Abstract

Hybrid sterility can prevent gene flow between diverging subpopulations and hence might contribute to speciation. The hybrid sterility 1(Hst1) gene was originally described in male progeny obtained from a cross between laboratory inbred mouse strains C57BL/10 and C3H, and wild Mus musculus musculus, sampled at localities in Prague, Czech Republic. This study asked whether the presence of sterility associated with the Hst1gene is limited to one local population or is extended over geographically distant regions. We studied the progeny derived from a wild population of M. m. musculussampled in Studenec, Czech Republic, 160 km south-east from Prague, crossed reciprocally to C57BL/10 mice. Spermatogenesis was examined in 251 hybrid males; among them 109 males (43.4%) were sterile and 142 (56.6%) were fertile. Sterile males had significantly lower testis mass and lower epididymis mass compared with fertile males. The size of the reproductive organs was dependent on cross reciprocity within the classes of sterile and fertile males. Although our phenotype data resemble those presented in the original description of Hst1, molecular analysis revealed incomplete segregation of sterility and fertility in male progeny and markers from the Hst1region in some families. Therefore, there are probably additional genes affecting hybrid sterility that are polymorphic in wild M. m. musculus.

INTRODUCTION

Two house mouse subspecies, Mus musculus musculusand M. m. domesticus, meet and form a narrow hybrid zone, which runs across Europe (Boursot et al., 1993; Sage, Atchley & Capanna, 1993; Macholán,Kryštufek & Vohralík, 2003; see also Božíkováet al., 2005, this issue; Dod et al., 2005, this issue; Raufaste et al., 2005, this issue). Despite long-standing interest in studying this hybrid zone, there is no direct evidence from wild mice of the mechanisms which keep the two mouse genomes separate (Boursot et al., 1993; Sage et al., 1993). Hybrid sterility is one of the reproductive isolating barriers that can prevent gene flow between diverging populations (Coyne & Orr, 1998; Howard et al., 2002) and is also well-documented in the laboratory mouse (Forejt, 1996 and references therein). In particular, the study of the hybrid sterility 1(Hst1) gene could be of great importance in understanding the genetics of mouse speciation because it was described from crosses between M. m. musculusand M. m. domesticus.

Hst1was the first genetically dissected gene to be described in mammals (Forejt & Iványi, 1975). When the laboratory inbred strain C57BL/10 (herein referred to as B10) is crossed with some wild M. m. musculus, the resulting male F1 hybrids are sterile. The phenotypic effect of sterility is characterized by complete arrest of spermatogenesis at the pachytene stage of primary spermatocytes indicating a cell autonomous, germ-cell specific defect (Iványi et al., 1969; Yoshiki et al., 1993). Sterile males have reduced mass of testes and no sperm in the ductus epididymis, but normal levels of testosterone in the blood (Forejt & Iványi, 1975; Forejt, 1985). The F1 hybrid females from the same cross are fertile (Forejt & Iványi, 1975); this means the sterility obeys Haldane's rule (Haldane, 1922). Another laboratory inbred strain, C3H/Di, produced fertile progeny when crossed with wild M. m. musculus. The difference between the C3H/Di and B10 mice with respect to the fertility phenotype was attributed to a gene, mapped to mouse chromosome 17 within the region delimited by the Tand H2loci in the wild-type form of the t-complex region (Forejt & Iványi, 1975; Forejt, 1981; Forejt et al., 1991).

The nomenclature of Hst1alleles reflects the outcome of crosses between wild and laboratory mice. Chromosome 17 in the B10 strain was suggested to carry the Hst1s(sterility ensuring) allele, while chromosome 17 in the C3H strain was suggested to carry the Hst1f(fertility ensuring) allele. The gene located on chromosome 17 of wild M. m. musculusmice was named Hstw, with two alleles, Hstwsand Hstwf.The Hstwgene, polymorphic in wild males, has been located in the same linkage group as Hst1; nevertheless, there has been no direct evidence as yet that this gene occupies the same locus, i.e. that it is identical to the Hst1gene (Forejt & Iványi, 1975).

Allelic interactions at the Hst1locus define the phenotypic effect: only the combination Hst1s/Hstwsensures sterility; all other homozygous or heterozygous forms are fertile (Forejt & Iványi, 1975; Forejt, 1985; Forejt et al., 1991; Forejt, 1996). The single-gene inheritance of hybrid sterility is true only for crosses between laboratory and wild (W) mice: W × (B10 × C3H). If, however, the fertile hybrid females from crosses of wild and laboratory mice (B10 × W) are used for further crosses ((B10 × W) × B10), then genetic control of male sterility is much more complex. The male progeny delivered from such backcrosses display almost continuous variation, from full spermatogenetic arrest to complete fertility, suggesting the interaction of about three nonallelic genes (Forejt & Iványi, 1975).

The Hst1gene causes male sterility only between M. m. musculusand laboratory strains considered to be derived from M. m. domesticus. Other hybrid sterility genes in mice have been described from crosses between laboratory strains and M. spretus(Guénet et al., 1990; Pilder, Hammer & Silver, 1991; Pilder et al., 1993; Pilder, 1997), taxa which hybridize only rarely in nature (Orth et al., 2002).

This report is part of a three-step project we designed to study the potential effects of hybrid sterility on the pattern and dynamics of the hybrid zone between M. m. musculusand M. m. domesticus. In the first step, individuals from a wild population of M. m. musculuswere crossed with B10. Using this standard laboratory strain, we basically repeated the experiment performed in Forejt's laboratory in the 1960s and 1970s (Forejt & Iványi, 1975). Although the B10 strain has traditionally been considered to represent predominantly the M. m. domesticusgenome, molecular analyses have shown genomes of most old (‘classical’) inbred strains (including B10) to be mosaics of a number of various segments derived from domesticus, musculusand castaneussources (Bishop et al., 1985; Bonhomme et al., 1987; Nagamine et al., 1992; Wade et al., 2002). Therefore, the results of this first experiment may be of limited value for assessing the evolutionary significance of male sterility upon hybridization in natural populations of mice. In the second step, we aimed to study hybrid sterility in completely natural populations of mice using the M. m. musculusmice tested in the first step and replacing the B10 mice with wild M. m. domesticussampled near the hybrid zone. Finally, we will determine the extent of hybrid sterility polymorphism by testing geographically distant populations of M. m. domesticus.

In this paper we report on data obtained during the first step of the project. We specifically asked if sterile males were produced irrespective of the direction of the cross. Also, as little effort had been put into evaluating the geographical extent of hybrid sterility within M. m. musculuspopulations, we tested whether male sterility associated with the Hst1gene is a unique trait of local Prague populations. Therefore, a sample of wild mice from a geographically distant population in Moravia, Czech Republic, was used instead of wild or inbred mice originating in Prague (Forejt & Iványi, 1975; Gregorová & Forejt, 2000). The analysis of spermatogenesis in male offspring derived from crosses between these mice and the B10 strain proved that hybrid sterility could be found widely in populations of M. m. musculusand that spermatogenetic breakdown was independent of the direction of the cross. Sterile males had significantly lower testis mass and lower epididymis mass compared with fertile males. These phenotypic data correspond closely with those ascertained in the original description of the Hst1gene (Forejt & Iványi, 1975). Molecular analysis using six DNA markers which map within and around the Hst1region on chromosome 17 failed to reveal complete association between these markers and the presence/absence of sperm in hybrid males. Further studies will be necessary to show whether the genes causing hybrid sterility in mice of the two M. m. musculuspopulations are identical.

MATERIAL AND METHODS

Mice

Wild mice were sampled in the village of Studenec, Czech Republic (49°11′N, 16°03′E) in November 2000. The population was located within the M. m. musculusrange for the Czech Republic, as delineated by Munclinger et al. (2002). To confirm that these mice were of M. m. musculusorigin, we genotyped individuals for four diagnostic markers: the presence/absence of (1) the BamHI restriction site in mitochondrial DNA (Boursot et al., 1996; Božíkováet al., 2005, this issue); (2) an 18-bp deletion located within the Zinc finger protein 2gene on the Y chromosome (Nagamine et al., 1992; Boissinot & Boursot, 1997); (3) the B1 insertion in the Bruton agammaglobulinemia tyrosine kinasegene on the X chromosome (Munclinger, Boursot & Dod, 2003); (4) the PCR product using subspecies-specific primers within the Androgen-binding protein alphagene on chromosome 7 (Dod et al., 2005, this issue; B. Bímová, unpubl. data). All markers were of the musculustype with no sign of introgression (data not shown). The mice of the B10 strain were purchased from a local provider (Velaz, Praha). All mice were kept under standard conditions in a breeding facility in Studenec (perspex cages measuring 30 × 15 × 15 cm, food and water available ad libitum, 14 : 10 photoperiod, with light on between 06:30 h and 20:30 h).

Experimental mating

Experimental crosses with the mice were started after 2 weeks of keeping them in isolation following the design shown in Figure 1. Assuming a roughly bimodal distribution for the presence or absence of sterility in the offspring if the parental individuals were heterozygotes for the hybrid sterility gene, we planned to use at least eight individuals per cross. Because of unknown age of wild animals we first derived progeny from ten pairs propagated within the sampled population. The two generations are referred to here as G0 and  G1 generations  (Fig. 1).  Eleven  G1 males  and  30 G1 females were mated with B10 individuals. To distinguish the direction of each cross we always present the female first (for example, female G1 × male B10 is abbreviated to G1 × B10). Usually, one male was paired with several females in one large cage and the females were examined for pregnancy every third day. Pregnant females were removed, housed singly in a standard cage and checked daily for the presence of a litter. F1 hybrids were weaned at 20 days of age. After weaning all males were housed individually or with two per cage. In the latter case, the males were separated after 55 days and housed singly for five days to remove the effect on sperm count of social structure among cage mates.

Figure 1.

Scheme of experimental crosses used to derive males scored for sterility.

Figure 1.

Scheme of experimental crosses used to derive males scored for sterility.

Phenotype scoring

All males were phenotyped at 60 days of age. Animals were sacrificed, three external measurements were taken (body mass and length and tail length), and the spleen was removed and preserved in 96% ethanol for molecular analysis. In addition, the left testis and epididymis were weighed immediately after dissection; the length and width of the left testis were also measured. The whole left epididymis was transferred into 1 mL 1% sodium citrate solution in a watchglass, cut with scissors into tiny pieces, and squashed with a pair of tweezers. Another 1 mL 1% sodium citrate solution was added and the suspension was mixed thoroughly using a pipette and left for 15 min. After maceration, the suspension was pipetted again and applied to a Bürker haematocytometer. If sperm were present, we counted sperm heads in five chambers selected arbitrarily. The number of sperm characterizing each hybrid male is presented as the average sperm count (ASC) over five chambers.

Molecular analysis

A small piece of ethanol-preserved tissue was digested in extraction buffer (100 m m EDTA, 100 m m NaCl, 50 m m Tris pH 8, 1% SDS) and proteinase K (20 µg/mL). DNA was extracted using a standard phenol–chloroform procedure (Hoelzel, 1992).

Two markers located outside and four markers located within the Hst1region on chromosome 17 (Fig. 2) were scored to find the association between the Hst1gene and sterility in hybrid progeny. The presence of male sterility can be masked by the detrimental effect of thaplotypes on male fitness. Most thaplotypes carry recessive lethal mutations (Bennett, 1975). Mice homozygous for the same lethal thaplotype die at early stages of development, while individuals carrying two different thaplotypes with complementing lethal or semilethal factors are viable, but male-sterile (Silver, 1985). To distinguish between these two alternative causes of male sterility, we genotyped mice for the presence of thaplotype-specific microsatellite marker Hba-ps4, located in the distal part of the t-complex, using the protocol by Schimenti & Hammer (1990). From the proximal part of the t-complex we used microsatellite marker D17Mit164(Dietrich et al., 1996).

Figure 2.

Diagram of the t-complex region of wild-type (non-t) origin on the proximal part of chromosome 17. Boxes In1–In4 indicate four inversions associated with the thaplotype. Distances of D17Mit164, Hst1and Hba-ps4are in centiMorgans (cM) relative to the centromere (ellipse at the left). Map positions of four markers spanning the Hst1region are shown above in the enlargement and their distances are in cM relative to the Hst1gene.

Figure 2.

Diagram of the t-complex region of wild-type (non-t) origin on the proximal part of chromosome 17. Boxes In1–In4 indicate four inversions associated with the thaplotype. Distances of D17Mit164, Hst1and Hba-ps4are in centiMorgans (cM) relative to the centromere (ellipse at the left). Map positions of four markers spanning the Hst1region are shown above in the enlargement and their distances are in cM relative to the Hst1gene.

The markers from the Hst1region were designed in the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic. In a pilot study, 17 markers from the Hst1region were tested for length polymorphism in M. m. musculusDNA samples using PCR. One marker, DB3, found within this region has been  presented  elsewhere  (Trachtulec et al.,  2005, this issue) and three other suitable markers, CH07, M334 and Ph4, are presented here. The forward and reverse primers of the first two markers were CH07F (5′-TTGCTATAAAAGGACTGTTTGAT) and CH07L (5′-ACACAAAGACAGAAGAAGAGGA); M334F (5′-TGGTTACTGGTTATCATCCTC) and M334L (5′-GGCTTGGTATTTTCTCCTTAG). The last primer flanked an inserted element RLTR10 in the intron of the D17Ph4egene. Because the original primers amplified long fragments, not suitable for routine scoring, new primers flanking the RLTR10 element were designed by Pavel Munclinger using the programme Primer3 (Rozen & Skaletsky, 2000). The sequences of these new primers were: Ph4INF (5′-CTGGGTCCTC CAATCTAGCA) and Ph4INR (5′-GATTGAGGTGA GCCCAAGAG).

An aliquot of 50 ng genomic DNA was amplified using the gradient RoboCycler thermal cycler (Stratagene) in a 10-µL PCR reaction mix with 2 m m MgCl2, 200 µm dNTPs, 0.5 U Taqpolymerase and 0.3 µm each primer. The reactions were amplified for 38 cycles of 94 °C for 30 s, 58 °C for 40 s and 72 °C for 40 s. The only exception was DB3, which was amplified for 38 cycles of 94 °C for 30 s, 58 °C for 1 min and 72 °C for 2 min. PCR products were separated on 5% agarose gels, except for the Ph4 fragments, which were separated on 2% agarose gels. Figure 2 shows a map of all markers.

Statistical analysis

To analyse the effects of measured variables, we grouped the male offspring according to the original wild-caught G0 females. In the case of the G1 × B10 cross, the descendants of as many as four G1 females were pooled together, while in the B10 × G1 cross, the descendants of one wild-derived male were usually grouped. Descriptive statistics were calculated for these groups.

Logistic regression was used to fit the probability of the presence of sterile and fertile males to a linear model which included the effects of phenotypic variables. The Newton iteration process was applied to fit a model; this process stops when the log-likelihood (LL) reaches its minimum. When finished, it counts the difference in LL between the full model, which includes regressing variables, and the reduced model, which has only intercepts. Twice the value of the difference in negative log-likelihoods, 2LL, approximates a chi-square distribution, and this value was used to test the significance of the full model.

The effects of the direction of the cross (G1 × B10 vs. B10 × G1) in sterile and fertile males were estimated by Student's t-tests. Due to the design of crosses in which we mated one male with several females, we introduced additional genetic variability into the cross between G1 females and B10 males. This might result in unequal variance of the measured variables between the two types of crosses. Therefore, in all t-tests we assumed unequal variances among reciprocal tests and accounted for this factor in statistical analyses. JMP statistical software (SAS Institute Inc., 2002) was used for all analyses.

RESULTS

Defining male sterility

Forejt & Iványi (1975) described a close relationship between the paired testes mass and fertility of hybrid males. The fertility was estimated from the number of offspring sired by single males (wild M. m. musculus) paired with single females (B10) for 1 month. The authors showed that the number of offspring in such matings is dependent on the testes mass and determined that hybrid males with a testes mass lower than 75 mg are sterile, whereas normal fertility was ensured by testes heavier than 120 mg. A testes mass in the range of 75–120 mg reflects continuous variation from complete sterility to fertility.

We used the relationship described above to define sterility of hybrid males in our crosses. Because we did not measure fitness of males directly in terms of the numbers of offspring produced by hybrid males, we related testes mass to ASC. The rationale behind this approach was that fertility has been shown repeatedly to be a function of the number of sperm produced in the testes and stored in the epididymides (e.g. Searle & Beechey, 1974; Forejt & Iványi, 1975); therefore, we expected that distinct groups of fertile and sterile males would be clustered separately. These two clusters can be visualized either in a morphospace defined by testis mass and ASC or in histograms showing the distribution for the scored traits.

The relationship between left testis mass and ASC are plotted in Figure 3 for progeny of both crosses. Both panels indicate the presence of two clusters: first, there were numerous males with low testis mass and ASC = 0, and second, there were males with both heavy testis and high ASC scores. However, a small proportion of males displayed rather larger testes but with low scores of ASC, contrary to expectation (we discuss this pattern below).

Figure 3.

Relationship between average sperm count and testis mass plotted for both reciprocal crosses: B10 ¥ Mus musculus musculus(A) and M. m. musculus¥ B10 (B). White circles depict males which could be scored for phenotype traits only; black circles indicate males involved in molecular analysis of segregation between sterility and the Hst1region.

Figure 3.

Relationship between average sperm count and testis mass plotted for both reciprocal crosses: B10 ¥ Mus musculus musculus(A) and M. m. musculus¥ B10 (B). White circles depict males which could be scored for phenotype traits only; black circles indicate males involved in molecular analysis of segregation between sterility and the Hst1region.

To define hybrid male sterility in our crosses we looked at the distributions of single traits. As reasoned above we expected to find two peaks, one each associated with sterility and fertility. Defining a nonoverlapping region, if the two distributions are separated, or a point of intersection, if the distributions overlap, would help define the limits between sterility and fertility. We found the deepest trough in values for distributions of ASC at level ASC = 5 (Fig. 4, upper panels). Based on these findings we defined sterile males as those with ASC < 5, while we considered fertile males to be those with ASC ≥ 5.

Figure 4.

Distributions of average sperm counts, left testis mass and left epididymis mass plotted separately for both reciprocal crosses B10 ¥ Mus musculus musculus(left) and M. m. musculus¥ B10 (right). Arrows in the average sperm count (ASC) distributions depict the borderline between sterility and fertility. Black bars in the remaining panels indicate the distributions of the organs measured among sterile males (ASC < 5) and white bars show distributions among fertile males (ASC ≥ 5).

Figure 4.

Distributions of average sperm counts, left testis mass and left epididymis mass plotted separately for both reciprocal crosses B10 ¥ Mus musculus musculus(left) and M. m. musculus¥ B10 (right). Arrows in the average sperm count (ASC) distributions depict the borderline between sterility and fertility. Black bars in the remaining panels indicate the distributions of the organs measured among sterile males (ASC < 5) and white bars show distributions among fertile males (ASC ≥ 5).

Several lines of evidence have indicated that some males having few sperm in their epididymides are sterile. We observed a high proportion of abnormal sperm heads in a category of males with low ASC scores. Furthermore, histological studies have revealed that few spermatogonia can escape the spermatogenic block and differentiate to sperm (Yoshiki et al., 1993). Finally, Searle & Beechey (1974) reported that reduced fertilization is likely in mice whenever sperm counts fall to less than 10% of normal. All these data suggest that the limiting value of the sperm count estimated from the trough in histograms of ASC represents a reliable estimator of sterility in hybrid males.

Phenotypic data

Of ten pairs of wild mice used to derive the G1 generation, all pairs produced at least one male and nine pairs produced at least one female, which could have been used for mating with the B10 mice. When more offspring were produced we selected individuals for experimental breeding arbitrarily. In total, we obtained 251 F1 males in reciprocal crosses between G1M. m. musculusand B10 mice. Of these, 138 males were produced in crosses between B10 females and G1 males from Studenec and 113 males were produced in the reciprocal cross. The proportion of sterile males (50.4%) was equal to that of fertile males in the G1 × B10 progeny, but it was skewed towards fertile males in the B10 × G1 progeny (37.7% sterile males). The proportion of sterile males in the whole dataset was 43.4%.

We did not find any predictable relationship between external morphological traits and the occurrence of sterility in the whole set of F1 hybrids. Nominal logistic regression (NLR) revealed that the presence of fertile and sterile males was distributed independent of body mass (2LL < 0.016, P= 0.901, N= 251) or body length (2LL = 0.139, P= 0.71, N= 250). The only external trait showing association with sterility/fertility was tail length (2LL = 6.216, P= 0.013, N= 240), with a negative relationship between fertility and tail length (slope = −0.084, P= 0.015). However, when this trait was regressed on ASC, this relationship disappeared (linear regression, F1,238 = 3.79, P= 0.053). On the other hand, the presence of fertile and sterile males was significantly dependent on testis mass (NLR, 2LL = 255.382, P< 0.001, N= 251) and epididymis mass (NLR, 2LL = 206.854, P< 0.001, N= 251).

Values of variables differing between sterile and fertile F1 hybrids are presented in Table 1. There was a significant effect of reciprocity of mating on testis mass of sterile males (t= 4.29, N= 109, P< 0.001). Males from crosses of female M. m. musculus× B10 males displayed larger testes (average left testis mass = 43.3 mg) compared with males from reciprocal crosses (average left testis mass = 34.6 mg). Sterile males had, on the other hand, the same mass of the left epididymis irrespective of cross reciprocity (t= 1.72, N= 109, P< 0.088; average mass = 16.6 mg).

Table 1.

Results of phenotype scores summarized over G0 females and reciprocal crosses

G0 mice G1 mice Sterile males
 
Fertile males
 
N Left testis mass Left epididymis ASC N Left testis mass Left epididymis ASC 
(B10 × G1) F1 hybrid male progeny 
2802 3010 33.3(4.1) 14.0(1.5) >0.4(0.6) 88.4(12.5) 26.7(3.7) 35.5(4.6) 
2803 3370 31.2(8.5) 14.3(1.6) >0.1(0.1) 77.9(5.3) 23.9(1.5) 26.1(8.0) 
2809 2998 39.0 18.2 0.2 17 78.8(7.9) 25.9(2.9) 25.5(5.0) 
2810 3660 30.9(4.1) 19.4(1.6) 86.9(13.9) 26.4(2.2) 26.9(9.4) 
2813 3006 34.5(6.1) 17.0(1.8) >0.4(0.5) 13 62.7(12.0) 21.2(2.8) 19.0(5.7) 
2814 4337 51.6(7.1) 17.5(1.1) >3.2(1.0) 65.2(8.1) 19.3(3.2) 13.0(7.7) 
2818 3578 28.9(4.8) 17.1(1.3) >0.6(0.7) 16 71.6(7.2) 23.2(3.3) 30.1(8.5) 
2819 4269 31.3 15.1 75.0(7.7) 23.0(3.2) 25.0(6.9) 
2821 3112 10 29.1(2.7) 15.9(1.4) –    
3507 29.0(1.7) 16.7(1.1) –    
2826 3112 50.8(11.0) 17.7(1.1) >1.6(1.7) 65.4(7.1) 20.1(2.2) 11.5(4.7) 
Mean  52 34.6(9.9) 16.2(2.0) >0.6(1.2) 86 73.6(12.3) 23.3(3.7) 23.9(9.5) 
(G1 ¥ B10) F1 hybrid male progeny 
2802 3220  5 38.3(4.0) 16.9(1.9) 57.8(6.1) 23.5(3.5)  6.5 (0.5) 
3221 –    116.6(8.6) 28.4(2.1) 27.6(7.3) 
3222 51.4(8.2) 16.9(1.9) >1.8(2.0) 90.5(7.1) 26.5(2.3) 30.7(8.9) 
2809 3001 47.5(9.4) 17.2(0.5) >0.1(0.2) 87.8(2.0) 24.6(0.4) 20.3(7.3) 
3148 42.4 15.7 88.8 22.0 33.0 
3353 –    110.2(10.2) 29.0(2.2) 47.1(8.2) 
2810 3413 –    131.7 30.4 73.4 
2813 3436 –    75.4(22.6) 21.0(3.3) 20.6(15.4) 
3437 –    108.1(14.3) 26.0(1.0) 43.2(1.0) 
3438 –    94.6(5.1) 25.4(2.7) 47.1(3.8) 
3668 55.9 17.9 114.1 28.3 39.6 
2814 4700 –    125.6 28.5 41.2 
4701 50.5 28.5 –    
4702 56.3(6.8) 17.6(1.7) >0.7(1.0) 99.1(7.7) 25.2(2.2) 31.6(12.8) 
2818 3971 43.8(0.5) 17.8(1.6) >0.3(0.5) –    
3972 –    86.3(13.4) 26.3(4.4) 36.9(6.7) 
2819 3100 –    76.2(6.0) 24.4(0.4) 13.1(3.1) 
3295 53.2(8.7) 20.2(0.7) >1.4(1.4) 80.6(12.0) 21.2(3.3) 17.0(6.5) 
3740 –    98.6(5.0) 23.7(1.9) 14.8(8.4) 
3741 68.5 21.0 113.9 31.2 52.0 
4014 –    97.7 27.6 30.2 
4015 37.7 17.9 92.8(5.4) 26.0(1.1) 41.0(4.4) 
2821 2987 32.0(0.9) 14.6(1.0) –    
3988 33.5(3.0) 15.7(0.8) –    
3114 31.5 16.4 –    
3510 36.0(3.3) 14.8(2.2) –    
2826 2995 41.7(2.6) 16.6(3.6) –    
3124 61.1(15.4) 18.3(2.6) >2.3(2.3) 105.2(7.6) 19.8(4.2) 17.9(4.4) 
3125 49.1(12.0) 19.7(1.9) >0.8(0.9) 88.0 25.3 17.8 
3126 44.8 15.6 87.8(18.2) 22.0(1.2) 14.7(9.5) 
Mean  57 43.3(11.3) 16.9(2.3) >0.4(1.1) 56 97.2(18.3) 25.4(3.7) 29.8(15.3) 
G0 mice G1 mice Sterile males
 
Fertile males
 
N Left testis mass Left epididymis ASC N Left testis mass Left epididymis ASC 
(B10 × G1) F1 hybrid male progeny 
2802 3010 33.3(4.1) 14.0(1.5) >0.4(0.6) 88.4(12.5) 26.7(3.7) 35.5(4.6) 
2803 3370 31.2(8.5) 14.3(1.6) >0.1(0.1) 77.9(5.3) 23.9(1.5) 26.1(8.0) 
2809 2998 39.0 18.2 0.2 17 78.8(7.9) 25.9(2.9) 25.5(5.0) 
2810 3660 30.9(4.1) 19.4(1.6) 86.9(13.9) 26.4(2.2) 26.9(9.4) 
2813 3006 34.5(6.1) 17.0(1.8) >0.4(0.5) 13 62.7(12.0) 21.2(2.8) 19.0(5.7) 
2814 4337 51.6(7.1) 17.5(1.1) >3.2(1.0) 65.2(8.1) 19.3(3.2) 13.0(7.7) 
2818 3578 28.9(4.8) 17.1(1.3) >0.6(0.7) 16 71.6(7.2) 23.2(3.3) 30.1(8.5) 
2819 4269 31.3 15.1 75.0(7.7) 23.0(3.2) 25.0(6.9) 
2821 3112 10 29.1(2.7) 15.9(1.4) –    
3507 29.0(1.7) 16.7(1.1) –    
2826 3112 50.8(11.0) 17.7(1.1) >1.6(1.7) 65.4(7.1) 20.1(2.2) 11.5(4.7) 
Mean  52 34.6(9.9) 16.2(2.0) >0.6(1.2) 86 73.6(12.3) 23.3(3.7) 23.9(9.5) 
(G1 ¥ B10) F1 hybrid male progeny 
2802 3220  5 38.3(4.0) 16.9(1.9) 57.8(6.1) 23.5(3.5)  6.5 (0.5) 
3221 –    116.6(8.6) 28.4(2.1) 27.6(7.3) 
3222 51.4(8.2) 16.9(1.9) >1.8(2.0) 90.5(7.1) 26.5(2.3) 30.7(8.9) 
2809 3001 47.5(9.4) 17.2(0.5) >0.1(0.2) 87.8(2.0) 24.6(0.4) 20.3(7.3) 
3148 42.4 15.7 88.8 22.0 33.0 
3353 –    110.2(10.2) 29.0(2.2) 47.1(8.2) 
2810 3413 –    131.7 30.4 73.4 
2813 3436 –    75.4(22.6) 21.0(3.3) 20.6(15.4) 
3437 –    108.1(14.3) 26.0(1.0) 43.2(1.0) 
3438 –    94.6(5.1) 25.4(2.7) 47.1(3.8) 
3668 55.9 17.9 114.1 28.3 39.6 
2814 4700 –    125.6 28.5 41.2 
4701 50.5 28.5 –    
4702 56.3(6.8) 17.6(1.7) >0.7(1.0) 99.1(7.7) 25.2(2.2) 31.6(12.8) 
2818 3971 43.8(0.5) 17.8(1.6) >0.3(0.5) –    
3972 –    86.3(13.4) 26.3(4.4) 36.9(6.7) 
2819 3100 –    76.2(6.0) 24.4(0.4) 13.1(3.1) 
3295 53.2(8.7) 20.2(0.7) >1.4(1.4) 80.6(12.0) 21.2(3.3) 17.0(6.5) 
3740 –    98.6(5.0) 23.7(1.9) 14.8(8.4) 
3741 68.5 21.0 113.9 31.2 52.0 
4014 –    97.7 27.6 30.2 
4015 37.7 17.9 92.8(5.4) 26.0(1.1) 41.0(4.4) 
2821 2987 32.0(0.9) 14.6(1.0) –    
3988 33.5(3.0) 15.7(0.8) –    
3114 31.5 16.4 –    
3510 36.0(3.3) 14.8(2.2) –    
2826 2995 41.7(2.6) 16.6(3.6) –    
3124 61.1(15.4) 18.3(2.6) >2.3(2.3) 105.2(7.6) 19.8(4.2) 17.9(4.4) 
3125 49.1(12.0) 19.7(1.9) >0.8(0.9) 88.0 25.3 17.8 
3126 44.8 15.6 87.8(18.2) 22.0(1.2) 14.7(9.5) 
Mean  57 43.3(11.3) 16.9(2.3) >0.4(1.1) 56 97.2(18.3) 25.4(3.7) 29.8(15.3) 

Average values for measured variables (plus standard deviations in parentheses) are averaged over males produced by individual G1 females; means show averaged values over G0 females. ASC, average sperm count.

We restricted the analysis of the effect of reciprocity in fertile males to families that produced more than five males in both types of cross. This restricted dataset included 111 males with 53 and 58 hybrid males from crosses G1 × B10 and B10 × G1, respectively. We found significant differences in testis mass (t= 8.53, P< 0.001; hybrid males from the G1 × B10 cross had an average mass of 97.0 mg, while those from B10 × G1 had an average mass of 72.2 mg), epididymis mass (t= 3.11, P< 0.002; hybrid males from G1 × B10 cross had an average mass of 25.2 mg, while those of B10 × G1 had an average mass of 23.0 mg), and ASC (t= 3.14, P< 0.002; hybrid males from G1 × B10 cross had on average 28.68 sperm, while those of B10 × G1 had on average 21.61 sperm).

The distributions of sperm count, epididymis and testis mass were bimodal in both types of cross (Fig. 4), the only exception being the distribution of ASC in the M. m. musculus× B10 cross, in which the number of fertile males was rather uniform over the classes of sperm counts. The overlaps of values for sterile and fertile males were higher for left epididymis mass than they were for left testis mass.

Molecular data

To assess the role of the Hst1gene in the sterility described in the previous section, a segregation analysis was performed using molecular markers on G1 and F1 mouse DNA. The results of the molecular analysis are summarized in Table 2. Three new markers were introduced to map genes from the Hst1region: CH07, M334 and Ph4. Two of them, CH07 and M334, as well as the already published marker DB3, were found to be highly polymorphic with up to six detectable alleles on the gel. The remaining three markers displayed only two different alleles. One of the Hba-ps4alleles was preferentially associated with the presence of thaplotypes. The remaining two markers, D17Mit164and Ph4, displayed one allele with the same length as the B10 allele and the second allele was found in some M. m. musculus.

Table 2.

The genotypes of D17Mit164, four markers characterized at the Hst1region (DB3, Ph4, CH07, M334), and thaplotype (Hba-ps4) of nine G1 mice and 76 hybrid males with corresponding data for left testis mass and average sperm count (ASC)

G0 mice G1 mice F1 no. Left testis mass ASC Markers
 
Mit164 DB3 Ph4 CH07 M334 Hba 
2802 3010    a/a a/f b/b a/c a/e +/+ 
88.4(12.5) 35.5(4.6) +/+ 
33.3(4.1) >0.4(0.6) +/+ 
3222    a/a a/b a/a b/b a/b +/+ 
95.5(0.1) 24.9(4.5) +/+ 
51.4(8.2) >1.8(2.0) +/+ 
2809 3001    a/b b/c a/a b/f a/e +/+ 
87.8(2.0) 20.3(7.3) +/+ 
47.5(9.4) >0.1(0.2) +/+ 
2813 3006    a/b c/f a/b a/d b/e +/+ 
68.1(10.2) 22.9(3.0) +/+ 
34.4(6.1) >0.3(0.4) +/+ 
2814 4702    a/b c/d a/a a/b e/e +/+ 
102.9(7.7) 35.3(14.1) +/+ 
96.3(6.4) 28.9(11.0) +/+ 
65.9 2.2 +/+ 
51.5(1.1) 0 (0) +/+ 
2818 3578    a/a c/e b/b c/d c/d +/t 
10 70.3(5.8) 30.9(7.4) +/t 
30.3(5.5) >0.8(0.8) +/+ 
26.2 0.2 +/t 
2819 3295    a/a b/d a/b b/e d/e +/t 
98.6 27.2 +/t 
69.7(0.2) 12.8(3.6) +/+ 
44.4 +/+ 
2821 3112    a/a b/c a/b b/c a/e +/+ 
30.9(2.0) 0 (0) +/+ 
27.4(2.3) 0 (0) +/+ 
3507    a/a b/c a/b b/c a/e +/+ 
29.8(1.2) 0 (0) +/+ 
28.0(1.9) 0 (0) +/+ 
G0 mice G1 mice F1 no. Left testis mass ASC Markers
 
Mit164 DB3 Ph4 CH07 M334 Hba 
2802 3010    a/a a/f b/b a/c a/e +/+ 
88.4(12.5) 35.5(4.6) +/+ 
33.3(4.1) >0.4(0.6) +/+ 
3222    a/a a/b a/a b/b a/b +/+ 
95.5(0.1) 24.9(4.5) +/+ 
51.4(8.2) >1.8(2.0) +/+ 
2809 3001    a/b b/c a/a b/f a/e +/+ 
87.8(2.0) 20.3(7.3) +/+ 
47.5(9.4) >0.1(0.2) +/+ 
2813 3006    a/b c/f a/b a/d b/e +/+ 
68.1(10.2) 22.9(3.0) +/+ 
34.4(6.1) >0.3(0.4) +/+ 
2814 4702    a/b c/d a/a a/b e/e +/+ 
102.9(7.7) 35.3(14.1) +/+ 
96.3(6.4) 28.9(11.0) +/+ 
65.9 2.2 +/+ 
51.5(1.1) 0 (0) +/+ 
2818 3578    a/a c/e b/b c/d c/d +/t 
10 70.3(5.8) 30.9(7.4) +/t 
30.3(5.5) >0.8(0.8) +/+ 
26.2 0.2 +/t 
2819 3295    a/a b/d a/b b/e d/e +/t 
98.6 27.2 +/t 
69.7(0.2) 12.8(3.6) +/+ 
44.4 +/+ 
2821 3112    a/a b/c a/b b/c a/e +/+ 
30.9(2.0) 0 (0) +/+ 
27.4(2.3) 0 (0) +/+ 
3507    a/a b/c a/b b/c a/e +/+ 
29.8(1.2) 0 (0) +/+ 
28.0(1.9) 0 (0) +/+ 

Allele b is identical with the B10 allele. In the notation of genotypes of F1 hybrids, the B10 alleles were removed for simplicity from the first five markers. Relative sizes of alleles are for D17Mit164: b = B10 > a; DB3: d > c > a > f > b = B10 > e; Ph4: a > b = B10; CH07: d > e > b = B10 > f > a > c; M334: e > c > a > b = B10 > d.

For technical reasons, we could not score all offspring so we had to restrict our analysis to families in which sons possessed alleles differing substantially in length of the scored microsatellites. In total, we analysed 76 males representing nine segregating families. (Note  that  in  Table 2 two  families  descended  from  a G0 female 2821 were included; however, only sterile males were produced in these families. This reduced the number of families suitable for mapping of sterility to seven.)

The t-haplotype associated allele at the Hba-ps4locus was detected in only two families. In one case it segregated with both sterile and fertile males (in the family derived from 3578 G1 female). In the second case this allele was present in a fertile male. Thus, the presence of thaplotypes did not contribute to male sterility in this study and was excluded from further analysis.

Because of the close proximity of all molecular markers used here we assumed a priori that only two haplotypes would be found in segregating families. In fact, this assumption was confirmed in all nine families. No recombination was found among 76 males scored. We found 15 different haplotypes among the progeny and there were two different haplotypes in the family producing only sterile males.

Within each family, males were grouped according to either their inherited haplotype or fertility estimates based on sperm count. Sterility (in males with ASC < 5) was found to segregate with microsatellite markers in three of six families (females G0 2802, 2809, 2813; 34 males in total). No such association could be revealed in three families (females G0 2814, 2818, 2819; 27 males).

DISCUSSION

Phenotypic data

Since the discovery of mouse hybrid male sterility in the 1960s (Iványi et al., 1969; Forejt & Iványi, 1975) research has focused largely on mapping the Hst1gene (Forejt et al., 1991; Trachtulec et al., 1994, 1997, 2004; Gregorováet al., 1996). This study extends the knowledge of hybrid sterility in a different way. By crossing wild M. m. musculus, sampled from a population geographically separated from that originally studied by Forejt & Iványi (1975), to B10 mice we have found that some phenotypic traits of F1 males depend on the direction in which the parents are crossed. In addition, we have demonstrated that the production of sterile males associated with the Hst1gene is not a unique trait of Prague mice and that this phenomenon is more widespread in wild populations of M. m. musculus. It is worth mentioning here that the presence of male sterility has also been reported in one out of three males sampled in Denmark (Forejt & Iványi, 1975); unfortunately, no detailed information has been provided on the genetic basis underlying sterility in Danish males (but see Britton-Davidian et al., 2005, this issue, for new data).

The phenotypic manifestation of sterility observed in this study closely resembles the pattern described earlier in crosses between wild M. m. musculusand B10 mice (Forejt & Iványi, 1975), in that sterile males had low testis mass and the frequency of sterile males was not correlated with body mass. In addition to these findings, we have shown for the first time that sterile males have significantly lower epididymis mass when compared with fertile males.

Because only wild M. m. musculusmales were used in the original study by Forejt & Iványi (1975), describing hybrid sterility, we designed experiments to test for differences in reciprocal crosses. The results confirmed that the occurrence of sterility in hybrid males is independent of the direction of the cross (Figs 3, 4). However, we found statistically significant effects of reciprocity in ASC, testis and epididymis mass in fertile males (Table 1). The differences were consistent: males from the G1 × B10 cross were shown to surpass those from the reciprocal cross in all measured traits. Unfortunately, the scarcity of similar data in the literature prevents us from generalizing our results. Only one of the traits studied here has been analysed and discussed as a criterion of sterility (Forejt & Iványi, 1975). These authors found that in crosses between (B10 × C3H)F1 × W the mass of paired testes was lower than 90 mg in sterile males, while it was higher in fertile males. They also found a bimodal distribution of testes mass with a gap between classes of sterile and fertile males in B10 × W crosses. While, on average, our results matched theirs for both types of cross (nearly 70 mg for paired mass of testes in B10 × G1 and 87 mg in G1 × B10), we found that limiting values of paired testis mass for sterile males can, especially in the G1 × B10 crosses, exceed those reported by Forejt & Iványi (1975). The maximum value of paired testis mass in a sterile male was 137 mg (Table 1, G1 × B10 cross, a descendant of the 2819 G0 female). Differences in the distribution of testis mass also depended on the direction of the cross: this distribution was clearly bimodal in the B10 × G1 progeny but rather uniform in the G1 × B10 progeny (Fig. 4), with higher variation of ASC in the G1 × B10 males. Unfortunately, we are unable to propose any reason that might explain this pattern of distribution.

We also observed an unequal proportion of sterile males in reciprocal crosses in our data. The most plausible explanation for this inequality is that it resulted from the experimental design adopted in this study: we crossed one G1 male with four genetically uniform B10 females, but one B10 male with four genetically heterogeneous G1 females. This design introduced higher variability in the latter case and is documented in Figure 3.

Despite the paucity of comparable data, two general conclusions based on these results can be drawn. First, the overlap of bimodally distributed variables is more restricted in testis mass than it is in epididymis mass. Although we found significant relationships between these two variables and sterility, the more pronounced bimodality in histograms of testis mass supports the finding that this parameter is a reliable predictor of hybrid male sterility. Second, we have shown that the phenotype associated with hybrid sterility is dependent on the direction of crosses. Hence, the limiting value of any parameter used to distinguish fertile and sterile males, such as testis mass, can vary and must be estimated for both types of cross.

Molecular data

Male sterility has been associated with thaplotypes in numerous variants in Mus(Bennett, 1975). The presence of thaplotypes in our study was determined using the Hba-ps4molecular marker whose reliability has been proven recently in numerous pure and hybrid populations of wild mice (Dod et al., 2003). Our results allowed us to exclude unequivocally the possibility that the sterility is confounded by the presence of the sterility factors in the thaplotype of M. musculusorigin and its interaction with the B10 genome.

The high level of allelic diversity revealed in this study at three microsatellite markers in the Hst1region provided a solid tool for haplotype analysis of sterility gene(s) presumably located in this region. The results of these molecular analyses indicate that hybrid male sterility is, at least in three out of seven families, associated with a gene or genes in the Hst1region. Surprisingly, in three families, fertility of males did not segregate with the Hst1region; although the low number of males analysed in one family (2819 G0 female, Table 2) prevents us from drawing strong conclusions, data for the other two families (2814 and 2818 G0 females, Table 2) suggest that the breakdown of spermatogenesis in these two families is not associated with a gene or genes located in the Hst1region. This finding suggests that these two families possess allelic variation at different loci underlying hybrid sterility and stresses the need for studying geographical variation and genetic complexity of hybrid sterility. Recently, two independent papers reported the presence of male sterility caused exclusively by genes located on the X chromosome (Oka et al., 2004; Storchováet al., 2004). In these two studies, sterile males were observed in the chromosome substitution strain C57Bl6-XMSM, in which the X chromosome of the C57Bl6 strain was substituted by the X chromosome of the MSM strain derived from M. m. molossinus(Oka et al., 2004), or in the C57Bl6-XPWDstrain, in which the X chromosome was derived from the PWD strain representing M. m. musculus(Storchováet al., 2004).

Finally, one family produced only sterile males. We are currently generating two wild-derived lines of M. m. musculus: one producing only fertile males when crossed to B10 mice, the second producing only sterile males (J. Piálek, unpubl. data). Backcrosses ((sterile line × fertile line) × B10) should facilitate genetic mapping and confirm whether male sterility in this family is associated with the Hst1region or with the newly described genetic system. The discovery of two different Hst1haplotypes in the family descending from G0 female 2821 producing only sterile males indicates possible problems in searching for sterility-specific haplotypes in populations of M. m. musculus. It appears that recombination and/or microsatellite expansion has removed the association between marker alleles and the gene causing hybrid sterility observed in this study.

Summarizing the results of our phenotypic and molecular analyses, the most plausible explanation for our observations is that at least two genes polymorphic in wild M. m. musculussampled in Studenec contribute to hybrid male sterility. Their phenotypic expression is significantly different between reciprocal crosses. Both the Prague and Studenec populations were found to be polymorphic for genes underlying sterility, and these independent observations might indicate a recent origin of genes causing spermatogenetic breakdown.

ACKNOWLEDGEMENTS

This work was supported by the Academy of Sciences of the Czech Republic (IAA6093201) and Grant Agency of the Czech Republic 204/98/KO15. Pavel Munclinger is acknowledged for designing primers Ph4IN from the D17Ph4egene. We thank Dana Havelková, Lída Malá and Jana Piálková for keeping mice and Jan Zima for keeping M.V. and J.P. in Studenec. MilošMacholán, Michael Nachman, Kate Teeter, Janice Britton-Davidian and two anonymous reviewers greatly improved an earlier version of this paper.

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