Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean Society of London, 2005? 2005
843
407416
Original Article
FERTILITY IN CHROMOSOMAL RACES OF HOUSE MOUSE
N. CHATTI
Et al.
Biological Journal of the Linnean Society, 2005, 84, 407–416.
The genus Mus as a model for evolutionary studies
Edited by J. Britton-Davidian and J. B. Searle
Reproductive trait divergence and hybrid fertility
patterns between chromosomal races of the house mouse
in Tunisia: analysis of wild and laboratory-bred males and
females
NOUREDDINE CHATTI1*, JANICE BRITTON-DAVIDIAN2, JOSETTE CATALAN2,
JEAN-CHRISTOPHE AUFFRAY2 and KHALED SAÏD1
1
Laboratoire de Génétique, Biodiversité et Environnement, UR09/30, Institut Supérieur de Biotechnologie
de Monastir, 5000, Monastir, Tunisia
2
Laboratoire Génétique et Environnement, Institut des Sciences de l’Evolution (UMR 5554 CNRS),
Université Montpellier II, cc65, Place Eugène-Bataillon, 34095 Montpellier Cedex 05, France
Received 29 October 2003; accepted for publication 7 October 2004
The divergence in reproductive features and hybrid fertility patterns between two chromosomal races (2n = 40, 40St,
and 2n = 22, 22Rb) of the house mouse in Tunisia were re-assessed on a larger sample of wild and laboratory-bred
individuals than studied hitherto. Results showed that litter sizes were significantly smaller in 40St than in 22Rb
mice, contrary to previous analyses. This suggests that variation in litter size between the two chromosomal races
is more likely related to selective and/or environmental factors acting locally than to interracial reproductive trait
divergence. However, the significantly reduced litter size of F1 hybrids compared with parental individuals was confirmed, and further highlighted a sex difference in hybrid infertility, as F1 females produced fewer litters and of
smaller size than males. Histological analyses of F1 and backcrosses showed a breakdown of spermatogenesis in
males and a significantly reduced primordial follicle pool in females. The degree of gametogenic dysfunction was not
related to the level of chromosomal heterozygosity per se, but a significant effect of two Rb fusions on follicle number
was observed in hybrid females. These results suggest that genetic incompatibilities contribute to primary gametogenic dysfunction in hybrids between the chromosomal races in Tunisia. © 2005 The Linnean Society of London,
Biological Journal of the Linnean Society, 2005, 84, 407–416.
ADDITIONAL KEYWORDS: gametogenic dysfunction – genetic incompatibilities – litter size – oogenesis –
Robertsonian fusion – spermatogenesis.
INTRODUCTION
Recent models of chromosomal speciation have highlighted the role of chromosomal changes as strong
barriers to gene flow through their effect on recombination rather than fertility reduction (Rieseberg,
2001; Navarro & Barton, 2003). The attractiveness of
the recombinational model of chromosomal speciation
lies in the resolution of the paradox associated with
*Corresponding author. E-mail: [email protected]
the traditional model, which is burdened by the low
probability of fixation of highly underdominant rearrangements. Theoretical and empirical data supporting the recombinational model have involved
rearrangements such as reciprocal translocations,
inversions and tandem fusions (Noor et al., 2001;
Rieseberg, 2001; Navarro & Barton, 2003), whereas
Robertsonian (Rb) fusions/fissions have often not been
considered as convincing candidates for models of
chromosomal speciation. The reasons for this are twofold: single Rb fusions usually have little deleterious
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 407–416
407
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N. CHATTI ET AL.
impact on reproductive fitness (Winking, Dulic¢ &
Bulfield, 1988; Britton-Davidian et al., 1990; Scriven,
1992; Viroux & Bauchau, 1992; Wallace, Searle &
Everett, 1992; Wallace, 2003) and thus on gene flow,
and, when patterns of recombination are modified
(Davisson & Akeson, 1993; Bidau et al., 2001; Castiglia & Capanna, 2002; Dumas & Britton-Davidian,
2002), they affect centromeric regions that are known
to be gene-poor (Navarro & Barton, 2003). The exception to the rule is the monobrachial speciation model
(Baker & Bickham, 1986, but see Britton-Davidian,
Catalan & Belkhir, 2002, and Searle & Wójcik, 1998)
in which the independent fixation in two populations
of a few Rb fusions with one arm in common will lead
to a high level of chromosomal incompatibility in
hybrids between them. However, another situation
exists in which populations have successively accumulated a large number of Rb fusions. Contact between
these and ancestral populations results in the formation of a hybrid zone in which chromosomal hybrids
show a considerable reduction in fertility (Saïd et al.,
1993; Searle, 1993; Hauffe & Searle, 1998; Castiglia &
Capanna, 1999). When such a barrier to gene flow is
present, genic divergence of the parapatric populations is expected (Capanna, 1982; Britton-Davidian
et al., 1989), ultimately leading to speciation (Saïd
et al., 1999).
The effects on fertility of chromosomal structural
heterozygosity have been extensively investigated in
house mice. The standard karyotype of the house
mouse, Mus musculus domesticus , consists of 20 pairs
of acrocentric chromosomes, but many chromosomal
races with a low diploid number have been found in
Europe and North Africa (Nachman et al., 1994; see
also Piálek, Hauffe & Searle, 2005, this issue). The
formation of these races is due to the fixation of Rb
translocations formed by centric fusion of acrocentric
chromosomes (Capanna, 1982). Contact between
ancestral and Rb races produces hybrids showing
trivalent Rb configurations at meiosis. The decrease in
fertility is mainly due to malsegration of the trivalents
leading to the production of aneuploid gametes, and
germ cell death, the rates of which are related to the
number and type of heterozygous fusions (Winking
et al., 1988; Britton-Davidian et al., 1990; Scriven,
1992; Viroux & Bauchau, 1992; Wallace et al., 1992;
Saïd et al., 1993; Hauffe & Searle, 1998; Castiglia &
Capanna, 1999). In some cases, premeiotic perturbations leading to a reduction in germ stem cell number
has been observed (Garagna et al., 1990). As not only
the number but also the type and geographical origin
of Rb fusions has an effect on hybrid fertility, several
authors have suggested that both chromosomal and
genic factors influence reproductive fitness of chromosomal heterozygotes in house mice (Garagna et al.,
1990; Wallace, Searle & Everett, 2002; Wallace, 2003).
In addition, the occurrence of primordial germ cell
depletion in Rb heterozygotes has led Capanna & Redi
(1994) to argue that Rb rearrangements modify the
spatial distribution of interphasic chromosomes, altering the expression of genes involved in early stages of
cytodifferentiation. Despite the large underdominance
observed in multiple Rb heterozygotes, which is
expected to provide a strong barrier to gene flow, few
direct indications of genic divergence have been
revealed (Britton-Davidian et al., 1989; Saïd & Britton-Davidian, 1991; Nachman et al., 1994; Hauffe
et al., 2002; Castiglia, Annesi & Capanna, 2005, this
issue; Tryfonopoulos, Chondropoulos & FraguedakisTsolis, 2005, this issue). This has been attributed to
the recent origin of these races (Britton-Davidian
et al., 1989), since the colonization of the Mediterranean Basin and Europe by the house mouse occurred
in the last 5000 years (Auffray, 1993; but see indications for an even later date in Cucchi, Vigne & Auffray,
2005, this issue). In Tunisia, however, a number of
studies have shown that the two chromosomal races
present (2n = 22 and 2n = 40) have diverged for several traits. In laboratory crosses between individuals
from the two races, the mean litter size of the 2 n = 22
(22Rb) race was found to be significantly smaller than
that of the all-acrocentric (40St) mice (Saïd et al.,
1993), indicating that differential demographic strategies between the races may have evolved. In addition,
analysis of the testicular histology of laboratory
hybrid males showed several sterile phenotypes
that in one case was unrelated to chromosomal
heterozygosity, suggesting that genic incompatibilities
between the two genomes might be involved. This was
supported by the significant decrease in developmental stability in wild hybrids compared with that in
parental populations (Chatti et al., 1999b).
In the present work, the divergence in reproductive
features between the two Tunisian races was reassessed by the analysis of a larger sample of laboratory-bred mice. In addition, fertility patterns in both
male and female wild and laboratory-born individuals
involving parental, F1 and backcross hybrids were
determined, and their relationship to chromosomal
heterozygosity and/or genic incompatibilities was
evaluated.
MATERIAL AND METHODS
MICE
Mice were caught during three separate field sessions
(April–May 1994, October 1995 and May–July 1996)
from different localities in central Tunisia (see Chatti
et al., 1999a, for details on geographical distribution).
The fertility of 83 males and 42 females, 22Rb, 40St
and hybrids, was assessed (see Tables 2, 3 for details).
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 407–416
FERTILITY IN CHROMOSOMAL RACES OF HOUSE MOUSE
For direct estimates of fertility (breeding success,
number of litters per pair and litter size), crosses were
established with the first generation progeny of wild
house mice live-trapped in Monastir (2 n = 22, 22Rb)
and Teboulba (2n = 40, 40St) (36 km apart) and reared
under standard conditions (temperature: 20–25 ∞C,
light cycle: 12 : 12 h). Five series of reciprocal crosses
were performed: intraracial: (1) 22Rb¥22Rb (2)
40St¥40St, interracial: (3) 22Rb¥40St, and backcrosses: (4) 22Rb¥F1 (that is ‘BC22’), (5) 40St¥F1 (that
is ‘BC40’). All pairs were maintained for an average of
12 months (12 ± 1) and checked every 2 days for birth
of litters; the number of pups per litter at birth was
recorded. An indirect approach was also performed by
histological analyses of gametogenesis of 90 male and
63 female progeny of these crosses (see Tables 1–3 for
details).
CHROMOSOME
PREPARATION
Chromosomes were prepared from a suspension of
yeast-stimulated bone marrow cells (Lee & Elder,
1980). G-banding was performed according to the
method of Seabright (1971). The diploid number and
the karyotype of all wild 22Rb, 40St and hybrid
individuals were determined by conventional and
G-banded metaphases. In laboratory-born mice, the
karyotypes of 54 backcrosses and 28 F1 animals were
G-banded as well as that of a subsample of the 22Rb
and 40St individuals (n = 30). The 22Rb race carried
nine pairs of Rb fusions previously described as the
‘Monastir race’: Rb(1.11), Rb(2.16), Rb(3.12), Rb(4.6),
Rb(5.14), Rb(7.18), Rb(8.9), Rb(10.17) and Rb(13.15)
(Saïd et al., 1986). The meiotic configuration of F1 individuals (2n = 31) consisted of nine trivalents formed
by the pairing of the Rb fusions with the homologous
acrocentrics, and two bivalent pairs: the sex chromosomes and autosome 19. The karyotypes of all wild
hybrids and laboratory-bred backcrosses are available
upon request.
HISTOLOGICAL
ANALYSIS
Animals were killed by cervical dislocation and the
testes or ovaries were immediately removed. The testes were weighed. The left testis or the two ovaries
were placed in Bouin’s fixative for 24 h. After paraffin
embedding, 6-mm-thick sections were prepared and
stained with haematoxylin and eosin and viewed
under the light microscope. A sample of 48 wild and 72
laboratory-bred males was used for histological preparations of testis (see Table 2 for details). One hundred transverse cross-sections of seminiferous tubules
were examined per animal using the testicular histopathology interpretation score (TMI; Lecornu et al.,
1984). The TMI score is a semiquantitative method,
409
commonly used in the quantification of human male
sterility; it attributes a value varying from 0 (normal)
to 3 (highly perturbed) to the biopsy according to the
histological state of each of the following components:
the seminiferous tubules, the peritubular membrane,
the interstice, density and differentiation of germ cells
(see details in Saïd et al., 1993). Thus, as the TMI
score is the sum of the individual trait values, it
ranges from 0 to 15. A sample of 42 wild and 63 laboratory-bred females was used for the histological preparations of ovaries (see Table 3 for details). For each
female, only the larger of the two ovaries was considered. All follicles and corpora lutea were counted in
the five widest sections; from these counts, the mean
number of follicles at different stages was calculated
per section for each individual. Follicles were classed
following Peters & McNatty (1980): (i) primordial follicles with one layer of flattened granulosa cells surrounding the oocyte, (ii) very early growing and early
growing follicles with 1–2 and 2–5 layers of cuboidal
granulosa cells, respectively, and (iii) preantral and
antral follicles characterized by many layers of granulosa cells hollowed by antral fluid-filled cavities. In
antral follicles, there is one large cavity composed of
such fluid.
STATISTICAL
ANALYSES
Differences in breeding success were evaluated by c2
and Fisher’s exact tests. We used ANOVAs to test for
differences in testis weight and in the number of follicles between chromosomal groups. The relationship
between the number of heterozygous Rb fusions and
testis weight, and the number of follicles was investigated using Spearman rank correlations. ANOVAs followed by planned comparisons were used to test the
effect of the chromosome state (homozygous or heterozygous) of each individual Rb fusion on the fertility
parameters. The decision to reject the null hypothesis
was based on probabilities lower than or equal to a
level adjusted to the number of tests performed
according to the sequential Bonferroni method (Rice,
1989). All statistical analyses were performed with the
Statistica 3.5 program.
RESULTS
BREEDING
SUCCESS AND LITTER SIZE
Breeding success is expressed as the ratio of reproducing pairs to the total number of pairs (Table 1). Our
results indicated that all series of crosses yielded progeny. Backcrosses showed the lowest breeding success
rate (47%) when compared with interracial (50%) or
intraracial (65%) crosses. Although the 40St¥40St had
the highest value of breeding success, statistical comparison with the other crosses showed that these dif-
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 407–416
410
N. CHATTI ET AL.
Table 1. Breeding success, number and size of litters in the different crosses
No. of pairs
Cross
Intraracial
Interracial
Backcrosses
22Rb¥22Rb
40St¥40St
m22Rb¥f40St
m40St¥f22Rb
m22Rb¥f31
f22Rb¥m31
m40St¥f31
f40St¥m31
with
litter
with no
litter
Breeding
success
(%)
No. of
litters
No. of litters
per pair
Litter size
5
10
3
4
5
10
4
5
7
1
6
1
7
9
5
6
41.7
90.9
33.3
80
41.7
52.6
44.4
45.5
27
29
10
16
7
36
6
16
3.4 ± 1.6
2.9 ± 1.4
3.3 ± 1.5
4.0 ± 0.0
1.4 ± 0.9
4.0 ± 2.1
1.5 ± 1.0
3.2 ± 2.2
5.0 ± 1.7
3.1 ± 1.3
3.5 ± 1.4
3.6 ± 1.3
1.1 ± 0.4
1.7 ± 0.9
1.0 ± 0.0
2.2 ± 1.0
m, male; f, female; 22Rb, 2n = 22; 40St, 2n = 40.
Table 2. Sample size, diploid number, mean age in months, mean TMI score and mean relative testis weight per
chromosomal group in wild and laboratory-bred mice. The number of mice used for histological preparations is indicated
in parentheses
Mice
No.
2n
Wild
22Rb
40St
Hybrids
35 (17)
30 (20)
18 (11)
22
40
23–39
Laboratory-bred
22Rb
40St
F1
BC22
BC40
16
22
14
28
10
22
40
31
23–30
33–39
(12)
(14)
(12)
(26)
(8)
Mean age ± SD
7±2
8±1
10 ± 2
7±2
7±2
ferences were not significant. As all pairs were
maintained for the same duration, the number of litters per pair between crosses was compared. The
ANOVAs showed no differences between any of the
crosses (Tukey HSD for unequal sample size,
P >> 0.05 ), but planned comparisons on the same
analysis indicated a significantly higher number of litters in crosses involving an F 1 male than those including an F1 female (F1,42 = 9.675, P = 0.0033).
Mean litter sizes at birth for all mating types are
reported in Table 1. Before comparing litter size
between crosses, an effect of the rank of the litter and
of the pair was checked and was not found to be significant. The ANOVA analyses showed that 22Rb
crosses yielded the highest litter size, whereas the
lowest was found in the backcrosses (Tukey HSD for
unequal sample size, F4,145 = 21.03, P < 0.001). No significant difference in mean litter size was detected
either between interracial and 40St crosses or
between the two series of backcrosses (P > 0.05). When
TMI ± SD
Testis weight (¥10-3) ± SD
0.94 ± 1.3
0.15 ± 0.37
8.36 ± 3.67
7.46 ± 1.17
9.38 ± 2.26
5.29 ± 1.58
0.41 ± 0.67
0.57 ± 1.28
6.08 ± 2.78
5.73 ± 2.42
6.25 ± 2.55
9.83 ± 1.64
9.02 ± 2.04
5.65 ± 1.24
6.40 ± 1.51
6.44 ± 1.86
data were compared between sexes within each karyomorph, the only significant difference was that
between F1 females and males (planned comparison,
F1,88 = 8.086, P = 0.0055). The latter produced more
progeny per litter regardless of the mate’s karyotype.
MALE
FERTILITY
Testis weight is expressed here as the ratio of testis to
body weight (Table 2). The mean relative testis weight
was significantly different between wild chromosomal
groups: the highest value was recorded in the 40St
group and the lowest in hybrids, the 22Rb group being
intermediate (Tukey HSD for unequal sample size,
F2,80 = 32.14, P < 0.001). Statistical analysis showed no
correlation between relative testis weight and the
number of heterozygous Rb fusions (P > 0.05). In laboratory-bred mice, relative testis weight was significantly reduced in the F 1 and backcross individuals
compared with parental mice (Tukey HSD for unequal
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 407–416
1.44 ± 2.46
2.16 ± 3.35
6.53 ± 3.61
4.22 ± 1.67
3.15 ± 3.80
51.66 ± 14.43
36.09 ± 13.32
16.30 ± 6.99
37.43 ± 11.7
31.93 ± 10.5
0.61 ± 0.62
0.55 ± 0.69
0.83 ± 0.79
1.02 ± 0.76
1.10 ± 0.70
6.90 ± 3.66
4.18 ± 2.20
1.50 ± 1.23
4.58 ± 2.66
3.02 ± 1.70
6.33 ± 4.22
5.05 ± 3.20
2.30 ± 1.67
6.08 ± 2.66
4.32 ± 1.90
6.59 ± 2.20
8.65 ± 4.02
4.96 ± 2.93
13.13 ± 6.35
10.27 ± 5.52
14
11
14
12
12
Laboratory-bred
22Rb
40St
F1
BC22
BC40
22
40
31
23–29
31–38
18
12
12
Wild
22Rb
40St
Hybrids
22
40
23–31
7±2
8±1
10 ± 2
7±2
7±2
31.23 ± 10.57
17.65 ± 6.59
6.71 ± 2.81
12.62 ± 6.93
13.23 ± 5.86
3.26 ± 2.00
2.93 ± 2.16
3.36 ± 2.14
32.26 ± 7.09
28.49 ± 8.14
20.65 ± 7.18
1.06 ± 0.78
1.36 ± 0.88
0.72 ± 0.80
2.83 ± 1.24
3.49 ± 1.59
3.99 ± 1.80
3.41 ± 1.15
4.19 ± 0.99
3.68 ± 1.93
4.90 ± 2.24
4.90 ± 2.51
3.40 ± 1.40
20.06 ± 5.39
14.55 ± 6.23
8.87 ± 3.37
Total
Antral
Preantral
E. growing
V.e. growing
No. of follicles/section (mean ± SD)
Primordial
2n
No.
The mean number of follicles at different stages was
calculated (Table 3). In wild hybrids, the total number
of follicles was significantly reduced when compared
with the parental races (22Rb and 40St) (Tukey HSD
for unequal sample size, F2,39 = 8.85, P < 0.01). This
difference was essentially due to the decrease of primordial follicles in hybrids. No significant differences
were detected between the two parental races
(P > 0.05). The number of corpora lutea was similar in
the three chromosomal groups. The sample size of wild
hybrid females was not sufficient to test the effect of
Rb heterozygosity on the number of follicles. Laboratory-born female mice were classed into five groups:
22Rb, 40St, F1, BC22 and BC40. Before making com-
Mice
FERTILITY
Mean age
± SD
FEMALE
Table 3. Number of mice, diploid number, mean age in months and mean follicle number at different stages in wild and laboratory-bred females
sample size, F4,85 = 20.42, P < 0.001). Although values
were higher in backcrosses than in F1, they were not
significantly different. No differences were detected
between the 22Rb and 40St races. A correlation test,
using F1 and backcross progeny, revealed no significant correlation between relative testis weight and
the number of heterozygous Rb fusions (P > 0.05).
Although individuals heterozygous for the Rb(4.6)
fusion had smaller testes than the corresponding
homozygous mice (F1,46 = 7.258, P = 0.0098), these
differences were not significant after Bonferroni
adjustment.
The histological analysis showed that the tubular
membrane and the interstitial space were not pathologically affected in any of the individuals. Spermatogenesis was normal in the wild and laboratory-bred
22Rb and 40St mice despite necrobiosis and exfoliation, which affected a few seminiferous tubules in
some individuals; all the TMI scores for these individuals were generally equal to zero. In wild hybrids as
well as in the F1 and backcross mice, the TMI scores
were always different from zero and in some cases
reached very high values (Table 2). We noted an alteration of the spermatogenic process that was interrupted at early stages. The tubular diameter, cellular
differentiation and density were reduced. Exfoliation
and necrobiosis were also usually observed within the
seminiferous tubules. The effect of Rb heterozygosity
on TMI in wild hybrids could not be tested owing to the
reduced sample size (N = 9). In F1 and backcross progeny, there was no correlation between Rb heterozygosity and TMI (P > 0.05). This is not surprising given
that the TMI scores in the F1 mice that had the highest
degree of heterozygosity (Rb heterozygosity = 9) did
not differ considerably from that of backcrosses (Rb
heterozygosity £ 9). By contrast, in the F1 and backcrosses, the TMI score was highly and negatively correlated with relative testis weight (N = 46, R = -0.452,
P = 0.0015).
C. lutea
FERTILITY IN CHROMOSOMAL RACES OF HOUSE MOUSE
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 407–416
411
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N. CHATTI ET AL.
parisons, the effect of age on the follicle number was
tested and not found to be significant in any of the five
chromosomal groups (P > 0.05). The 22Rb females had
the highest number of follicles and differed significantly from all other groups (Tukey HSD for unequal
sample size, F4,58 = 16.58, P < 0.001), whereas the 40St
were not significantly different from BC22 and BC40
(Table 3). The lowest values were recorded in F 1
females and increased in both BC22 and BC40 (F 1–
BC22: P < 0.001; F1–BC40: P < 0.05). F1 mice showed
the highest number of corpora lutea, 22Rb and 40St
the lowest, with backcross progeny being intermediate. The only significant differences were detected
between F1 and both 22Rb and 40St groups (Tukey
HSD for unequal sample size, F4,58 = 5.66, respectively,
P < 0.001 and P = 0.013). To test the effect of Rb heterozygosity on the number of follicles, we considered
only the backcross progeny. No significant correlation
was detected (P > 0.184). The effect of the different
heterozygous Rb fusions was analysed by ANOVA.
Two Rb fusions contributed significantly to the
decrease in the number of follicles in hybrids: Rb(1.11)
(F1,34 = 9.414; P = 0.0042) and Rb(8.9) (F1,34 = 13.935;
P = 0.0006).
DISCUSSION
COMPARISON OF
THE
REPRODUCTIVE FEATURES BETWEEN
22RB
AND
40ST
RACES
The 22Rb race showed a higher litter size and follicle
number than the 40St race, despite a lower breeding
success (Tables 1, 3). However, among the fertility
traits assessed, the only significant difference was the
mean litter size. This was consistent with the indirect
estimates, as the follicle pool in both wild and laboratory-bred females was larger in the 22Rb race than in
the 40St race. Similar results have been reported in a
study of Italian chromosomal races (Castiglia &
Capanna, 2000) in which the Rb race (2n = 22, CD
race) also exhibited a higher mean litter size than the
neighbouring standard mice. However, our data contrast with a previous analysis of the Tunisian races by
Saïd et al. (1993) (12 intraracial crosses) in which
opposite results were observed, as the 40St individuals had a higher mean litter size (6.0) than in the
present analysis (3.1), whereas values for the Rb race,
which originated from Monastir in both cases, were
lower (3.6 vs. 5.0). Such comparisons suggest that the
differences in litter size observed between races are
more related to intraracial variability than to interracial divergence. This is supported by recent crosses
involving a large geographical sample of both races,
and showing that a similar variability for this trait is
present in the 22Rb and the 40St mice, mean litter
size having the same value in both races (79 intraracial crosses from seven different localities; K. Ben-
zekri, pers. comm.). In south-eastern Australia,
Singleton et al. (2001) showed that litter size in house
mice changed seasonally, from highest in spring to
lowest in autumn and winter, and varied remarkably
between years; they concluded that this could be due
to variation in food, particularly protein, quality. In
addition, geographical variation in litter size among
populations of the cotton rat Sigmodon hispidus was
related to differences in ovulation rate (Oswald &
McClure, 1985). An indication of the effect of environmental factors on female fertility can be found in
the higher number of follicles in laboratory-reared
females than in wild females (see also Garagna et al.,
1990), although the number of corpora lutea was surprisingly slightly lower in the former. In all, our
results provide no support for a divergence in mean
litter size between chromosomal races, and agree with
Castiglia & Capanna (2000) that where differences
are recorded, they are most likely related to selective
or environmental factors acting locally. Thus, there is
no indication that these chromosomal races have
evolved differential reproductive strategies in relation
to the habitat segregation they display in Tunisia, as
suggested by Saïd et al. (1993). In effect, Chatti et al.
(1999a) showed that almost no demographic differences were found between populations of the two chromosomal races when they occurred in the same or in
different habitats. Likewise, Castiglia & Capanna
(2000) observed no significant shift of the contact zone
over a 20-year period between the Rb and standard
races in central Italy, as would have been expected had
large reproductive fitness differences been recorded
(Castiglia & Capanna, 1999).
HYBRID
FERTILITY PATTERNS
Among the fertility parameters measured, breeding
success of F1 hybrids was similar to that of parental
individuals involved in either intraracial or interracial
crosses. Although in agreement with the data of Castiglia & Capanna (2000) for the Italian chromosomal
races, these results contrast with those of Saïd et al.
(1993), who reported a lower breeding success of backcrosses. The lack of a difference in the present study
seems to be related to the low breeding success of pairs
involving a male 22Rb (intraracial: 40%; interracial:
33%; Table 1), which exhibited a value of 100% in the
previous analysis.
Despite the observed variation in litter size of
parental individuals, our results for F1 hybrids are
consistent with those of the previous analysis (Saïd
et al., 1993), as well as with other studies on chromosomal races of the house mouse (Hauffe & Searle,
1998; Castiglia & Capanna, 2000). In all cases, a significant decrease in litter size is observed in crosses
involving an F1 individual, although the extent of the
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 407–416
FERTILITY IN CHROMOSOMAL RACES OF HOUSE MOUSE
reduction is more pronounced in the Tunisian samples, even when compared with the central Italian Rb
hybrids, which have a similar degree of chromosomal
divergence (F1 individuals with 2n = 31). Interestingly,
this difference in hybrid fertility between the two sets
of races matches that in hybrid zone width, which
exceeds 14 km in central Italy (Spirito et al., 1980;
Castiglia & Capanna, 2000), compared with 0.5 km in
Tunisia (Chatti et al., 1999a). Such results support the
idea that the reduction in reproductive fitness of Tunisian hybrids contributes effectively to the isolation
between the 22Rb and 40St races. However, in addition to hybrid infertility, ecological and behavioural
factors should also be considered to explain the scarcity of hybrid individuals in the Tunisian hybrid zone
(Benzekri, Britton-Davidian & Ganem, 2002). In this
respect, it is of note that the litter size in the interracial crosses was significantly smaller than in
22Rb¥22Rb crosses. Although these results may be
ascribed to a maternal effect in the m22Rb¥f40St
crosses, i.e. litter size matched that of the least fertile
parental female (Table 1), such an explanation no
longer holds true for the reciprocal cross, suggesting
that inviability of F1 embryos may be involved to a certain extent in reducing litter sizes. The same effect
was detected in crosses between karyotypic races of
the house musk shrew Suncus murinus (Rogatcheva
et al., 1998), in which the less fertile individuals from
both sexes reduced the litter size of the more fertile
ones. Aulchenko et al. (1998) attributed this effect to
the genetic background of these laboratory strains.
A surprising finding from our data is the difference
in fertility parameters between male and female F 1
individuals. The analysis showed that crosses involving an F1 male had a higher breeding success, more litters (either total number or number per pair) and a
larger litter size than those involving an F 1 female.
None of the previous studies on litter size estimates
of fertility (Britton-Davidian et al., 1990; Viroux &
Bauchau, 1992; Hauffe & Searle, 1998; Castiglia &
Capanna, 2000) uncovered such consistent differences, although the same trend, albeit not significant,
was present in Saïd et al.’s (1993) previous assessment
for Tunisian races. Sex differences in the fertility of
Rb chromosomal hybrids have been observed for
anaphase I non-disjunction (NDJ) rates, which were
generally higher in females than in males, although
variation occurred with the type and number of Rb
fusions in a heterozygous state (Winking & Gropp,
1976; Gropp & Winking, 1981; Hauffe & Searle, 1998).
These results suggest that the higher infertility value
of the Tunisian F1 females may be related to the production of a larger number of aneuploid gametes, and
thus of embryo loss. Caution must be expressed, however, because Hauffe & Searle (1998) showed that the
values of germ cell death (GCD) and NDJ did not
413
always agree with litter size estimates. Unexpectedly,
a lower fertility of F1 female vs. male hybrids does not
follow Haldane’s rule, which assumes that, among the
F1 offspring between differentiated taxa, it is the heterogametic sex that is absent, rare or sterile (Haldane,
1922; Turelli, 1998). This suggests that hybrids
between house mice chromosomal races may be an
exception to this rule. The origin of the lower infertility values of female F1 chromosomal hybrids needs to
be assessed further by meiotic analyses.
The histological analysis of female and male Tunisian hybrids showed impairment of gametogenesis in
both sexes. In wild caught and laboratory-bred male
hybrids, the spermatogenic process was disturbed,
and in some cases was arrested at early stages, leading to complete sterility. The extent of this disturbance
was highly correlated with testis mass, which was lowest in the F1 individuals and increased slightly in
backcross individuals. Similar results for testis weight
have been observed in hybrids carrying seven to eight
heterozygous Rb fusions between Italian chromosomal
races (Hauffe & Searle, 1998). Evidence that premeiotic processes affect spermatogenesis in chromosomal
hybrids has previously been reported in house mice
(Burgoyne, Mahadeviah & Baker, 1985; Coerdt et al.,
1985; Redi et al., 1985; Chandley, 1988). However,
sterile phenotypes are usually restricted to chromosomal hybrids with complex meiotic chain configurations, as those carrying trivalent generally suffer from
subfertility (Redi & Capanna, 1988). Thus, hybrids
between the Tunisian chromosomal races represent an
exception to this pattern, as they show lower than
expected levels of hybrid fitness given their meiotic
configuration (see also Saïd et al., 1993). In addition, a
large number of studies have shown a positive relationship between NDJ rates and the number of segregating Rb fusions (Winking & Gropp, 1976; Redi &
Capanna, 1978; Gropp & Winking, 1981; Gropp, Winking & Redi, 1982; Harris, Wallace & Evans, 1986). In
the case of the Tunisian hybrids, no significant correlation was observed between either testis mass or
degree of spermatogenic disturbance and number of
heterozygous Rb fusions. Thus, this study confirms
that testis development and spermatogenesis are perturbed in these hybrids, and further indicate that the
degree of impairment is not related to chromosomal
heterozygosity per se. This suggests that genetic
incompatibilities between the 22Rb and 40St genomes
must be involved in generating the perturbed fertility
phenotypes. The existence of genic hybridity has also
been stated as a factor explaining variation in NDJ
rates in males heterozygous for different single Rb
translocations (Searle, 1988; Everett, Searle & Wallace, 1996). In the case of hybrid females, the total
oocyte pool was severely reduced in wild and laboratory-bred hybrids in comparison with 22Rb and 40St
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 407–416
414
N. CHATTI ET AL.
parents. In the F1 females, the important loss of primordial follicles most likely leads to a shortened
reproductive lifespan (see Burgoyne & Baker, 1984;
Garagna et al., 1990; Searle, 1993). In fact, there is a
marginally significant positive relationship between
the number of primordial follicles and that of litters
(N = 14, R = 0.525, P = 0.053). A decrease in oocyte
pool was also reported by Garagna et al. (1990),
whereas Hauffe & Searle (1998) found no differences
between parental and Rb heterozygotes, although
they indicated that ovary sections of pure-race individuals appeared healthier than those of hybrids.
Such a dysfunctional oogenesis most likely contributes
to the small-sized and infrequent litters, although an
increase in aneuploidy rates, which were not measured in this study, is probably also involved, as suggested by Hauffe & Searle (1998). Interestingly, the
number of corpora lutea was much higher in F1
females than in wild and backcross hybrids, which
indicates that a compensation effect may be occurring,
as suggested by Bengtsson (1980). The relationship
between the number of corpora lutea and ovulation
rate remains to be ascertained, but, if confirmed, these
results would suggest that both aneuploidy and oogenesis dysfunction participate in female hybrid infertility, because litter sizes are reduced despite a higher
ovulation rate. As observed for males, the extent of
oogenesis impairment was not related to the number
of heterozygous Rb fusions, but in this case was associated with heterozygosity of two individual fusions,
Rb(1.11) and Rb(8.9).
In summary, this study shows that the gametogenic
process is severely impaired in both male and female
chromosomal hybrids from Tunisia, to a larger extent
than in similar hybrids between European chromosomal races of the house mouse. Hybrid dysfunction in
litter size is more pronounced in females than in
males, probably owing to higher levels of embryo loss
due to aneuploidy in the former than in the latter.
Although this primary infertility is not related to chromosomal heterozygosity per se, two Rb fusions were
found to be significantly associated with oogenesis disturbance. These results suggest that genic incompatibilities contribute to hybrid dysfunction in the
Tunisian chromosomal hybrids. The observation that
specific Rb fusions are related to female infertility
suggests that these chromosomes carry genes, the
sequence or expression of which has diverged between
the chromosomal races. Previous assessments of
genetic differentiation have shown that the Tunisian
Rb race had a lower genic diversity than the neighbouring 40St populations (Saïd & Britton-Davidian,
1991). However, a recent allozymic analysis (see Ould
Brahim et al. 2005, this issue) has evidenced similar
heterozygosity rates and no genetic differentiation in
an additional sample of the Tunisian Rb race that oth-
erwise displays the same characteristics as recorded
elsewhere (i.e. narrow hybrid zone, habitat segregation; Chatti et al., 1999a). The existence of genic
incompatibilities affecting reproductive and developmental traits (Chatti et al., 1999b) supports the model
of chromosomal speciation in which a reproductive
barrier initiated by chromosomal underdominance
builds up by accumulation of genic differences
(Capanna, 1982; Saïd et al., 1999). Such results should
stimulate additional research on the nature of the
divergence (sequence or expression) between the
genes and their chromosomal location.
ACKNOWLEDGEMENTS
We are grateful to G. Ganem for invaluable help in the
field. This project was financed by Franco-Tunisian
CMCU (no. 95/F0931) and PICS (no. 01/0906) collaborations. This is contribution ISEM 2004-018.
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