C57BL/6J-T-Associated Sex Reversal in Mice Is Caused by Reduced Expression of a Mus domesticus Sry Allele

Corresponding author: Eva M. Eicher, The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609., eme{at}jax.org (E-mail)

Communicating editor: N. A. JENKINS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

C57BL/6J-T-associated sex reversal (B6-TAS) in XY mice results in ovarian development and involves (1) hemizygosity for Tas, a gene located in the region of Chromosome 17 deleted in T^hp and T^Orl, (2) homozygosity for one or more B6-derived autosomal genes, and (3) the presence of the AKR Y chromosome. Here we report results from experiments designed to investigate the Y chromosome component of this sex reversal. Testis development was restored in B6 T^Orl/+ XY^AKR mice carrying a Mus musculus Sry transgene. In addition, two functionally different classes of M. domesticus Sry alleles were identified among eight standard and two wild-derived inbred strains. One class, which includes AKR, did not initiate normal testis development in B6 T^Orl/+ XY mice, whereas the other did. DNA sequence analysis of the Sry ORF and a 5' 800-bp segment divided these inbred strains into the same groups. Finally, we found that Sry is transcribed in B6 T^Orl/+ XY^AKR fetal gonads but at a reduced level. These results pinpoint Sry as the Y-linked component of B6-TAS. We hypothesize that the inability of specific M. domesticus Sry alleles to initiate normal testis development in B6 T^Orl/+ XY^AKR mice results from a biologically insufficient level of Sry expression, allowing the ovarian development pathway to proceed.

TWO inherited sex reversal conditions in mice depend on the presence of a Mus domesticus Y chromosome on a C57BL/6J genetic background. In C57BL/6J-Y^POS (B6-Y^POS) sex reversal, ovarian tissue develops if autosomal testis-determining genes are homozygous B6 and the Sry gene is from M. d. poschiavinus (EICHER et al. 1982 , EICHER et al. 1996 ; EICHER and WASHBURN 1983 ; NAGAMINE et al. 1987 ; EICHER 1988 ; BIDDLE et al. 1994 ). In B6-T-associated (B6-TAS) sex reversal, the focus of this study, ovarian tissue develops if the dominant brachyury mutation T-hairpin tail (T^hp) or T-Orleans (T^Orl) is present, the M. domesticus Y chromosome is from the AKR/J inbred strain, and the remainder of the genome is of B6 origin (WASHBURN and EICHER 1983 , 1989; WASHBURN et al. 1990 ). [T^hp and T^Orl are partially overlapping deletions (MOUTIER 1973A , MOUTIER 1973B ; BENNETT et al. 1975 ; ERICKSON et al. 1978 ).] Although B6-Y^POS and B6-TAS sex reversals share the requirement for homozygosity for B6-derived genes and the presence of an Sry allele of M. domesticus origin, they differ in that B6-TAS requires the presence of T^hp or T^Orl. We have hypothesized that a gene necessary for testicular development, designated T-associated sex reversal (Tas), resides in the deleted region common to T^hp and T^Orl (WASHBURN and EICHER 1989 ).

Here we present results from experiments designed to identify the Y chromosome component of B6-TAS:

1. We utilized a transgenic rescue approach to determine if Sry, the genetic switch for the testis pathway (GUBBAY et al. 1990 ; SINCLAIR et al. 1990 ), was the Y-linked component. Testis development was restored in B6 T^Orl/+ XY^AKR transgenic mice, indicating that the AKR Sry allele is the Y-linked gene responsible for B6-TAS.

2. We then investigated whether ovarian development in B6 T^Orl/+ XY^AKR mice results from the lack of, or reduction in, Sry transcription. Results indicated that Sry is transcribed but at a significantly reduced level. Inferred from this finding is that Tas affects the expression level of Sry.

3. We tested whether the AKR Sry allele is unique among M. domesticus Sry alleles carried by several other inbred strains. We found that the Y chromosome (i.e., Sry allele) of strains LEWES/Ei, MA/MyJ, PL/J, RF/J, and WSB/Ei causes sex reversal whereas the Y chromosome of strains BUB/BnJ, SJL/J, ST/bJ, or SWR/J allows normal testis development.

4. We sequenced the Sry open reading frame (ORF) and 800 bp 5' to the ORF from each of the Y chromosomes noted above. All were identical except for the number of glutamine residues in glutamine repeat cluster 3 and a 10-bp deletion in the 5' region. On the basis of the differences found, these Y chromosomes sort into the same two groups that caused sex reversal or normal testis development. These findings are discussed in terms of the evolutionary relationship of the two groups of M. domesticus Y chromosomes identified.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

B6 T^Orl congenic and Y chromosome consomic strains:
The T^Orl mutation and the M. domesticus Y chromosomes carried by standard inbred strains AKR/J, BUB/BnJ, MA/MyJ, RF/J, SJL/J, ST/bJ, and SWR/J and two wild-derived strains, LEWES/Ei and WSB/Ei, each were transferred onto the B6 strain (subline, C57BL/6JEi) by standard backcross methods for a minimum of 10 generations. The T^Orl congenic B6 strain is designated B6.T^Orl and the Y chromosome consomic B6 strains are designated B6.Y^AKR, B6.Y^BUB, etc. We also used a C57BL/6By consomic strain developed by Donald Bailey (The Jackson Laboratory) that contained the PL/J Y chromosome (also of M. domesticus origin).

Sry transgenic lines:
Transgenic mice were produced using a 14.6-kb genomic DNA segment that contains the Y-linked Sry gene derived from a 129 inbred strain (GUBBAY et al. 1990 ) and is capable of completely sex reversing XX mice (i.e., causes testicular development) when present as a transgene (KOOPMAN et al. 1991 ; EICHER et al. 1995 ). DNA was injected into the male pronucleus of fertilized eggs obtained from mating B6 females to B6 XY^AKR males using standard procedures (WAGNER et al. 1981 ). Four of 23 offspring recovered carried the transgene. One was an XX male, thus sex reversed by the transgene but sterile due to the presence of two X chromosomes and absence of a Y chromosome. The remaining three were XY males, each of which contained a transgene proven functional by the fact that all XX transgenic offspring were completely sex-reversed males. A transgenic line was established from each founder, hereafter designated C57BL/6JEi-Y^AKR TgN(Sry-129)1Ei; ... 2Ei; and ... 3Ei. The transgene carried by each line appears genetically stable because no transgenic females or hermaphrodites (having both ovarian and testicular tissue) have been observed since these lines were established in 1992. We used line Tg1Ei for the work presented here and refer to it hereafter as Tg.

Gonad development in B6 T^Orl/+ Tg mice:
Two groups of T^Orl/+ Tg offspring were analyzed for gonad development. In both cases, mice were produced by mating B6 T^Orl/+ females to B6 XY^AKR Tg males. The first group consisted of 80 weanling mice analyzed at 3 to 4 weeks of age. Each mouse was phenotypically sexed by inspection of external and internal genitalia, classified for normal (+/+) or shortened tail length (T^Orl/+), and genotyped for the presence of the Y chromosome and Tg (see below). The second group consisted of 21 fetuses analyzed at 14.5 to 15 days of fetal development. This time of gonadal development was chosen because a small amount of ovarian tissue is easily detected in an ovotestis (EICHER et al. 1980 ). (Later in development, ovarian tissue can be compressed by the growth of testicular tissue and thus be difficult to detect.) Each fetus was classified for gonad development (ovary, testis, or ovotestis), tail length, and the presence of the Y chromosome and Tg.

Ability of M. domesticus Y chromosomes to direct normal testis development in T^Orl/+ XY mice:
The M. domesticus-derived Y chromosome carried by each B6.Y consomic strain was tested to determine if B6 T^Orl/+ XY individuals developed as females (as when an AKR Y chromosome is present) or as males (as when a M. musculus B6 or C3H Y chromosome is present). Males from each consomic line were mated to B6 T^Orl/+ females. T^Orl/+ offspring were classified at weaning as female, male, or hermaphrodite by the appearance of external and internal genitalia. Non-T^Orl (+/+) offspring served as controls. Presence of a Y chromosome was determined by analysis of G-banded mitotic chromosome preparations from bone marrow (EICHER and WASHBURN 1978 ) or a PCR assay (see below) that detects Y chromosome sequences.

Detection of Y chromosome and Tg:
Presence of the Y chromosome and the Tg were determined by Southern blot analysis or a multiplex PCR protocol. For Southern blot analysis, genomic DNA was digested with TaqI and restriction fragments were separated and blotted using standard methods. Filters were probed with a 380-bp NotI/PstI insert isolated from clone p422.04 (GUBBAY et al. 1990 ) and labeled with ³²P. A 4.7-kb TaqI fragment identified the Sry transgene and a 2.1-kb TaqI fragment identified the AKR Sry allele. The multiplex PCR protocol is given in CAPEL et al. 1999 .

Identifying T^Orl carriers:
T^Orl/+ fetuses 12.5 days post-coitum (dpc) and older were distinguished from their +/+ sibs by a shortened tail. For younger fetuses, tail length is not a reliable method to distinguish the two genotypes so a PCR method was used that amplifies D17Tu1, producing an 230-bp product if T^Orl is absent and a slightly larger product if T^Orl is present (HIMMELBAUER and SILVER 1993 ). Fetal tissue was digested in 200 µl of PCR buffer supplemented with nonionic detergents and Proteinase K and 2 µl of this mixture was used as PCR template. Primers were (5'-GGGGAACAGTAATAAAGCTGA-3') and (5'-TCTGCTTCATCTGAGGGTCCA-3') and PCR conditions were 40 cycles of 94°, 30 sec; 55°, 30 sec; and 72°, 30 sec + 1 sec/cycle. PCR reactions were analyzed on 5% NuSieve 3:1 gels.

RNA extraction and RT-PCR:
Paired mesonephros/gonadal ridges were dissected from individual fetuses at 10.5 to 12.5 dpc and immediately lysed in Buffer RLT (QIAGEN, Valencia, CA; RNeasy total RNA miniprep kit) and stored at -80°. Total RNA was purified and DNased using RNeasy miniprep columns and eluted in 30 µl H₂O. Alternatively, RNA was DNased after purification using the DNA-free protocol (Ambion, Austin, TX).

Ten microliters of denatured RNA was used for first-strand cDNA synthesis in a 20-µl reaction using MuLV reverse transcriptase (RT) and oligo(dT) primers incubated at 42° for 1 hr. For each RNA template, a control reaction without RT was included. For each RT reaction condition, an H₂O no-template reaction was included as an additional negative control. Four microliters (Sry) or 1 µl (Hprt) of the reverse transcription reaction served as template for subsequent PCR. PCR amplification of Sry used primers Y34A (5'-CTGGCACTACTGGACTTCTAAG-3') and cs-1B (5'-(T)18GGGATGG-3'), which are RNA specific, and the method of JESKE et al. 1995 except that Applied Biosystems (Foster City, CA) buffer II and 1.5 mM MgCl₂ were substituted. PCR amplification of Hprt used primers (5'-CCTGCTGGATTACATTAAAGCACTG-3') and (5'-GTCAAGGGCATATCCAACAACAAAC-3'; KOOPMAN et al. 1989 ), Applied Biosystems buffer II, 1.5 mM MgCl₂, 200 µM each nucleotide, 0.2 µM each primer, and 1 unit Taq (Applied Biosystems). Thermal cycling conditions were 40 cycles of 94°, 30 sec; 59°, 30 sec; and 72°, 30 sec. A control reaction without cDNA template was included for each PCR reaction condition. Ten microliters of each PCR reaction was analyzed on a 4% NuSieve 3:1 gel. Although this RT-PCR assay is not quantitative, it is specific for the presence of Sry RNA. One of the primers overlaps and depends on the presence of a poly(A) tail at the minor polyadenylation site (JESKE et al. 1995 ). An Sry PCR product is not detected on an agarose gel if 200 µl of genomic DNA is used as template (data not shown). RNA from a B6 T^Orl/+ XY^AKR fetus and a +/+ XY^AKR sib were analyzed for the presence of an Sry transcript at 10.5 dpc (11- to 12-tail somite stage), 11.5 dpc (16- to 17-tail somite stage), and 12 dpc (23-tail somite stage).

Sry expression levels were compared to the expression levels of LIM homeobox protein 1 (Lhx1) using a semiquantitative RT-PCR assay. Lhx1 is expressed only in the mesonephric component of the urogenital ridge (BARNES et al. 1994 ). Lhx1-specific primers were designed to span a region containing a 97-bp intron and no NlaIV restriction sites and to amplify a 139-bp fragment using primers Lhx1-1660 (5'-GGCGAGGAGCTCTACATCATAG-3') and Lhx1-1798 (5'-CTTGGGAATCCGGAGATAAAC-3'). These primers were combined with Sry-9431 (5'-TGGTGAGCATACACCATACC-3') and Sry-9808 (5'-TTGCTGTCTTTGTGCTAGCC-3') in a multiplex PCR reaction containing [-³²P]dCTP and 2 µl of the reverse transcription reaction as outlined above. (Sry primers are designated by the 5' base using numbering in GenBank entry X67204 where positions 8304 and 9491 represent the beginning and end, respectively, of the M. musculus ORF.) Thermal cycling conditions were 29 cycles of 94°, 30 sec; 57°, 30 sec; and 72°, 30 sec. The PCR reaction was digested with NlaIV, separated on 3% agarose gels, and Southern blotted using standard methods. The amount of radioactivity in each band was determined using Phosphor imaging plates and Image Gauge software (Fuji Medical Systems, Stamford, CT). Primers Sry-9431 and Sry-9808 amplify a 377-bp DNA fragment from the Sry gene. When this PCR product is digested with NlaIV, a single 377-bp undigested fragment is diagnostic for M. musculus alleles and two comigrating fragments (189 and 188 bp) are diagnostic for M. domesticus alleles.

Sry sequence analysis:
The Sry ORF and portions of the 5'- and 3'-untranslated regions (UTRs) were analyzed by direct sequencing of PCR products. A total of 100–150 ng of genomic DNA served as template for primers Sry-8212 (5'-TTGATTTTTAGTGTTCAGCCCTACAGCC-3') and Sry-9791 (5'-AGCTGTTTGCTGTCTTTGTGCTAGCC-3') in a 100-µl reaction. PCR was performed by conventional techniques using Taq DNA polymerase (Applied Biosystems) and 1.5 mM MgCl₂ employing 35 cycles of 94° for 30 sec; 59° for 30 sec; and 72° for 90 sec. A sample of each PCR was assayed for specificity on a 1% agarose gel (the expected PCR product size is 1.6 kb) and the remainder purified for sequencing using either Wizard PCR preps (Promega, Madison, WI) or QIAquick spin columns (QIAGEN). A total of 45 ng of purified Sry ORF PCR product was directly sequenced using primers Sry-8212, Sry-9791, Sry-8653 (5'-GGAGTAGAGCTGCACACCTGTACTCC-3'), and Sry-9475 (5'-CCAGTGTCATGAGACTGCCAACC-3') and the PRISM Ready Reaction DyeDeoxy terminator cycle sequencing kit (manufacturer's recommendations, Applied Biosystems) on an ABI 373 Stretch automated sequencer.

The 5' UTR and proximal promoter region was PCR amplified and sequenced using a strategy similar to that given above and employing primers Sry-7436 (5'-CAGAAATGAACTACTGCATCCC) and Sry-8371 (5'-AACTTGTGCCTCTCACCACG).

DNA sequence analysis and alignment were performed using the GeneWorks computer program (IntelliGenetics, ver. 2.4) with subsequent hand editing. The primer strategy used allowed for double-strand coverage and multiple-pass sequence for most regions. Any ambiguities detected in the single-strand/single-pass regions were resolved or confirmed by resequencing. Genomic DNA from at least two individual males from each Y chromosome consomic strain served as PCR template and the reactions were combined prior to purification and sequencing to control for any individual variation. No sequence heterozygosity was detected in any of the templates analyzed. The nucleotide sequences for the 10 Sry alleles listed in Table 2 are deposited in GenBank under accession nos. U70642, AF009519, and AF337043, AF337044, AF337045, AF337046, AF337047, AF337048, AF337049, AF337050.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Testing the Sry transgene:
We analyzed gonadal development in B6 T^Orl/+ XY^AKR mice carrying a functional M. musculus Sry transgene of strain 129 origin to gain insight into the Y chromosome component of B6-TAS. Development of testicular tissue would indicate that Sry is the sole Y-linked component. Development of ovarian tissue would eliminate Sry as the Y-linked component. The data from these experiments are presented in Table 1. At both developmental stages, T^Orl/+ XY^AKR mice presented as normal females with ovaries in the absence of the Tg and as normal males with testes in the presence of the Tg. These findings provide strong evidence that Sry is the Y-linked gene responsible for ovarian tissue development in B6 T^Orl/+ XY^AKR mice.

Testing other Sry alleles:
To determine if the development of ovarian tissue was unique to the Sry allele carried on the AKR Y chromosome, we mated B6 mice carrying one of nine other M. domesticus-derived Y chromosomes to B6 T^Orl/+ females and analyzed the T^Orl/+ XY offspring. We found that the Y chromosomes fell into two groups based on gonadal development (Table 2). Group A was like AKR and caused sex reversal whereas group B was like B6 or C3H and resulted in normal male development.

Sequence analysis within and 5' to the Sry ORF:
We sequenced the ORF of the M. domesticus Sry gene carried on the Y chromosomes used in the experiments noted above and compared the predicted amino acid sequences to each other and to that published for the Sry¹²⁹ allele (M. musculus, GenBank accession no. X67204, which was the Sry allele used in the transgenic experiments). The predicted amino acid sequence of these 10 alleles was identical except for a polymorphism in glutamine repeat cluster 3 (GRC-3; Fig 1). Group A Sry alleles contained 13 glutamines in this cluster, whereas group B Sry alleles contained 12 glutamines. Compared to the Sry¹²⁹ allele, the M. domesticus alleles harbor nine additional shared changes. The most striking is the one first reported by COWARD et al. 1994 , which involves a stop codon in GRC-8 and results in a greatly shortened M. domesticus SRY protein compared to the SRY protein coded for by M. musculus alleles (see Fig 1).

View larger version (41K): In this window In a new window Download PPT slide   Figure 1. Comparison among predicted SRY proteins from M. musculus (129 inbred strain) and M. domesticus group A and group B. For discussion purposes, SRY is depicted to contain five regions: unique region 1 (first two amino acids), HMG domain (solid underline), unique region 2 (dashed underline), glutamine repeat region (dotted underline), and unique region 3 (dashed/dotted underline). The amino acid sequence of group A and B Sry alleles is identical except for the number of glutamines in glutamine repeat cluster 3, which is 13 and 12, respectively (GRC-3, boxed). The eight amino acid differences between the group A/B and 129 alleles are shaded. The ninth polymorphism creates the M. domesticus stop codon in GRC-8. The predicted 129 SRY protein is 395 amino acids (aa), group A SRY protein is 232 aa, and group B SRY protein is 231 aa. The number of CAG repeats in cluster 3 also was determined for strains AKR, BUB, PL, MA, RF, SJL, ST, and SWR by COWARD et al. 1994 , and ALBRECHT and EICHER 1997 reported partial DNA sequence for the open reading frame for strains AKR, BUB, and WSB.

The DNA sequence immediately 5' of the ORF also was determined for the 10 M. domesticus alleles. This 800-bp region contains all of the 5' UTR and part of the proximal promoter. All alleles were identical except for a 10-bp deletion in a single polymorphic region (GRAVES and ERICKSON 1995 ). The absence or presence of the deletion also defines groups A and B, respectively. This 10-bp repeat does not contain a recognized transcription factor binding site [TRANSFAC v3.3, Transcription Element Search Software (TESS); http//www.cbil.upenn.edu/tess]. Compared to M. musculus (Sry¹²⁹), this 800-bp region contains three polymorphisms: G-T at position 7656, G-A at 7768, and A-G at 7928. The polymorphisms at 7656 and 7928 are located in transcription factor binding sites identified by TESS (CREB and OCT, respectively). However, these sites are fairly ubiquitous and the biological relevance is unknown. Significantly, the variant sequences are shared by all 10 M. domesticus alleles, thus eliminating them as causative elements in B6-TAS.

Sry expression in B6 T^Orl/+XY^AKR fetal gonads:
The fact that B6 T^Orl/+ XY^AKR mice develop bilateral ovaries suggests that the male sex determination pathway fails early. To determine if this failure involved activation of Sry, we used a qualitative RT-PCR assay to analyze Sry expression in paired urogenital ridges isolated from 10.5- to 13.5-dpc B6 T^Orl/+ and +/+ XY^AKR mice. Sry expression was detected in both genotypes at all time points sampled (data not shown). Because this result did not eliminate the possibility that Sry was expressed at a reduced level in T^Orl/+ XY^AKR gonads, a semiquantitative radioactive RT-PCR assay was used. To accomplish this, we determined the level of Sry transcript in the genital ridge and compared this to the level of Lhx1 transcript in the mesonephros (Fig 2A). Normally, Sry transcription begins at 10.5 dpc (8-tail somite stage), peaks at 11.5 dpc (18-tail somite stage), and is absent by 13.0 dpc. Our results indicate that Sry expression is significantly delayed in B6 T^Orl/+ XY^AKR gonads compared to B6 +/+ XY^AKR gonads (Fig 2B). For example, the Sry/Lhx1 ratio was 0.04 at the 15- to 16-tail somite stage in T^Orl/+ fetuses, whereas the ratio was 0.63 in same aged +/+ fetuses. In fact, of the six pairs of T^Orl/+ urogenital ridges analyzed from fetuses at the 12- to 16-tail somite stage of development, only one pair had detectable Sry expression. To determine if Sry was present but below the level of detection of the semiquantitative assay, we increased the number of PCR cycles. When 35 PCR cycles were used, Sry was detectable in the T^Orl/+ samples using ethidium bromide-stained gels (Fig 2C).

View larger version (18K): In this window In a new window Download PPT slide   Figure 2. Sry expression is significantly delayed in B6 TOrl/+ XYAKR urogenital ridges. (A) Representative semiquantitative radioactive RT-PCR results for B6 TOrl/+ XYAKR urogenital ridges. Samples labeled + and - identify those with and without reverse transcriptase, respectively. Fetal age is indicated by tail somite number. (B) Average Sry/Lhx1 expression vs. developmental age in B6 +/+ XYAKR and TOrl/+ XYAKR urogenital ridges. The numbers above each data point indicate the number of paired urogenital ridges analyzed. (C) Five of the six TOrl/+ XYAKR 12- to 16-tail somite samples were reanalyzed for Sry expression using an increased number of PCR cycles. Ctrl indicates the DNA positive control, RB the no RNA template (water) reverse transcription reaction negative control, and PB the no template (water) PCR negative control.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Gonad development in B6 T^Orl/+ XY^AKR mice is corrected by an Sry transgene:
Previously, we reported that ovarian tissue development in C57BL/6J-T-associated sex reversal (B6-TAS) depends upon the simultaneous inheritance of three genetic components: a B6 inbred strain background, the presence of the brachyury deletion alleles T^hp or T^Orl, and a M. domesticus Y chromosome derived from the AKR/J inbred strain (WASHBURN and EICHER 1983 , WASHBURN and EICHER 1989 ; WASHBURN et al. 1990 ). These three conditions are absolute requirements. For example, if a C3H/HeSnJ or (B6 x C3H)F₁ genome is substituted for the B6 genome in T^hp/+ mice (WASHBURN et al. 1990 ), or if certain other T-deletion mutations are used (E. M. EICHER and L. L. WASHBURN, unpublished data), or if a B6- or C3H-derived Y chromosome (of M. musculus origin) is substituted for the AKR-derived Y chromosome in T^Orl/+ mice, normal testicular tissue is formed (WASHBURN et al. 1990 ).

The experiments reported here were designed to provide insight into the Y-linked aspects of B6-TAS. We used a transgenic approach (i.e., genetic complementation) to determine if the Y-linked gene was Sry. We found that by adding a functional M. musculus-derived Sry transgene to the genome, B6 T^Orl/+ XY^AKR mice develop normal testes. Because the genomic DNA used as a transgene encodes only the Sry gene, these results provide strong evidence that the Sry gene present on the AKR Y chromosome is functionally deficient in the B6 T^Orl/+ genetic environment and cannot initiate normal testis differentiation. Further support for this conclusion was obtained from experiments using a set of B6 Y chromosome consomic strains together with DNA sequence data from the Sry allele carried by these Y chromosomes.

An earlier study by PALMER and BURGOYNE 1991 provided evidence that the timing of testis differentiation was dependent upon the specific Y chromosome present. They reported that testicular differentiation began as much as 14 hr later in XY mice carrying an AKR Y chromosome than in XY mice carrying a B6 Y chromosome. This difference in timing of gonad differentiation must be specific to the Y chromosome because the rest of the genetic background was uniform. Our finding that the AKR/J Sry gene is the Y-linked component responsible for ovarian tissue development in B6-TAS implies that the Palmer and Burgoyne finding was due to the different Sry alleles they tested. Together, these observations suggest that under genetically specific circumstances, the AKR Sry gene is inappropriately controlled.

Evolution of M. domesticus Sry alleles studies:
TUCKER et al. 1992 reported that the M. domesticus Y chromosomes of standard inbred mouse strains fall into two major groups based on an analysis of Southern blots containing EcoRI restriction fragments probed with YB10, a highly repetitive sequence present on the long arm of the Y chromosome (EICHER et al. 1989 ). In the present work we tested if the Sry gene carried by these inbred strains separated into the same two groups based on gonadal development. We found that the Sry alleles carried by group A mice (see Table 2) caused sex reversal whereas the Sry alleles carried by group B mice did not and that these groups were the same groups identified by TUCKER et al. 1992 .

To explore these findings further, DNA sequence analysis was performed on the Sry alleles carried by the inbred strains representing group A and group B. Again we found concordance between ovarian vs. testicular development and the DNA sequence of the Sry gene. In addition, this data further supports Tucker and co-workers, who concluded that the laboratory strains AKR/J, PL/J, MA/MyJ, and RF/J are likely related to descendants of a population of M. domesticus mice that colonized the mid-Atlantic seaboard (i.e., the ancestors of LEWES/Ei and WSB/Ei were caught in Delaware and Maryland, respectively).

Although the functional differences between groups A and B correlate with specific Sry sequence similarities in the ORF and 5' to the ORF, we do not contend that these sequence differences cause ovarian development in B6 T^Orl/+ mice carrying a group A Y chromosome. Further comparative analyses of Sry alleles from these two groups of Y chromosomes may identify the functionally important controlling sequences. The proof of speculation rests with engineering presumptive causative sequences from a group A Sry allele into a group B or M. musculus Sry allele (or vice versa).

Expression of Sry in B6 T^Orl/+ XY^AKR mice:
To investigate the improper functioning of the AKR/J Sry gene, we determined the timing and level of Sry transcripts in B6 T^Orl/+ XY^AKR fetal gonads. We found that Sry transcription is initiated at the correct time, but the level of transcription is severely reduced compared to non-T^Orl (+/+) controls. Of relevance is the report by NAGAMINE et al. 1999 that the level of Sry transcript is reduced in B6 mice carrying an AKR Y chromosome compared to those carrying an FVB Y chromosome. Our data indicate that Tas is involved in controlling Sry transcript levels, given that the Sry transcript level is further reduced in B6 Y^AKR fetal gonads when T^Orl is present. We suggest that the level of Sry expression is so severely reduced in B6 T^Orl/+ XY^AKR gonads that the next steps in the testicular pathway are not initiated, allowing the ovary-determining pathway to proceed and ovarian tissue to develop. Further support for this suggestion requires cloning of the Tas gene.

Cause of ovarian tissue development in B6 T^Orl/+ XY^AKR mice:
Two other observations correlate with the finding that B6 XY^AKR mice have a reduced level of Sry transcription (NAGAMINE et al. 1999 ). First, migration of mesonephric cells into the undifferentiated gonads in B6 XY^AKR mice is delayed (ALBRECHT et al. 2000 ). Previous work indicates that migration of mesonephric cells is dependent on the presence of a functional Sry gene and is required for the formation of testicular cords (MARTINEAU et al. 1997 ; CAPEL et al. 1999 ; TILMANN and CAPEL 1999 ). Second, B6 XY^AKR mice have delayed testis cord formation (WASHBURN and EICHER 1983 , WASHBURN and EICHER 1989 ; NAGAMINE et al. 1987 ; WASHBURN et al. 1990 ). This delay is apparent at the cranial and caudal ends of the gonads at 14 dpc but absent 12 to 24 hr later when cords have formed at the polar regions. Delayed cord formation is not observed in B6 mice carrying the M. domesticus-derived FVB Y chromosome (NAGAMINE et al. 1999 ) or the BUB/BnJ Y chromosome (L. L. WASHBURN and E. M. EICHER, unpublished data). In addition, as shown in the present study, the BUB/BnJ Y chromosome initiates normal testis development in B6 T^Orl/+ mice. (The case for the FVB Y chromosome has not been tested.) Because the FVB Sry gene has 12 CAG repeats in GRC-3 (NAGAMINE et al. 1999 and reported here), it probably belongs to group B, which includes BUB/BnJ. We suggest that the level of Sry transcripts in B6 mice carrying a group B Y chromosome is higher than the level present in B6 mice carrying a group A Y chromosome.

We hypothesize that reduction in Sry transcript levels in B6 XY^AKR fetal gonads causes a delay in mesonephric cell migration into the gonad and this, in turn, causes a delay in cord formation. If this is correct, there are two possible causes of the failure of B6 T^Orl/+ XY^AKR mice to form testicular cords during gonad differentiation: (1) The number of Sry-expressing cells is normal but the level of Sry transcription per cell is severely reduced or (2) the level of Sry transcription is normal per Sry-expressing cell but the number of Sry-expressing cells per gonad is significantly reduced. Either of these possibilities could severely affect the signaling pathway that is required for mesonephric cell migration into the developing gonad. The outcome is that few if any mesonephric cells migrate into the gonad, testis cords fail to form, and the ovarian pathway begins. This explanation is similar to what has been proposed for the cause of B6-Y^POS sex reversal (ALBRECHT et al. 2000 ).

	ACKNOWLEDGMENTS

We thank Dr. Peter Hoppe for his expertise in making Sry transgenic animals on the C57BL/6J inbred strain, Douglas McMinimy for his sequencing skills, and Michelle Higgins and Leona Gagnon for helping to maintain the B6 Y chromosome consomic strains. We also thank Tim O'Brien, Beverly Richards-Smith, and John Schimenti for helpful suggestions concerning this report. This work was funded by National Institutes of Health research grant GM-20919 (to E.M.E.) and the National Cancer Institute cancer core grant CA34196 (to The Jackson Laboratory).

Manuscript received March 12, 2001; Accepted for publication May 15, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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