Genetics, Vol. 154, 803-811, February 2000, Copyright © 2000

BALB/c Alleles at Modifier Loci Increase the Severity of the Maternal Effect of the "DDK Syndrome"

Stéphanie Le Brasa, Michel Cohen-Tannoudjia, Chantal Kressa, Sandrine Vandormael-Pournina, Charles Babineta, and Patricia Baldaccia
a Unité de Biologie du Développement, URA CNRS 1960, Département d'Immunologie, Institut Pasteur, 75724 Paris Cedex 15, France

Corresponding author: Patricia Baldacci, Unité de Biologie du Développement, Département d'Immunologie, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France., baldacci{at}pasteur.fr (E-mail)

Communicating editor: D. M. KINGSLEY


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

The Om locus was first described in the DDK inbred mouse strain: DDK mice carry a mutation at Om resulting in a parental effect lethality of F1 embryos. When DDK females are mated with males of other (non-DDK) inbred strains, e.g., BALB/c, they exhibit a low fertility, whereas the reciprocal cross, non-DDK females x DDK males, is fertile (as is the DDK intrastrain cross). The low fertility is due to the death of (DDK x non-DDK)F1 embryos at the late-morula to blastocyst stage, which is referred to as the "DDK syndrome." The death of these F1 embryos is caused by an incompatibility between a DDK maternal factor and the non-DDK paternal pronucleus. Previous genetic studies showed that F1 mice have an intermediate phenotype compared to parental strains: crosses between F1 females and non-DDK males are semisterile, as are crosses between DDK females and F1 males. In the present studies, we have examined the properties of mice heterozygous for BALB/c and DDK Om alleles on an essentially BALB/c genetic background. Surprisingly, we found that the females are quasi-sterile when mated with BALB/c males and, thus, present a phenotype similar to DDK females. These results indicate that BALB/c alleles at modifier loci increase the severity of the DDK syndrome.

FEMALE mice of the DDK inbred strain have a striking phenotype, which was first described by Wakasugi. DDK intrastrain crosses are fertile, as are crosses between DDK males and females from other inbred strains. However, when DDK females are mated with males from other inbred strains, viable offspring are rarely obtained (WAKASUGI et al. 1967 Down; WAKASUGI 1973 Down). For example, matings between DDK females and BALB/c males are considered to be "quasi-sterile," since only 2 pups were obtained from 27 females presenting a vaginal plug (RICHOUX 1991 Down). This is due to the majority of (DDK x BALB/c)F1 embryos dying around the time of implantation and is referred to as the "DDK syndrome" (RENARD et al. 1994 Down). This embryonic lethality can also be observed in vitro, where ~90% of (DDK x BALB/c)F1 embryos fail to reach the blastocyst stage (BALDACCI et al. 1992 Down). It should be noted that the rare survivors of the DDK syndrome are normal.

To define the genetic determination of this embryonic lethality, Wakasugi studied the in vivo fertility of F1 heterozygous mice obtained from several crosses between DDK and NC, KK, or C57Bl/6 inbred mouse strains (non-DDK strains are referred to hereafter as "alien" strains). These studies showed that matings between heterozygous F1 females and DDK males were fertile, and the average litter size of these females was reduced by half when mated with alien males (semisterile cross). The cross alien females x heterozygous F1 males was fertile, but the cross DDK females x heterozygous F1 males was semisterile (see Table 1 for summary). The analysis of backcross matings showed the following: (1) Alien females x heterozygous F1 males produced, in equal proportions, two categories of female offspring: when mated with alien males, they were either semisterile (phenotype of F1 females) or fertile (phenotype of alien females). (2) Heterozygous F1 females x DDK males produced female offspring, in equal proportions, that were either quasi-sterile (phenotype of DDK females) or semisterile (phenotype of F1 females) when mated with alien males. Thus, the data using F1 females or males was compatible with a single locus controlling the DDK syndrome. (3) The backcross F1 females x alien males was semisterile, and the phenotypes of the resulting female offspring were either semisterile or fertile, in equal proportions, when mated with alien males. This showed that the survival of an embryo was independent of the allele (alien or DDK) inherited from the mother. (4) Finally, matings between F1 females and F1 males produced female offspring that were phenotypically like DDK, F1, and alien females in proportions slightly different than expected from Mendel's law for a single locus (WAKASUGI 1974 Down).


 
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  		Table 1. Summary of crosses between DDK and alien strains

On the basis of these studies, Wakasugi proposed that the embryonic lethality is determined by a single locus called "Ovum mutant" (Om). In the majority of mouse strains, Om is postulated to contain genes responsible for the synthesis of a cytoplasmic factor O in the egg and a sperm-carried factor S. In DDK mice, the alleles of these genes produce the factors o and s. The death of (DDK x alien)F1 embryos is caused by an incompatibility between the factors o and S (WAKASUGI 1974 Down). To explain the results from the backcross heterozygous F1 females x alien males, which was semisterile and produced females phenotypically like F1 and alien females, it was proposed that heterozygous females produce eggs with equivalent amounts of o and O cytoplasmic factors. Upon fertilization, these egg factors, o and O, interact independently, stochastically, and irreversibly with the sperm factor S. The embryos that have undergone the o-S interaction die around implantation, thus accounting for the semisterility, and the surviving embryos (50%) receive either the DDK or alien maternal Om allele, thereby explaining the phenotypes of the female offspring.

Although Wakasugi's model was formulated long before genotyping of Om became feasible, several of his postulates have been confirmed. Om has been mapped as a single locus to chromosome 11 (BALDACCI et al. 1992 Down; SAPIENZA et al. 1992 Down), within an interval of ~2 cM between the gene Scya2 and the microsatellite marker D11Mit36 (BALDACCI et al. 1996 Down). The genes for the maternal egg factor and the paternal sperm factor cosegregate with this locus (COHEN-TANNOUDJI et al. 1996 Down; PARDO-MANUEL DE VILLENA et al. 1997 Down). Pronuclear transplantation experiments performed in this laboratory (RENARD and BABINET 1986 Down) and independently by MANN 1986 Down show that the interaction between DDK egg cytoplasm and the alien paternal pronucleus severely compromises embryonic development. This indicates, as suggested by Wakasugi, that the sperm factor is either a gene on the paternal genome or a product synthesized during spermatogenesis and that it is associated with the paternal pronucleus. Furthermore, there is evidence from cytoplast fusion experiments that the DDK cytoplasmic factor is still present at the eight-cell stage (BABINET et al. 1990 Down). Finally, the DDK oocyte cytoplasmic factor has been shown to be present as RNA and, thus, is of maternal origin (RENARD et al. 1994 Down).

During the establishment of three BALB/c congenic lines carrying the DDK alleles of Om (hereafter called C.D-Omd strains), an unexpected observation was made. According to Wakasugi's observations, the distribution of Om genotypes in the progeny from matings between mice heterozygous for BALB/c and DDK Om alleles was expected to be homozygous BALB/c (Omc/c), homozygous DDK (Omd/d), and heterozygous (Omc/d) in the proportions 1:2:3 (see Figure 1). When matings were performed with mice heterozygous at Om, but having an essentially BALB/c genome, we observed a significant departure from this distribution; specifically, very few homozygous Omc/c offspring were obtained. The discrepancy between our data and Wakasugi's predictions could be due to the different inbred strains used to create the heterozygous mice and/or to the degree of heterozygosity in the genome.



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  		Figure 1. Schematic representation of a cross between Omc/d heterozygous mice according to Wakasugi's observations. The maternal alleles Omd and Omc are shown on the left, and the paternal alleles on the top. Both maternal and paternal alleles segregate 1:1. The genotypes of the resulting progeny are shown in the open boxes. Hatched areas represent the expected embryonic lethality (50%), which is similar to that observed when F1 females are mated with alien males (here shown as BALB/c males), resulting in the semisterility of the cross. The ratio of genotypes Omc/c:Omd/d:Omc/d is thus expected to be 1:2:3.

To distinguish between these possibilities and gain a better understanding of the genetic determination of the DDK syndrome, we have analyzed, in this article, the segregation of genotypes in crosses involving (BALB/c x DDK)F1, (DDK x BALB/c)F1, and (BALB/c x C.D-Omd)F1 mice, and we compared the results to Wakasugi's predictions. Only the data obtained with (BALB/c x DDK)F1 mice gave the expected distribution of genotypes. On the contrary, data from both (DDK x BALB/c)F1 and (BALB/c x C.D-Omd)F1 mice were significantly different. We then tested which elements of Wakasugi's observations may not be valid for (BALB/c x C.D-Omd)F1 mice: the equal transmission of Om alleles via the female and male germline and/or the semisterile phenotype of F1 females when mated with alien (in this study BALB/c) males. Our results show that (BALB/c x C.D-Omd)F1 mice do indeed transmit Om alleles in equal proportions. However, the crosses between (BALB/c x C.D-Omd)F1 females and BALB/c males are quasi-sterile. Thus, contrary to expectation, these females have a DDK phenotype. We propose that the discrepancy between the Om genotype and phenotype of these females is due to the presence of BALB/c alleles at modifier genes affecting the expression of Om during oogenesis.


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Mouse strains:
DDK/Pas mice were from our breeding colony at the Pasteur Institute. BALB/cByJ mice were obtained from IFFA CREDO (L'Arbresle, France) and R. Janvier (Le Genest-St-Isle, France).

C.D-Omd congenic strains: Three males heterozygous at Om (Omc/d) had been selected during experiments to map Om. They were backcrossed to BALB/c females, Omc/d male offspring were identified by genotyping for markers flanking Om, and their Om phenotype was determined using an in vitro test described previously (BALDACCI et al. 1992 Down). This procedure was repeated for six backcross generations. Three congenic BALB/c strains carrying DDK Om alleles were obtained by sib matings of Om heterozygous mice at the N6 backcross. The genotypes of the three congenic strains are as follows:

  • C.D-Omd1: Scya2d/d, Scya1d/d, D11Mit66d/d, D11Mit36d/d, and D11Mit211d/d

  • C.D-Omd2: Scya2d/d, Scya1d/d, D11Mit66d/d, D11Mit36c/c, and D11Mit211c/c

  • C.D-Omd3: Scya2c/c, Scya1d/d, D11Mit66d/d, D11Mit36d/d, and D11Mit211d/d,

where d represents the DDK allele and c the BALB/c allele.

Both males and females of these strains behave as DDK mice with respect to the DDK syndrome. Heterozygous mice were obtained for analysis by mating BALB/c females to C.D-Omd1, C.D-Omd2, or C.D-Omd3 males.

Genotype analysis:
DNA was extracted from tail biopsies, and genotypes were determined by polymerase chain reaction and gel electrophoresis, as described previously (BALDACCI et al. 1992 Down). Oligonucleotide primers were obtained from Research Genetics (Huntsville, AL), Genset (Paris), or Life Technologies (Cergy Pontoise, France). Offspring from crosses involving (BALB/c x DDK)F1 and (DDK x BALB/c)F1 mice were genotyped for the locus Scya2 (~0.5 cM proximal to Om). Offspring from BALB/c x C.D-Omd parents were typed with Scya2, Scya1 (which cosegregates with Om), and D11Mit211 (this marker cosegregates with D11Mit36 and is ~2 cM distal to Om; our unpublished data). All pups were genotyped at day 10–12 after birth. Due to recombination events between the markers Scya2, D11Mit211, and Om, there can be an error of ~0.5 and 2%, respectively, for the deduced Om genotype.

Evaluation of the fertility of females in different crosses:
DDK and (BALB/c x C.D-Omd)F1 females (aged between 2 and 4 mo) were caged with either DDK or BALB/c fertile males. Females were examined daily for vaginal plugs and separated from males when positive. The number of pups born and those viable at day 10–12 postpartum were recorded. The fertility of a cross was defined as the total number of viable pups at day 10–12 (when genotyping was performed) divided by the total number of females with vaginal plugs.

Statistical analysis:
The litter sizes from the crosses DDK females x BALB/c males and (BALB/c x C.D-Omd)F1 females x BALB/c males did not follow a normal distribution, thereby excluding a comparison of the crosses by a {chi}2 test. A Mann-Whitney nonparametric test was performed using Stat View F-4.5 software (Abacus Concepts, Berkeley, CA).


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

To establish BALB/c congenic strains carrying DDK alleles at the Om locus (C.D-Omd strains), sib matings were performed at the N6 backcross, as described in MATERIALS AND METHODS. According to Wakasugi's observations, the distribution of genotypes was expected to be Omc/c:Omd/d:Omc/d in the ratio 1:2:3. Surprisingly, these proportions were not observed. However, Wakasugi had studied F1 mice obtained from crosses between DDK and NC, KK, or C57Bl/6J inbred strains. Thus, the discrepancy between our data and Wakasugi's predictions could be related to the different inbred strains used to create the Om heterozygous mice and/or to the degree of heterozygosity in the genome. The F1 mice studied previously were heterozygous at 100% of loci, whereas our mice were homozygous for BALB/c alleles at >95% of loci. This suggested to us that BALB/c alleles in the genome were in some way modifying the distribution of Om genotypes and/or influencing the severity of the DDK syndrome. Therefore, a series of crosses was undertaken to compare the properties of (BALB/c x DDK)F1, (DDK x BALB/c)F1, and (BALB/c x C.D-Omd)F1 mice with the F1 mice studied by Wakasugi.

Departure from Wakasugi's predictions:
The following crosses were set up with reciprocal F1 heterozygotes of DDK and BALB/c strains: (i) (BALB/c x DDK)F1 females x (BALB/c x DDK)F1 and (DDK x BALB/c)F1 males; (ii) (DDK x BALB/c)F1 females x (BALB/c x DDK)F1 and (DDK x BALB/c)F1 males. Sib matings of (BALB/c x C.D-Omd)F1 mice were also performed. The offspring from these crosses were genotyped as described in MATERIALS AND METHODS, and the results of this analysis are presented in Table 2.


 
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  		Table 2. Segregation of genotypes in crosses between mice heterozygous at the Om locus

The crosses concerning (BALB/c x DDK)F1 females mated to either (BALB/c x DDK)F1 or (DDK x BALB/c)F1 males gave distributions for the Omc/c:Omd/d:Omc/d genotypes in the 1:2:3 ratio expected from Wakasugi's predictions. On the contrary, results obtained from sib matings of (BALB/c x C.D-Omd)F1 mice were significantly different from this ratio ({chi}2 values between 35.1 and 52.3). The distribution from the cross (DDK x BALB/c)F1 females x (BALB/c x DDK)F1 males was also significantly different from that expected ({chi}2 value 7.9), and the distribution from the cross (DDK x BALB/c)F1 females x (DDK x BALB/c)F1 males gave a {chi}2 value (5.3) at the limit of significance.

Two striking features were common to these crosses: First, the percentage of Omc/c offspring was less than the expected 16.6%, ~10% with (DDK x BALB/c)F1 females, and 5% or less with (BALB/c x C.D-Omd)F1 females; second, there were more Omd/d genotypes than the 33% expected, ~45% for (DDK x BALB/c)F1 and 55% for (BALB/c x C.D-Omd) females. A reduction of Omc/c offspring and an increase in the proportion of Omd/d offspring could be achieved in at least two ways: a transmission ratio distortion in favor of the Omd allele through the female or male germline and if 90%, rather than 50%, of embryos containing the paternal Omc allele die from the DDK syndrome. Operationally, this would imply that (DDK x BALB/c)F1 and (BALB/c x C.D-Omd)F1 females are quasi-sterile, not semisterile, when mated with BALB/c males, i.e., they have a DDK phenotype.

To test these hypotheses, a further series of crosses was undertaken. It should be noted that (DDK x BALB/c)F1 mice are extremely difficult to obtain, since they are the rare survivors of the DDK syndrome. For this reason, we were unable to perform all possible crosses using these mice, and our analysis has focused on the (BALB/c x C.D-Omd)F1 mice.

Absence of transmission ratio distortion of Om alleles in heterozygous males and females:
If the segregation of genotypes, seen in Table 2, were the result of a transmission ratio distortion (TRD) in favor of the Omd allele via the female or male germline, then >80% of the gametes would have to carry the Omd allele to account for the results observed. The transmission of Om alleles through the (BALB/c x C.D-Omd)F1 male and female germlines was tested in a series of fertile crosses in which there was no loss of embryos due to the DDK syndrome. (BALB/c x C.D-Omd)F1 males were mated with BALB/c females, and (BALB/c x C.D-Omd)F1 females were mated with DDK males. Results of the genotypes of offspring are presented in Table 3. The (BALB/c x DDK)F1 males transmitted Omc and Omd alleles in equal proportions, as expected for a single locus. These results confirmed our previous data obtained from the backcross BALB/c females x (BALB/c x DDK)F1 males, which had been performed to map Om (BALDACCI et al. 1992 Down, BALDACCI et al. 1996 Down). The (BALB/c x C.D-Omd)F1 males also transmitted the alleles in equal proportions. Similarly, all the F1 females studied transmitted Omc and Omd alleles in a ratio not significantly different from 1:1.


 
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  		Table 3. Transmission of Om alleles through heterozygous male and female germlines

Since both (BALB/c x C.D-Omd)F1 males and females transmit the alleles as anticipated for a single locus, the results presented in Table 2 cannot be explained by a TRD during gametogenesis. Therefore, we envisaged the second hypothesis: (BALB/c x C.D-Omd)F1 females have a DDK phenotype.

The (BALB/c x C.D-Omd)F1 females are phenotypically like DDK females:
If (BALB/c x C.D-Omd)F1 females have a DDK phenotype, they should be fertile when mated with DDK males, whereas the cross with BALB/c males should be quasi-sterile. To test this (BALB/c x C.D-Omd)F1 females were caged with either DDK or BALB/c males of proven fertility.

The distributions of litter sizes from these crosses are shown in Figure 2. A comparaison of these distributions was performed using a nonparametric analysis (Mann-Whitney test). The results showed that there was a significant difference (P = 0.003) between the distribution from the cross DDK x DDK compared to DDK x BALB/c. The distributions observed in the crosses (BALB/c x C.D-Omd1)F1, (BALB/c x C.D-Omd2)F1, and (BALB/c x C.D-Omd3)F1 females mated with DDK males compared to crosses with BALB/c males were also significantly different (P < 0.0001, < 0.0001, and = 0.004, respectively). There was no significant difference (see Figure 2) between the distributions obtained with (BALB/c x C.D-Omd2)F1 or (BALB/c x C.D-Omd3)F1 females compared to DDK females mated with DDK males. The distribution obtained with (BALB/c x C.D-Omd1)F1 females x DDK males was significantly different. However, when the distributions from the three categories of (BALB/c x C.D-Omd)F1 females mated with BALB/c males was compared to DDK females x BALB/c males, no significant difference was observed.



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  		Figure 2. Distribution of litter sizes from different crosses. Histograms show the number of females with vaginal plugs (y axis) that gave litters of a given size (x axis). Values obtained from crosses with DDK males are shown as black columns, and white columns are the data obtained with BALB/c males. The comparison of the distributions from different crosses was performed using the Mann-Whitney test. p(DDK male) corresponds to the value of P calculated from the comparison of the distributions obtained from crosses C.D-Omd females x DDK males with the cross DDK females x DDK males. p(BALB/c male) is the value obtained from the comparison between crosses C.D-Omd females x BALB/c males and DDK females x BALB/c males.

The fertility of females in the different crosses was measured as the total number of viable pups at day 10–12 divided by the number of females presenting a vaginal plug (see MATERIALS AND METHODS). The results were compared to breeding data obtained from crosses with DDK females and are presented in Table 4. The fertility of the cross DDK females x DDK males was 1.57 and decreased ~20-fold when DDK females were mated with BALB/c males. The crosses (BALB/c x C.D-Omd)F1 females x DDK males were more fertile, with values ranging from 3.19 to 4.32. As with DDK females, when (BALB/c x C.D-Omd)F1 females were mated with BALB/c males, the fertility values decreased 10- to 40-fold.


 
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  		Table 4. Fertility of (BALB/c x C.D-Omd)F1 heterozygous females

We conclude from these in vivo data that (BALB/c x C.D-Omd)F1 females have the same phenotype as DDK females; i.e., they are fertile when mated with DDK males and quasi-sterile with BALB/c males.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

In this article, we have performed a genetic analysis of the Om locus in (BALB/c x DDK)F1, (DDK x BALB/c)F1, and (BALB/c x C.D-Omd)F1 mice. The results show that the properties of (DDK x BALB/c)F1 and (BALB/c x C.D-Omd)F1 females differ from those of (BALB/c x DDK)F1, and they suggest the existence of other loci that affect the severity of the DDK syndrome.

Wakasugi had previously studied F1 mice obtained from crosses between DDK and C57Bl/6, NC, or KK inbred strains. Two major observations were made: (1) a 1: 1 segregation of Om alleles via the female and male germline; (2) F1 females are semisterile when mated with alien males. As a result, the offspring from crosses between F1 mice is expected to result in a distribution of genotypes Omalien/alien:Omddk/ddk:Omalien/ddk in the ratio 1:2:3 (see Figure 1, where Omalien is shown as Omc). This was indeed observed in our crosses between (BALB/c x DDK)F1 females and F1 males. However, it was not the case with (DDK x BALB/c)F1 and (BALB/c x C.D-Omd)F1 mice. In these crosses, there was a significant reduction in the proportion of Omc/c individuals and an increase in Omd/d progeny. This suggested that Wakasugi's observations were not applicable to these F1 mice.

A first possible explanation for the biased segregation of genotypes observed would be the existence of a significant TRD in favor of the Omd allele via the male or female germline. This hypothesis was ruled out, since (BALB/c x DDK)F1, (DDK x BALB/c)F1, and (BALB/c x C.D-Omd)F1 mice transmitted Omc and Omd alleles in equal proportions when fertile crosses were performed. However, we cannot formally exclude the existence of a sperm-mediated segregation distortion of the type described previously for homogeneously staining regions on chromosome 1 (RUVINSKY 1995 Down). If such were the case, it would require that the paternal Omc allele provokes the preferential exclusion of the Omc maternal allele to the polar bodies upon fertilization.

Although we have not observed a TRD in F1 mice obtained from DDK and BALB/c strains, it should be noted that two other groups have reported this phenomenon on chromosome 11. F1 females, from reciprocal crosses of DDK and C57Bl/6 strains, backcrossed to C57Bl/6 males, gave a small but significant (64%) TRD in favor of DDK alleles in the Om region (PARDO-MANUAL DE VILLENA et al. 1996 Down; PARDO-MANUEL DE VILLENA et al. 1997 Down). A similar distortion in favor of C57Bl/6 alleles near Om was reported recently in reciprocal crosses of (C57Bl/6 x DBA2)F1 and C57Bl/6 mice (SHENDURE et al. 1998 Down). These TRDs could be controlled by the same locus, either by Om itself or by a closely associated locus.

Having ruled out the possibility of a TRD as an explanation for the reduction in the proportion of Omc/c offspring in our crosses, we then considered the hypothesis that, contrary to Wakasugi's observations (BALB/c x C.D-Omd)F1 females were not semisterile, but quasi-sterile, when mated with BALB/c; i.e., these females are phenotypically like DDK females. To test this, the fertility of (BALB/c x C.D-Omd)F1 females was compared with DDK females in crosses with DDK and BALB/c males. The (BALB/c x C.D-Omd)F1 females, like DDK females, were indeed found to be fertile with DDK males, and their fertility decreased dramatically with BALB/c males. Furthermore, there was no significant difference between the distribution of litter sizes obtained with (BALB/c x C.D-Omd)F1 females and DDK females mated with BALB/c males. The (BALB/c x C.D-Omd)F1 females, therefore, have a DDK phenotype.

It is interesting to compare (DDK x BALB/c)F1 and (BALB/c x C.D-Omd)F1 females, since they both gave a distribution of genotypes that departed from Wakasugi's predictions. Although we have not been able to compare, due to their scarcity, the fertility of (DDK x BALB/c)F1 females with DDK and BALB/c males, we suppose that they gave rise to an unusual genotype distribution via the same mechanisms as (BALB/c x C.D-Omd)F1 females. It is then noteworthy that (DDK x BALB/c)F1 females, which have 50% BALB/c alleles, gave a less biased distribution than (BALB/c x C.D-Omd)F1 females, which are homozygous for BALB/c alleles at >95% of loci. This results in a seemingly paradoxical situation in which the greater the BALB/c genetic contribution of Omc/d females, the more they have a DDK phenotype. This suggests an additive effect of BALB/c alleles at loci that interact with Om.

It seems very likely that the differences observed between (BALB/c x DDK)F1 and (DDK x BALB/c)F1 females are related to their parental chromosomal contributions. Indeed, no modifier effect of BALB/c alleles was observed in (BALB/c x DDK)F1 females, which have DDK paternal chromosomes, whereas (DDK x BALB/c)F1 females, which present a modifier effect, have paternal BALB/c chromosomes. This suggests that there is parental imprinting of the modifier loci. Another laboratory has analyzed a series of crosses with F1 mice obtained from C57Bl/6 and DDK inbred strains. Although they did not report any difference in the properties of their reciprocal F1 females (PARDO-MANUAL DE VILLENA et al. 1996 Down), they have recently described a phenotypic variability of the DDK syndrome in heterozygous females, which is determined by the C57Bl/6 alleles at modifier loci (PARDO-MANUEL DE VILLENA et al. 1999 Down). The differences between (BALB/c x DDK)F1 and (DDK x BALB/c)F1 females provide a probable explanation for an observation made during the establishment of recombinant inbred (RI) strains of DDK and BALB/c. When (BALB/c x DDK)F1 mice were intercrossed, as many RI strains homozygous for BALB/c Om alleles (Omc/c) as for DDK alleles (Omd/d) were established. However, the crosses between (DDK x BALB/c)F1 mice gave rise to only Omd/d strains (BALDACCI et al. 1992 Down). From the data presented herein, it is very likely that Omc alleles were rapidly counterselected and Omd alleles were favored when (DDK x BALB/c)F1 founders were used.

The demonstration that (BALB/c x C.D-Omd)F1 females have a DDK phenotype makes it necessary to reevaluate Wakasugi's Om model. It was proposed that all the oocytes produced by F1 females contain both Omd and Omc cytoplasmic factors (WAKASUGI 1974 Down). Upon fertilization, these factors interact stochastically and irreversibly with an incoming sperm factor. The presence of both cytoplasmic factors in the oocytes implies a biallelic expression of Om during oogenesis. In the context of this model, how could (BALB/c x C.D-Omd)F1 females acquire a DDK phenotype? A possible explanation could be a greater affinity of the Omd cytoplasmic factor for the sperm factor. The modifier genes referred to above would then change the affinity of the Omd cytoplasmic factor for the sperm factor. Alternatively, the modifiers could provoke a differential expression in favor of the Omd allele during oogenesis. In this scenario, Omd and Omc alleles are expressed equally in all oocytes of (BALB/c x DDK)F1 females, whereas in (BALB/c x C.D-Omd)F1 females, there is a greater expression of Omd compared to Omc, resulting in an excess of the Omd factor.

However, an alternative model can be considered to explain both Wakasugi's data and the results of this study. We propose that during oogenesis, Om expression is subject to allelic exclusion, a mechanism that has been described for expression at several loci: imprinted genes (for review see BARTOLOMEI and TILGHMAN 1997 Down), Xist and most X-chromosome genes in somatic cells (HEARD et al. 1997 Down), genes coding for immunoglobulins (CHEN and ALT 1993 Down), T cell receptor (MALISSEN et al. 1992 Down), Ly49 receptor (HELD et al. 1995 Down), and odorant receptor genes (CHESS et al. 1994 Down). We and others have already suggested this mode of expression to explain the genetic behavior of some (alien x DDK)F1 females (COHEN-TANNOUDJI et al. 1996 Down; PARDO-MANUAL DE VILLENA et al. 1996 Down). In this model, 50% (not 100%) of oocytes from a (BALB/c x DDK)F1 female express Omd and the other 50% express Omc independently of the Om allele remaining in the zygote after fertilization (see Figure 3). The DDK phenotype of (BALB/c x C.D-Omd)F1 females can then be explained by an unbalanced allelic exclusion in favor of Omd; i.e., 90% of the oocytes express Omd. This results in a distribution of genotypes in the ratio 1:10:10. It is interesting to note that unbalanced allelic exclusion has already been reported for X-chromosome inactivation (CATTANACH and WILLIAMS 1972 Down). When two X chromosomes carrying different alleles at the Xce locus are present in the same cells, <=65% of the cells will inactivate the X chromosome carrying a particular Xce allele. In this framework, the BALB/c alleles at modifier loci would control the balance of allelic exclusion at Om. When the paternally inherited alleles of a F1 female are of DDK origin for the modifier loci, a balanced allelic exclusion occurs at Om during oogenesis, whereas paternal BALB/c alleles provoke an unbalanced allelic exclusion in favor of the Omd allele.



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  		Figure 3. Model of allelic exclusion at Om during oogenesis. An example of a cross between F1 mice is shown. The two boxes in the first column correspond to the Om allele expressed during oogenesis in an individual oocyte. An example of balanced exclusion is illustrated with 50% of oocytes expressing Omc and 50% Omd. The second column shows the Om allele remaining in the egg after fertilization. It should be noted that this is independent of the Om allele expressed during oogenesis. Thus, four categories of eggs are produced, in equal propotions, by a F1 female. The last two columns show the Om alleles carried by fertilizing sperm. The hatched area corresponds to embryos that fail to develop as a result of the DDK syndrome. The resulting ratio of genotypes Omc/c:Omd/d:Omc/d is 1:2:3, as observed in crosses between (BALB/c x DDK)F1 mice.

This model of allelic exclusion at Om during oogenesis could be tested by determining which Om alleles are expressed in individual oocytes of F1 females. For this purpose, it will be necessary to identify genes at the Om locus that are expressed in oocytes and for which there is a polymorphism between DDK and BALB/c alleles. The search for such genes is in progress.


*  ACKNOWLEDGMENTS

We thank Professor Wakasugi for interesting discussions, as well as Drs. Edith Heard, Philip Avner, Benoit Robert, Xavier Montagutelli and Jean-Louis Guénet for their comments concerning this manuscript. We are also extremely grateful to Dr. Jean-Francois Bureau for his help with the statisitical analysis. We also acknowledge Christelle Maintoux for excellent technical assistance. This work was funded by A. C. C-SV 4 from the Ministère de l'Education Nationale, de la Recherche et de la Technologie, Institut Pasteur and Centre National de la Recherche Scientifique.

Manuscript received August 15, 1999; Accepted for publication October 28, 1999.


*  LITERATURE CITED
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
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