TESTIS DEVELOPMENT IN THE XY SEX REVERSED MOUSE

长期目标是了解人类胎儿性腺 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， 性腺分化成卵巢，形成雌性表型。 的 这个项目的健康相关性是，在某些情况下，Y失败 诱导睾丸产生具有XY核型的表型雌性 (XY性逆转）。 了解参与的生物学机制 睾丸发育将导致更好地了解病因 和XY女性的治疗。 这项提案的目的是阐明生物学机制 通过利用小鼠模型参与正常睾丸发育 称为XYd性逆转的睾丸发育异常。 在XYD性爱 反转，M. m. Yd，d= Atomicus）的Y染色体在它们的 在实验室菌株C57 BL/6中诱导睾丸的能力。 在极端 例，XY女性出生。 为什么一个Yd染色体诱导正常睾丸 而另一种诱导XY卵巢或卵睾丸的方法尚不清楚。 XYd性爱 反转模型将用于检验工作假设，即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性逆转株中的等位基因导致异常Sry proteins. 这将通过对来自以下的Sry cDNA进行测序来完成： XYd性逆转菌株并分析其编码的蛋白质， 可能影响Sry的睾丸决定功能的突变。 的 一种假说是，Sry基因突变产生的蛋白质 在睾丸测定中不理想。2)鉴定Sry替代mRNA 在XYd性逆转菌株中。 这将通过RACE来实现。 的 假设Xyd性逆转株利用替代mRNA 其特性导致睾丸发育异常。第三章 表征XYd性逆转菌株中Sry的表达。 这 将使用原位杂交、RT-PCR和 免疫组化 假设是， 在XYd性逆转株中Sry表达异常。4)量化 Sry在XYd性逆转株中的表达。 这将是 使用RNase保护测定和cRNA内标完成 定量RT-PCR分析。 假设XY性别中的Sry 逆转菌株的表达水平相对于 正常睾丸测定。 5)识别和表征基因 上游和下游的Sry使用XYd性逆转模型。 这 将按照Sry的概述完成。

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.