Function in Germ Cells of c-kit Encoded at the W Locus of Mice

W和S1基因座的基因分别编码酪氨酸激酶受体及其配体。W和S1突变导致原始生殖细胞和后来的生殖细胞以及造血细胞和黑素细胞发育缺陷。Bachvarova博士已经证明，c-kit(编码在W位点处)在卵母细胞的表面表达，从原始卵母细胞的保存阶段到卵母细胞的生长、减数分裂成熟、发育到2-细胞期。本研究的主要目的是阐明c-kit在卵母细胞生长、存活、发育和/或黏附中的作用。卵巢中配体的表达将通过原位杂交、免疫细胞化学和功能分析来检测。这些结果可能有助于阐明是什么功能参与了该过程。她将使用免疫沉淀和自动磷酸化试验来确定卵母细胞上是否存在功能性c-kit。使用体外培养系统，她将测试添加配体是否促进卵母细胞发育的各个方面。将测试在次优条件下培养的卵母细胞中添加KL的效果，以及培养对表达KL转基因或基因突变形式的细胞系的影响。将检测对卵母细胞生长、存活和减数分裂成熟时间的影响。她还将确定添加KL是否可以诱导卵母细胞在体外开始生长。最后，她将确定针对配体或受体的阻断抗体是否可以抑制这些过程。

The genes at the W and S1 loci encode a tyrosine kinase receptor and its ligand, respectively. W and S1 mutations cause defects in development of primordial germ cells and later germ cells, as well as of hematopoietic cells and melanocytes. Dr. Bachvarova has shown that c-kit (encoded at the W locus) is expressed on the surface of oocytes from the stage of stored primordial oocytes through oocyte growth, meiotic maturation, and in development to the 2-cell stage. The principal aim here is to elucidate the role of c-kit in oocyte growth, survival, development and/or adhesion. Expression of ligand in the ovary will be examined by in situ hybridization, immunocytochemistry and functional assays. These results may elucidate what function is involved. She will determine whether functional c-kit is present on oocytes using an immunoprecipitation and autophosphorylation assay. Using in vitro culture systems, she will test whether addition of the ligand promotes aspects of oocyte development. The effect of adding KL to oocytes cultured under suboptimal conditions will be tested, as well as the effect of culture on cell lines expressing transfected or genetically mutated forms of KL. Effects on oocytes growth, survival, and time to undergo meiotic maturation will be examined. She will also determine whether added KL can induce the initiation of oocyte growth in vitro. Finally, she will determine whether blocking antibodies directed against either ligand or receptor can inhibit these processes.