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C57BL/6J-T-Associated Sex Reversal in Mice Is Caused by Reduced Expression

of a

Mus domesticus Sry

Allele

Linda L. Washburn, Kenneth H. Albrecht and Eva M. Eicher

The Jackson Laboratory, Bar Harbor, Maine 04609 Manuscript received March 12, 2001 Accepted for publication May 15, 2001

ABSTRACT

C57BL/6J-T-associated sex reversal (B6-TAS) in XY mice results in ovarian development and involves (1) hemizygosity forTas, a gene located in the region of Chromosome 17 deleted inThpand TOrl, (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 B6TOrl/XYAKRmice carrying aMus musculus Srytransgene. 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 B6TOrl/XY mice, whereas the other did. DNA sequence analysis of theSryORF and a 5⬘800-bp segment divided these inbred strains into the same groups. Finally, we found thatSryis transcribed in B6TOrl/XYAKRfetal gonads but at a reduced level. These results pinpointSryas the Y-linked component of B6-TAS. We hypothesize that the inability of specificM. domesticus Sry alleles to initiate normal testis development in B6TOrl/XYAKRmice results from a biologically insufficient level ofSryexpression, allowing the ovarian development pathway to proceed.

T

WO inherited sex reversal conditions in mice 1. We utilized a transgenic rescue approach to deter-depend on the presence of a Mus domesticus Y mine ifSry, the genetic switch for the testis pathway chromosome on a C57BL/6J genetic background. In (Gubbay et al. 1990; Sinclair et al. 1990), was the C57BL/6J-YPOS(B6-YPOS) sex reversal, ovarian tissue de- Y-linked component. Testis development was re-velops if autosomal testis-determining genes are homo- stored in B6TOrl/⫹

XYAKRtransgenic mice, indicating zygous B6 and theSrygene is from M. d. poschiavinus that the AKRSryallele is the Y-linked gene responsi-(Eicheret al.1982, 1996;EicherandWashburn1983; ble for B6-TAS.

Nagamineet al.1987;Eicher1988;Biddleet al. 1994). 2. We then investigated whether ovarian development In B6-T-associated (B6-TAS) sex reversal, the focus of in B6TOrl/⫹XYAKRmice results from the lack of, or this study, ovarian tissue develops if the dominant reduction in,Srytranscription. Results indicated that brachyury mutation T-hairpin tail (Thp) or T-Orleans Sryis transcribed but at a significantly reduced level. (TOrl) is present, the M. domesticus Y chromosome is Inferred from this finding is that

Tasaffects the ex-from the AKR/J inbred strain, and the remainder of pression level ofSry.

the genome is of B6 origin (Washburn and Eicher 3. We tested whether the AKRSryallele is unique among 1983, 1989; Washburn et al. 1990). [Thp and TOrl are

M. domesticus Sry alleles carried by several other in-partially overlapping deletions (Moutier1973a,b;Ben- bred strains. We found that the Y chromosome (i.e., nett et al. 1975; Erickson et al. 1978).] Although Sryallele) of strains LEWES/Ei, MA/MyJ, PL/J, RF/J, B6-YPOSand B6-TAS sex reversals share the requirement

and WSB/Ei causes sex reversal whereas the Y chro-for homozygosity chro-for B6-derived genes and the presence mosome of strains BUB/BnJ, SJL/J, ST/bJ, or SWR/J of anSryallele ofM. domesticusorigin, they differ in that allows normal testis development.

B6-TAS requires the presence of Thp or TOrl. We have

4. We sequenced the Sry open reading frame (ORF) hypothesized that a gene necessary for testicular devel- and 800 bp 5⬘to the ORF from each of the Y chromo-opment, designatedT-associatedsex reversal (Tas), re- somes noted above. All were identical except for the sides in the deleted region common to Thp and TOrl

number of glutamine residues in glutamine repeat

(WashburnandEicher1989). cluster 3 and a 10-bp deletion in the 5⬘region. On

Here we present results from experiments designed the basis of the differences found, these Y chromo-to identify the Y chromosome component of B6-TAS: somes sort into the same two groups that caused sex reversal or normal testis development. These find-ings are discussed in terms of the evolutionary rela-tionship of the two groups ofM. domesticusY

chromo-Corresponding author:Eva M. Eicher, The Jackson Laboratory, 600

Main St., Bar Harbor, ME 04609. E-mail: eme@jax.org somes identified.

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MATERIALS AND METHODS chromosome and the Tg were determined by Southern blot analysis or a multiplex PCR protocol. For Southern blot analy-B6TOrlcongenic and Y chromosome consomic strains:The

sis, genomic DNA was digested withTaqI and restriction frag-TOrlmutation and theM. domesticus Y chromosomes carried

ments were separated and blotted using standard methods. by standard inbred strains AKR/J, BUB/BnJ, MA/MyJ, RF/J, Filters were probed with a 380-bp NotI/PstI insert isolated SJL/J, ST/bJ, and SWR/J and two wild-derived strains, from clone p422.04 (Gubbay et al. 1990) and labeled with LEWES/Ei and WSB/Ei, each were transferred onto the B6 32P. A 4.7-kbTaqI fragment identified theSrytransgene and strain (subline, C57BL/6JEi) by standard backcross methods a 2.1-kb TaqI fragment identified the AKR Sry allele. The for a minimum of 10 generations. TheTOrlcongenic B6 strain

multiplex PCR protocol is given inCapelet al.(1999). is designated B6.TOrland the Y chromosome consomic B6

IdentifyingTOrlcarriers:TOrl/fetuses 12.5 days post-coitum strains are designated B6.YAKR, B6.YBUB, etc. We also used a

(dpc) and older were distinguished from their⫹/⫹sibs by a C57BL/6By consomic strain developed by Donald Bailey (The shortened tail. For younger fetuses, tail length is not a reliable Jackson Laboratory) that contained the PL/J Y chromosome method to distinguish the two genotypes so a PCR method was (also ofM. domesticusorigin). used that amplifiesD17Tu1, producing an230-bp product if Srytransgenic lines:Transgenic mice were produced using TOrlis absent and a slightly larger product if TOrlis present a 14.6-kb genomic DNA segment that contains the Y-linked (Himmelbauerand Silver1993). Fetal tissue was digested Srygene derived from a 129 inbred strain (Gubbayet al.1990) in 200␮l of PCR buffer supplemented with nonionic deter-and is capable of completely sex reversing XX mice (i.e., causes gents and Proteinase K and 2␮l of this mixture was used as testicular development) when present as a transgene (Koop- PCR template. Primers were (5⬘-GGGGAACAGTAATAAAG

man et al.1991; Eicheret al.1995). DNA was injected into CTGA-3⬘) and (5⬘-TCTGCTTCATCTGAGGGTCCA-3⬘) and the male pronucleus of fertilized eggs obtained from mating PCR conditions were 40 cycles of 94⬚, 30 sec; 55⬚, 30 sec; and B6 females to B6 XYAKR males using standard procedures 72, 30 sec1 sec/cycle. PCR reactions were analyzed on 5%

(Wagneret al.1981). Four of 23 offspring recovered carried NuSieve 3:1 gels.

the transgene. One was an XX male, thus sex reversed by the RNA extraction and RT-PCR: Paired mesonephros/go-transgene but sterile due to the presence of two X chromo- nadal ridges were dissected from individual fetuses at 10.5 to somes and absence of a Y chromosome. The remaining three 12.5 dpc and immediately lysed in Buffer RLT (QIAGEN, were XY males, each of which contained a transgene proven Valencia, CA; RNeasy total RNA miniprep kit) and stored at⫺80⬚. Total RNA was purified and DNased using RNeasy functional by the fact that all XX transgenic offspring were

completely sex-reversed males. A transgenic line was estab- miniprep columns and eluted in 30␮l H2O. Alternatively, RNA was DNased after purification using the DNA-free protocol lished from each founder, hereafter designated C57BL/

6JEi-YAKRTgN(Sry-129)1Ei; . . . 2Ei; and . . . 3Ei. The transgene (Ambion, Austin, TX).

Ten microliters of denatured RNA was used for first-strand carried by each line appears genetically stable because no

transgenic females or hermaphrodites (having both ovarian cDNA synthesis in a 20-␮l reaction using MuLV reverse tran-scriptase (RT) and oligo(dT) primers incubated at 42⬚ for and testicular tissue) have been observed since these lines

were established in 1992. We used line Tg1Ei for the work 1 hr. For each RNA template, a control reaction without RT was included. For each RT reaction condition, an H2O no-presented here and refer to it hereafter as Tg.

Gonad development in B6TOrl/⫹Tg mice:Two groups of template reaction was included as an additional negative con-trol. Four microliters (Sry) or 1␮l (Hprt) of the reverse tran-TOrl/Tg offspring were analyzed for gonad development.

In both cases, mice were produced by mating B6TOrl/fe- scription reaction served as template for subsequent PCR. PCR amplification ofSryused primers Y34A (5⬘-CTGGCACTACTG males to B6 XYAKRTg males. The first group consisted of 80

weanling mice analyzed at 3 to 4 weeks of age. Each mouse was GACTTCTAAG-3⬘) and cs-1B (5⬘-(T)18GGGATGG-3⬘), which are RNA specific, and the method ofJeskeet al.(1995) except phenotypically sexed by inspection of external and internal

genitalia, classified for normal (⫹/⫹) or shortened tail length that Applied Biosystems (Foster City, CA) buffer II and 1.5 mmMgCl2were substituted. PCR amplification ofHprtused (TOrl/), and genotyped for the presence of the Y

chromo-some and Tg (see below). The second group consisted of 21 primers (5⬘-CCTGCTGGATTACATTAAAGCACTG-3⬘) and (5⬘-GTCAAGGGCATATCCAACAACAAAC-3⬘;Koopmanet al. fetuses analyzed at 14.5 to 15 days of fetal development. This

time of gonadal development was chosen because a small 1989), Applied Biosystems buffer II, 1.5 mmMgCl2, 200 ␮m each nucleotide, 0.2␮meach primer, and 1 unitTaq(Applied amount of ovarian tissue is easily detected in an ovotestis

(Eicher et al. 1980). (Later in development, ovarian tissue Biosystems). Thermal cycling conditions were 40 cycles of 94⬚,

30 sec; 59⬚, 30 sec; and 72⬚, 30 sec. A control reaction without can be compressed by the growth of testicular tissue and thus

be difficult to detect.) Each fetus was classified for gonad cDNA template was included for each PCR reaction condition. Ten microliters of each PCR reaction was analyzed on a 4% development (ovary, testis, or ovotestis), tail length, and the

presence of the Y chromosome and Tg. NuSieve 3:1 gel. Although this RT-PCR assay is not quantita-tive, it is specific for the presence of Sry RNA. One of the Ability ofM. domesticus Y chromosomes to direct normal

testis development in TOrl/⫹ XY mice: The M. domesticus- primers overlaps and depends on the presence of a poly(A) tail at the minor polyadenylation site (Jeskeet al.1995). An derived Y chromosome carried by each B6.Y consomic strain

was tested to determine if B6TOrl/XY individuals developed SryPCR product is not detected on an agarose gel if 200l of genomic DNA is used as template (data not shown). RNA as females (as when an AKR Y chromosome is present) or as

males (as when aM. musculus B6 or C3H Y chromosome is from a B6TOrl/XYAKRfetus and a/XYAKRsib were ana-lyzed for the presence of anSrytranscript at 10.5 dpc (11- to present). Males from each consomic line were mated to B6

TOrl/females.TOrl/offspring were classified at weaning as 12-tail somite stage), 11.5 dpc (16- to 17-tail somite stage), and 12 dpc (23-tail somite stage).

female, male, or hermaphrodite by the appearance of external

and internal genitalia. Non-TOrl (/) offspring served as Sryexpression levels were compared to the expression levels of LIM homeobox protein 1 (Lhx1) using a semiquantitative controls. Presence of a Y chromosome was determined by

analysis of G-banded mitotic chromosome preparations from RT-PCR assay. Lhx1 is expressed only in the mesonephric component of the urogenital ridge (Barneset al.1994). Lhx1-bone marrow (EicherandWashburn1978) or a PCR assay

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139-bp fragment using primers Lhx1-1660 (5⬘-GGCGAGGAG Development of testicular tissue would indicate thatSry CTCTACATCATAG-3⬘) and Lhx1-1798 (5⬘-CTTGGGAATCC is the sole Y-linked component. Development of ovarian GGAGATAAAC-3⬘). These primers were combined with

Sry-tissue would eliminateSry as the Y-linked component. 9431 (5⬘-TGGTGAGCATACACCATACC-3⬘) and Sry-9808

The data from these experiments are presented in Table (5⬘-TTGCTGTCTTTGTGCTAGCC-3⬘) in a multiplex PCR

re-action containing [␣-32P]dCTP and 2l of the reverse tran- 1. At both developmental stages,TOrl/⫹XYAKRmice pre-scription reaction as outlined above. (Sryprimers are desig- sented as normal females with ovaries in the absence nated by the 5⬘ base using numbering in GenBank entry of the Tg and as normal males with testes in the presence X67204 where positions 8304 and 9491 represent the

begin-of the Tg. These findings provide strong evidence that ning and end, respectively, of theM. musculusORF.) Thermal

Sryis the Y-linked gene responsible for ovarian tissue cycling conditions were 29 cycles of 94⬚, 30 sec; 57⬚, 30 sec;

and 72⬚, 30 sec. The PCR reaction was digested withNlaIV, development in B6TOrl/⫹XYAKRmice.

separated on 3% agarose gels, and Southern blotted using Testing otherSryalleles:To determine if the develop-standard methods. The amount of radioactivity in each band ment of ovarian tissue was unique to theSryallele carried was determined using Phosphor imaging plates and Image

on the AKR Y chromosome, we mated B6 mice carrying Gauge software (Fuji Medical Systems, Stamford, CT). Primers

one of nine otherM. domesticus-derived Y chromosomes Sry-9431 and Sry-9808 amplify a 377-bp DNA fragment from

to B6TOrl/⫹ females and analyzed theTOrl/⫹ XY off-theSrygene. When this PCR product is digested withNlaIV,

a single 377-bp undigested fragment is diagnostic forM. muscu- spring. We found that the Y chromosomes fell into two lusalleles and two comigrating fragments (189 and 188 bp) groups based on gonadal development (Table 2). Group are diagnostic forM. domesticusalleles.

A was like AKR and caused sex reversal whereas group Sry sequence analysis: The Sry ORF and portions of the

B was like B6 or C3H and resulted in normal male 5⬘- and 3⬘-untranslated regions (UTRs) were analyzed by direct

development. sequencing of PCR products. A total of 100–150 ng of genomic

DNA served as template for primers Sry-8212 (5⬘-TTGATTTT Sequence analysis within and 5ⴕto theSryORF:We TAGTGTTCAGCCCTACAGCC-3⬘) and Sry-9791 (5⬘-AGCTG sequenced the ORF of theM. domesticus Srygene carried TTTGCTGTCTTTGTGCTAGCC-3⬘) in a 100-␮l reaction. PCR on the Y chromosomes used in the experiments noted was performed by conventional techniques using Taq DNA

above and compared the predicted amino acid se-polymerase (Applied Biosystems) and 1.5 mm MgCl2

em-quences to each other and to that published for the ploying 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 Sry129allele (M. musculus, GenBank accession no. X67204, 1% agarose gel (the expected PCR product size isⵑ1.6 kb) which was theSryallele used in the transgenic experi-and the remainder purified for sequencing using either Wiz- ments). The predicted amino acid sequence of these ard PCR preps (Promega, Madison, WI) or QIAquick spin

10 alleles was identical except for a polymorphism in columns (QIAGEN). A total of 45 ng of purifiedSryORF PCR

glutamine repeat cluster 3 (GRC-3; Figure 1). Group A product was directly sequenced using primers 8212,

Sry-Sry alleles contained 13 glutamines in this cluster, 9791, Sry-8653 (5⬘

-GGAGTAGAGCTGCACACCTGTACTCC-3⬘), and Sry-9475 (5⬘-CCAGTGTCATGAGACTGCCAACC-3⬘) whereas group B Sry alleles contained 12 glutamines. and the PRISM Ready Reaction DyeDeoxy terminator cycle Compared to the Sry129allele, the M. domesticus alleles sequencing kit (manufacturer’s recommendations, Applied

harbor nine additional shared changes. The most strik-Biosystems) on an ABI 373 Stretch automated sequencer.

ing is the one first reported byCowardet al. (1994), The 5⬘UTR and proximal promoter region was PCR

ampli-which involves a stop codon in GRC-8 and results in a fied and sequenced using a strategy similar to that given above

and employing primers Sry-7436 (5⬘-CAGAAATGAACTACTG greatly shortenedM. domesticusSRY protein compared CATCCC) and Sry-8371 (5⬘-AACTTGTGCCTCTCACCACG). to the SRY protein coded for byM. musculusalleles (see

DNA sequence analysis and alignment were performed us- Figure 1). ing the GeneWorks computer program (IntelliGenetics, ver.

The DNA sequence immediately 5⬘of the ORF also 2.4) with subsequent hand editing. The primer strategy used

was determined for the 10 M. domesticus alleles. This allowed for double-strand coverage and multiple-pass

se-quence for most regions. Any ambiguities detected in the ⵑ800-bp region contains all of the 5⬘UTR and part of single-strand/single-pass regions were resolved or confirmed the proximal promoter. All alleles were identical except by resequencing. Genomic DNA from at least two individual for a 10-bp deletion in a single polymorphic region males from each Y chromosome consomic strain served as

(GravesandErickson1995). The absence or presence PCR template and the reactions were combined prior to

puri-of the deletion also defines groups A and B, respectively. fication and sequencing to control for any individual variation.

This 10-bp repeat does not contain a recognized tran-No sequence heterozygosity was detected in any of the

tem-plates analyzed. The nucleotide sequences for the 10Sryalleles scription factor binding site [TRANSFAC v3.3, Tran-listed in Table 2 are deposited in GenBank under accession scription Element Search Software (TESS); http// nos. U70642, AF009519, and AF337043–AF337050.

www.cbil.upenn.edu/tess]. Compared to M. musculus

(Sry129), this800-bp region contains three polymor-phisms: G-T at position 7656, G-A at 7768, and A-G at RESULTS

7928. The polymorphisms at 7656 and 7928 are located

Testing theSrytransgene:We analyzed gonadal devel- in transcription factor binding sites identified by TESS opment in B6TOrl/⫹ XYAKRmice carrying a functional (CREB and OCT, respectively). However, these sites are

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TABLE 1

Results from matingTOrl/⫹females to B6.YAKRTg males

Tg1Ei Sex

chromosomes Genotype Present Absent

Weanling data XX ⫹/⫹ 12 males 21 females

TOrl/7 males 8 females

XY ⫹/⫹ 10 males 12 males

TOrl/6 males 4 females

Fetal data XX ⫹/⫹ 2 males 1 female

TOrl/ 2 males 1 female

XY ⫹/⫹ 4 males 1 male

TOrl/4 males 6 females

B6 TOrl/mice have an increased probability of developing extreme spina bifida and die perinatally, accounting for the less-than-expected 1:1 ratio of normal and short-tailed mice.

by all 10M. domesticusalleles, thus eliminating them as at a reduced level inTOrl/⫹XYAKRgonads, a semiquanti-tative radioactive RT-PCR assay was used. To accomplish causative elements in B6-TAS.

Sry expression in B6TOrl/XYAKRfetal gonads: The this, we determined the level of Sry transcript in the

genital ridge and compared this to the level of Lhx1

fact that B6TOrl/⫹XYAKRmice develop bilateral ovaries

suggests that the male sex determination pathway fails transcript in the mesonephros (Figure 2A). Normally,

Sry transcription begins at ⵑ10.5 dpc (ⵑ8-tail somite early. To determine if this failure involved activation of

Sry, we used a qualitative RT-PCR assay to analyze Sry stage), peaks at 11.5 dpc (ⵑ18-tail somite stage), and is absent by 13.0 dpc. Our results indicate thatSry expres-expression in paired urogenital ridges isolated from

10.5- to 13.5-dpc B6 TOrl/⫹ and ⫹/⫹ XYAKR mice. Sry sion is significantly delayed in B6TOrl/⫹XYAKRgonads compared to B6 ⫹/⫹ XYAKRgonads (Figure 2B). For expression was detected in both genotypes at all time

points sampled (data not shown). Because this result example, the Sry/Lhx1 ratio was 0.04 at the 15- to 16-tail somite stage inTOrl/⫹fetuses, whereas the ratio was did not eliminate the possibility that Srywas expressed

0.63 in same aged⫹/⫹fetuses. In fact, of the six pairs ofTOrl/⫹urogenital ridges analyzed from fetuses at the TABLE 2 12- to 16-tail somite stage of development, only one pair had detectableSryexpression. To determine ifSrywas TOrl/⫹offspring from B6TOrl/⫹females mated to B6 males

present but below the level of detection of the semiquan-carrying aMus domesticusY chromosome

titative assay, we increased the number of PCR cycles. When 35 PCR cycles were used, Sry was detectable in TOrl/offspring

theTOrl/⫹samples using ethidium bromide-stained gels

Female Male

(Figure 2C).

Groupa Males XX XY XY

A B6.YAKR 8 4 0

DISCUSSION

B6.YLEWES 12 8 0

Gonad development in B6TOrl

/XYAKR

mice is

cor-B6.YMA 11 5 0

B6.YPL 2 7 0 rected by anSrytransgene:Previously, we reported that

B6.YRF 2 4 0

ovarian tissue development in C57BL/6J-T-associated

B6.YWSB 9 5 0

sex reversal (B6-TAS) depends upon the simultaneous inheritance of three genetic components: a B6 inbred

B B6.YBUB 5 0 7

B6.YSJL 10 0 4 strain background, the presence of the brachyury

dele-B6.YST 11 0 15

tion allelesThporTOrl, and aM. domesticusY chromosome

B6.YSWR 11 0 5

derived from the AKR/J inbred strain (Washburnand Eicher1983, 1989;Washburnet al.1990). These three aGroup A males contain anSryallele with 13 CAG repeats

in the GRC-3 region. Group B males contain anSryallele with conditions are absolute requirements. For example, if 12 CAG repeats in the GRC-3 region and a 10-bp deletion in a C3H/HeSnJ or (B6C3H)F1genome is substituted the 5⬘ UTR region. Note thatCowardet al. (1994) also re- for the B6 genome in Thp/⫹ mice (Washburn et al. ported the number of CAG repeats in cluster 3 for strains

1990), or if certain otherT-deletion mutations are used AKR, BUB, MA, PL, RF, SJL, ST, and SWR. In addition,Graves

(E. M.Eicherand L. L.Washburn, unpublished data),

andErickson(1995) also reported the presence/absence of

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muscu-Figure1.—Comparison among predicted SRY proteins fromM. musculus(129 inbred strain) andM. domesticusgroup 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 BSryalleles 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 theM. domesticusstop 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 byCowardet al. (1994),

andAlbrechtandEicher(1997) reported partial DNA sequence for the open reading frame for strains AKR, BUB, and WSB.

lusorigin) is substituted for the AKR-derived Y chromo- provide strong evidence that the Sry gene present on the AKR Y chromosome is functionally deficient in the some inTOrl/⫹mice, normal testicular tissue is formed

(Washburnet al.1990). B6TOrl/⫹genetic environment and cannot initiate

nor-mal testis differentiation. Further support for this con-The experiments reported here were designed to

pro-vide insight into the Y-linked aspects of B6-TAS. We clusion was obtained from experiments using a set of B6 Y chromosome consomic strains together with DNA used a transgenic approach (i.e., genetic

complementa-tion) to determine if the Y-linked gene was Sry. We sequence data from the Sry allele carried by these Y chromosomes.

found that by adding a functionalM. musculus-derived

Sry transgene to the genome, B6 TOrl/⫹ XYAKR mice An earlier study by Palmer and Burgoyne (1991) provided evidence that the timing of testis differentia-develop normal testes. Because the genomic DNA used

as a transgene encodes only theSrygene, these results tion was dependent upon the specific Y chromosome

Figure 2.—Sry expression is

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present. They reported that testicular differentiation Nagamine et al. (1999) that the level ofSry transcript is reduced in B6 mice carrying an AKR Y chromosome began as much as 14 hr later in XY mice carrying an

AKR Y chromosome than in XY mice carrying a B6 Y compared to those carrying an FVB Y chromosome. Our data indicate thatTasis involved in controllingSry

chromosome. This difference in timing of gonad

differ-entiation must be specific to the Y chromosome because transcript levels, given that the Sry transcript level is further reduced in B6 YAKRfetal gonads when TOrl is the rest of the genetic background was uniform. Our

finding that the AKR/JSrygene is the Y-linked compo- present. We suggest that the level of Sryexpression is so severely reduced in B6TOrl/⫹XYAKRgonads that the nent responsible for ovarian tissue development in

B6-TAS implies that the Palmer and Burgoyne finding was next steps in the testicular pathway are not initiated, allowing the ovary-determining pathway to proceed and due to the different Sry alleles they tested. Together,

these observations suggest that under genetically spe- ovarian tissue to develop. Further support for this sug-gestion requires cloning of theTasgene.

cific circumstances, the AKRSrygene is inappropriately

controlled. Cause of ovarian tissue development in B6 TOrl

/XYAKRmice: Two other observations correlate with the

Evolution ofM. domesticus Sryalleles studies:Tucker

et al.(1992) reported that theM. domesticusY chromo- finding that B6 XYAKR mice have a reduced level ofSry transcription (Nagamine et al. 1999). First, migration somes of standard inbred mouse strains fall into two

major groups based on an analysis of Southern blots of mesonephric cells into the undifferentiated gonads in B6 XYAKR mice is delayed (Albrecht et al. 2000). containing EcoRI restriction fragments probed with

YB10, a highly repetitive sequence present on the long Previous work indicates that migration of mesonephric cells is dependent on the presence of a functionalSry

arm of the Y chromosome (Eicheret al.1989). In the

present work we tested if theSrygene carried by these gene and is required for the formation of testicular cords (Martineauet al. 1997;Capel et al.1999; Til-inbred strains separated into the same two groups based

on gonadal development. We found that theSryalleles mann and Capel 1999). Second, B6 XYAKR mice have delayed testis cord formation (WashburnandEicher carried by group A mice (see Table 2) caused sex

rever-sal whereas theSryalleles carried by group B mice did 1983, 1989; Nagamine et al. 1987; Washburn et al.

1990). This delay is apparent at the cranial and caudal not and that these groups were the same groups

identi-fied byTuckeret al.(1992). ends of the gonads at 14 dpc but absent 12 to 24 hr later when cords have formed at the polar regions. De-To explore these findings further, DNA sequence

analysis was performed on theSryalleles carried by the layed cord formation is not observed in B6 mice carrying the M. domesticus-derived FVB Y chromosome ( Naga-inbred strains representing group A and group B. Again

we found concordance between ovarian vs. testicular mineet al.1999) or the BUB/BnJ Y chromosome (L. L. Washburn and E. M. Eicher, unpublished data). In development and the DNA sequence of the Sry gene.

In addition, this data further supports Tucker and co- addition, as shown in the present study, the BUB/BnJ Y chromosome initiates normal testis development in workers, who concluded that the laboratory strains

AKR/J, PL/J, MA/MyJ, and RF/J are likely related to B6TOrl/⫹mice. (The case for the FVB Y chromosome has not been tested.) Because the FVB Sry gene has descendants of a population ofM. domesticusmice that

colonized the mid-Atlantic seaboard (i.e., the ancestors 12 CAG repeats in GRC-3 (Nagamine et al. 1999 and reported here), it probably belongs to group B, which of LEWES/Ei and WSB/Ei were caught in Delaware

and Maryland, respectively). includes BUB/BnJ. We suggest that the level ofSry tran-scripts in B6 mice carrying a group B Y chromosome is Although the functional differences between groups

A and B correlate with specificSrysequence similarities higher than the level present in B6 mice carrying a group A Y chromosome.

in the ORF and 5⬘to the ORF, we do not contend that

these sequence differences cause ovarian development We hypothesize that reduction inSrytranscript levels in B6 XYAKRfetal gonads causes a delay in mesonephric in B6TOrl/⫹ mice carrying a group A Y chromosome.

Further comparative analyses ofSry alleles from these cell migration into the gonad and this, in turn, causes a delay in cord formation. If this is correct, there are two groups of Y chromosomes may identify the

func-tionally important controlling sequences. The proof of two possible causes of the failure of B6 TOrl/⫹ XYAKR mice to form testicular cords during gonad differentia-speculation rests with engineering presumptive

caus-ative sequences from a group A Sryallele into a group tion: (1) The number ofSry-expressing cells is normal but the level of Sry transcription per cell is severely B orM. musculus Sryallele (or vice versa).

Expression ofSryin B6TOrl/XYAKRmice:To investi- reduced or (2) the level of Srytranscription is normal

perSry-expressing cell but the number ofSry-expressing gate the improper functioning of the AKR/JSrygene,

we determined the timing and level of Sry transcripts cells per gonad is significantly reduced. Either of these possibilities could severely affect the signaling pathway in B6TOrl/⫹XYAKRfetal gonads. We found thatSry

tran-scription is initiated at the correct time, but the level that is required for mesonephric cell migration into the developing gonad. The outcome is that few if any of transcription is severely reduced compared to

(7)

in C57BL/6J-YPOSmice corrected by aSrytransgene. Philos. Trans. fail to form, and the ovarian pathway begins. This

expla-R Soc. Lond. B Biol. Sci.350:263–269.

nation is similar to what has been proposed for the Eicher, E. M., L. L.Washburn, N. J.Schork, B. K.Lee, E. P.Shown

et al., 1996 Sex-determining genes on mouse autosomes

identi-cause of B6-YPOSsex reversal (Albrechtet al.2000).

fied by linkage analysis of C57BL/6J-YPOSsex reversal. Nat. Genet.

We thank Dr. Peter Hoppe for his expertise in makingSrytransgenic 14:206–209.

animals on the C57BL/6J inbred strain, Douglas McMinimy for his Erickson, R. P., S. E.Lewisand K. S.Slusser, 1978 Deletion map-ping of the t complex of chromosome 17 of the mouse. Nature sequencing skills, and Michelle Higgins and Leona Gagnon for

help-274:163–164. ing to maintain the B6 Y chromosome consomic strains. We also thank

Graves, P. E., and R. P.Erickson, 1995 Sequence variation in the Tim O’Brien, Beverly Richards-Smith, and John Schimenti for helpful

5⬘, putative promoter ofSry and its possible relevance to the suggestions concerning this report. This work was funded by National C57BL/6J-YDOMsex reversal. Biochem. Biophys. Res. Commun.

Institutes of Health research grant GM-20919 (to E.M.E.) and the 208:624–628.

National Cancer Institute cancer core grant CA34196 (to The Jackson Gubbay, J., J.Collignon, P.Koopman, B.Capel, A.Economouet al., 1990 A gene mapping to the sex-determining region of Laboratory).

the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature346:245–250.

Himmelbauer, H., and L. M.Silver, 1993 High-resolution compar-ative mapping of mouse chromosome 17. Genomics17:110–120.

Jeske, Y. W., J.Bowles, A.Greenfieldand P.Koopman, 1995

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Figure

TABLE 1

References

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