karyotype analysis ofascaris lumbricoides var.suum

Chromosoma (Berl.)58, 101- 111 (1976) CHROMOSOMA �9 by Springer-Verlag 1976

Karyotype Analysis of Ascaris lumbricoides var. suum

Male and Female Pachytene Nuclei by 3-D Reconstruction from Electron Microscopy of Serial Sections

Paul Goldstein and Peter B. Moens

Department of Biology, York University, Downsview, Ontario, Canada M3J lP3

Abstract. Twelve synaptonemal complexes are present in both oocyte and spermatocyte pachytene nuclei of Ascaris lumbricoides var. suum, as determined by 3-D reconstruction of the nuclear contents from electron microscopy of serial sections and therefore, n = 12 in the strain of Ascaris described here. In the female the heterochromatic end of each synaptonemal complex is attached to the nuclear envelope and the other end is free in the nucleoplasm. In the male neither end ot the synaptonemal complex is attached, but there is a heterochromatic knob at one end of each complex. Five additional large heterochromatic masses are present in the spermatocyte nucleus and these may be the sex chromosomes described by earlier workers.

Introduction

Interest in the karyotype of nematode worms was generated by Boveri (1887) who showed that in Ascaris megalocephala the two chromosomes present at meiosis appeared as many minute chromosomes during mitosis in the early embryo. Boveri also observed that in the early embryo the loss of chromosomal material occurred only during divisions of future somatic cells and not during divisions of the germ line. Chromosome diminution has since been verified and characterized in A. megalocephala (Zur Strassen, 1899; Lin, 1954) and Ascaris lumbricoides var. suum (Bonnevie, 1902; Goldstein, 1976), but the actual chromsome number has remained obscure.

Many different chromosome numbers have been reported for A. lunbricoides var. suum, a common parasitic worm in pig intestine. Koidzumi (1962) reports n= 13; Kurashvili (1965) n=8 ; and Vassilev and Mutafova (1974) n=24. The differences may be due to real karyotypic variation between strains, but the techniques used, such as tissue homogenization and squash preparation may have introduced some error. Furthermore, the small nuclei, 5 gm in diameter, have minute chromosomes which are difficult to analyse with the light microscope. Under similar circumstances Moens and Perkins (1969) showed that

102 P. Goldstein and P.B. Moens

the chromosome number can be determined from a count of the synaptonemal complexes in pachytene nuclei of meiosis prophase I. The accuracy of this method, which is based on three-dimensional reconstruction of the nuclear contents from electron microscopy of serial sections, has been verified in Neuros- pora and Zea (Gillies, 1973, 1975) and in Locusta (Moens, 1973).

This report shows that spermatocyte nuclei of male A. lumbricoides var. suum have 12 synaptonemal complexes and possibly 5 unpaired sex chromosomes; the complexes are not associated with the nuclear envelope but have differentiated ends. The oocyte nuclei also have 12 synaptonemal complexes which are all attached with one end to the nuclear envelope, and no sex chromosomes are evident.

Materials and Methods

Ascaris lumbricoides var. suum were collected from a local abattoir, The worms were placed into a phosphate buffered 2% glutaraldehyde solution, pH 7.2, and the ovaries and testes were immediate- ly removed, transferred to fresh fixative and kept at 4 ~ C overnight. Post-fixation was in Dalton's osmium-chromic acid (Zickler and Olson, 1975) for 2 h at room temperature, followed by dehydra- tion through an alcohol and propylene oxide series, embedded in Epon, and stained with uranyl acetate and Fiske lead citrate (Fiske, 1966). Serial sections were cut on a Porter-Blum ultramicrotome and examined with a Philips EM 200.

All stages of meiosis prophase I are present at successive intervals from the distal end of the male and female reproductive tract. Oocytes and spermatocytes in the pachytene of meiosis were located by examination of successive 5 cm portions of testis and ovary starting from the blind distal end.

The lengths of the synaptonemal complexes were calculated from Z c = X ~ , where a is the projected distance from the position in one section to the position of the complex in the next section; b is the thickness of the section; and c is the real lenght of the complex from one section to the next (Moens, 1973). The position of the synaptonemal complex in a section was recorded in terms of x and y coordinates. Successive coordinates formed the data for a PLC computer program which calculated projected and real lengths. Two oocyte and two spermatocyte pachytene nuclei were completely reconstructed from serial sections. The karyotypes are compar- ed in Table 1. Nuclear volumes were determined by recording the number of boxes from a grid within each serial section of a nucleus. The volume of each box was then multiplied by the total number of boxes within an entire nucleus.

Observations

L Synaptonemal Complexes

In A. lumbricoides var. suum the synaptonemal complex (SC) appears as a tripartite structure similar to SCs reported in other organisms (for review see Westergaard and von Wettstein, 1972). The dimensions are: lateral element (275 N); central element (250 A); and the central region (650 ]~). The central element is striated while the lateral elements are amorphous (Fig. 1). There is no specific organization of the heterochromatin and euchromatin associated with the synaptonemal complex (Fig. 4a, b). In the female a heterochromatic mass is present at the end of the SC that is attached to the inner membrane

Karyotype of Ascaris lumbricoides var. suum 103

Fig. 1. The synaptonemat complex ~u an Ascaris lumbricoides var. suum oocyte or spermatocyte pachytene nucleus is a tripartite structure. The lateral elements (LE) are amorphous and the central element (CE) is striated. Condensed heterochromatin (CH) is apparent along the synaptonemal complex and at times is in close association with the nucleolus (NO). Nuclear envelope (NE). Bar equals 0.1 gm

of the nuc lea r enve lope while the oppos i t e end is free in the nuc leop lasm (Fig. 2). Both ends o f the SC are u n a t t a c h e d in the male (Fig. 3), bu t there is a d is t inct h e t e r o c h r o m a t i c k n o b at one end o f the SC (Figs. 3, 5, 7 a - f ) and , in some cases, a smal l he t e roch roma t i c mass a t the oppos i t e end. This served to d i f ferent ia te the ends o f the SC.


9 1 1 21,6 C ~ 3

2. 17.1 11 3 12.5

6 L, 11.9 5 / 5. 11.6 ... /

6 1 .2 - - !;ji!? 7 11.0 :i::ii: /

::!:i:

8. 9.3

9 8.8 ;i 10. 83

11 6.6 1 % 12 t..3

0.5/z

12 Fig. 2. Right. Oocyte pachytene nucleus reconstructed from 85 consecutive serial sections. The 12 synaptonemal complexes are all attached at one end to the nuclear envelope, and the opposite end is free in the nucleoplasm (open circle). The synaptonemal complexes are numbered according to length (as in Fig. 2, left). The centriole (C) is attached to the nuclear envelope. The nucleoli (stippled) are localized on synaptonemal complexes no. 5 and no. 7. Left. Reconstructed karyotype of oocyte pachytene nucleus as in Figure 2, right. The lengths are in lam. The nucleoli (stippled) on synaptonemal complexes no. 5 and no. 7 are localized as to their distance from the point of attachment of the synaptonemal complex to the nuclear envelope

13.9 1 2- 12.0 3 11.6 4 10.5

5 9.8 1 6 9.8

7 9.7

8 8.9 9 6.3 10 5.8

1~ s----2---5 4.2 0.5..__..~

Fig. 3. Right. Spermatocyte pachytene nucleus reconstructed from 70 consecutive serial sections. The 12 synaptonemal complexes are unattached at both ends which may be differentiated by the presence of a large heterochromatic knob (darkened circles) and the absence of this mass at the opposite end (open circle), The synaptonemal complexes are numbered according to length as in Figure 3, left. Five heterochromatic masses are present and are not associated with SCs. The centriole (C) is attached to the nuclear envelope. The nucleolus (stippled) is localized on synaptonemal complex no. 6. Left. Reconstructed karyotype of spermatocyte pachytene nucleus as in Figure 3, right. The lengths are in gm. The nucleolus (stippled) on synaptonemal complex no. 6 is localized as to the distance from the terminal heterochromatic knob


Table 1. Pachytene chromosome lengths in lain of male and female Ascaris lurnbricoides var. suum

f rom reconstruction of synaptonemal complexes

Chromosome Oocyte Spermatocyte number

Nucleus Avg. Nucleus Avg.

1 II I II

1 21.6 18.2 19.9 14.0 13.3 13.6 2 17.1 15.8 16.5 12.0 9.3 10.6 3 12.5 14.9 13.7 i1.6 9.2 10.4 4 11.9 13.9 12.9 10.5 9.1 9.8 5 11.6 12.9 12.3 9.8 7.8 8.9 6 11.2 12.4 11.8 9.8 7.5 8.7 7 11.0 10.8 10.9 9.7 7.3 8.5 8 9.3 10.6 9.9 8.9 6.8 7.8 9 8.8 10.1 9.4 6.3 6.7 6.5

10 8.3 8.5 8.4 5.8 6.5 6.1 11 6.5 7.3 6.9 5.5 6.1 5.8 12 4.3 5.9 5.1 4.2 4.2 4.2

Total karyotype 134.1 141.3 137.7 108.1 93.8 100.9 length

Nuclear volume 106.2 93.6 99.9 70.5 56,2 63.4 (gm 3)

In both oocyte and spermatocyte pachytene nuclei 12 synaptonemal complexes are present. Total karyotype length varies slightly from cell to cell. The lengths of the SCs in the oocyte nuclei range from 4.3 u r n to 21.6 u r n and in the spermatocyte nuclei from 4.2 u r n to 14.0 u r n (Table 1). Differences in the length of the SCs between two oocytes or two spermatocytes may be due, in part, to the shortening of SCs at late pachytene (Carpenter, 1975).

Fig. 4a and b. Spermatocyte pachytene nucleus. The arrangement of chromatin around the synaptonemal complex as observed in individual sections suggests that the condensed chromatin ( C H ) occurs in pairs and that some regions along the synaptonemal complex are devoid of chromatin (a). However, reconstruction from serial sections of a portion of a synaptonemal complex shows that there are no extensive regions on the synaptonemal complex lacking chromatin and there is no specific organization of chromat in (b). Lateral element (LE). Central element (CA). Nucleolus (Nu). Centriole (C). Stippled chromat in located on top or to the side of the SC. Bar equals 0.2 gm

Fig. 5. Sex chromosomes in A. lumbricoides var. suum may be present in the spermatocyte nucleus as five large heterochromatic masses. One of these masses (arrow) is localized near the nuclear envelope but the others are dispersed throughout the nucleus (see Fig. 3). Terminal heterochromatic knob of a synaptonemal complex (HK). Nuclear envelope (ARE). Mitochondria (3//). Bar equals 1 lain

Fig.6a-d. Four consecutive sections through one of the heterochromatic masses observed in the spermatocyte pachytene nucleus. Axial cores are not present and there is no association with synaptonemal complexes. Bar equals 0.1 pm


LE ~ . ~


Fig. 7a-f. Six consecutive sections through the terminal heterochromatic knob of a synaptonemal complex in the spermatocyte pachytene nucleus. The synaptonemal complex is not attached to the nuclear envelope. Terminal heterochromatic knob (HK). Lateral element (LE). Central element (CE). Bar equals 0.1 gm


Fig. 8. The synaptonemal complex remains unaltered as it passes through the nucleolus (NO). The central element (CE) is striated. Bar equals 0.1 pm

The terminal heterochromatic knobs in the male are occasionally fused. No centromeres can be recognized along the bivalents, but may be located in the basal knobs, as was the case in mouse spermatocytes (Woollam, et at., t967).

Two nucleoli are present in the oocyte and are localized on bivalents no. 5 and no. 7 (Fig. 2). The SC is unaltered where it passes through the nucleolus (Fig. 8). In the spermatocyte one nucleolus is present and is localized on bivalent no. 6 (Fig. 3) which may well correspond to either no. 5 or no. 7 described above. The nucleolus is not associated with the nuclear envelope in either oocytes or spermatocytes.

I1. Supernumerary Chromosomes

A. lumbricoides does not have a pair of heteromorphic sex chromosomes. Rather, the existence of five univalents has been proposed as sex determinants (Boring, 1909; Edwards, 1910; McClung, 1902; Montgomery, 1909). Five large heterochromatic masses have been observed in this study in spenlaatocyte but not in oocyte pachytene nuclei (Figs. 3, 5, 6a-d). These masses have no obvious axial cores and no apparent association with SCs.


Discussion

Chromosome number has been widely used in classifying organisms. A major problem in the classification of Ascaris species is that several different chromosome values have been assigned to A. lunbricoides var. suum. For example, light microscopic analysis has yielded values of n = 13 (Koidzumi, 1962); n= 8 (Kurashvili, 1965); and n=24 (Vassilev and Mutafova, 1974). Some of the metaphase chromosomes are less than 1 gm and difficult to observe with the light microscope. Additional errors may have been introduced by the techniques used which included tissue homogenization and squash preparation. However, these differences in A. lumbricoides vat. suum may also be due, in part, to real karyotypic variation in different strains of the same species. Five different varieties of A. megalocephala have reported haploid chromosome values, ranging from n= 1 to n=9 (Walton, 1959). These values too may reflect real differences as well as artifacts.

Three-dimensional reconstruction of pachytene nuclei by serial sectioning and electron microscopic analysis shows the presence of 12 synaptonemal complexes in the male and in the female and therefore, n = 12 in the strain of A. lumbricoides var. suum described in this study.

The five heterochromatic masses present in the spermatocyte nuclei may be similar to sex chromosomes reported in other organisms that are associated with large amounts of dense heterochromatin. However, axial cores are not obvious in the heterochromatic masses which suggests they may not be univalents. Jeffrey and Haertl (1938) argue that the heterochromatic masses may have nothing to do with sex determination and may, in fact, be .supernumerary or accessory chromosomes.

Two nucleoli are present in the oocyte although only one nucleolus is present in the spermatocyte. The oocyte is active in RNA synthesis for the production of yolk (Kaulenas and Fairbairn, 1968) but the male genome is not activated until fertilization at which time there is a great proliferation of ribosome synthesis (Clark et al., 1972; Foor, 1968; Kaulenas and Fairbairn, 1968).

Acknowledgements. We thank Dr. Kathleen Church for her helpful discussions and interest in this project. Financial assistance was provided by the National Research Council of Canada, Grant 1901 to P.B. Moens.

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Received April 4-June 27, 1976 / Accepted July 5, 1976 by H. Bauer Ready for press July 7, 1976

karyotype analysis ofascaris lumbricoides var.suum

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