Ovarian failure in LH/hCG receptor knockout animals

LH/hCG 受体敲除动物的卵巢衰竭

基本信息

批准号：
6943637
负责人：
Ch V RAO
金额：
$ 29.77万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-01 至 2008-08-31
项目状态：
已结题

项目摘要

Both IIpo Huhtaniemi's and our group recently succeeded in generating LH receptor knockout mice by gene targeting in embryonic stem cells. Although this gene knockout was not lethal, it rendered animals infertile. While ovaries of wild- type and heterozygous animals contained LH receptors, ovaries of homozygous littermates contained none. Also, while ovaries of wild-type and heterozygous animals were normal in size and contained preovulatory follicles and corpora lutea, the ovaries of homozygous littermates were small and pale with an arrest of follicular growth at the antral stage. Preliminary studies indicated this arrest could, at least partly, be due to a decrease in telomerase levels and a consequent increase in apoptosis. In homozygous animals, LH levels were markedly elevated, FSH levels were moderately elevated, and estradiol and progesterone levels decreased but were not totally suppressed. Knockout animals can be extremely useful in answering a number of unknowns in LH biology. For example, we could learn: 1) whether LH actions are required for the presence of normal numbers of primordial, primary, preantral and antral follicles; 2) whether FSH can induce follicular growth and ovulation in the total absence of LH actions; 3) what role LH signaling plays in ovarian development and function from one week after birth through one year of age; 4) what ovarian actions of LH are mediated by estradiol, progesterone and testosterone; 5) identify and characterize previously unidentified ovarian genes that are regulated by estradiol, progesterone, testosterone, LH or by their combination; and 6) whether using gene therapy to introduce LH receptors into ovaries of null animals makes them cyclic and ovulate but still not get pregnant because they do not have LH receptors in the uterus. These are only a few examples of how the use of null animals could advance our current understanding on the role of LH in different ovarian functions. We propose three specific aims in this application: 1) Investigate structural and functional defects in ovaries of 7-day, 25-day, 60-day and 1-year old null mice to compare with their age- matched, wild-type and heterozygous siblings. 2) Investigate whether estradiol, progesterone and testosterone replacement therapy can correct structural and functional defects in ovaries of LH receptor knockout animals. 3) Determine whether retroviral mediated LH receptor gene transfer can correct structural and functional defects in ovaries of null animals so they become cyclic and ovulate even though pregnancy may not occur due to the absence of LH receptors in the uterus. There are several strengths in this proposal. Foremost is studying LH biology using knockout technology. Second is using steroid hormone replacement and gene therapies. Third is using cDNA expression arrays, a powerful technique in gene expression analysis. All techniques to be used in the proposed studies have already been established to obtain preliminary data presented in the proposal.

IIPO HUHTANIEMI和我们的组最近通过基因靶向胚胎干细胞成功地产生了LH受体敲除小鼠。尽管这种基因敲除不是致命的，但它使动物不育。虽然野生型和杂合动物的卵巢含有LH受体，但纯合同窝仔的卵巢却没有。同样，虽然野生型和杂合动物的卵巢大小正常，并且含有排卵前的卵泡和lutea，但纯合的同窝仔的卵巢很小，很苍白，在粪便阶段捕获了卵泡生长。初步研究表明，至少部分可能是由于端粒酶水平降低和凋亡增加所致。在纯合动物中，LH水平明显升高，FSH水平适度升高，雌二醇和孕酮水平降低，但并未完全抑制。敲除动物在回答LH生物学中的许多未知数方面可能非常有用。例如，我们可以学习：1）是否存在正常数量的原始，原发性，疗程和肛门卵泡的正常数量； 2）FSH在完全没有LH作用的情况下是否可以诱导卵泡生长和排卵； 3）LH信号在出生后一周到一岁从卵巢发育和功能中发挥了什么作用； 4）LH的卵巢作用是由雌二醇，孕酮和睾丸激素介导的； 5）识别和表征由雌二醇，孕酮，睾丸激素，LH或通过其组合调节的先前未识别的卵巢基因； 6）是否使用基因疗法将LH受体引入零动物的卵巢会导致它们循环和排卵，但由于子宫中没有LH受体，但仍未怀孕。这些只是几个例子，说明使用无效动物如何能够提高我们当前对LH在不同卵巢功能中的作用的理解。我们在此应用中提出了三个具体目标：1）研究7天，25天，60天和1岁的零小鼠的卵巢中的结构和功能缺陷，以与它们的年龄相匹配，野生型和杂合兄弟姐妹进行比较。 2）研究雌二醇，孕酮和睾丸激素替代疗法是否可以纠正LH受体敲除动物卵巢中的结构和功能缺陷。 3）确定逆转录病毒介导的LH受体基因转移是否可以纠正零动物卵巢中的结构和功能缺陷，因此即使由于子宫中缺乏LH受体而不会出现妊娠，它们即使不会发生妊娠。该提议中有几个优势。最重要的是使用敲除技术研究LH生物学。第二是使用类固醇激素替代和基因疗法。第三是使用cDNA表达阵列，这是基因表达分析中的强大技术。已经在提出的研究中建立了所有用于获取提案中提出的初步数据的技术。