Principles of the Highly Ordered Arrangement of Metaphase I Bivalents in Spermatocytes of Agrodiaetus (Insecta, Lepidoptera)
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Chromosome Research 10: 5^20, 2002. 5 # 2002 Kluwer Academic Publishers. Printed in the Netherlands Principles of the highly ordered arrangement of metaphase I bivalents in spermatocytes of Agrodiaetus (Insecta, Lepidoptera) Vladimir A. Lukhtanov & Alexander V. Dantchenko Faculty of Biology, St. Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia; E-mail: [email protected] Received 21 May 2001; received in revised form and accepted for publication by Herbert Macgregor 9 September 2001 Key words: chromosome, chromosome centripetal movement, chromosome number, holokinetic chromosome, karyotype, kinetochore, Lycaenidae, meiosis, metaphase plate, mitosis, spindle, translocation Abstract We have investigated the nature of highly ordered bivalent arrangement in lepidopteran spermatocytes by ana- lysingandcomparingthepatternsofbivalentdistributioninintactmetaphaseIplatesof24closelyrelatedspecies of the genus Agrodiaetus (Lycaenidae). The studied species greatly differed in haploid chromosome numbers (from n 13 to n 90) and in the structure of their karyotypes. We found that the larger the bivalent, the closer tothecentreofthemetaphaseplateitwassituated.Inspecieswithahighchromosomenumberandasymmetrical karyotype structure, the largest bivalent was located in the centre of the circular metaphase plate. Bivalents of equal size were approximately equidistant from the centre of the metaphase plate and formed concentric circles around the largest bivalent. These principles are diametrically different from those known in the majority of other animals and plants, in which the smallest elements of the chromosome set are situated in the centre of metaphase plate. The only exception from the above principles was observed in spermatocytes of A.surakovi which were heterozygous for reciprocal translocation involving two or three chromosome pairs. In addition to one large bivalent, the heterozygous cells had a multivalent, the size of which was comparable to or even exceededthatofthelargestbivalentinthekaryotype.Inspiteofthelargesize,themultivalentwasalwayssituated at the periphery of metaphase plate. This indicated that the chromosome size itself is not the only factor determining the bivalent position. We also found that the structure of the metaphase plate is fundamentally different in mitotic and meiotic cells of Agrodiaetus. In spermatogonial metaphase, chromosomes were tightly brought together, forming a dense compact disk, whereas during metaphase I of spermatocytes, all bivalents were clearly separated from each other, and the distance between adjacent bivalents varied from 0.4 to 1.5 mm. Based on the above ¢ndings, we proposed a model of bivalent distribution in the Lepidoptera. According to the model, during congregation in the prometaphase stage there is a centripetal movement of bivalents made by a force directed to the centre of the metaphase plate transverse to the spindle. This force is proportional to the kinetochore size of a particular bivalent. The Lepidoptera have a special near-holokinetic type of chromo- some organisation. Therefore, large bivalents having large kinetochores are situated in the central part of metaphase plate. Another possible factor affecting the bivalent position is the interaction of bivalents with the cisternae of the membrane system compartmentalising the intraspindle space. 6 V. A. Lukhtanov & A. V. Dantchenko Introduction The hollow and `reptilian' con¢gurations are not the only types of metaphase plate organisation. In The spatial distribution and arrangement of chro- most plants (Lewitsky 1976) and in some animals mosomes may be highly ordered during interphase (White 1973), mitotic and meiotic plates have and mitotic divisions of plant and animal cells the con¢guration of a disk in which all chromo- (Chiarelli et al. 1977, Bennett 1982, Qumsiyeh somes are located inside the spindle. According 1995, Watson et al. 1996, Joffe et al. 1998, Klein to White (1973), the small and the large chromo- et al. 1998, Visser et al. 1998). In mitotic meta- somes are distributed randomly inside the disk-like phase cells, the order resides often in the fact that metaphases. However, on some published draw- metaphase plate has a hollow (i.e. ring) con¢gur- ings and micrographs showing disk-like meiotic ation in which chromosomes are assembled into metaphases in the lepidopteran genera Leptidea, regular wheel-like rosettes around the periphery Erebia, Lysandra and Agrodiaetus (de Lesse of the spindle. Numerous cases of the metaphase 1960a, 1960b, Lukhtanov 1989, Munguira et al. plates of this type are illustrated in early cytol- 1995), one can see a distinct non-random chromo- ogical works using the standard paraf¢n-cut some arrangement with the largest elements in method (see reviews: White 1963, 1973) and more the middle of the metaphase I and metaphase II recently using different modern cytological tech- plates. These observations contradict White's niques (Slijepcevic et al. 1997, Nagele et al. 1998). opinion as well as the pattern of the chromosome In the mitotic rosettes, the chromosome cen- distribution in other animals. Nevertheless, the tromeric domains were shown to be positioned central position of the larger bivalents and centrally with chromosome arms projecting chromosomes in Lepidoptera was not the focus radially. A special (`reptilian' according to White of the papers cited. 1973) type of the hollow metaphase plate is found In this work, we have investigated the phenom- in taxa with an asymmetrical karyotype including enon and some general principles of highly groups of micro- and macrochromosomes, e.g. ordered bivalent arrangement in spermatocytes in many reptiles, amphibians, birds, insects of of 24 closely related species of the lepidopteran the orthopteran family Tettigoniidae and in some genus Agrodiaetus (Lycaenidae), which is an ideal plants (see reviews: White 1973 on animal cytology model for such a study. Species of the genus are and Lewitsky 1976 on plant cytology). In the last very similar morphologically but extremely dif- taxa, the microchromosomes occupy a position ferent in their karyotypes (de Lesse 1960a, 1960b, in the centre of the metaphase rosette inside the Lukhtanov 1989, Lukhtanov et al. 1998). They spindle, and the macrochromosomes are situated show enormous interspeci¢c variation of chromo- at the periphery and form a ring around the some numbers, ranging from 2n 14 to 2n 250. spindle. This range is unique not only for Lepidoptera The spatial distribution of chromosomes during but also for the entire animal kingdom (White the ¢rst and second meiotic divisions is less well 1973). In addition, most Agrodiaetus species known. Non-random centromeric associations possess one or more morphologically well- were found in mouse meiotic cells (Brinkley et differentiated chromosome pairs which can be al. 1986). It was noted that, in some amphibians, used as markers. reptiles and birds, small bivalents occupy the We used three different approaches. Firstly, we central region of the metaphase I plate, whereas carried out a comparative analysis of the spatial large bivalents are situated near the edge (White order of metaphase I bivalents in a series of 1973). Some representatives of the insect orders taxonomically related species, very different in Hemiptera and Homoptera have a hollow-like their chromosome numbers and karyotype struc- metaphase I con¢guration, which were termed ture. Secondly, we compared distribution patterns radial metaphase plate (Nokkala 1986a). In the of bivalents in normal spermatocytes of A. sura- radial metaphase plate, all chiasmatic bivalents kovi with those heterozygous for reciprocal form a ring, while univalents and pseudobivalents translocation involving two or three pairs of are placed in its centre (Nokkala 1986a, Kuznet- non-homologous chromosomes. Thirdly, we com- sova et al. 1997). pared metaphase plates in spermatogonial mitotic Ordered arrangement of metaphase I bivalents in Agrodiaetus 7 cells with metaphase I plates in spermatocytes of of 40% lactic acid. During the metaphase I stage, different Agrodiaetus species. each spermatocyst is a regular sphere and consists It should be noted that the male meiosis in of 64 spermatocytes. Intact spermatocysts were Lepidoptera is a dichotomous process leading to studied and photographed, at ¢rst by using 40Â eupyrene (fertile) and apyrene (anucleate, non- and 60Â objectives (Figure 1) and then a 100Â fertile) spermatozoa (FriedlÌnder 1997). The objective. In the second phase, different stages eupyrene and apyrene primary spermatocytes dif- of chromosome spreading were observed using a fer fundamentally in structure and function (for slight, gradually increasing pressure on the cover- areview,seeWolfet al. 1987, Wolf 1994). Our slip. The second phase was very useful for studying study deals only with eupyrene spermatocytes. the bivalent structure, identifying bivalents and multivalents, and solving controversial cases of touched or overlapped bivalents. By scaling up Materials and methods thepressureonthecoverslip,wewereableto manipulate the chromosomes by changing their Insects position and orientation on the slide. Population samples of 24 species of the genus Agrodiaetus were collected in the period 1984^ 2000. In each sample, the haploid chromosome Results number was assessed in squashed preparations of metaphase I bivalents from males (Table 1). Bivalent spatial arrangement in the intact metaphase I cells Chromosome preparation and karyotyping