Around 1967-1968 it became obvious that research on H-2 immunogenetics had to be extended to wild mice populations. At that time, several functional aspects of the H-2 system began to receive attention. To understand better the biological role of the H-2 system, it was desirable to have at least initial information about the degree of polymorphism of the H-2 system in wild mouse populations. Such data would also be essential for speculations about the mechanisms involved in the generation and maintenance of this kind of polymorphisms and complexity. The initiation of these studies was promoted by a successful definition of MHS antigens of non-inbred populations in other species such as human, chicken, and rabbit (for review see 1, 2). In other words, if MHS serology became possible in other non-inbred populations, why should all work in mice remain restricted to inbred strains? The available inbred strains have frequently a common genetic origin. They might represent a selection of the most suitable (and interesting) genes for some specialized laboratory studies, but they are certainly not fully representative of the natural degree of variation either at the structural or the functional levels. The laboratory conditions might have enabled us to study genes which had no chance to spread (and to be found) under natural conditions.
When H-2 studies in wild mice began, there was a temptation to extend the number of thoroughly studied H-2 haplotypes (alleles at that time) because the number of "independent" inbred H-2 haplotypes was less than ten. The only information about H-2 antigens of non-inbred mice came from the work of Rubinstein and Ferrebee ( 3). Serotyping of 47 Swiss-Webster mice of a random-bred colony showed a high degree of polymorphism. New H-2 alleles and H-2 antigens were to be expected.
In this short survey only the summary of our work performed until 1975 will be given. All data have been published and the interested reader should consult the original articles. The extensive data of Dr. Jan Klein's group are summarized in a separate communication in this volume. The remarkable series of Dr. Klein's new H-2w haplotypes now available for laboratory work will doubtless open new horizons in H-2 immunogenetics.
Wild males were mated with inbred females of the C57BL/10 strain. Skin grafts were exchanged among the progeny of one wild male. In such a situation the number of surviving grafts is proportional to the number of H loci which were in a heterozygous state in the original wild male. It was found that, while 20-30 histocompatibility loci occur in heterozygous state in F1 hybrids of two inbred strains, only 3-7 histocompatibility loci can be supposed to occur in a heterozygous state in individual wild males. This indicates that wild mice are relatively homozygous in respect to their H loci ( 4, 5, 6).
This study represents a general approach for the estimation of the degree of heterozygosity at H loci in any noninbred individual. However, two points should be considered. 1) The number of surviving grafts can be biased if there are antigens shared between the outbred individual and the inbred strain. Therefore, any one outbred individual should be tested by mating with two or more inbred strains of diverse genetic background. When a greater number of non-inbred individuals are tested with the same inbred strain, the average values thus obtained can be considered as real because it is highly improbable that different non-inbred individuals would share histocompatibility genes with the same inbred strain. 2) The number of surviving grafts is influenced by the sex of the recipients and possibly other physiological factors. Pre-immunization of the recipient with donor tissue can result in graft rejection due to weak H systems otherwise undetected. Thus, the above-mentioned estimates have only a relative value obtained in an operationally comparable methodological assay ( 6).
1. The H-2 phenotype of 226 wild mice was tested by the PVP hemagglutination test with anti-H-2 sera prepared by immunization of inbred strains. Mice from 16 localities were tested (13 from central Bohemia). Wild mice within any given locality (with the exception of two or three localities) did not show much similarity in their H-2 phenotypes. This contrasted sharply with the phenotypes of 100 wild mice captured at Great Gull Island (a small rocky island in the Atlantic Ocean) where only three different H-2 phenotypes were detected. In all localities the phenotypes observed suggested the presence of new, thus far unknown, H-2 haplotypes. Some wild mice reacted with a very high number of anti-H-2 sera, others were negative for all H-2 antigenic specificities tested ( 4, 7, 8).
2. Sera against public H-2 specificities had a high reaction frequency on wild mice. The most frequent reactions were observed with anti-H-2 sera defining so-called long public specificities of the inbred mouse strains (anti-H-2.5, 3, 28, 11, 1). These data suggest extensive sharing of public antigens among inbred and wild mice or, in other words, they illustrate the extent of sharing of cross-reactive structures among gene products on a large sample of individuals of the given species.
Sera against private H-2 antigens also reacted positively although less frequently. Absorption experiments, however, showed that the H-2w haplotypes did not share identical private antigens with the inbred strains ( 7, 8, 9). The positive reactions of anti-H-2-inbred-private sera with wild mice were rather due to cross reactions with similar antigenic products of the H-2w haplotypes.
3. Three new H-2 congenic strains were prepared on the C57BL/10 background by introducing three new H-2w haplotypes by successive backcross matings (B10.W44 with H-2w7p, B10.W67 with H-2w8p, B10.W627 with H-2w9p). Those were characterized for some of the inbred public specificities, Ss phenotypes, three new private specificities, and three new public specificities detected by antisera prepared against the respective H-2w haplotypes ( 8).
The haplotype designations must be considered as provisional because the new H-2wp haplotypes have not been compared with the extensive series of the H-2w haplotypes of Dr. Jan Klein. The addition of letter "p" to the haplotype symbol is proposed to designate the origin (Prague) of these new haplotypes until direct serological comparison will allow a final nomenclature.
A number of functional, structural, and genetic analogies emerge from the comparison of the H-2 and HLA system. In general, the H-2K and H-2D loci are considered as analogous with HLA-A and HLA-B loci. However, the H-2 system, after preliminary insight into the degree of its polymorphism, seems to be much more polymorphic than the HLA system. Several monospecific anti-HLA sera can be completely absorbed by cells obtained from different populations. Although several HLA antigen splits are yet to be expected, the frequencies of several HLA genes in a large area (e.g., Caucasoid populations) are estimated to be around 0.05-0.10. The H-2 gene frequency estimates are more difficult, but prediction for a large enough population sample is <0.01. To explain this difference we suggest that mutations arising in a mouse population can be maintained because of the social structure, generation time, and number of offspring in mice. Furthermore, the distribution of the mutant H-2 alleles may be affected by the alleles at the T/t region. We presume that the difference in polymorphism is not "inherent" to the MHS of the two species (e.g., different mutation rates) but the consequence of a number of circumstances "outside" the MHS of both species ( 8).
When wild males were mated with inbred females, we noticed that a) some wild males mated with C57BL/10 (B10) females produced sterile sons; b) the same wild males produced fertile sons with C3H females; and c) the daughters from all matings were fertile ( 4, 7, 10, 11, 12, 13).
Because both parents were fully fertile and sterile sons occurred only in certain combinations, the observed type of sterility was designated as (male)-hybrid sterility (Hst).
A major gene responsible for the observed hybrid sterility was located on chromosome 17 between the T/t and the H-2 system. This was shown by a three-point test cross (B10-TxC3H)T/+ female x W male. The hybrid sterility gene ( Hst-1) mapped 6 cM distally from dominant T.
Some wild males were heterozygous for the Hst gene. Serotyping of the two H-2w haplotypes in the male progeny from B10 female x W male matings showed that the recombination fraction between H-2 and Hst was 8-13%.
Fifty-three wild males were tested for the presence of Hst gene; of these, 23 males yielded only sterile sons (Hst homozygotes), 10 wild males yielded sterile and fertile sons (Hst heterozygotes), and 20 wild males sired only fertile sons. The majority of wild males with the Hst gene were captured in the Prague zoological garden, but five were captured in different localities (four in Bohemia, one in Denmark).
Wild males with the Hst gene sired sterile sons with C57Bl/10, A, BALB/c, DBA/1, and AKR/J females, whereas the same wild males sired fertile males with inbred females of C3H/Di, CBA/J, P/J, I/St, and F/St strains.
The cause of the observed male-hybrid-sterility was a spermatogenesis arrest at the stage of spermatogonia or primary spermatocytes; the sterile males had small testis (90 mg). Examination of meiotic and mitotic chromosomes of sterile hybrids did not reveal any gross chromosomal rearrangements which would point to a chromosomal type of sterility. Blood testosterone level, vesicular gland weight (an indicator or target organ sensitivity to testosterone), and gonadotropin activity were within ranges of physiological variation ( 13).
The sterile males were healthy individuals with signs of "hybrid vigor" for quantitative traits identical to that of fertile wild x inbred male hybrids.
The mechanism of the male-hybrid-sterility as well as its consequences for possible incipient reproductive isolation of Mus musculus subspecies is not known and its clarification will require more data.
The identification of the Hst gene clearly shows a hitherto unknown type of polymorphism on the 17th chromosome of the inbred strains (e.g., B10 versus C3H) which would have remained undetected without combining the inbred chromosome with the wild chromosome.
A nomenclature for the hybrid sterility genetic system ( Hst) was proposed by Forejt and Iványi ( 13).
A short summary of studies on histocompatibility antigens and hybrid sterility in wild mice was presented. It was found that 1) wild mice are relatively homozygous for the numerous histocompatibility loci when compared with F1 hybrids of two inbred strains; 2) the H-2 system is highly polymorphic and serologically complex in wild mice populations; 3) three new congenic strains that contain H-2w haplotypes on the C57BL/10 background have been established and characterized; and 4) hybrid sterility gene was located on chromosome 17 between the T/t and H-2 region.
The experiments described in this communication were performed at the Institute of Experimental Biology and Genetics in close cooperation with Drs. Milada Micková, Jiri Forejt, Peter Démant, Marta Vojtisková, and Dagmar Iványi. Requests for B10.Wwp mice and wild mice with the Hst gene should be addressed to Drs. Milada Micková and Jiri Forejt, respectively (Institute of Molecular Genetics, Czechoslovac Academy of Sciences, Prague 4).
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