The role of WEE-1.3 in oocyte maturation

为了探索染色体如何为减数分裂做准备，我们正在研究C。Myt 1直系同源物。 Myt 1属于Wee 1激酶家族，被认为在细胞周期中下调Cdk 1。 用Myt 1直系同源物wee-1.3进行的RNAi研究导致不育。注射dsRNA的母亲很快变得不育;卵母细胞染色体在减数分裂I的终变期不再暂停。这些染色体具有许多有丝分裂的特征，它们被染色成 有丝分裂标记抗体的数量。 卵母细胞的成熟似乎也是早熟的。 我们认为WEE-1.3在卵子发生过程中通常起到保持母体CDK-1不活跃的作用，而在受精后，CDK-1被激活以允许胚胎的减数分裂和有丝分裂。在缺乏WEE-1.3的情况下，CDK-1变得早熟活跃并驱动卵母细胞 未成熟卵母细胞中未完全分化的成熟和染色体成熟。 这些卵母细胞不能受精，大概是因为它们没有合成所需的所有适当的卵母细胞/胚胎产物。 进一步发展，或者因为他们过早地触发了他们对多精受精的阻碍。 我们最近构建并表达了编码WEE-1.3：：GFP融合蛋白的转基因。 这些转基因在生殖系和核膜中表达，并将用于鉴定干扰这种表达模式的突变体。 这些GFP转基因还拯救了wee-1.3缺失突变体。 为了确定与wee-1.3相互作用的基因，我们利用OpenBiosystems RNAi文库中的1874个胚胎致死克隆进行了RNAi抑制剂筛选。从这个筛选中，我们确定了149个基因，当与wee-1.3共耗尽时，会导致生育力的恢复。我们已经证实了这些抑制基因的身份，并开始利用RNAi方法和可用的突变体来表征它们抑制wee-1.3 RNAi不育性的机制。 这种方法的优势在于它有可能识别细胞周期和卵母细胞减数分裂成熟的新成分。我们目前正专注于表征两个特定的基因，这两个基因与人类疾病有关，但以前并不知道它们在卵母细胞成熟或生殖系发育过程中起作用。

To explore how chromosomes prepare for the meiotic divisions, we are examining the function of the C. elegans Myt1 ortholog. Myt1 belongs to the Wee1 family of kinases and is thought to down regulate Cdk1 during the cell cycle. RNAi studies with the Myt1 ortholog, wee-1.3, result in infertility. Mothers injected with dsRNA quickly become infertile; the oocyte chromosomes are no longer paused in diakinesis of meiosis I. These chromosomes have many hallmarks of being mitotic; they stain with a number of mitotic marker antibodies. Oocyte maturation also appears to be precocious. We propose that WEE-1.3 normally functions to keep maternal CDK-1 inactive during oogenesis, and that upon fertilization, CDK-1 becomes activated to allow for the meiotic and mitotic divisions of the embryo. In the absence of WEE-1.3, CDK-1 becomes precociously active and drives oocyte maturation and chromosome maturation in immature oocytes that are not fully differentiated. These oocytes fail to be fertilized presumably because they have not synthesized all the proper oocyte/embryo products they need for further development or because they have precociously triggered their block to polyspermy. We have recently constructed and expressed transgenes coding for WEE-1.3::GFP fusion proteins. These transgenes are expressed in the germline and nuclear envelope and will be useful for identifying mutants that perturb this expression pattern. These GFP transgenes also rescue a wee-1.3 deletion mutant. To determine genes that interact with wee-1.3, we performed an RNAi suppressor screen utilizing 1874 embryonic lethal clones from the OpenBiosystems RNAi library. From this screen we identified 149 genes that when co-depleted with wee-1.3 result in a restoration of fertility. We have confirmed the identity of these suppressor genes and begun to characterize the mechanisms through which they suppress the sterility of wee-1.3 RNAi utilizing both RNAi approaches and available mutants. The strength of this approach is its potentiality for identifying novel components of both the cell cycle and oocyte meiotic maturation. We are currently focusing on the characterization of two specific genes that have been implicated in human disease, but that have not been previously known to function during oocyte maturation or germline development.