Copyright 2006 by the Genetics Society of America

DOI: 10.1534/genetics.106.061424

The Mechanism of Secondary Nondisjunction in

Drosophila melanogaster Females

Youbin Xiang* and R. Scott Hawley*,,1

*Stowers Institute for Medical Research, Kansas City, Missouri 64110 and Department of Physiology,
University of Kansas Medical Center, Kansas City, Kansas 66160
Manuscript received June 1, 2006
Accepted for publication June 20, 2006

ABSTRACT
Bridges (1916) observed that X chromosome nondisjunction was much more frequent in XXY females
than it was in genetically normal XX females. In addition, virtually all cases of X nondisjunction in XXY
females were due to XX 4 Y segregational events in oocytes in which the two X chromosomes had failed to
undergo crossing over. He referred to these XX 4 Y segregation events as secondary nondisjunction.
Cooper (1948) proposed that secondary nondisjunction results from the formation of an X-Y-X trivalent,
such that the Y chromosome directs the segregation of two achiasmate X chromosomes to opposite poles on
the first meiotic spindle. Using in situ hybridization to X and YL chromosomal satellite sequences, we dem-
onstrate that XX 4 Y segregations are indeed presaged by physical associations of the X and Y chromosomal
heterochromatin. The physical colocalization of the three sex chromosomes is observed in virtually all
oocytes in early prophase and maintained at high frequency until midprophase in all genotypes examined.
Although these XXY associations are usually dissolved by late prophase in oocytes that undergo X chromo-
somal crossing over, they are maintained throughout prophase in oocytes with nonexchange X chromo-
somes. The persistence of such XXY associations in the absence of exchange presumably facilitates the
segregation of the two X chromosomes and the Y chromosome to opposite poles on the developing meiotic
spindle. Moreover, the observation that XXY pairings are dissolved at the end of pachytene in oocytes that do
undergo X chromosomal crossing over demonstrates that exchanges can alter heterochromatic (and thus
presumably centromeric) associations during meiotic prophase.

I N the article that began this journal in 1916, Calvin

Bridges observed that X chromosome nondisjunc-
tion was much more frequent in XXY females than it
was in genetically normal XX females (Bridges 1916).
He further observed that nearly all cases of nondis-
junction in XXY females involved achiasmate X chro-
mosomes and that nondisjunction was caused by the
segregation of the two achiasmate X chromosomes from
the Y chromosome (XX 4 Y segregations). Realizing
that this nondisjunctional process was mechanistically
different from whatever nondisjunctional processes
occurred in genetically normal females, he dubbed the
nondisjunction observed in XXY females secondary
nondisjunction.
Bridges initial observation that the vast majority
(97%) of secondary nondisjunction events involved
nonexchange (E0) X chromosomal bivalents has been
further supported by two lines of evidence. First, a
thorough analysis of exchange and nondisjunction in
This article is dedicated to Kenneth W. Cooper, who inspired the senior
author by the high quality of his science throughout his career and by
his unparalleled role as a teacher and mentor over 30 years ago at the
University of California at Riverside.
1
Corresponding author: Stowers Institute for Medical Research, 1000 E.
50th St., Kansas City, MO 64110. E-mail: rsh@stowers-institute.org
XXY females performed by both Sturtevant and
Beadle (1936) and by OTousa (1982) confirmed
Bridges original finding that 9096% of secondary
nondisjunctional events involved E0 tetrads. [However,
Carpenter (1973) obtained a somewhat lower fre-
quency of E0 tetrads (75%) among the secondary non-
disjunctional events observed in her study]. Similarly,
the limited cytological evidence available suggests that
the X chromosomes that undergo nondisjunction in
XXY females are usually, although not invariably,
achiasmate (Puro and Nokkala 1981). The cases
of secondary nondisjunction that do involve exchange
X chromosomes predominantly involve X chromo-
somal bivalents that carry a single very distal exchange
(Carpenter 1973; Luning 1982; OTousa 1982).
Second, the frequency of secondary nondisjunction is
greatly elevated in females in which X chromosomal
exchange has been reduced, either as a consequence of
the presence of recombination-defective meiotic mu-
tants (Carpenter and Sandler 1974) or as a conse-
quence of heterozygosity for paracentric inversions
(Sturtevant and Beadle 1936; Cooper 1948). How-
ever, the presence of structural heterology alone does
not appear to influence the probability that an oocyte
with two achiasmate X chromosomes and a Y chromo-
some will undergo nondisjunction; the proportion of E0

Genetics 174: 6778 (September 2006)

68 Y. Xiang and R. Scott Hawley
tetrads that undergo nondisjunction in FM7/X/Y fe-
males (7080%; Cooper 1948) is roughly similar to the
fraction of E0 tetrads that nondisjoin in XXY females
bearing two normal-sequence X chromosomes (66
83%; OTousa 1982). Thus, the ability of the Y chro-
mosome to induce two X chromosomes to segregate to
the opposite pole at anaphase I depends on the ex-
change status of the X chromosome bivalent, and not on
structural heterozygosity. And the probability that two
nonexchange X chromosomes will undergo secondary
nondisjunction in an XXY female is not affected by
sequence divergence. The frequency of secondary
nondisjunction in otherwise genetically normal XXY
females carrying isogenic X chromosomes is similar to
that observed in females that carry different normal-
sequence X chromosomes (Rutherford and Carpenter
1988).
One could imagine two causes for elevated X chro-
mosomal nondisjunction in XXY females that do not
involve X and Y chromosomal pairing. First, the Y might
reduce the amount or distribution of X chromosomal
exchange. Such a reduction in exchange would then
lead to an increase in nondisjunction, as is commonly
observed for recombination-defective mutants. How-
ever, Y chromosomes do not reduce the frequency of
exchange in XXY females bearing normal-sequence X
chromosomes (cf. Bridges 1916; Sturtevant and
Beadle 1936; OTousa 1982; Ashburner et al. 2005).
Indeed, the presence of the Y chromosome usually
slightly increases X exchange, as measured among reg-
ular gametes, in XXY females with normal-sequence X
chromosomes. Similarly, although the Y chromosome
can induce small increases and decreases in the resid-
ual exchange observed in inversion heterozygotes and
some inversion homozygotes (Grell 1962; Merriam
1967), these changes are too small to account for the
high levels of nondisjunction observed in XXY females
heterozygous for these inversions. Second, the kinds of
exceptional offspring recovered from XXY females
demonstrate that the ability of the Y chromosome to
induce secondary nondisjunction is not simply a nega-
tive effect of the presence of a Y chromosome in the
female germline on the fidelity of X chromosome
disjunction. If this were true, then the segregation of
two X chromosomes to the same pole should be
concomitant with the random segregation of the Y
chromosome, and XXY oocytes and oocytes carrying
no sex chromosomes would be as common as XX- or
Y-bearing oocytes. Such XXY- or 0-bearing oocytes are
only rarely observed (see below).
To explain this phenomenon, Bridges (1916) ini-
tially suggested that XX 4 Y segregational events oc-
curred as a consequence of competitive pairing and
synapsis of the Y chromosome with one of the two X
chromosomes. He proposed that one X paired with and
segregated from the Y chromosome, while the remain-
ing X chromosome segregated at random (see Figure 1A).
Figure 1.Models of secondary nondisjunction in XXY fe-
males. (A) Bridges (1916) model of secondary nondisjunc-
tion in which the Y chromosome pairs with, and segregates
from, only one of the two X chromosomes. Because the un-
paired X is free to segregate at random, it will segregate only
from the Y in 50% of the cases. Thus, the maximum frequency
of secondary nondisjunction cannot exceed half the fre-
quency of nonexchange X chromosomes. (B) Coopers
(1948) model of secondary nondisjunction in which each
of the two arms of the Y chromosome pairs with the hetero-
chromatin of one of the two X chromosomes, creating an X-Y-
X trivalent. The Y chromosome then directs the segregation
of the two X chromosomes to opposite poles. This model al-
lows the frequency of secondary nondisjunction to be sub-
stantially higher than half the frequency of nonexchange X
chromosomes.
To quote Bridges (1916, p. 116), That synapsis in an
XXY female does not involve all three chromosomes at
once, but is between two of them with the third
chromosome left unsynapsed, is proved by the fact that
the chromosomes in XX eggs are never crossovers, while
the Xs of the X and XY eggs are crossovers in about the
usual percent. A difference in the paths followed by
these two chromosomes originated before the stage at
which crossingover became possible. As shown in
Figure 1, a crucial prediction of this model is that the
frequency of secondary nondisjunction cannot exceed
50% of the frequency of E0 bivalents because the un-
synapsed X chromosome will cosegregate only with the
X chromosome in 50% of the cases. Thus, the maximum
observed frequency of nondisjunction will be 33% of
the E0 frequency because of the inviability of XXX
and 0Y embryos.
The studies of secondary nondisjunction in inver-
sion heterozygotes by both Sturtevant and Beadle
(1936) and Cooper (1948) necessitated a modification
of Bridges model. These studies obtained observed
frequencies of secondary nondisjunction of 60% in fe-
males in which the X chromosomes were heterozygous
for one or more inversions that strongly suppressed the
actual occurrence of exchange. When these measure-
ments are corrected for the inviability of XXX and 0Y
embryos, they yield estimates of the true frequency of
secondary nondisjunction in the range of 70%. Both
 Secondary Nondisjunction in D. melanogaster Females
Sturtevant and Beadle (1936) and Cooper (1948)
realized that these frequencies of nondisjunction sug-
gested that the vast majority (8090%) of E0 bivalents
underwent secondary nondisjunction in XXY females.
Similar observations of high frequencies of second-
ary nondisjunction in inversion and balancer hetero-
zygotes have been made by others (cf. Gershenson
1935; Zitron and Hawley 1989; Zhang and Hawley
1990). These observations clearly contradict Bridges
original competitive pairing model, which predicts a
maximum frequency of secondary nondisjunction of
50% (when corrected for the inviability of XXX and 0Y
embryos), even in a population of oocytes in which the
E0 frequency was 100%. Cooper proposed instead that
secondary nondisjunction reflects the formation of an
X-Y-X trivalent such that each arm of the entirely het-
erochromatic Y chromosome pairs with and thus co-
orients the heterochromatic regions of one of the two
X chromosomes (see Figure 1B). This trivalent then
directs the XX 4 Y segregations that are observed as
secondary nondisjunction.
Cooper (1948) proposed that structural dissimilar-
ities within the euchromatin caused by inversion het-
erozygosity created a situation where the strongest X-X
pairings would involve the heterochromatin, a region
with substantial homology to the Y chromosome. Thus,
in the absence of proper euchromatic synapsis and
subsequent crossing over, pairing would be deter-
mined primarily by the heterochromatic and Y homol-
ogous regions of the X chromosome. To quote Cooper
(1948, p. 183), a moments reflection will bring convic-
tion that were the euchromatic lengths of the two X
chromosomes to be made wholly dissimilar, but the so-
called inert or chromocentral regions to remain essen-
tially unaltered, then conjunction between the two Xs
would become a primarily heterochromatic affaira
process occurring almost exclusively between the chro-
mocentral regions . . . those regions of the X wherein
pairing with the Y normally occurs. Indeed, Cooper
proposed, that both arms of the Y chromosome share
homology with the X and thus may conjoin with an X.
Thus, Coopers model differs from Bridges explana-
tion in two crucial ways. First, Bridges proposes that
the Y actually competes with the second X chromosome
to pair with the first, and in doing so prevents X chro-
mosome synapsis and pairing, while Cooper proposes
that the Xs associate with the Y chromosome only
when euchromatic synapsis and crossing over fails or is
prevented by structural heterozygosity. Second, while
Bridges proposes that one X segregates from the Y and
the remaining X segregates at random, Cooper propo-
ses that the three sex chromosomes form a simple tri-
valent that usually segregates the two Xs to one pole and
the Y to the other (see Figure 2 of Cooper 1948).
Consistent with this hypothesis of an X-Y-X trivalent,
Cooper (1948) demonstrated that a one-armed Y chro-
mosome derivative (Y S) shows a much reduced ability to
69
induce secondary nondisjunction when compared to a
structurally normal metacentric Y chromosome [i.e., the
frequency of secondary nondisjunction in In(1)dl-
49,BM1/1/YS females is 36% compared to 78% in
In(1)dl-49,BM1/1/Y females]. Moreover, Y chromosomes
differ in their effects on the frequency of secondary
nondisjunction (Neuhaus 1941; Cooper 1948). These
differences appear to be related to the size of the Y.
Deleted Y chromosomes [i.e., R(YL), scV1.YS] have a
much weaker effect than normal-size Y chromosomes
(Chadov 1981). This observation supports the inter-
pretation that the cause of secondary nondisjunction is
a physical interaction between the two X chromosomes
and both arms of the Y chromosome.
Coopers trivalent model was given credence by the
finding of similar heterochromatic sequences, most
notably the AATAT and AAGAG satellites as well as the
rDNA, on both the X and Y chromosomes (Lohe et al.
1993). More important, the suggestion that such ho-
mologies might facilitate both stable heterochromatic
pairings and achiasmate segregation is given real cre-
dence by the observations of Dernburg et al. (1996),
who demonstrated that meiosis in Drosophila melano-
gaster females exhibits a modified diplotene-like stage
in which heterochromatic associations are maintained
until the end of prophase while euchromatic pairings
are dissolved at the end of pachytene. These hetero-
chromatic pairings can be maintained even during pro-
metaphase (Dernburg et al. 1996 and below) and
Hawley et al. (1993) and Karpen et al. (1996) demon-
strated that these heterochromatic pairings were both
necessary and sufficient to ensure the segregation of
achiasmate homologs. Finally, the ability of a meta-
centric partner of a trivalent to direct the segregation of
its two acrocentric homologs to the opposite poles is
well documented by classic and modern cytogenetic
studies (cf. MacKnight and Cooper 1944; Haaf et al.
1989). Thus, it seems fully reasonable to propose that
the heterochromatic regions of X chromosomes, which
surround the centromere, actually do pair with the en-
tirely heterochromatic Y chromosome and that, in the
absence of exchange, these pairings are sufficient to
ensure segregation.
Finally, Grell (1962, 1976) has proposed a model of
secondary nondisjunction that is similar to Coopers in
suggesting that the X chromosomes that failed to sy-
napse and crossover were free to form a trivalent with a
metacentric Y chromosome, or indeed with any meta-
centric chromosome, during a postulated second round
of so-called distributive pairing that was presumed to
be homology independent. It is, however, clear from
numerous cytological studies that no such secondary
round of pairing exists (Hawley and Theurkauf 1993;
Dernburg et al. 1996). Moreover, it is equally clear
that the homology-independent segregations on which
Grell based her model for secondary nondisjunction
(such as those involving compound autosomes) do not
70 Y. Xiang and R. Scott Hawley
involve the physical interaction of segregating chromo-
somes (Dernburg et al. 1996).
For those reasons, Coopers 1948 model for second-
ary nondisjunction has been the predominant explana-
tion for this phenomenon and has been widely accepted
and taught for the last six decades. However, until now it
has never been directly tested. Here we use the tech-
nique of fluorescence in situ hybridization (FISH) to X
and Y chromosomal satellite sequences developed by
Dernburg et al. (1996) to study the association of X
and YL heterochromatin in meiotic prophase in XXY
oocytes in which exchange has or has not occurred. In
these studies we suppress exchange by using either the
highly rearranged balancer chromosome FM7 or an X
chromosome bearing the euchromatic inversion In(1)dl-
49, which suppresses X exchange without disrupting the
homology of the pericentric X heterochromatin. These
chromosomes are diagrammed in Figure 2.
We demonstrate that the XXY associations occur at
high frequency in early prophase and persist through
midmeiotic prophase for all genotypes studied. These
XXY associations are usually lost by late prophase in oo-
cytes that are free to undergo X chromosomal crossing
over. However, XXY associations are maintained until
late prophase in XXY oocytes in which X chromosomal
exchange fails to occur. We presume that those XXY
associations that persist until prometaphase create the
trivalent proposed by Cooper and thus facilitate the
segregation of the two X chromosomes and the Y chro-
mosome to opposite poles on the developing meiotic
spindle. Thus, the XX 4 Y segregations observed in
exchange-suppressed oocytes are indeed presaged by
physical associations of the X and Y chromosomal het-
erochromatin that persist throughout meiotic prophase.
However, our data disagree with Coopers view that
XXY associations are formed following failure of the two
X chromosomes to properly synapse and crossover. First,
Gong et al. (2005) demonstrated that in FM7/X females
the FM7 balancer and the normal-sequence X chromo-
some pair and synapse normally. Second, our data argue
that XXY associations are visible from the beginning of
meiotic prophase in FM7/X/Y females and are usually
maintained until the end of prophase.
The fact that the early XXY associations observed in
oocytes that undergo X chromosomal exchanges are
dissolved following the end of the pachytene demon-
strates that exchanges can alter heterochromatic (and
thus presumably centromeric) associations long before
nuclear envelope breakdown (NEB). These observa-
tions are consistent with those of Kemp et al. (2004) and
Tsubouchi and Roeder (2005) in yeast, which demon-
strated a role of exchange in controlling centromeric
associations prior to NEB.
MATERIALS AND METHODS
Drosophila stocks: In this study, the normal X, which carries
the markers y and w, was obtained from a stock that also carries
the y1Y chromosome. The FM7a,w chromosome (denoted as
FM7), which is marked with y sc w v and B, is derived from a
stock that is homozygous for FM7a and also carries y1Y. In(1)dl-
49 is marked with y Hw m and g. The normal Y chromosome
was obtained from our copy of the Oregon-R stock.
Crossing schemes: To obtain FM7/FM7/y1Y females, rare fe-
males that were phenotypically non-yellow, white, and strong-
Bar were selected from the FM7 stock. These FM7/FM7/
y1Y exceptional females were then continuously crossed with
FM7/y1Y males to create a stock. Similarly, rare exceptional yw/
yw/ y1Y were obtained from our male yw/y1Y stock. These yw/
yw/ y1Y females were then crossed with yw/y1Y males to
continuously produce yw/yw/y1Y female offspring. These
females are denoted by the designation X/X/y1Y. Both
stocks were selected from every generation to maintain X/X/
y1Y females. To obtain In(1)dl-49/X/y1Y females, FM7/yw/y1Y
females were crossed with In(1)dl-49, y Hw m g/Y males and the
yellow-plus, white-plus Hairy-wing non-Bar females with the
genotype of In(1)dl-49, y/yw/y1Y [denoted In(1)dl-49/X/Y]
were collected. To create XXY females bearing a normal-
sequence Y chromosome, we crossed either y w/y w/y1Y or
FM7/y w/y1Y females with w1/Y (Oregon-R) males and
obtained the desired yellow y w/y w/Y (denoted simply as
XXY) or FM7/y w/Y offspring (denoted FM7/X/Y).
Secondary nondisjunction assays: To measure the fre-
quency of secondary nondisjunction, females for each geno-
type were crossed individually to attached-XY, y1 v f B; C(4)RM, ci
eyR males. For details of this cross and its analysis, see Hawley
et al. (1993) and Harris et al. (2003). Although this cross does
allow us to measure fourth chromosome nondisjunction as
well as X chromosome nondisjunction, fourth chromosome
exceptions were rare in all genotypes examined and are not
considered here.
Probes for in situ hybridization: The 1.686 satellite sequen-
ces (also known as the 359-bp repeats) on the X chromosome
and AATAC repeats on the Y chromosome were chosen
as probes for in situ hybridization (Dernburg et al. 1996;
Dernburg 2000). The 1.686 satellite is located in the
pericentromeric region on both normal X chromosomes and
the euchromatic inversion X chromosome, In(1)dl-49 (Figure
2). The FM7 balancer chromosome displays two blocks of
hybridization of the 1.686 sequence. Due to In(1)sc8, one of
the three inversions that composes FM7, the 1.686 satellite
sequence was separated into two parts on this balancer chro-
mosome: the larger region of satellite sequence located near
the distal tip of the chromosome and the smaller region of
1.686 satellite sequences located at the centromere region (see
Figure 2). Thus, as shown in Figure 3, in prometaphase oocytes
the FM7 and X chromosomes can be easily differentiated
because the normal-sequence X chromosome has one large
block of 1.686 signal near its centromere, while FM7 has a
small block of 1.686 signal at its base (near the centromere)
and a larger block of 1.686 signal near its distal tip. The
position of the long arm of the Y chromosome (YL) was
assessed by hybridization using the AATAC repeats, which are
located medially on the long arm on the Y chromosome
(Figure 2). The y1Y chromosome also bears the tip of the
In(1)sc8 chromosome appended to the distal tip of YL. This
fragment of X contains a large block of X heterochromatin,
including a large block of 1.686 satellite sequence, resulting in
both X and Y signals being observed for the y1Y chromosome.
The 359-bp sequence of 1.686 satellite and (AATAC)6 were
used for probe preparation. The 30-nucleotide oligos for both
strands of the 359-bp sequence and (AATAC)6 for AATAC
repeats were generated by Integrated DNA Technologies.
The synthesized oligos for the 359-bp sequence were pooled
in equal molar amounts and a total 10 mg of pooled oligos
or (AATAC)6 were used for fluorescent probe labeling. The
 Secondary Nondisjunction in D. melanogaster Females 71

TABLE 1
Comparision of nondisjunction in X/X and X/X/Y Drosophila females

Sperm
Oocyte genotype genotypea X/Xb X/X/Y X/X/y1Y FM7/X FM7/X/Y FM7/X/y1Y dl-49/Xb dl-49/X/y1Y FM7/FM7/y1Y
Normal
X; 4 XY; 44 970 582 743 614 244 684 3761 127 792
X; 4 0; 44 1707 573 1333 584 232 672 2111 231 363
X nondisjunction
0; 4 XY; 44 1 7 19 1 270 409 16 94 7
XX; 4 0; 44 2 12 21 2 307 668 12 68 11
Total progeny 2680 1174 2128 1201 1054 2433 5900 521 1181

Adjusted total 2683 1193 2168 1204 1631 3502 8039 683 1199

% nullo-X 0.1 1.2 1.8 0.2 33.1 23.4 0.4 19.9 1.2
% diplo-X 0.1 2 1.9 0.3 37.7 37.9 0.3 27.5 1.8
Total % X 0.2 3.2 3.7 0.5 70.8 61.3 0.7 47.4 3.0
nondisjunction
a
The genotype of male tester is attached-XY, y1 v f B; C(4), ci eyR.
b
The control data are from Zitron and Hawley (1989).

probes were labeled by using terminal deoxynucleotidyl trans-
ferase (Roche) to incorporate 5-(3-aminoallyl)-dUTP (Molec-
ular Probes, Eugene, OR) onto the 39-terminus as described
in Dernburg et al. (1996) and Dernburg (2000). The 5-(3-
aminoallyl)-labeled oligos were conjugated to a reactive
fluorescent dye by using the ARES Alexa Fluor DNA labeling
kit (Molecular Probes) followed by column purification as
described in the kit. For probes of the 359-bp sequence on the
X chromosome, Alexa Fluor 488 dye was used. For probes of
(AATAC)6 on the Y chromosome, Alex Fluor 647 dye was used.
The labeled probes were diluted in TE buffer at a concen-
tration of 50100 ng/ml and kept at 80 for in situ hybridiza-
tion use.
Egg chamber dissection and fixation: Approximately 3040
females, which had enclosed 23 days previously, were mated
to 510 males and fed on yeast for 3 days prior to egg chamber
dissection. These females were then anesthetized and the
abdomens were ruptured one by one with forceps. Whole
ovaries were collected and kept in 13 Robbs solution during
the dissection. The ovaries were then transferred to 1.0 ml
prewarmed fixation solution (4% formaldehyde, 100 mm Na
cacodylate, pH 7.2, 10 mm EGTA) on a dissecting plate to fix
for 4 min. Oocytes were staged according to their morphology
as described in Spradling (1993). Basically, the oocytes that
composed less than one-third of the volume of their egg
chamber were deemed to be in midprophase (stages 29). The
oocytes that composed one-half or more of the volume of the
egg chambers, but which lacked dorsal filaments, were con-
sidered to be in late prophase (stages 1012). The oocytes in
which the dorsal filaments were visible, and for which few
or no nurse-cell nuclei remained, were considered to be in
prometaphasemetaphase (stages 1314). During the fixa-
tion, ovaries were teased apart using forceps. After fixation,
the egg chambers were quickly transferred to 23 SSCT (0.3 m
NaCl, 0.03 m Nacitrate, pH 7.2, 0.1% Tween-20) and washed
with 23 SSCT four times for 10 min each wash.
Fluorescent in situ hybridization and microscopy: FISH
was performed as described in Dernburg (2000) with only
the following slight modifications. Fixed egg chambers were
incubated successively in 20, 40, and 50% formamide-containing
23 SSCT for 10 min each. The egg chambers were then
transferred to fresh 23 SSCT with 50% formamide (Fluka)
and incubated at 37 for at least 1 hr. The solution was as-
pirated carefully and the egg chambers were left at the bottom.
Two microliters of each X and Y chromosome fluorescence-
labeled DNA probe solution was combined with 36 ml of hy-
bridization solution containing 10% dextran sulfate (Fluka),
33 SSCT, and 50% formamide (Fluka) and the solution was
added to the egg chambers. The egg chambers with probes
were denatured at 94 for 2 min followed by incubation for hy-
bridization at 30 overnight. When hybridization was finished,
the egg chambers were washed in 23 SSCT with 50% formam-
ide three times at 37 for 10 min/wash, followed by two washes
in 23 SSCT with 40 and 20% formamide, respectively, at room
temperature. The egg chambers were then washed twice in 23
SSCT for a total of 30 min, stained for 10 min in 23 SSCT
with 0.5 mg/ml DAPI, and rewashed four times in 23 SSCT
for a total of 40 min. The egg chambers were mounted on
slides in Vectashield (Vector Laboratories, Burlingame, CA)
for analysis.
Microscopy was conducted using a DeltaVision microscopy
system (Applied Precision, Issaquah, WA) equipped with an
Olympus IX70 inverted microscope and high-resolution CCD
camera. The image data were deconvolved using the softWoRx
v.25 software (Applied Precision) and projected with multiple
stacks. In all prophase oocytes examined, the two X chromo-
somes were observed as a single bright mass of hybridization,
and thus, as previously shown by Dernburg et al. (1996),
X heterochromatin remains paired throughout prophase.
(Individual X chromosomes were not observed as separate
entities until prometaphase; see Figure 3.) However, as shown
in Figure 4, the Y chromosome can be shown to be either
physically associated or not associated with the X chromosome
during prophase I by examining the colocalization of the
1.686 and AATAC hybridization signals.
RESULTS
Genetic analysis of secondary nondisjunction in the
genotypes used in this study: The results of measuring
X chromosomal nondisjunction in both XX and XXY
females are reported in Table 1. The crosses reported
72 Y. Xiang and R. Scott Hawley
Figure 2.Sites of hybridization for the heterochromatic
probes used in this study. The 1.686 satellite sequences on
the X chromosome and the AATAC repeats on the Y chromo-
some were chosen as probes for in situ hybridization (Dernburg
2000). The 1.686 satellite is located in the pericentric hetero-
chromatin on both the normal-sequence X chromosome and
the X chromosome carrying the euchromatic inversion
In(1)dl-49. The 1.686 satellite sequence is separated into
two parts on the FM7 balancer chromosome; the larger block
of satellite sequences is near the distal tip of the chromosome
and the smaller region of 1.686 satellite sequences is located
near the centromere. Thus, as shown in Figure 3, in prome-
taphase oocytes the FM7 and X chromosomes can be easily dif-
ferentiated because the normal-sequence X chromosome has
one large block of 1.686 signal near its centromere, while FM7
has a small block of 1.686 signal near the centromere and a
larger block of 1.686 signal near its distal tip. The AATAC re-
peats are located medially on the long arm of the Y chromo-
some. The y1Y chromosome also carries the tip of the In(1)sc8
chromosome appended to the distal tip of YL. This fragment
of the X chromosome contains a large block of 1.686 satellite
sequence, resulting in both X and Y signals being observed for
the y1Y chromosome.
here involve three different X chromosomes, whose
structures are portrayed in Figure 2: a normal-sequence
X chromosome, the FM7 balancer chromosome, and a
chromosome bearing the entirely euchromatic inver-
sion In(1)dl-49. Multiple lines of evidence suggest that
FM7 fully blocks exchange when heterozygous, while
heterozygosity for In(1)dl-49 appears to reduce the total
frequency of X chromosomal exchange to 7080% of
normal (Novitski and Braver 1954; Roberts 1962;
Theurkauf and Hawley 1992; Gong et al. 2005).
We also used two separate Y chromosomes: a normal-
sequence Y chromosome derived from our Oregon-R
stock and the y1Y chromosome.
As shown in Table 1, the frequency of secondary
nondisjunction is low (3%) in XXY females in which
the X chromosomes are free to undergo crossing over,
but quite high in FM7/X/Y females in which exchange is
absent. The frequencies of X nondisjunction that are
observed in XXY and X/X/y1Y females are 3.2 and 3.7%,
respectively. Twenty-fold higher frequencies of X non-
disjunction are observed in FM7/X/Y and FM7/X/y1Y
females (70.8 and 61.3%, respectively). These frequen-
cies of Y-induced nondisjunction can be compared to
the frequency of X chromosomal nondisjunction in
either genetically normal XX females or exchange-
suppressed FM7/X females, which are usually in the
range of 0.10.2% (Ashburner et al. 2005; Gong et al.
2005). The presence of the marked y1Y chromosome in
X/X/y1Y and FM7/X/y1Y females allows us to conclude
that nearly all cases of X nondisjunction in XXY females
were due to XX 4 Y segregations. For example, 100%
of the instances of X nondisjunction observed in X/
X/y1Y females and 99.6% instances of X nondisjunc-
tion observed in FM7/X/y1Y females reflected XX 4 Y
segregations.
Similarly, in females heterozygous for In(1)dl-49,
which reduces exchange by 7080%, the frequency
of X nondisjunction is quite low (0.7%) (Novitski
and Braver 1954; Roberts 1962; Zitron and Hawley
1989; Whyte et al. 1993). However, a 50-fold higher
frequency of X nondisjunction is observed in In(1)dl-49/
X/y1Y females (47.4%). The presence of the marked y1Y
chromosomes reveals that all of the 162 instances of X
nondisjunction observed in In(1)dl-49/X/y1Y females
were the result of XX 4 Y segregations, and no off-
spring derived from XXY- or 0-bearing oocytes were
obtained.
We should note that while the frequency of second-
ary nondisjunction in In(1)dl-49/X/y1Y females observed
here (47.4%) is similar to the frequency of secondary
nondisjunction observed in In(1)dl-49/X/Y females by
Sturtevant and Beadle (1936) and by Zitron and
Hawley (1989) (45.6 and 56.4%, respectively); it is sub-
stantially lower than the frequency of 70% obtained for
In(1)dl-49/X/Y females by Cooper (1948). However,
Cooper also obtained a frequency of secondary non-
disjunction of 80% in In(1)dl-49/In(1)AM/Y females,
exceeding the values of 6170% that we observe in FM7/
X/Y females, in which exchange is fully suppressed. The
basis for these differences is not understood, but it pre-
sumably reflects the effect of genetic background on the
efficacy of the Y chromosome in directing XX 4 Y segre-
gations. Indeed, Neuhaus (1941) noted that different
wild-type Y chromosomes yielded different frequencies
of secondary nondisjunction for the same X chromo-
somal pairs.
Thus our observations reprise those of Bridges
(1916), Gershenson (1935), Sturtevant and Beadle
(1936), and Cooper (1948) in demonstrating that the
frequency of secondary nondisjunction increases with
the fraction of E0 tetrads (achiasmate X chromosomes)
and that virtually all cases of secondary nondisjunction
reflect XX 4 Y segregations. Finally, on the basis of esti-
mates of E0 frequencies in XX, FM7/X, and dl-49/X fe-
males (810, 100, and 7080%, respectively; Ashburner
et al. 2005), it seems clear that a high fraction of
 Secondary Nondisjunction in D. melanogaster Females
nonexchange X chromosome pairs participate in sec-
ondary nondisjunction.
Direct cytological visualization of XX 4 Y co-
orientation in prometaphase and metaphase: In oocytes
in which the X chromosomes are free to undergo
crossing over, an analysis of chromosome orientation
in stage 13 (prometaphase) to stage 14 (metaphase)
oocytes reveals that the two X chromosomes are usually
oriented toward opposite poles of the meiotic spindle
73
with the Y or y1Y chromosome usually located on one of
the two half-spindles (see Figure 3, A and B, and Table
2). In all of the XXY and X/X/y1Y oocytes examined, the
two centromeric regions of the two X chromosomes are
observed at opposite ends of the main mass of chiasmate
chromosomes, with the Y or y1Y chromosome usually
located between the main mass and one of the two poles
(see Figures 3, A and B, and Table 2). No cases were
observed in which the X chromosomes appeared to be
segregating from the Y chromosome (N 72). How-
ever, given that the observed frequency of secondary
nondisjunction in these crosses is only 3.0% (see Ta-
ble 1), our failure to observe such events is perhaps not
surprising.
However, cases in which the two X chromosomes were
oriented to one pole with the Y chromosome oriented
toward the opposite pole were frequently observed
when X chromosome exchange is prevented by hetero-
zygosity for FM7 (see Figures 3, C and D, and Table 2). As
noted in Table 2, the frequencies of XX 4 Y orienta-
tions in FM7/X/Y and FM7/X/y1Y oocytes (69.1 and
67.1%, respectively) correspond well to the frequencies
of XX 4 Y segregations observed genetically (70.8 and
61.3%). To ensure that the high levels of XX 4 Y co-
orientation events observed in FM7/X/Y females were
not simply a consequence of the structural aberrations
associated with the FM7 balancer chromosome, we also
examined prometaphase orientation in FM7/FM7/y1Y
oocytes, which have near normal levels of recombina-
tion (Zhang and Hawley 1990). In all 23 FM7/FM7/y1Y
oocytes imaged, the centromeric regions of the two FM7
Figure 3.Centromere co-orientation in prometaphase/
metaphase oocytes as detected with FISH. In XXY (A) and
X/X/y1Y (B) oocytes, in which the X chromosomes were free
to crossover, the centromeres of two X chromosomes were al-
ways at the ends of the main mass of chiasmate chromosomes,
with the two homologous centromeres oriented toward oppo-
site poles. The Y chromosome is virtually always observed be-
tween the main mass and one of the two poles of the spindle.
However, in oocytes in which X chromosomal exchange is
fully suppressed by the presence of the FM7 balancer chromo-
some, both the X and FM7 chromosomes are often oriented
toward one pole with the Y chromosome oriented toward the
other. Such orientations will result in XX 4 Y segregations.
(C) Depicts such a metaphase figure from an FM7/X/Y oocyte.
(D) Depicts a similar metaphase figure from an FM7/X/y1Y
oocyte. In both figures, the two Xs and the Y chromo-
some are oriented toward opposite poles. The frequencies
of XX 4 Y orientations for each genotype studied are pre-
sented in Table 2. As shown in E, XX 4 y1Y segregational
events are also commonly observed in In(1)dl-49/X/Y oocytes.
As shown in F, in FM7/FM7/y1Y females in which the FM7
chromosomes were free to crossover (Zhang and Hawley
1990), the centromeres of two X chromosomes were always
oriented toward opposite poles. However, as indicated by
the arrow in F, the distal heterochromatic blocks of the two
homologs usually remained associated at the metaphase plate
(see text). The Y chromosome is seen associated with either of
the two poles. Red bar, 2 mm.
74 Y. Xiang and R. Scott Hawley
TABLE 2
Centromere co-orientation during secondary nondisjunction
Genotype Total XY 4 X
FM7/X/Y 68 21
X/X/Y 37 37
FM7/X/y1Y 76 24
X/X/y1Y 35 35
In(1)dl-49/X/y1Y 31 17
FM7/FM7/Y 23 23
chromosomes are associated with opposite poles of the
spindle (see Figure 3F).
Moreover, high levels of XX4Y co-orientation events
were also observed in X exchange-suppressed females
that carried homologous blocks of structurally normal
X heterochromatin [In(1)dl-49/X/y1Y]. As shown in
Figure 3E and Table 2, XX 4 Y co-orientations were
commonly observed in In(1)dl-49/X/y1Y oocytes. This cy-
tologically observed frequency of XX 4 Y co-orientations
is comparable to the frequency of secondary nondis-
junction (47.4%) obtained genetically. While the fre-
quencies of XX 4 Y co-orientation and segregation
events observed in In(1)dl-49/X/y1Y oocytes are lower
than the frequencies with which such events are observed
in FM7/X/Y and FM7/X/y1Y, it must be remembered
that, unlike FM7, the In(1)dl-49 chromosome still allows
a substantial frequency of X exchange (Sturtevant
and Beadle 1936; Novitski and Braver 1954; Roberts
1962). Thus, the nondisjunctional events observed by
genetic tests appear to be an accurate reflection of the
real frequency of XX 4 Y segregational events.
To determine whether or not these XX 4 Y segrega-
tions were presaged by XXY pairings during meiotic
prophase as predicted by Cooper (1948), we set out to
examine X and Y chromosomal associations prior to nu-
clear envelope breakdown. As shown below, such associa-
tions are common in oocytes of all the tested genotypes
in early to late prophase, but are maintained only until
the end of prophase in exchange-suppressed oocytes.
The heterochromatic regions of the X and Y chro-
mosomes are physically associated during early to
midprophase in all genotypes studied: To examine XY
associations in early prophase, we asked whether or not
the Y-specific AATAC probe colocalized with the 1.686 X
chromosomal probe in the germarial cells of FM7/X/Y,
FM7/X/y1Y, and X/X/y1Y females (N 91, 99, and 89,
respectively). In each of these cases, the Y chromosome
was physically associated with the X chromosomes in at
least 93% of the cells (data not shown). Cases in which
the Y chromosome signal was clearly separated from the
% XX 4 Y segregation
As assayed As assayed
XX 4 Y cytologically genetically
47 69.1 70.8
0 0.0 3.2
51 67.1 61.3
0 0.0 3.7
14 45.2 47.4
0 0.0 3.2
X chromosomes were observed in ,7% of the oocytes
for all three genotypes.
As noted above, following synaptonemal complex
dissolution at the end of pachytene, meiotic chromo-
somes in Drosophila enter an extended and unusual
diplotene-like phase where euchromatic regions desyn-
apse but heterochromatic regions remain tightly paired
(Dernburg et al. 1996). To determine whether the X
and Y chromosomes remained associated during this
period of middle prophase, we examined X and Y chro-
mosomal associations during stages 29 of oogenesis.
As shown in Figure 4 and quantified in Table 3, XXY
associations during midprophase were observed in at
least 50% of the oocytes in all genotypes studied. In
oocytes in which X chromosomal exchange has been
suppressed [those of FM7/X/Y, FM7/X/y1Y, and In(1)dl-
49/X/y1Y females], the observed frequencies of such
associations (6769%) correlate well with the observed
frequencies of secondary nondisjunction (7173%).
However, for exchange-competent oocytes (XXY, X/X/
y1Y), the observed frequencies of XXY associations in
mid-prometaphase (5055%) are far higher than would
be predicted on the basis of observed frequencies of
secondary nondisjunction (34%). To test the possi-
bility that XXY associations might be dissolved prior to
the end of prophase in oocytes in which the X chro-
mosomes had undergone crossing over, but maintained
in oocytes in which X chromosomal exchange had been
suppressed, we next examined XXY associations in late
prophase oocytes (stages 1012), which precede nu-
clear envelope breakdown.
The association of X and Y chromosomes is greatly
diminished by the end of prophase in exchange-
competent oocytes but maintained in oocytes in which
X chromosomal exchange has been suppressed: As
shown in the right column of Figure 4 and quantified in
Table 3, in oocytes in which X chromosomal crossing
over was suppressed by inversion heterozygosity [either
FM7a or In(1)dl-49], the frequencies of XXY associa-
tions in late prophase (stages 1012) remained virtually
 Secondary Nondisjunction in D. melanogaster Females
Figure 4.XXY associations in mid- and late meiotic pro-
phase. FISH was used to determine the positions of the X and
Y chromosomes in both midprophase (left column) and late
prophase (right column) oocytes. The frequency of XXY asso-
ciations for each genotype at mid- and late prophase is quan-
tified in Table 3. Although the X and Y chromosomes were
associated as a single mass in more than half of the mid-
prophase oocytes examined for all genotypes, the highest
frequencies of XXY associations (70%) were observed in oo-
cytes heterozygous for the FM7 balancer chromosome. These
XXY associations persisted until late prophase at high fre-
quencies in oocytes in which X chromosomal crossing over
was suppressed by inversion heterozygosity [i.e., in FM7/X/
Y, FM7/X/y1Y, or In(1)dl-49/X/y1Y oocytes]. Indeed, in either
FM7 or In(1)dl-49 heterozygotes the frequency of XXY associ-
ations in late prophase remained virtually unchanged from
the frequency observed in the mid-prometaphase. However,
when exchange-competent oocytes were examined in late
prophase, the frequency of XXY associations had diminished
by approximately fivefold, demonstrating that XXY associa-
tions are frequently dissolved in exchange-competent oocytes.
Red bar, 2 mm.
75
unchanged from the frequencies observed in midpro-
phase. This demonstrates that in the absence of ex-
change these XXY associations are stable throughout
prophase. However, when XXY associations were ob-
served in late prophase in X chromosome exchange-
competent oocytes, their frequency had diminished
substantially (see Table 3). Indeed, in X/X/Y or X/X/y1Y
oocytes the frequency of such associations diminished
by approximately fivefold to 1112%. Since many of
our oocytes were in stage 10, we suspect that this value
may be an overestimate of the true frequency of such
associations that will still exist at nuclear envelope
breakdown.
The fact that XXY associations are maintained in
In(1)dl-49/X/y1Y oocytes suggests that the mainte-
nance of stable XXY associations observed in FM7/X/
y1Y oocytes is not simply a consequence of the hetero-
chromatic rearrangements associated with the FM7
chromosome. By the same token, the dissolution of
XXY associations in FM7/FM7/y1Y females, in which the
homozygous balancer chromosomes are free to un-
dergo exchange, reveals that even in this genotype the
frequency of XXY associations diminishes as prophase
continues. Thus, the maintenance of XXY associations
throughout prophase that is observed in FM7/X/Y and
FM7/X/y1Y oocytes cannot be a consequence of hetero-
chromatic rearrangement, but rather must reflect the
fact that XXY associations are stabilized throughout pro-
phase only in the absence of crossing over.
The X chromosomal sequences at the distal tip of y1Y
often remained associated with the X chromosomes
even when the X and Y chromosomes were no longer
paired: In FM7/X/y1Y or X/X/y1Y oocytes in which the
medial region of YL was not associated with the two X
chromosomes (as judged by the failure of the AATAC
probe to colocalize with the X chromosomal 1.686
signal), the 1.686 sequences located on the distal tip
of the y1Y chromosome often remained paired with the
X chromosomes. Indeed, for all y1Y-bearing genotypes
examined, the 1.686 signal located on the y1Y tip could
be resolved only from the main X signal in 1417% of
the oocytes in which the AATAC YL-specific sequences
were clearly separated from the X chromosomes. Thus,
although the association of Y-specific heterochromatic
sequences with the X chromosomes appears to be dis-
solved in the presence of chiasmata, pairing between
the X chromosomal 1.686 sequences located at the tip
of the Y and the homologous 1.686 sequences on the X
chromosomes appears to persist.
The continued association of these blocks of 1.686 sat-
ellite sequence confirms the observations of Dernburg
et al. (1996) that heterochromatic pairings can per-
dure until metaphase. However, the fact that the fre-
quency of secondary nondisjunction observed in FM7/
X/ y1Y females is in fact somewhat lower than the
frequency of secondary nondisjunction observed in
FM7/X/Y females (see Table 1) argues that such pairings
76 Y. Xiang and R. Scott Hawley
TABLE 3
XXY association during mid- to late prophase
Midprophase
Total XY association
FM7/X/Y 82 60
X/X/Y 91 50
FM7/X/y1Y 76 54
X/X/y1Y 88 44
dl-49/X/y1Y 45 23
FM7/FM7/y1Y 56 37
are not sufficient to interfere with the segregation of
nonexchange X chromosomes or to promote XX 4 Y
segregational events. Moreover, the presence of these
1.686 sequences on the tip of the y1Y chromosome also
appears to have little or no effect on the maintenance of
XXYL associations as assayed by hybridization using
the Y chromosome-specific AATAC probe (see Table 3).
Thus, we conclude that the expulsion of the Y chromo-
some from the XXY trivalent in crossover oocytes oc-
curs in such a fashion as to allow the 1.686 sequences
appended to the tip of YL to maintain their association
with the X chromosome.
DISCUSSION
A new model of secondary nondisjunction: Our
data suggest that XXY pairings are common, if not uni-
versal, in early meiotic prophase. As first reported by
Dernburg et al. (1996), the pairing of homologous
regions of X chromosomal heterochromatin is main-
tained throughout prophase, regardless of whether or
not crossing over occurs. We saw no instances in which
the two X chromosomes were visible as separate entities
prior to NEB in any of the genotypes reported here.
However, the association of the two X chromosomes
with the Y chromosome is maintained only at high fre-
quency in those oocytes in which the X chromosomal
crossing over is suppressed. To explain these data, we
propose, contrary to previous models, that XXY pairings
occur early in prophase, coincident with or even prior
to, the initiation of synapsis. We further propose that
the occurrence of X chromosomal crossing over alters
the spatial relationship between the heterochromatin
of the Y and X chromosomes, and more specifically,
their centromeres, in a fashion that limits the associa-
tions of the X centromeric regions with each other and
dissolves their association with the less homologous Y
chromosome.
Support for exactly such a role of crossing over in
restricting centromeric associations comes from re-
cently published work in yeast by Tsubuchi and Roeder
(2005). These authors demonstrated that during early
Late prophase
% Total XY association %
73.2 69 48 69.6
54.9 95 11 11.6
71.1 88 60 68.2
50.0 68 8 11.8
51.5 39 18 46.2
66.1 44 11 25.0
meiotic prophase in wild-type yeast, centromeric pair-
ings usually involve the centromeres of nonhomologous
chromosomes. However, during the process of synapsis
and exchange these nonhomologous centromere cou-
plings undergo switching until all couples involve homo-
logs. The transition to purely homologous centromere
pairings requires Spo11, a protein required for the
initiation of meiotic recombination. Similar observa-
tions supporting a role for centromeric associations in
mediating the segregation of nonexchange chromo-
somes in yeast cells carrying either two homologous or
two homeologous chromosomes and a competing but
nonhomologous, CEN-bearing, yeast artificial chromo-
some (YAC) have also been published by Kemp et al.
(2004). In the absence of exchange in cells bearing the
two homeologous chromosomes, the YAC centromere
paired at high frequency with the centromeres of two
homeologous chromosomes. Pairings involving the YAC-
born CEN sequence were much less frequent in cells
carrying two homologous chromosomes that were free
to crossover. In that case, the plasmid or YAC-born
centromere was excluded from pairing with the homol-
ogous centromeres.
We propose that a similar set of processes occur in
XXY females in Drosophila oocytes, which is to say that
XXY pairing (and perhaps synapsis) is common in early
prophase, but that the process of X exchange promotes
the co-orientation of the two X centromeres prior to
spindle formation and dissolves the association of the X
chromosome centromeric region with the Y chromo-
some. Unlike Coopers model, which suggests that the
association of the Y with the X chromosomes occurs
subsequent to their failure to undergo crossing over, we
propose that the XXY associations occur in early pro-
phase in all oocytes and that crossing over between the X
chromosomes acts to dissolve the connection between
the Y and the two X chromosomes. However, as did
Cooper (1948), we propose that the XXY trivalents that
are maintained until prometaphase can and do direct
XX 4 Y segregational events.
Why dont all nonexchange X bivalents participate in
secondary nondisjunction? It is curious that only some
 Secondary Nondisjunction in D. melanogaster Females
(6080%) of the E0 tetrads undergo secondary non-
disjunction. Cooper (1948) envisioned a model in
which the trivalent could occasionally line up as a linear
structure on the metaphase spindle, with the two X
chromosomes pointed toward opposite poles and un-
oriented Y in the middle (see Figure 2b of Cooper
1948). However, our observation that the fractions of
XXY associations in late prophase are similar to the
frequencies of secondary nondisjunction as assayed
genetically and cytologically in both FM7/X/Y and
In(1)dl-49/X/Y oocytes (see Tables 1 and 2) suggests
that most XXY associations that persist until the end of
prophase do result in the formation of classic trivalents
and that in these trivalents the metacentric Y chromo-
some orients the two acrocentric X chromosomes to
opposite poles of the spindle. Thus, we propose that 20
40% of nonexchange X chromosomes that fail to
segregate from the Y do so because of an occasional
failure of the Y chromosome to maintain its association
with the achiasmate X chromosomes during early to
midprophase.
One could argue that Coopers proposal might apply
to XXY females bearing normal-sequence X chromo-
somes, since the frequency of XXY associations observed
in late prophase (1112%) is threefold higher than the
observed frequency of spontaneous nondisjunction (3
4%). However, unlike the frequency of XXY associations
in X exchange-suppressed oocytes, which is similar in
both mid- and late prophase oocytes, the frequency of
XXY associations appears to continuously decline dur-
ing the progression of prophase in XXY females. The
frequency of such associations is 93% in early prophase,
but declines to 5055% in midprophase and 1112%
in late prophase oocytes. Because our estimate for the
frequency of XXY associations in late prophase in-
cludes oocytes throughout this interval, we propose
that the observed frequency of XXY associations (11
12%) is likely to be an overestimate of the frequency of
XXY associations that will indeed persist beyond NEB.
Thus, even in this genotype it seems likely that the
XXY associations that do survive past NEB will direct
XX4Y segregational events.
Carpenters two-step model of centromere co-
orientation in XXY females: Carpenter (1973) pro-
posed that the co-orientation of the X chromosomes by
the Y chromosome involves two separate steps: first, a
mechanism that committed the two X chromosomes to
cosegregate (i.e., to move to the same pole, perhaps as a
single entity), and second, the co-orientation of that set
of locked X chromosomes from the Y chromosome.
She based this hypothesis of the analysis of secondary
nondisjunction in females homozygous for a loss-of-
function mutation (noda) in the nod gene. The nod gene
encodes a chromokinesin-like protein that serves to
hold achiasmate chromosomes on the developing
spindle and prevent their precocious migration to the
poles (Carpenter 1973; Cui et al. 2005). In the absence
77
of functional Nod protein, achiasmate X chromosomes
nondisjoin at high frequency and thus appear to seg-
regate at random from their homolog (Carpenter 1973;
Theurkauf and Hawley 1992).
Carpenter (1973) observed that the frequency of X
chromosomal nondisjunction in noda/noda/Y females
was much higher than that observed in noda/noda
females and was, in fact, identical to that observed in
nod1/nod1/Y controls. However, in the controls, the
nondisjunctional progeny were of only two gamete
types, namely XX and Y, while noda/noda/Y females
produced ova with XX, XXY, Y, and 0 at equal frequen-
cies. Carpenter interpreted these results to mean that
the noda mutant allowed the proper commitment of the
X chromosomes to cosegregate to the same pole, fol-
lowed by defective disjunction of this pair from the Y
chromosome, such that the two X chromosomes still
cosegregate to the same pole while the Y disjoins at
random.
In the images of XX4Y co-orientation in Figure 3, we
do not observe a physical association between two Xs
that are segregating toward the same pole. Thus, if the
Xs truly are somehow locked together during the pro-
cess of setting up XX 4 Y segregations, such associa-
tions do not usually persist into late prometaphase or
metaphase oocytes. However, we do note that hetero-
chromatic associations between the proximally located
blocks of X heterochromatin can persist into prometa-
phase (Dernburg et al. 1996; but see also Gilliland
et al. 2005). Moreover, associations between the distally
located blocks of heterochromatin often persist even
into late prometaphase/metaphase in FM7/FM7/y1Y
females. Perhaps the persistence of such pairings, at
least until early prometaphase, helps to facilitate the
commitment of the X chromosomes to co-segregate
step proposed by Carpenter (1973). In other words,
when not acted on by forces that would normally drag
the two X centromeres to opposite poles in XX oocytes,
perhaps such pairings can persist long enough in nod
oocytes to facilitate the observed cosegregation events.
Summary: Our data both provide strong evidence for
the XXY trivalent postulated by Cooper (1948) and
disprove Bridges (1916) model of competitive pair-
ing between the X and Y chromosomes. But of greater
importance, our data suggest a new model of second-
ary nondisjunction in which XXY associations are ubiq-
uitous during early prophase in all oocytes of this
genotype and then dissolve in mid- to late prophase
following the occurrence of X chromosomal crossing
over. These data suggest that that the association of
homologous centromeres, and perhaps even their co-
orientation, can be influenced or directed by the oc-
currence of crossing over long before NEB.
We gratefully acknowledge Abby Dernburg for consultation and
guidance during the early phases of this project. We also thank mem-
bers of the Hawley lab (especially Cathleen Lake and Susan Flynn) for
valuable discussion and comments on the manuscript. This research
78 Y. Xiang and R. Scott Hawley
was supported by funds from the Stowers Institute for Medical Re-
search and by an American Cancer Society Research Professor Award
to R.S.H.
LITERATURE CITED
Ashburner, M., K. Golic and R. S. Hawley, 2005 Drosophila: A Lab-
oratory Handbook, Ed. 2. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
Bridges, C. B., 1916 Non-disjunction as proof of the chromosome
theory of heredity. Genetics 1: 152, 107163.
Carpenter, A. T. C., 1973 A mutant defective in distributive disjunc-
tion in Drosophila melanogaster. Genetics 73: 393428.
Carpenter, A. T. C., and L. Sandler, 1974 On recombination-
defective meiotic mutants in Drosophila melanogaster. Genetics
76: 453475.
Chadov, B. F., 1981 Behavior of chromosomes during mitosis and
meiosis and chromocentral organization of nuclei in Drosophila
melanogaster. Proceedings of the XIV International Congress of
Genetics, Vol. 3, Ithaca, NY, pp. 343356.
Cooper, K. W., 1948 A new theory of secondary non-disjunction
in female Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 34:
179187.
Cui, W., L. R. Sproul, S. M. Gustafson, H. J. G. Matthies, S. P.
Gilbert et al., 2005 Drosophila Nod protein binds preferentially
to the plus ends of microtubules and promotes microtubule
polymerization in vitro. Mol. Biol. Cell 16: 54005409.
Dernburg, A. F., 2000 In situ hybridization to somatic chromo-
somes, pp. 2556 in Drosophila Protocol, edited by W. Sullivan,
M. Ashburner and R. S. Hawley. Cold Spring Harbor Labora-
tory Press, Cold Spring Harbor, NY.
Dernburg, A. F., J. W. Sedat and R. S. Hawley, 1996 Direct evi-
dence of a role for heterochromatin in meiotic chromosome
segregation. Cell 86: 135146.
Gershenson, S. M., 1935 The mechanisms of non-disjunction in
the ClB stock of Drosophila melanogaster. J. Genet. 30: 115125.
Gilliland, W. D., S. M. Wayson and R. S. Hawley, 2005 The
meiotic defects of mutants in the Drosophila mps1 gene reveals
a critical role of Mps1 in the segregations of achiasmate homo-
logs. Curr. Biol. 15: 672677.
Gong, W. J., K. S. McKim and R. S. Hawley, 2005 All paired up
with no place to go: pairing, synapsis, and DSB formation in a
balancer heterozygote. PLoS Genet. 1: 589602.
Grell, R. F., 1962 A new model for secondary nondisjunction: the
role of distributive pairing. Genetics 47: 17371754.
Grell, R. F., 1976 Distributive pairing, pp. 435486 in Genetics and
Biology of Drosophila, edited by E. Novitski and M. Ashburner.
Academic Press, New York.
Haaf, T., H. Winking and M. Schmid, 1989 Immunocytogenetics.
III. Analysis of trivalent and multivalent configurations in mouse
pachytene spermatocytes by human autoantibodies to synaptone-
mal complexes and kinetochores. Cytogenet. Cell Genet. 50: 14
22.
Harris, D., C. Orme, J. Kramer, L. Namba, M. Champion et al.,
2003 A deficiency screen of the major autosomes identifies a
single gene (matrimony) that is haplo-insufficient for achiasmate
segregation in Drosophila oocytes. Genetics 165: 637652.
Hawley, R. S., and W. Theurkauf, 1993 Requiem for distributive
segregation: achiasmate segregation in Drosophila melanogaster.
Trends Genet. 9: 310317.
Hawley, R. S., H. Irick, A. E. Zitron, D. A. Haddox, A. Lohe et al.,
1993 There are two mechanisms of achiasmate segregation in
Drosophila females, one of which requires heterochromatic ho-
mology. Dev. Genet. 13(6): 440467.
Karpen, G. H., M. H. Le and H. Le, 1996 Centric heterochromatin
and the efficiency of achiasmate disjunction in Drosophila female
meiosis. Science 273: 118122.
Kemp, B., R. M. Boumil, M. N. Stewart and D. S. Dawson, 2004 A
role for centromere pairing in meiotic chromosome segregation.
Genes Dev. 18: 19461951.
Lohe, A. R., A. J. Hilliker and P. A. Roberts, 1993 Mapping simple
repeated DNA sequences in heterochromatin of Drosophila mela-
nogaster. Genetics 134: 11491174.
Luning, K.G., 1982 Genetics of inbred Drosophila melanogaster.
VI. Crossing-over in secondary non-disjunction exceptionals.
Hereditas 96: 161174.
MacKnight, R. H., and K. W. Cooper, 1944 The synapsis of the sex
chromosomes of Drosophila Miranda in relation to their directed
segregation. Proc. Natl. Acad. Sci. USA 30: 384387.
Merriam, J. R., 1967 The initiation of nonhomologous chromo-
some pairing before exchange in female Drosophila melanogaster.
Genetics 57: 409425.
Neuhaus, M., 1941 The influence of separate arms of the Y chromo-
some on secondary nondisjunction in the females of D. mela-
nogaster. Dros. Inf. Ser. 15: 16.
Novitski, E., and G. Braver, 1954 An analysis of crossing over within
a heterozygous inversion in Drosophila melanogaster. Genetics 39:
197209.
OTousa, J., 1982 Meiotic chromosome behavior influenced by
mutation-altered disjunction in Drosophila melanogaster females.
Genetics 102: 503524.
Puro, J., and S. Nokkala, 1981 Secondary nondisjunction in mul-
tiple inversion heterozygotes as revealed by direct cytological
and genetic tests. Proceedings of the 7th European Drosophila
Research Conference, p. 87.
Rutherford, S. L., and A. T. C. Carpenter, 1988 The effect of se-
quence homozygosity on the frequency of X-chromosomal ex-
change in Drosophila melanogaster females. Genetics 120: 725732.
Spradling, A. C., 1993 Development genetics of oogenesis, pp.
171 in The Development of Drosophila melanogaster, edited by
M. Bate and A. M. Arias. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
Sturtevant, A. H., and G. W. Beadle, 1936 The relations of inver-
sions in the X chromosome of Drosophila melanogaster to crossing
over and disjunction. Genetics 21: 554604.
Theurkauf, W. E., and R. S. Hawley, 1992 Meiotic spindle assem-
bly in Drosophila females: behavior of non-exchange chromo-
somes and the effects of mutations in the nod kinesin-like
protein. J. Cell Biol. 116: 11671180.
Tsubouchi, T., and G. S. Roeder, 2005 A synaptonemal complex
protein promotes homology-independent centromere coupling.
Science 308: 870873.
Whyte, W., H. Irick, T. Arbel, G. Yasuda, R. L. French et al.,
1993 The genetic analysis of distributive pairing in Drosophila
melanogaster. III. The wild-type product of the Axs locus is re-
quired for achiasmate meiotic segregation. Genetics 134: 825
835.
Zhang, P., and R. S. Hawley, 1990 The genetic analysis of distrib-
utive segregation in Drosophila melanogaster. II. Further genetic
analysis of the nod locus. Genetics 125: 115127.
Zitron, A. E., and R. S. Hawley, 1989 The genetic analysis of
distributive pairing in Drosophila melanogaster. I. Isolation and
characterization of Aberrant X segregation, Axs, a mutant defective
in chromosome partner choice. Genetics 122: 801821.
Communicating editor: B. S. Ganetzky