Chromosoma (Berl.) 83, 289-293 (1981) CHROMOSOMA 9 Springer-Verlag 1981 Behaviour of the ZW Sex Bivalent in the Snake Bothrops jararaca Maria Luiza Be~ak* and Willy Be~ak Servigo de Gen~tica, Instituto Butantan, Silo Paulo, Brasil Abstract. The behavior of the ZW sex bivalent was investigated in female meiosis of the poisonous snake Bothrops jararaca. The Z is euchromatic and synapses end to end with the W. The W chromosome shows a heterochromatic segment distally in the short arm. Pairing occurs between the long arm of the W and the slightly longer arm of the mediocentric Z. A sex vesicle, similar to the one found in the XY placental mammals, does not occur in snakes. The Z and W chromosomes segregate reductionally in the first meiotic division and equationally in the second. Introduction The sub-order Serpentes has a basic karyotype of 36 chromosomes including 8 pairs of macrochromosomes and 10 pairs of microchromosomes. Different stages of sex chromosome differentiation have been found in the families Boidae, Colubridae and Viperidae (Begak et al., 1962a, b; Be~ak and Be~ak, 1965). Well differentiated sex chromosomes with female digamety of the ZW type occur in the venomous snakes of the family Viperidae (Crotalinae) and in some non venomous snakes of the family Colubridae. In both families the Z is metacentric and the 4th largest member of the karyotype, the W being in general acrocentric and the smallest of the macrochromosomes. In some Colubridae, however, both sex chromosomes have the same size differing only by a pericentric inversion in the W which is again acrocentric. In the Boidae sex chromosomes heteromorphism has, so far, been found in only one species, namely Acrantophis dumereli (Mengden and Stock, 1980 and compare Begak et al., 1962b and Begak and Be~ak, 1969). Heteromorphism of the ZW type has since been confirmed in the sub-family Viperinae (Baker et al., 1972; Guti6rrez et al., 1979; Kobel, 1962; Singh, 1972). In snakes the size of the Z chromosome is uniform representing ca. 10% of the haploid set, which has a nuclear DNA content about half that of placental mammals (Be~ak et al., 1964; Atkin et al., 1965). In female somatic cells the W is condensed and shows positive * This work is dedicated to the memory of my father Lino Pires de Camargo 0009-5915/81/0083/0289/$01.00
290 M.L. Begak and W. Be~ak heteropycnosis giving rise to the sex chromatin which shows positive fluorescence by quinacrine staining (Be~ak and Be~ak, 1972). The short arm of the W chromosome shows late replication (Bianchi et al., 1964). In the male somatic cells the ZZ pair is euchromatic (Be~ak, 1966) and its DNA replication is synchronous with that of the autosomes. These aspects of the homogametic sex imply the absence of a mechanism of Z dosage compensation equivalent to that found in the X chromosome of placental mammals (Be~ak, 1966; Bianchi, 1969; Ohno, 1967). The ZZ pair is also euchromatic, like the autosomes, in the male germ cells (Be~ak, 1966). As yet, however, no reports are available on the behavior of the ZW pair in female germ cells. In this paper we describe the mode of pairing and segregation of the Z and W chromosomes in the female meiotic cycle of the crotalid Bothropsjararaca (3n=36). Additionally the pachytene stage of this snake has been compared to that of the colubrid Waglerophis merremii (2n = 30) and the boid Boa constrictor amarali (2n = 36). Material and Methods The snakes used in this study include four adult females of Bothrops jararaca (Wied) (2n=36) belonging to the family Viperidae subfamily Crotalinae (Fig. la, b); two adult females and one male of Waglerophis merremii (Wagler) (2n=30), previously described as Xenodon merremii, of the family Colubridae, subfamily Xenodontinae, and an adult male and a female of Boa constrictor amarali (Stull) (2n : 36) of the family Boidae, subfamily Boinae. Meiotic chromosomes were obtained from oocytes using the squash technique. Small fragments of about 0.5 cm of the ovarian duct were immersed in cold distilled water (15 rain) fixed in 50% glacial acetic acid (15 rain) and squashed. The coverslips were removed in a mixture of dry ice and methanol. The material was hydrolyzed in HC1 N (7 min) and stained in Giemsa (5-10 min). The preparations were analysed by contrast phase microscopy and photographed in a Zeiss photomicroscope using Kodak Panatomic X film. Mitotic metaphase chromosome preparations were obtained from short term cultures of peripheral blood (Be~ak et al., 1962a). Results In Bothrops jararaca female meiosis was first detected during February and March. Pachytene was found in about twenty nuclei per slide in each of the four females examined. At this stage the ZW chromosomes are synapsed end to end. With the exception of an apparent heterochromatic block at the free extremity of the W, the sex pairs is similar to the autosomes regarding heteropycnosis (Fig. 2a). We did not find first metaphase nuclei but first anaphase nuclei containing a well defined Z half bivalent have been seen (Fig. 2b). This sex chromosome is easily identified as a comparison with the mitotic karyotypes of the male (Fig. 1 a) and female (Fig. lb) of this species indicates. The Z macrochromosome is metacentric and, as in other snakes, is the 4th in order of size (Be~ak et al., 1962a). The existence of a Z half bivalent at first meiotic anaphase indicates a reductional segregation of the ZW bivalent at first division. During April and May we found mainly the more advanced stages of the meiotic division and, again, the Z chromosome could be identified at second metaphase and anaphase (Fig. 2c and d). The appearance of the Z at second division confirms that the segregation of the sex pair is reductional in the first division and equational at the second. The early stages of female meiosis in Boa constrictor amarali and Waglerophis
ZW in Female Meiosis of Bothrops jararaca 291 Fig. la and b. Male (a) and female (b) karyotypes of B. jararaca to identify the sex chromosomes. The bar in this and Figure 2 represents 10 ~.tm merrernii were detected during August and October. The pachytene nuclei of both sexes showed only euchromatic bivalents although the latter display heteromorphic sex chromosomes in the female (Fig. 2e and f). Discussion Among vertebrates, differentiated sex chromosomes with female digamety of the type ZZ/ZW occurs in birds and most of the ophidians. In contrast to the abundance of data on sex chromosome behavior in somatic cells, literature on the behaviour of the ZW bivalent in female meiosis in scarce. Recently it has been shown by electron microscopy that the chicken ZW chromosomes
292 M.L. Be~ak and W. Begak Fig. 2a-L Female meiosis in snakes a A pachytene nucleus of B. jararaca showing a ZW bivalent with terminal pairing between euchromatic arms. b A single anaphase-i group from B. jararaca with a well defined Z half bivalent (compare with Fig. 1). e Metaphase II of B. jararaca including a Z half bivalent, d Anaphase II of B. jararaca showing the Z dividing equationally, e, f Pachytene nuclei of Boa constrictor amarali (e) and Waglerophis merremii (t) are synapsed along most of the length of the W and do not exhibit positive heteropycnosis (Solari, 1977). Even so the lateral element of the Z axis forms a large numbers of twists, which is unusual, and these result in a false impression of the relative lengths of the two chromosomes. Solari is of the opinion that this atypical behaviour may be indicative of non-homologous pairing.
ZW in Female Meiosis of Bothropsjararaca 293 Our observations with the light microscope on the behavior of the sex bivalent in Bothrops reveal apparent end to end association of the Z and W at pachytene. Although there is a clear association between end segments in most cells we could not see a strictly terminal association. In this species the Z is euchromatic while th the W has a heterochromatic block located distally in the non paired arm. Based on the pattern of late replication described for the short arm of the W (Bianchi et al., 1969), we assume that this chromosome synapses with the Z by its long arm. As the Z is a metacentric it is difficult to determine which of the two arms actually pairs with the W, but we are of the opinion that it is the slightly longer arm. The ZW pair does not give rise to a sex vesicle in Bothrops. This is also the case in the chicken (Solari, 1977). In Bothrops, again as in chicken, positive heteropycnosis of the sex chromosomes does not appear to play any role in preventing crossing over. The appearance and behaviour of the Z chromosome at first anaphase and second metaphase indicates that ZW segregation is reductional at first division and equational at the second. The Ayes, in general, are very similar to the Serpentes regarding DNA content, Z proportional size (Atkin et al., 1965; Begak et al., 1964; Ohno et al., 1964), female digamety (Begak et al., 1962a, b; Ohno, 1961), presence of microchromosomes, high chiasma frequency (Be9ak, 1962a, b), absence of Z sex chromatin in the homogametic sex (Be9ak, 1965) and presence of W sex chromatin in the heterogametic sex (Begak and Be9ak, 1972). The absence of a sex vesicle in snake oocytes is a further point of similarity. Acknowledgements. This research was supported by grants from the Brazilian CNPq-FINEP, FAPESP, PNUD-UNESCO and FEDIB. References Atkin, N.B, Mattinson, G., Be~ak, W., Ohno, S.: The comparative DNA content of 19 species of placental mammals, reptiles and birds. Chromosoma (Berl.) 17, 1-10 (1965) Baker, R.J., Mengden, G.A., Bull, J.J. : Karyotypic studies of thirty-eight species of North American snakes. Copeia (Wash.) 2, 257-265 (1972) Begak, W., Begak, M.L.: Cytotaxonomy and chromosomal evolution in Serpentes. Cytogenetics 8, 247-262 (1969) 1 Begak, W., Begak, M.L. : W-sex chromatin fluorescence in snakes. Experientia 28, 228 229 (1972) 1 Bianchi, N.O., Begak, W., Bianchi, M.S.A., Be9ak, M.L., Rabello, M.N. : Chromosome replication in four species of snakes. Chromosoma (Berl.) 26, 188-200 (1964) Guti6rrez, J.M., Taylor, R.T., Bolafios, R. : Cariotipos de diez especies de serpientes costarricenses de la familia Viperidae. Rev. Biol. Trop. 27, 309 319 (1979) Kobel, H.R. : Heterochromosomen bei Vipera berus L. (Viperidae, Serpentes). Experientia (Basel) 18, 173-174 (1962) Mengden, G.A., Stock, A.D.: Chromosomal evolution in Serpentes; a comparison of G and C banding patterns of some colubrid and boid genera. Chromosoma (Berl.) 79, 53-64 (1980) Ohno, S. : Sex chromosomes and sex-linked genes. Berlin, Heidelberg, New York: Springer Verlag (1967) 1 Singh, L. : Evolution of karyotypes in snakes. Chromosoma (Berl.) 38, 185-236 (1972) Solari, A.J.: Ultrastructure of the synaptic autosomes and the ZW bivalent in chicken oocytes. Chromosoma (Berl.) 64, 155 165 (1977) Received September 29-October 30, 1980 / Accepted by B. John Ready for press February 15, 1981 1 Citations in the text of earlier publications of the authors are to be found in their later publications included here