The behavior during pachynema of a normal and an inverted Y chromosome in Microtus agrestis

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1 Hereditas 111: (1989) The behavior during pachynema of a normal and an inverted Y chromosome in Microtus agrestis TERRY ASHLEY, MAARIT JAAROLA and KARL FREDGA2 Department of Zoology, University of Tennessee, Knoxville, TN 37996, USA 2Department of Genetics, Uppsala University, Uppsala, Sweden ASHLEY, T., JAAROLA, M. and FREDGA, K The behavior during pachynema of a normal and an inverted Y chromosome in Microtus agres?is.--heredi?as I I I: Lund, Sweden. ISSN Received August -22, Accepted October 16, 1989 The pachytene behavior of the chromosomes of Microrus agresris (L.) (Rodentia, Arvicolidae) males carrying either the standard, or the pencentrically inverted Lund Y chromosome have been examined by electron microscopy of microspread spermatocytes. There is no synapsis between the X and either the standard or the Lund Y chromosomes during any substage of pachynema. Since synapsis is generally considered a prerequisite for crossing over, there appears to be no opportunity for crossover or chiasma formation between the X and Y in this species. The G-, C- and NOR-banded mitotic karyotypes of animals carrying the standard and Lund Y are also presented. Terry Ashley, Department of Zoology, University of Tennessee, Knoxville, TN 37996, USA KOLLER and DARLINGTON (1934) observed a region of synapsis between the X and Y chromosomes of the Norwegian rat during pachynema and end association between them during metaphase I of meiosis. Since it was (and is) assumed that the mammalian X and Y arose from an ancestral pair of homologues, they concluded that there is a region of homology between the X and Y in all mammalian species and that this region regularly synapses and crosses over during meiosis in a manner analogous to the autosomes. This assumption is still widely accepted (BURGOYNE 1982; POLANI 1982). Indeed, the idea has gained recent momentum from the molecular data for regular exchange of DNA between the X and Y in humans (ROUYER et al. 1986). None the less, there is ample evidence that synapsis and subsequent crossover of the X and Y as a mechanism of disjunction of the sex chromosomes is not a universal phenomenon in mammals, or even in eutherians (cf. ASHLEY 1985, 1987 for review). One species which does not fit this general pattern is the Egyptian sand rat (Psammomys obesus), in which there is clearly no synapsis between the X and Y during pachynema (SOLARI and ASHLEY 1977; ASHLEY and MOSES 1980). Since synapsis is generally assumed to be a prerequisite for crossing over, it can be presumed that the asynapsis of the X and Y provides no opportunity for crossover. Consequently, disjunction of the sex chromosomes must be assumed to be achiasmatic in this species. The most prominent feature of the karyotype of the sand rat is formed by large blocks of heterochromatin on both the X and Y. SOLARI and ASHLEY (1977) suggested that this heterochromatin might be involved in an unspecified manner in the asynaptic behavior of the sex chromosomes of this species. It was therefore of interest to determine whether other species with large blocks of heterochromatin on their sex chromosomes are also asynaptic, or whether they synapse with a potential for chiasmatic disjunction. Microtus agrestis, the European field vole, has the distinction of having more constitutive heterochromatin on its sex chromosomes than any other species so far examined. Heterochromatin accounts for approximately 25 YO of the total female haploid genome (Hsu and ARRIGHI 1971; GAMPERL et al. 1982) of which 20 Yo is located on the X chromosome. Approximately three fourths of the submetacentric X is heterochromatic; the rest is euchromatic (WOLF et al. 1965). The Y contains a similar amount of heterochromatin. The synaptic and disjunctional behavior of this species is therefore of considerable interest, especially since there have been conflicting reports on the meiotic behavior of the sex chromosomes. MATTHEY (1949, 1950) and SACHS (1953) reported bivalents, while FREDGA (1970) and ZENZES and WOLF (1971) reported univalents at metaphase I of the X and the standard Y.

2 282 T. ASHLEY ET AL. Hereditas Ill (1989) In all populations of M. agrestis, except for those in the southwestern region of Sweden, the Y is acrocentric. FREDGA and HANSEN-MELANDER (1970) have reported that animals in this region have a subtelocentric Y, termed the Lund Y. The Lund Y is the result of a pericentric inversion (FREDGA and JAAROLA, in prep.). Whether this rearranged Y differs in meiotic behavior in the males carrying it from males carrying the normal (standard) Y was unknown. Spermatocytes of males carrying either the standard or the Lund Y have been examined and the pachytene behavior of both is reported. Materials and methods Animals. - Microtus agrestis carrying the Lund Y were originally trapped 20 km east of Lund, Sweden, while those carrying the standard Y were trapped around the Instititute of Genetics, Uppsala. Sweden. Meiotic preparations were made and examined from each of 3 males carrying the standard Y and each of 6 males carrying the Lund Y. Mitotic preparations were made from these animals, as well as from additional males of both chromosome types. Mitotic preparations. - Mitotic chromosome preparations were made from bone marrow as described by FREDGA (1987). The preparations were G-banded Mitosis. - As previously described (Matthey 1949), according to the procedure of WAX and FEDOROFF M. agrestis has 48 autosomes. The smallest pair is (1972), C-banded according to SUMNER (1972), or metacentric; the rest are acrocentric and, with the stained for nucleolus organizer regions (NORs) exception of the longest, which is significantly longer, according to BLOOM and GOODPASTURE (1976). exhibit a continuous gradation in length. The G- Meiotic preparations. - Animals were killed with banded mitotic autosomes (Fig. la) of the males an overdose of ether. The testes were removed and examined appeared as previously described (PERA minced in Minimum Essential Medium (without 1972; COOPER and Hsu 1972). No evidence of any glutamine). The preparation was centrifuged and autosomal rearrangements were observed in anithe cells were resuspended in a small quantity of the mals carrying either the standard or the Lund Y. medium. The suspension was dropped onto a solu- Distinct pericentric C-bands were seen in 1C12 Some material was stained in fresh 1 '/O PTA (phosphotungstic acid) in ethanol for 5 min within 38 hours of the initial preparation. Other slides were incubated in 70 YO silver nitrate at 60 C for two hours. (Time of silver staining after prepara- tion varied, but was not crucial.) Areas of high concentrations of well prepared cells were located by phase microscopy and marked. The plastic was floated off the slide and electron microscope grids were placed over the marked areas and picked up. The material was examined on either a Philips 300 (Uppsala) or a Hitachi 9000 (Knoxville, TN). Micrographs were made at the highest magnification consistent with inclusion of all synaptonemal complexes from one nucleus in a single electron micrograph. Micrographs of whole nuclei used for measurements were printed on 20 x 25 cm paper and measurements of each synapsed autosomal bivalent and the axes of each sex chromosome in the nucleus were made on a Hipad digital graphics tablet interfaced with an IBM PC and processed with a Bioquant Analytical software package. We also include some pachytene and metaphase I cells (Fig. 5 and 9) from preparations made in 1963 and 1970 (FREDGA, unpublished) prepared by the direct acetic-orcein squash technique of WELSHONS et al. (1962). Results Autosomes tion of 0.5M sucrose (FLETCHER 1979) on a plastic pairs of autosomes (Fig. lb), which is in agreement coated slide (FELLUGA and MARTINUCCI 1976; DRESSE-.R with the observations of PERA (1972). One pair of and MOSFS 1979). The cells were allowed to settle autosomes (No. 8 in Fig. lb) has additional C- for about ten min. The excess liquid was poured off bands in the proximal portion of the long ams, and the preparation was left standing for another 5 separated from the pericentric C-band by an euchromin, then placed in 4 YO paraformaldehyde ph 8.2 matic segment. This characteristic feature is also with 0.05g SDS (sodium dodecasulfate) per 100 nil seen in Fig. lh of GAMPERL et al. (1982). The remaiof solution where the cells were fixed for 5 min. The ning chromosomes exhibited a barely detectable slides were then briefly rinsed in 0.4 YO Photoflo, amount of C-positive pericentric heterochromatin followed by distilled water, and allowed to air dry. on the autosomes (Fig. lb), also as previously de- scribed (ARRIGHI et al. 1970; PERA 1972; COOPER and Hsu 1972; GAMPERL et al. 1982). Although NOR staining was not carried out on mitotic preparations from the same specimens of males used for synaptonemal complex analysis,

3 Hereditus 111 (1989) SEX CHROMOSOMES OF MICROTUS AGRESTIS 283 Fig. la and b. a The G-banded karyotype of M. agreslis of a male carrying a Lund Y. Chromosomes arranged as by COOPER and Hsu (1972). The sex chromosomes from a male carrying a standard Y are inserted. b The C-banded karyotype of M. agrestis with Lund Y. Sex chromosomes from a male carrying a standard Y are inserted. preparations were made later from additional animals: one carrying the Lund and one the standard Y, as well as one female. In animals carrying the Lund Y, NORs were observed on the 4 or 5 longest chromosomes (Fig. 2). The other two specimens studied showed NOR activity on some of these and other chromosomes as well (Table 1). Meiosis. - In electron micrographs of microspread preparations all autosomal bivalents were seen to synapse completely during pachynema (Fig. 3). No evidence of heterozygosity for any rearrangement involving the autosomes was observed. The length of the synaptonemal complex of each bivalent in a nucleus was measured and ranked. The rankings were also used to identify the single metacentric, or the several nucleolus organizer-carrying chromosomes. PTA (phosphotungstic acid) used as an electron

4 284 T ASHLEY ET AL Herediras Ill (1989) Fig. 2. The mitotic karyotype of a male M. agrestis carrying the Lund Y. showing the chromosomes with active NORs (arrows). microscopic stain on microspread spermatocytes reveals the kinetochores as differentiated regions of the lateral elements of the synaptonemal complexes (COUNCE and MEYER 1973). When spermatocytes of M. agrestis were stained with PTA. and all bivalents measured and ranked from longest to shortest, the kinetochores of the smallest bivalent (the metacentric) could be identified (Fig. 3), as well as those of the acrocentrics. In silver stained microspreads nucleolar material can be detected in early pachytene nuclei as cometlike structures that appear to shoot from the ends of the bivalents from which they arise, or in later substages. as more amorphous blobs of material (DRESSEH and MOSES 1980). A variable number of bivalents (47) in silver stained preparations of one male of M. agrestis carrying the Lund Y exhibited such structures (Fig. 4). The bivalents with which each nucleolus was associated in seven silver stained nuclei, in which the bivalents had been measured and ranked in length from longest to shortest, are tabulated in Table 1. There may be some ambiguity in identification of each bivalent by length alone due to preparation artifacts, such as stretching. For this reason, a bivalent measured as sixth longest might in fact be the fifth or seventh longest. None the less, it is obvious from Table 1, that the longer bivalents carry most of the active nucleous organizer regions. There was no detectable tendency for bivalents with silver stained nucleolar material to Tuhle 1. The number and location of nucleolar material on the autosoma1 bivalents of 7 pachytene nuclei from one male of M. agrestis carrving the Lund Y. and presence of active NORs in mitotic metaphases from three specimens, one carryingthe Lund Y, one the standard Y. and one female.?he last two mentioned from the same locality (Uppsala). Chromosome number based on size only Bivalent rank1 Chromosome No Number of nucleoli (1 3 3 I 2 I associated with hivalent at pachynema Presenceor absence of NOR in mitotic cells Lund Y (+I + Stdndard Y Female t + + +

5 Hereditas 11 I (1989) SEX CHROMOSOMES OF MICROTUS ACRESTIS 285 Fig. 3. A PTA stained pachytene nucleus of a microspread spermatocyte of M. ugresris. The kinetochores are visible as differentiations of the axes (kinetochores of acrocentrics are indicated with light arrows; of the small metacentric with a heavy arrow). The peripheral location of the X and Y axes suggests this nucleus is in about mid pachynema. Bar = 5 pm. clump or otherwise associate in pachytene spermatocytes. Sex chromosomes Mitosis. - As previously described (MATHEY 1949; OHNO 1967), the sex chromosomes of M. agrestis are extraordinarily large with big blocks of heterochromatin on both the X and Y. In G-banded pre- parations the heterochromatic portions of the X and both the standard and Lund Y stain comparatively homogeneously and darkly with Giemsa (Fig. la). As shown by FREDGA and JAAROLA (in prep.) the Lund Y differs from the standard Y by a pericentric inversion. The break in the short arm occurred near the centromere, and the break in the long arm occurred in the heterochromatin a short distance below the centromere.

6 286 T. ASHLEY ET AL Hereditas 111 (1989) Fig. 4. A silver stained nucleus of a microspread sperinatocyte of M. agrestir. The bivalents have been measured and are numbered from longest to shortest. The silver staining nucleolar material indicated by arrows. Bar = 5 um. The C-banded karyotype of a male carrying the Lund Y is shown (Fig. lb), as are the sex chromo- 5omes of a male carrying the standard Y. As previ.- ously described (FERA 1972; COOPER and Hsu 1972) the distal three quarters of the short arm of the X 3re euchromatic while the proximal quarter, the centromeric region, and the entire long arm are constitutively heterochromatic. In the standard Y.&nost the entire chromosome is heterochromatic. However, also as previously described (PERA 1972: COOPER and Hsu 1972), we found the short arm of the standard Y and its centromeric region weakly stained. In the Lund Y the centronieric region and possibly the distal tip of the short arm are weakly stained, the rest is deeply stained. Meiosis. - The sex chromosome axes are readily identifiable. As is the case in most mammalian species, during the progression of pachynema the sex chromosomes become compartmented in a sex

7 Hereditas I I I (1989) SEX CHROMOSOMES OF MICROTUS ACRESTIS 287 Fig. 5. Two primary spermatocytes (p) in mid-late pachynema exhibiting prominent sex bodies. The micrograph also shows a spermatogonial cell (sg) in late interphaseearly prophase with precondensed sex chromosomes, a round spermatid (rs) and a sperm head (s). Acetic orcein squash. Bar = 10 pm. body which becomes progressively more densely stained than the rest of the nucleus (Fig. 5). Despite the fact that all substages of pachynema were examined by electron microscopy, synapsis between the X and Y was never observed in males carrying either the standard or the Lund Y. However, the unpaired axes of the two chromosomes always lay in proximity to one another. In some cases one end of the X appeared to be in physical association with one end of the Y (Fig. 6b, 7c), and in one case both ends of the X and Y were in contact (Fig. 7a). However, in other cases there was no discernable contact between the two chromosomes (Fig. 6a, 7c). In a few case the two ends of the Y (Fig. 7c), or the X, lay in contact (auto-association). The axes of the sex chromosomes never thickened, a phenomenon observed during the progression of pachynema in many species. However, a region of the axes of bdfhthe Wand Y often exhibited small knobs or bumps (Fig. 6c, d, 7a-c). In Fig. 8 the distribution of the knobs along the X and Y axes of 21 nuclei from males carrying the Lund Y is plotted. It is not possible to definitively distinguish the arms of the X and Y if the kinetochores are not stained. However, since approximately two thirds of the X is heterochromatic, the occurrence of knobs along two thirds of the X axis suggests that the knobs are associated with the heterochromatic portion of that chromosome. As pachynema progresses and the axes of the X and Y become more diffuse, knobs become increasingly difficult to identify. In males carrying the standard Y, most of the population of nuclei observed were in late pachynema and distribution of knobs could not be followed. However, in males carrying the Lund Y, knobs were visible on both the X and Y in many nuclei. Since there were prominent knobs on one end of the Lund Y, the distribution of knobs provided an orientation which allowed us to evaluate specificity of end association. In 7 of 11 nuclei the knobby ends of both the X and Lund Y were in contact (Fig. 6c). In 4 of 11 nuclei the non-knobby end of the X was associated with an end of the Lund Y. In two of these, the end of the Lund Y could not be identified with certainty, but in one the knobby end of the Lund Y was associated with the non-knobby end of the X (Fig. 7b), while in the other, the non-knobby end of the Lund Y was the one associated with the X. Assuming a fixed location of knobs on the axes there appears to be preference, but not an absolute specificity, of end association. Since there is no synapsis between the X and Y, it is important to try to sequence events within pachynema to determine whether or not there is a discernible progressive pattern of behavior of the sex chromosomes, i.e., no end association early, but end association later, a pattern observed in the sand rat (ASHLEY and MOSES 1980). Such specificity of association might account for their normal segregation at Anaphase I. In other species, the amount of synapsis between the X and Y figures prominently in staging. However, additional criteria, such as relative number and appearance of nucleolar material, presence and density of the sex body, etc. have also been used for staging of pachynema (humans by SOLARI 1980; Mus by MOSES 1981 and GUITAR? et al. 1985; Peromyscus by GREENBAUM et al. 1986). In mouse, a sex body is not visible early, but becomes increasingly prominent as pachynema progresses. Similarly, some nuclei in M. agrestis have no discrete sex body and the sex chromosomes are not located on the periphery of the nucleus (Fig. 4; 6c, d) and therefore are assumed to be in early pachynema. In other nuclei, the sex chromosomes are located on the periphery and are surrounded by

8 288 r 4SHLEY ET AL Hereditas I1 I (1989) Fig. 6 a4. The sex chromosome axes from Four M. agresfi.r spermatocytes in pachynema. a and b Males carrying the standard Y chromosome. c and d Males carrying the Lund Y. a and d No ends associated. b One end of the X associated with one end of the Y. Identification of all ends not possible. c The knobby end of the X associated with the knobby end of the Y. Arrows indicate associated ends. Bar = 1 pn.

9 SEX CHROMOSOMES OF MICROTUS AGRESTIS 289 Fig. 7 a-c. The sex chromosome axes from three M. agrestis spermatocytes canying the Lund Y. a Both ends of the X associated with both ends of the Y. b The non-knobby end of the X associated with the knobby end of the Y. Compare with the association in Fig. 6c. c The ends of the Y self associated. Arrows indicate associated ends. Bar = 1 km. a denser staining material, or sex body (Fig. 3; 6a, b). The latter group are assumed to represent a later substage of pachynema. In males carrying either the standard or the Lund Y, the appearance of the nuclei can be divided into two categories: those in which the axes of the sex chromosomes appear solid with a varying number of knobs, or bumps, (average number 10.5 on the X vs. 4.4 on the Y) and on one end only, and those in which the axes appear more diffuse and exhibit few knobs (Fig. 6a, b, d). The former are mostly in early pachynema, as defined above, while the latter are, by the same criteria, mostly in later substages of pachynema. In both categories there are many nuclei in which there is no contact between the ends of the X and Y axes. There is no evidence of progressive association of ends during the course of pachynema. In fact, there appears to be more end association of the X and Y axes in those nuclei judged to be in early pachynema. At metaphase I in squash preparations the sex chromosomes are located together at the periphery of the group of autosomal bivalents, but are not associated end-to-end, nor is there evidence of any direct contact between the X and Y (Fig. 9a-d). Discussion Autosornes. -It is possible to construct a karyotype from electron micrographs of microspread meiocytes

10 290 T ASHLEY ET AL Hereditas 111 (1989) 10 5 Fig. 8. The distribution of knobs along the axes of the sex chromosomes in 21 nuclei of M. agresris carrying the Lund Y. Location of knobs are piotted as distance from the most knobby end of the axes, calculated as percentage of the length of the axes. that parallels the information derived from mitotic ends of 4 or 5 (pairs) of the largest acrocentric karyotyping. As has been previously demonstrated autosomes in a cell line (MaS04) of M. agrestis, by COGNCE and MEVEK (1973) in Locusta. PTA stain- and those of SPERLING et al. (1987), who reported ing allows identification of kinetochores, while NORs on 8 autosomes. measurements (MOSES et al. 1977) of total lengths Fusion of acrocentrics bearing NORs has been and arm ratios of bivalents permits construction of proposed as a mechanism of generation of Roberta synaptonemat complex karyotype which can be sonian translocations. For example, STAHL et al. shown to correspond to one derived from mitotic (1983) have suggested that meiotic exchange metaphase chromosomes. The correspondence between closely associated homologous sequences between rank in both the mitotic and the synaptonemal of rdna of NORs from nonhomologous chromocomplex (SC) complement based on length and the somes at the fibrillar centers of nucleoli during identification of the autosomal metacentric (smallest) pachynema may often be the origin of Robertsoand the NOR-carrying chromosomes suggests a nian chromosomes in humans. Although we found, similar degree of correlation between mitotic and as did GOODPASTURE and BLOOM (1975), mitoticasso- SC karyotypes in M. agrestis. ciation of NORs in M. agrestis cells in culture, we DRFSSFR and MOSES (1980) showed in the Chinese found virtually no association of NOR-carrying hamster that those bivalents carrying the nucleolus bivalents in pachytene spermatocytes, and conorganizing regions (NORs) can be identified in sequently no evidence of opportunity for meiotic microspread preparations of meiocytes stained with exchange in this species. More than 273 wild trapped silver nitrate. In M. agrestis we identified several specimens from 78 localities, mainly in Sweden, bivalents that, by this criterion, carry NORs. We have been studied without finding a single specimen found NOR activity associated with the largest with fused chromosomes (FREDGA and JAAROLA, in bivalents (especially 1-4) in pachytene spermato- prep.). These observations are consistent with the cytcs in animals carrying the Lund Y (Table 1). stability of the karyotype of M agrestis. The only These findings are in close agreement with our report, of which we are aware, of a Robertsonian observations on a small sample of mitotic metaphases fusion in a wild population involves two of the small from another animal carrying the Lund Y (Table acrocentrics in one specimen from Czechoslovakia 1). Mitotic preparations from one male carrying the (ZIMA and KRAL 1984). There is therefore no evistandard Y and one female from a different localiry dence from the present or previous studies that showed more variability (Table 1). These observa- meiotic fusion of acrocentric NOR-bearing chrotions are similar to those of GOODPASTURE and mosomes has been a frequent occurrence in M. Bi 00u (1975). who found NORs at the centromeric agrestis.

11 Hereditas 11 I (1989) SEX CHROMOSOMES OF MICROTUS AGRESTIS 29 1 Fig. 9a-d. Cells in Metaphase I showing the peripheral location and physical proximity between the X and Y without any apparent physical contact (end-toend association). Acetic orcein squash. Bar = 10 pm. Sex chromosomes. -One of the most notable features of the pachytene spermatocytes of M. agrestis is the total lack of synapsis between the X and Y. This asynaptic condition was observed in animals carrying either the standard Y or the Lund Y. Asynapsis of the X and Lund Y also has been reported recently by WOLF et al. (1988). The pericentric inversion of the Lund Y, therefore, does not appear to alter the meiotic synaptic behavior of the sex chromosomes of males carrying it. In M. agrestis the euchromatic portion of the X amounts to approximately 5 % of the genome the size of the original X as defied by OHNO (1967). While the euchromatic region of the Y is very small, the same can be said for the Y in many other mammals. Therefore, if there is a conserved region of homology between the X and Y in mammals, there is no reason to presume it has been lost in M. agrestis. Since synapsis is most frequently initiated at, or near chromosome ends in animals (MOENS 1969), it might be argued that a chromosomal rearrangement has moved the region of conserved homology presumed to be required for synapsis to an interstitial location and that synaptic initiation has thereby been inhibited. However, interstitial synapsis of the X and Y has been noted in Peromyscus sitkensis (HALE and GFEENBAUM 1986) and in Micrornys minutus (SCHMID et al. 1987) providing evidence that terminal initiation need not be obligatory. Since synapsis is not initiated in the euchromatin of the sex chromosomes of this species, the degree of homology of the heterochromatic sequences on the X and Y is of interest. YASMINEH and YUNIS (1975)

12 292 I ASHLEYETAL Hereditas I ll (1989) isolated three repeat DNA sequences from ht. agrestis: two highly repeated sequences that comprise about 8 % of the total nuclear DNA, and one fast intermediate repeat sequence that comprises about 17 YO. When these sequences were in situ hybridized to metaphase chromosomes, each of them (but especially the fast intermediate repeat) preferentially labeled the heterochromatin of both the X and Y, suggesting these chromosomes share homologous sequences. Their results are consistent with the earlier in situ results and the conclusion of ARRIGHI et al. (1970) that There is good homology between the heterochromatin parts of the X and Y in respect to repetitious DNA. If synaptic homology is based on DNA homology, it would appear that the potential for homologous synapsis involving these repeat sequences within the heterochromatin of the X and Y exists. Yet, synapsis between the two was never observed. Both the pericentric autosomal heterochromatin and the sex chromosomal heterochromatin are Giemsa positive in G-banded preparations, a fact which suggests it is AT rich. This assumption is borne out by the staining of these regions with the fluorochrome Hoechst (GROPP et al. 1973). an AT base-specific binding agent. It has recently been suggested that GC-rich Giemsa negative bands may be preferentially involved in synapsis and crossing over (ASHLEY and RUSSELL 1986; ASHLEY 1988; CHA~DLEY 1986). It is tempting to speculate that the AT-rich nature of the heterochromation of the sex chromosomes of M. agrestis may be related to the lack of synapsis between the X and Y, despite their probable molecular homology. However, NANDA et al. (1988) have shown that differences do exist between sequences in the heterochromatic portions of the X and Y in M. agrestis, specifically a GATA sequence that is highly repeated and dispersed on the long arm of the X, which is virtually absent from the Y. Therefore, the idea of strict homology between the heterochromatin of the X and Y must be discarded as too simplistic, and an explanation of the asynapsis between the X and Y remains to be found. In the evolution of asynaptic sex chromosomes with large blocks of heterochromatin an interesting issue u~~~~ an asynaptic xy condition precede or fo~~ow acquisition of the blocks Of heterochromatin? The occurrence of asynaptic sex chromosomes in a related species, M, arvalis (ASHLEY et 1989) with sex chromosomes that are normal both in size and stainability, suggests asynapsis of the sex chromosomes in the genus Microtus mav have preceded acquisition of large blocks of heterochromatin. In the absence of synapsis and (presumably) crossing over, the mechanism of disjunction of the sex chromosomes of M. agrestis is of interest. The presence of knobs along a portion of the axes of the X and Y allowed us to tentatively identify ends. While there appeared to be a preference of end association (knobby end of X with knobby end of the Lund Y). there was not an absolute specificity, as other associations were observed. WOLF et al. (1988) found a similar preference without obligate association. Moreover, although ends lay in close proximity, there was no actual contact between ends in most nuclei in mid-to-late pachynema. This behavior of the sex chromosome ends differed from the sand rat, in which there appeared to be a progressive association of ends of the X and Y, until by late pachynema all four ends were frequently in physical contact (ASHLEY and MOSES 1980). In contrast, in M. agrestis, an end association between the X and Y was observed most frequently in early to mid pachytene nuclei, but as pachynema progressed there appeared to befewer, not more, close contacts. At Metaphase I no end-to-end association, characteristic of most mammals, was observed in squash preparations. It is assumed that meiotic synapsis is a necessary, but insufficient prerequisite for crossing over and subsequent chiasma formation. Although the actual mechanism of disjunction in M. agrestis remains an enigma, since there is no synapsis between the X and Y and no opportunity for crossing over, chiasma disjunction of the sex chromosomes in this species can be excluded. Acknowledgemenu. -We would like to thank Dr. Lennart Hansson for providing us with voles from southern Sweden, and Suzanne Veenhuizen. Marianne Ekwall, Asa Jansson, and Steve Burman for technical assistance. We also thank Dr. Irwin Greenblatt for reading the manuscript and for his constructive comments. This collaboration was made possible by a Thor Grey Travel Award to T. A. from the American Scandinavian Foundation. The financial support by the Erik Philip-SDrensen Foundation, the Nilsson Ehle Foundation, and the Swedish Natural Science Research Council to K. F. is gratefully acknowledged. References ARRIGHI, F. 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