TESTIS DEVELOPMENT IN THE XY SEX REVERSED MOUSE

长期目标是了解人类胎儿性腺是如何 发育成睾丸。人类生殖生物学的这一关键步骤是 取决于Y染色体的存在。如果没有Y， 性腺分化为卵巢，形成雌性表型。这个 这个项目与健康相关的是，在某些情况下，Y失败 诱导产生具有XY核型的表型雌性的睾丸 (XY性反转)。了解涉及到的生物机制 睾丸发育将有助于更好地了解病因。 以及对XY女性的治疗。 这项提议的目的是阐明生物机制。 通过利用小鼠模型参与正常的睾丸发育 睾丸发育异常称为XYD性反转。在XYD性爱中 反转，M.M.家蝇Y染色体(Yd，d=家蝇)在它们的 实验室菌株C57BL/6在极端情况下诱导睾丸的能力 在病例中，XY雌性出生。为什么一条Yd染色体能诱导正常的睾丸 另一种则诱导XY卵巢或卵巢，目前尚不清楚。XYD性别 逆转模型将被用来检验工作假说Yd 染色体携带睾丸决定基因性别的不同等位基因。 确定区域Y(Sry)。具体目标是1)识别Sry XYd性反转品系导致异常Sry的等位基因 蛋白质。这将通过对Sry cDNA进行测序来实现 XYD性别反转菌株及其编码蛋白的分析 可能影响Sry的睾丸决定功能的突变。这个 假说是Sry基因突变会产生起作用的蛋白质 在睾丸测定中，这是次优的。2)识别Sry替代mRNAs 在XYd中，性别颠倒的菌株。这将使用RACE来完成。这个 假设Xyd性别颠倒菌株利用替代的mRNAs 它们的特性会导致睾丸发育异常。3) 鉴定Sry在XYd性反转菌株中的表达。这 将使用原位杂交、RT-PCR和 免疫组织化学。假设是，时间或水平 在XYd性反转品系中，Sry表达异常。4)量化 Sry在XYd性反转品系中的表达这将是 使用RNase保护分析和CRNA内标完成 对RT-PCR检测进行量化。假设是XYd性爱中的Sry 反转菌株的表达水平低于 睾丸测定正常。5)识别和表征基因 Sry的上游和下游使用XYd性反转模型。这 将按照Sry概述的那样完成。

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.