Effects of sex chromosomes on the developmental potential of the mouse oocyte

我的长期目标是描述性染色体如何影响小鼠卵母细胞的发育潜力。在哺乳动物发育过程中，生殖细胞根据其性腺环境，睾丸或卵巢进行性分化，这是由y连锁Sry基因的存在与否决定的。因此，精子发生和卵子发生分别发生在XY和XX染色体存在的情况下。性腺性别也通过性类固醇激素的分泌来决定表现型性别。然而，当性腺性别反转时，生殖细胞性别与染色体性别就不一致了。人类的XX雄性和XY雌性都是不育的，而在某些啮齿类动物中，XY雌性是可生育的。在小家鼠中，XY型雌鼠的生育能力取决于性别逆转的原因和遗传背景。B6。YTIR小鼠发育成具有完整Y染色体的不育雌性小鼠，为研究性染色体对雌性生育能力的影响提供了良好的模型。我们以前报道过不孕的原因在B6。YTIR雌性是其卵母细胞固有的；它们可以进入中期II (MII)，但不能完成第二次减数分裂，受精后很少发育超过合子期。这种发育缺陷可归因于它们有缺陷的卵浆；当XY卵母细胞的细胞核转移到去核的XX卵母细胞后，重建的卵母细胞产生健康的后代。因此，卵母细胞中的Y染色体使卵浆有缺陷，这使得第一次减数分裂基本完整，但破坏了第二次减数分裂。在接下来的5年中，我们将研究XY卵母细胞第二次减数分裂缺陷的分子机制，并研究y连锁基因的参与。(1)为了表征卵浆缺陷的性质，无论是关键成分的缺乏还是有害成分的存在，我们将一小部分细胞质从XX卵母细胞转移到XY卵母细胞中，然后在体外成熟，并检查孤雌激活（或受精）后的第二次减数分裂。(2)为了阐明第二次减数分裂失败的分子机制，我们将比较XX和XY卵母细胞激活前后ARP2/3通路的活性。ARP2/3通路对维持细胞质分裂沟附近的mii纺锤体至关重要。(3)为了评估y编码转录因子ZFY2在卵浆缺陷中的作用，我们将通过体外sirna表达或体内转基因shRNAs表达来敲除其在XY卵母细胞中的转录本。本研究的结果将有助于揭示小家鼠XY雌性姐妹染色单体分离和第二极体挤压的协调机制，以及Y染色体的进化与生育能力丧失的关系。

My long-term objective is to delineate how sex chromosomes affect the developmental potential of mouse oocytes. In mammalian development, germ cells undergo sexual differentiation according to their gonadal environment, testis or ovary, which is determined by the presence or absence of the Y-linked Sry gene. Therefore, spermatogenesis and oogenesis take place in the presence of XY and XX chromosomes, respectively. The gonadal sex also dictates the phenotypic sex by secretion of sex steroid hormones. When the gonadal sex is reversed, however, the germ cell sex becomes discordant with the chromosomal sex. Both XX males and XY females in humans are infertile, while XY females are fertile in certain rodent species. In Mus musculus, XY females are variably fertile depending on the cause of sex-reversal and genetic background. The B6.YTIR mouse, which develops into an infertile female with an intact Y chromosome, provides an excellent model for studying the effects of sex chromosomes on female fertility. We previously reported that the cause of infertility in the B6.YTIR female is intrinsic to its oocytes; they can reach the Metaphase II (MII) but fail to complete the second meiotic division and rarely develop beyond the zytote-stage after fertilization. This developmental incompetence can be attributed to their defective ooplasm; when XY oocyte nuclei have been transferred into enucleated XX oocytes, the reconstructed oocytes generate healthy offspring. Thus, the Y chromosome in the oocyte makes the ooplasm defective, which leaves the first meiotic division largely intact but disrupts the second meiotic division. In the next 5 years, we will investigate the molecular mechanisms underlying the second meiotic defects in the XY oocyte and investigate the involvement of Y-linked gene(s). (1) To characterize the nature of ooplasmic defects, whether it lies in a deficiency in critical components or the presence of detrimental components, we will transfer a small volume of cytoplasm from an XX oocyte into an XY oocyte or vice versa, followed by maturation in vitro, and examine the second meiotic division after parthenogenic activation (or fertilization). (2) To clarify the molecular mechanism underlying the failure in the second meiotic cytokinesis, we will compare the activity of ARP2/3 pathways, which are essential for maintaining the MII-spindle near the cytokinesis furrow, in XX and XY oocytes before and after activation. (3) To evaluate the role of Y-encoded transcription factor ZFY2 in the ooplasmic defects, we will knock-down its transcripts in the XY oocyte by siRNAs expression in vitro or by shRNAs expression from a transgene in vivo. The results of proposed studies will shed light on the mechanism coordinating the sister-chromatid separation and the second polar body extrusion, and evolution of the Y chromosome associated with the loss of fertility in Mus musculus XY females.