Use of a Fragile X premutation knock-in mouse to study FXPOI

描述(由申请人提供)： FMR1是X染色体上的一个基因，在其5‘非翻译区含有一个易于扩展的CGGCCG重复序列。有55-200个重复的等位基因被认为是脆性X预突变(PM)等位基因。这些等位基因在女性中的频率从每113人中有1人到每250人中有1人。这些等位基因的携带者有患脆性X原发卵巢功能不全(FXPOI)的风险，高达28%的携带此类等位基因的女性会患上FXPOI。FXPOI占家族性不孕症的11.5%，占特发性不孕症的3.5%。即使没有FXPOI的诊断，所有携带前突变的女性的绝经平均年龄也比她们没有PM的兄弟姐妹早约5岁因此，女性PM携带者不仅生育问题增加，而且患心血管疾病、阿尔茨海默病、骨质疏松症和其他问题的风险也更大，这些问题在更年期女性中出现的频率更高。PM携带者的激素评估显示，FSH升高，抑制素和抗苗勒氏激素(均由颗粒细胞产生)减少。来自包含约130个CGG重复序列的敲入脆性X PM小鼠模型的初步数据表明，存在许多卵巢问题。这些包括未成熟卵泡的早期丢失和黄体的减少。PM小鼠晚期卵泡较小，闭锁率较高。这与颗粒细胞(GC)的数量比正常少有关。此外，PM卵母细胞表现出核形态异常，缝隙连接蛋白水平降低，透明带异常染色，正常细胞质脆性X智力低下蛋白(FMRP)主要位于核内，泛素化蛋白水平较高，通常聚集在核或核周围区域。PM小鼠也有较高的大型囊肿率。因此，我们的小鼠表现出与卵巢功能不全一致的迹象，并有异常的卵巢变化，这可能导致这种卵巢功能障碍。因此，这些小鼠可能提供了一个很好的FXPOI模型。目的1使用移植方法来解决卵巢中的PM是否足以产生FXPOI样和异常的卵巢特征，或者是否需要下丘脑/垂体单位的PM。目的研究卵泡发育相关蛋白FMRP、泛素、缝隙连接的异常以及卵透明带组成的异常，试图揭示卵泡下降的机制(S)。

DESCRIPTION (provided by applicant): FMR1 is a gene on the X chromosome that contains an expansion-prone CGGCCG repeat in its 5' untranslated region. Alleles with 55-200 repeats are considered to be Fragile X premutation (PM) alleles. The frequency of such alleles in women ranges from 1 in 113 to 1 in 250. Carriers of such alleles are at risk for Fragile X Primary Ovarian Insufficiency (FXPOI) that is seen in up to 28% of women who carry such alleles. FXPOI accounts for ~11.5% of familial cases of infertility and 3.5% of idiopathic cases. Even without a diagnosis of FXPOI, the average age at menopause of all women with the premutation is ~5 yrs earlier than their siblings without the PM. Thus, female PM carriers not only have increased fertility problems, but are at greater risk of cardiovascular disease, Alzheimer disease, osteoporosis and other problems that are seen at higher frequency in menopausal women. Hormonal assessment of PM carriers indicates that FSH is elevated, inhibins and anti-Mullerian hormone (both made by granulosa cells) are reduced. Preliminary data from a knock-in Fragile X PM mouse model containing ~130 CGG repeats suggest a number of ovarian problems. These include early losses of immature follicles and reductions in corpora lutea. Advanced follicles were smaller in PM mice and showed a higher rate of atresia. This was associated with a smaller than normal number of granulosa cells (GCs). In addition, PM oocytes showed abnormal nuclear morphology, reduced levels of gap junction proteins, abnormal staining of their zona pellucida, a primarily nuclear location of the normally cytoplasmic fragile X mental retardation protein (FMRP) and high levels of ubiquitinated proteins that often accumulated in the nucleus or perinuclear region. A high incidence of large cysts was also seen in PM mice. Thus our mice exhibit signs consistent with ovarian insufficiency and have unusual ovarian changes that could contribute to this ovarian dysfunction. These mice may therefore provide a good model of FXPOI. Aim 1 uses transplantation approaches to address whether the PM in the ovary alone is sufficient to produce the FXPOI-like and aberrant ovarian features or instead requires the PM in the hypothalamic/pituitary unit. Aim 2 focuses on the abnormal FMRP, ubiquitin, and gap junctions as well as the abnormalities in zona pellucida composition to attempt to uncover the mechanism(s) responsible for the follicle decline.