The long-term objective is to understand how the human fetal gonad develops into a testis. This key step in human reproductive biology is dependent on the presence of the Y chromosome. In the absence of a Y, the gonads differentiate into ovaries and a female phenotype forms. The health-relatedness of this project is that in certain cases the Y fails to induce testes resulting in phenotypic females with an XY karyotype (XY sex reversal). Understanding the biological mechanisms involved in testis development will lead to a better understanding of the etiology and treatment of XY females. The goal of this proposal is to elucidate the biological mechanisms involved in normal testis development by capitalizing on a mouse model of abnormal testis development called XYd sex reversal. In XYD sex reversal, M.m. domesticus Y chromosomes (Yd,d=domesticus) vary in their ability to induce testes in the laboratory strain C57BL/6. In extreme cases, XY females are born. Why one Yd chromosome induces normal testes while another induces XY ovaries or ovotestes is not known. The XYd sex reversal model will be used to test the working hypothesis that Yd chromosomes carry different alleles of the testis determining gene Sex- determining region-Y (Sry).
The Specific Aims are 1) Identify Sry alleles in XYd sex reversed strains that result in abnormal Sry proteins. This will be accomplished by sequencing the Sry cDNAs from XYd sex reversed strains and analyzing the encoded proteins for mutations that could affect the testis-determining function of Sry. The hypothesis is that mutations in Sry give rise to proteins that function suboptimally in testis determination. 2) Identify Sry alternative mRNAs in XYd sex reversed strains. This will be accomplished using RACE. The hypothesis is that Xyd sex reversed strains utilize alternative mRNAs whose properties contribute to abnormal testis development. 3) Characterize the expression of Sry in XYd sex reversed strains. This will be accomplished using in situ hybridization, RT-PCR and immunohistochemistry. The hypothesis is that the timing or level of expression of Sry is abnormal in XYd sex reversed strains. 4) Quantify the expression of Sry in XYd sex reversed strains. This will be accomplished using RNase protection assays and a cRNA internal standard to quantify RT-PCR assays. The hypothesis is that Sry in XYd sex reversed strains is expressed at a lower level relative to strains with normal testis determination. 5) Identify and characterize genes upstream and downstream of Sry using the XYd sex reversal model. This will be accomplished as outlined for Sry.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD028976-02
Application #
3330494
Study Section
Reproductive Biology Study Section (REB)
Project Start
1992-04-01
Project End
1996-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Nagamine, C M; Morohashi, K; Carlisle, C et al. (1999) Sex reversal caused by Mus musculus domesticus Y chromosomes linked to variant expression of the testis-determining gene Sry. Dev Biol 216:182-94
Nagamine, C M; Capehart, J; Carlisle, C et al. (1998) Ovotestes in B6-XXSxr sex-reversed mice. Dev Biol 196:24-32
Nagamine, C M; Carlisle, C (1997) Zfy2/1 fusion gene fails to replicate Zfy1 expression pattern in fetal gonads. Genomics 43:397-8
Carlisle, C; Winking, H; Weichenhan, D et al. (1996) Absence of correlation between Sry polymorphisms and XY sex reversal caused by the M. m. domesticus Y chromosome. Genomics 33:32-45
Nagamine, C M; Carlisle, C (1996) The dominant white spotting oncogene allele Kit(W-42J) exacerbates XY(DOM) sex reversal. Development 122:3597-605
Nagamine, C M (1994) The testis-determining gene, SRY, exists in multiple copies in Old World rodents. Genet Res 64:151-9
Nagamine, C M; Shiroishi, T; Miyashita, N et al. (1994) Distribution of the molossinus allele of Sry, the testis-determining gene, in wild mice. Mol Biol Evol 11:864-74