The Chromosome 11 Region from Strain 129 Provides Protection from Sex Reversal in XYPOS Mice
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Copyright Ó 2008 by the Genetics Society of America DOI: 10.1534/genetics.108.088088 The Chromosome 11 Region From Strain 129 Provides Protection From Sex Reversal in XYPOS Mice Ganka Nikolova,* Janet S. Sinsheimer,*,† Eva M. Eicher‡ and Eric Vilain*,§,1 *Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, California 90095, †Departments of Biostatistics and Biomathematics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, §Departments of Pediatrics and Urology, David Geffen School of Medicine, University of California, Los Angeles, California 90095 and ‡The Jackson Laboratory, Bar Harbor, Maine 04609 Manuscript received February 12, 2008 Accepted for publication February 27, 2008 ABSTRACT C57BL/6J (B6) mice containing the Mus domesticus poschiavinus Y chromosome, YPOS, develop ovarian tissue, whereas testicular tissue develops in DBA/2J or 129S1/SvImJ (129) mice containing the YPOS chromosome. To identify genes involved in sex determination, we used a congenic strain approach to determine which chromosomal regions from 129Sl/SvImJ provide protection against sex reversal in XYPOS mice of the C57BL/6J.129-YPOS strain. Genome scans using microsatellite and SNP markers identified a chromosome 11 region of 129 origin in C57BL/6J.129-YPOS mice. To determine if this region influenced testis development in XYPOS mice, two strains of C57BL/6J-YPOS mice were produced and used in genetic experiments. XYPOS adults homozygous for the 129 region had a lower incidence of sex reversal than XYPOS adults homozygous for the B6 region. In addition, many homozygous 129 XYPOS fetuses developed normal- appearing testes, an occurrence never observed in XYPOS mice of the C57BL/6J-YPOS strain. Finally, the amount of testicular tissue observed in ovotestes of heterozygous 129/B6 XYPOS fetuses was greater than the amount observed in ovotestes of homozygous B6 XYPOS fetuses. We conclude that a chromosome 11 locus derived from 129Sl/SvImJ essentially protects against sex reversal in XYPOS mice. A number of genes located in this chromosome 11 region are discussed as potential candidates. NITIATING the male-determining cascade in mam- origin direct normal male sex determination when pres- I mals begins with the expression of the Y chromo- ent in C57BL/6J mice. This is quite striking, consid- some gene Sry (Sinclair et al. 1990; Ross and Capel ering that the corresponding predicted SRY proteins 2005). In both humans and mice, mutations in Sry vary in size due to a difference in the number of cause XY sex reversal (Ja¨ger et al. 1990; Lovell-Badge polyglutamine repeats within mouse Sry genes (Bowles and Robertson 1990), and presence of exogenous Sry et al. 1999), but not their human ortholog, and M. (Fechner et al. 1993; Koopman et al. 1991) causes XX domesticus Sry genes encode a protein that is signifi- sex reversal. Because cases of human sex reversal are cantly shorter than the protein encoded for by M. relatively rare and the heterogeneity found within our musculus Sry genes (Albrecht and Eicher 1997). species makes genotype-to-phenotype correlations dif- Some M. domesticus Y chromosomes cause XY sex ficult, mouse models of inherited sex reversal are reversal when present on C57BL/6J background, an particularly useful. All identified nonsynonymous SRY inbred laboratory strain that normally carries a M. polymorphisms in humans are associated with varying musculus Y chromosome, i.e., M. musculus Sry gene degrees of XY gonadal dysgenesis or disrupted gonadal (Albrecht and Eicher 1997). This inherited phenom- development (Hawkins et al. 1992). Although there is enon, referred to as C57BL/6J-YDOM sex reversal, has selection bias when analyzing such affected individuals, been extensively studied for the M. domesticus poschiavi- to date the human genome project has not identified nus Y chromosome (YPOS)(Eicher et al. 1982), as well any benign nonsynonymous polymorphisms. By con- as for the related M. domesticus tirano Y chromosome trast, at least 15 distinct Sry alleles are present among (YTIR)(Lee and Taketo 1994, 2001). Since the current different species of Mus (Albrecht and Eicher 1997), genetic investigation is based on C57BL/6J-YPOS, and and most Sry alleles of Mus domesticus and M. musculus previous genetic experiments have been exclusively per- formed using that strain, we have concentrated only on it and not on other M. domesticus models. The failure to develop testes in these mice stems from an incompati- 1Corresponding author: Department of Human Genetics, David Geffen bility of the SryPOS gene to initiate normal testicular de- School of Medicine, Gonda Center, Room 5506, 695 Charles Young Dr. S., University of California, Los Angeles, CA 90095-7088. velopment when C57BL/6J autosomal and/or X-linked E-mail: [email protected] factors are present. All XYPOS C57BL/6J-YPOS individuals Genetics 179: 419–427 (May 2008) 420 G. Nikolova et al. develop some ovarian tissue (Eicher et al. 1982); half MATERIALS AND METHODS develop exclusively ovarian tissue, thus are completely Genome scans: The microsatellite genome scan was per- sex reversed, and the remainder develop ovarian and formed on DNA isolated from C57BL/6J.129-YPOS mice re- testicular tissue, thus are partially sex reversed (Eicher ceived at the University of California to detect 129 vs. B6 et al. 1982; Eicher and Washburn 2001). An investiga- microsatellite markers. PCR amplification was performed tion using linkage analysis concluded that C57BL/ using fluorescently labeled primer pairs consisting of labeled 6J-YPOS sex reversal is inherited as a complex trait and unlabeled oligonucleotides. Individual PCR conditions were optimized for each primer pair. PCR products were influenced by at least three different autosomal loci analyzed using an ABI 3700 capillary sequencer (Applied POS and the Sry gene (Eicher et al. 1996). Biosystems, Foster City, CA) at the University of California, Los Although protein isoform differences among M. Angeles (UCLA) Human Genetics Sequencing and Genotyp- domesticus Sry alleles have been demonstrated to play a ing Core Facility. Data analysis was performed using the ABI role in the precise gonadal phenotype each causes when PRISM Genotyper software (Applied Biosystems). One hun- lbrecht dred forty-nine markers were used in the microsatellite scan transferred to C57BL/6J (A et al. 2003), the (supplemental Table 1). molecular mechanisms through which those differen- Two genome scans were performed using SNP markers. ces arise are unknown. In addition, the SryPOS gene, Approximately 183 SNP markers using DNA obtained from when introduced as a transgene into XX C57BL/6J C57BL/6J.129-YPOS strain mice received at UCLA, with 37 mice, is capable of directing testicular development markers located on chromosome 11 (supplemental Table 2). lbrecht A genome scan also was performed on DNA obtained from (A et al. 2003). This latter result suggests that C57BL/6J.129-YPOS strain maintained at The Jackson Labora- POS overexpression of the Sry gene is capable of initiating tory. In this case, 404 SNP markers were used, 11 of which are testis development in C57BL/6J mice and it is the located on chromosome 11 (supplemental Table 3). SNP improper expression of the single copy of SryPOS carried genotyping was performed by The Jackson Laboratory JAX on the YPOS chromosome that is the major factor under- Services SNP-based genome scanning commercially available lying sex reversal, with the protein isoform difference service (The Jackson Laboratory, Bar Harbor, ME). The results from the University of California SNP scan were reported by contributing to a lesser degree. JAX services as SNP genotypes only for those SNPs that In mice, Sry is normally expressed between E10.5 and differed from C57BL/6J and for all SNPs on chromosome E12.5 (where E is the embryonic developmental age) in 11. For all other SNPs the data were reported as matching the XY genital ridge (Koopman et al. 1990; Jeske et al. C57BL/6J. The full combined data from all three scans for 1995; Bullejos and Koopman 2001), with peak Sry chromosome 11 are depicted in supplemental Table 4. The SNP scans also identified a single marker, rs13480100 expression occurring in normal XY C57BL/6J genital on chromosome 9 as being of non-C57BL/6J origin. In- ridges at E11.5, i.e., at the 16–18 tail somite stage of terestingly, C57BL/6J is the only strain reported to have a T development (Bullejos and Koopman 2005). In con- base pair at this locus, differing even from C57BL/6N, a closely trast, expression of the SryPOS gene peaks at 10–14 hr related strain (data not shown). During the reconstruction of POS later in the genital ridges of XYPOS C57BL/6J-YPOS fetuses the C57BL/6J.129-Y strain the chromosome 9 rs13480100 lbrecht ullejos oopman region became of C57BL/6J origin. (A et al. 2003; B and K 2005). Mouse strains: Animals were housed according to the POS However, Sry is expressed at the normal time and guidelines of UCLA’s Division of Laboratory Animal Medicine. normal levels in the genital ridges of XYPOS (C57BL/6J- All experiments were approved by the Institutional Animal POS lbrecht Y 3 DBA/2J)F1 mice (A et al. 2003), which Care and Use Committees of The Jackson Laboratory and supports the notion that upstream factors derived from UCLA. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal the C57BL/6J genome, when homozygous, delay and Care. reduce Sry expression levels and therefore contribute to Construction of the C57BL/6J.129-YPOS strain: The original C57BL/6J-YPOS sex reversal. C57BL/6J.129-YPOS strain (Whitney et al. 2000) was produced We used a congenic approach to identify loci that by mating a C57BL/6J female to a 129-YPOS male. The modify the C57BL/6J-YPOS sex-reversal phenotype with nomenclature used to define the 129 strain used in this study, ‘‘129S1/Sv’’ is not in use today, making the actual 129 strain the goal of gaining further insight into male sex deter- used unknown.