An extreme bias in the germ line of XY C57BL/6<->XY FVB/N chimaeric mice

Abstract

Chimaeric analysis is a powerful method to address questions about the cell-autonomous nature of defects in spermatogenesis. Symplastic spermatids (sys) mice have a recessive mutation that causes male sterility due to an arrest in germ-cell development during spermiogenesis. Chimaeric mice were generated by aggregation of eight-cell embryos from sys (FVB/N genetic background) and wild-type C57BL/6 (B6) mice to determine whether the male germ-cell defect is cell-autonomous. The resulting FVB/N<->B6 chimaeras (<-> denotes fusion of embryos) were mated with FVB/N mice and coat colour of offspring was used to identify transmission of FVB/N or B6 gametes. Regardless of the relative contribution of B6 to somatic tissues of the chimaeras, almost all (282 of 284; 99.3%) offspring of B6 XY<->XY FVB/N (+/+ or sys/+) males (n = 9) received a FVB/N-derived paternal gamete. After mating of female B6<->FVB/N chimaeras, 51 of 73 (69.9%) offspring received an FVB-derived maternal gamete. Southern blot analysis of different tissues from chimaeric males indicated that, despite the presence of balanced chimaerism in somatic tissues, the germ line in B6 XY<->XY FVB/N mice was essentially FVB/N in composition. Thus there is a strong selective advantage for FVB/N male germ cells over B6 male germ cells in B6<->FVB/N-aggregation chimaeras at some stage during development of the male germ line. Each of three male chimaeras that were either B6 XY<->XY FVB/N (sys/sys) or B6 XX<->XY FVB/N (sys/sys) in composition was sterile, and testis histology was essentially sysmutant. This finding indicates that the function of the gene(s) affected in the sys mutation may be required in the testis, although whether expression is required in germ cells, somatic cells or both remains unknown. The extreme bias in transmission of male gametes has implications for experimental design in studies that use chimaeric analysis to address questions regarding the cell-autonomous nature of germ-cell defects in mice.

Fig. 1

Chimaeric mice used in the present study. Dorsal views of 21 male and one female (1143, upper right) chimaeric mice. Animals were anaesthetized before photography. On the basis of coat colour, approximately half of the animals display balanced somatic chimaerism.

Fig. 2

Molecular genetic analysis of FVB/N<->B6 chimaeric mice. (a) Southern blot analysis of Y-chromosome content of tail DNA from chimaeras. The blot was hybridized with a probe specific for the Y-linked Zfy1 locus. The graphic on the right-hand side illustrates the hybridization patterns indicative of the presence of a Mus musculus domesticus (FVB/N) Y chromosome or a M. m. musculus (B6) Y chromosome. Lanes with numbers represent individual chimaeras. Control samples are shown on the right. (b) Southern blot analysis of tail DNA to determine genotype at sys locus of FVB/N component of chimaeras. The blot in (a) was stripped and re-hybridized with a probe from pZap1 (MacGregor et al., 1990), which identifies one flank of the transgene integration site in the sys allele on chromosome 14 (MacGregor et al., 1990). This probe detects a 2.3 kb band associated with the wild-type FVB allele (FVB (+)), a 0.9 kb band associated with the sys mutant FVB allele (FVB (sys)) and a 14 kb band associated with the C57BL/6 wild type allele (B6). The 0.9 kb band is relatively weak owing to deletion of much of the probe target sequences in the sys allele. Lanes with numbers represent individual chimaeras. Control samples are shown on the right. In chimaera 1145, the 0.9 kb (sys allele) band is extremely faint. However, confirmation of this genotype was made (Fig. 4). In chimaera 1207, the relative intensity of the FVB specific alleles (2.3 kb and 0.9 kb) is inverted when compared with chimaeras with a sys/+ component, indicating that the animal may have arisen from aggregation of more than two embryos, two of which were B6 XY and FVB (sys/sys) XY. Consequently, this animal was not considered further.

Fig. 3

Testis histology from FVB/N (sys)<->B6 chimaeric mice. (a) Testis histology of chimaera 1294 (B6 XY<->XY FVB/N (sys/sys). Note the presence of symplasts of spermatids (arrow indicates an example), indicative of a sys mutant phenotype, in most tubules. Staining was with periodic acid–Schiff–haematoxylin. (b) Testis histology of chimaera 1210 (B6 XY<->XY FVB/N (sys/sys)). As in panel (a), symplasts of spermatids are observed in most tubules. A tubule containing a mixture of symplasts and normally developing elongate germ cells is boxed. (c) Higher magnification of box in (b). Symplasts of spermatids (arrowheads) and normally developing elongate spermatids (arrow) are present in the same tubule. (d) Testis histology of chimaera 1208 (B6 XX<->XY FVB/N (sys/+)), showing normal spermatogenesis in all tubules. (e) Testis histology of chimaera 1145 (B6 XY<->XX FVB/N (sys/+)), showing both aspermatogenic (arrowheads) and tubules with normal seminiferous epithelium (arrows). (f) Testis histology of chimaera 1204 (B6 XY<->XX FVB/N (sys/+)). The majority of tubules are aspermatogenic (arrowheads) with relatively few tubules with normal seminiferous epithelium (arrows). Inset: Similar morphology of Sertoli cells (arrowheads) in tubules containing (upper), or lacking (lower) germ cells. Scale bars represent (a,b) 200 μm, (c) 50 μm, (d–f) 400 μm and (inset in (f)) 30 μm.

Fig. 4

Molecular genetic analysis of cellular composition of testis and spleen in male FVB/N<->B6 chimaeric mice. Southern blot analysis of genomic DNA extracted from testis (T) or spleen (S) from the individual (numbered) chimaeras, digested with Taq I and hybridized with the insert from pZap1, which identifies RFLPs that discriminate between B6 (14 kb; B6), the wild-type allele of FVB/N (2.3 kb; FVB (+)), and the sys allele of FVB/N (0.9 kb; FVB (sys)). Control DNA samples are shown on the right side of the lower blot. Photographs of the ethidium bromide-stained gels are shown on the left of each blot to indicate loading of DNA. In some lanes, an expected signal for the B6 allele (14 kb) cannot be clearly observed (for example, testis lane for chimaeras 1205 and 1292).