromosoma (Berl.) 71: Hennig, W., 1973 Molecular hybridization of DNA and RNA in situ. Int. Rev. Cytol. 36: Hsu, T. C., 1975 A possible function of constitutive heterochromatin: The bodyguard hypothesis. Genetics 79: Jones, K. W., 1973 Satellite DNA. J. Med. Genet. 10: haul, D., R. Chaturvedi, P. Gaur, and R. R. Tewari,1978 Cytogenetics of the genus Parasarcophaga (Sarcophagidae: Diptera). Chromosoma (Berl.) 68: Pardue, M. L., and J. G. Gall, 1970 Chromosomal localization of mouse satellite DNA. Science 168: Peacock, W. J., R. Appels, P. Dunsmuir, A. R. Lohe, and W. L. Gerlach, 1977 Highly repeated DNA sequences: Chromosomal localization and evolutionary conservatism. Int. Cell Biol. Congr The Rockfeller University Press, New York. Rae, P. M. M., 1972 The distribution of repetitive DNA sequences in chromosomes. Advanc. Cell Molec. Biol. 2: Samols, D., and H. Swift, 1979 Genomic organization in the flesh fly Sarcophaga bullata. Chromosoma (Berl.) 75: Swift, H., 1973 The organization of genetic material in eukaryotes: Progress and Prospects. Cold. Spr. Harb. Symp. Quant. Biol. 38: Walker, P. M. B., 1972 "Repetitive" DNA in higher organisms. Progr. Biophys. Molec. Biol. 23:

8 274 D. KAUL, P. GAUR AND R. R. TEWARI White, M. J. D., 1973 "Animal Cytology and Evolution" 3rd ed. Cambridge University Press. London. Yamamoto, M., and G. L. G. Miklos, 1977 Genetic dissection of heterochromatin in Drosophila: The role of basal X heterochromatin in meiotic sex chromosome behaviour. Chromosoma (Ben.) 60: Yamamoto, M., and G. L. G. Miklos, 1978 Genetic studies on heterochromatin in Drosophila melanogaster and their implications for the functions of satellite DNA. Chromosoma (Berl.) 66: Yunis, J. J., L. Roldan, W. G. Yasmineh, and J. C. Lee, 1971 Staining of satellite DNA in metaphase chromosomes. Nature 231: