Ovarian failure in LH/hCG receptor knockout animals

LH/hCG 受体敲除动物的卵巢衰竭

• 批准号: 6780868
• 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
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6780868
• 关键词: aging; biological signal transduction; chorionic gonadotropin; corticosterone; estradiol; follicle stimulating hormone; gene targeting; genetically modified animals; histogenesis; hormone receptor; hormone regulation /control mechanism; hormone therapy; immunocytochemistry; in situ hybridization; laboratory mouse; leptin; luteinizing hormone; morphometry; northern blottings; ovary; ovary disorder; polymerase chain reaction; progesterone; southern blotting; terminal nick end labeling; testosterone; western blottings

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‘s和我们的团队最近都成功地通过胚胎干细胞的基因靶向培育出了黄体生成素受体基因敲除小鼠。虽然这种基因敲除不是致命的，但它使动物不育。野生型和杂合子动物的卵巢含有黄体生成素受体，而纯合子产仔的卵巢不含黄体生成素受体。此外，野生型和杂合型动物的卵巢大小正常，含有排卵前卵泡和黄体，而纯合子产仔的卵巢小而苍白，卵泡生长受阻于胃窦期。初步研究表明，这种停滞可能至少部分是由于端粒酶水平的降低和随之而来的细胞凋亡的增加。在纯合子动物中，黄体生成素水平显著升高，卵泡刺激素水平中度升高，雌二醇和孕酮水平下降，但并未完全抑制。在回答黄体生成素生物学中的一些未知问题时，基因敲除动物非常有用。例如，我们可以了解：1)原始卵泡、初级卵泡、腔前卵泡和腔前卵泡的正常数量是否需要黄体生成素作用；2)在完全没有黄体生成素作用的情况下，卵泡刺激素是否能诱导卵泡生长和排卵；3)从出生后一周到一岁，黄体生成素信号在卵巢发育和功能中起着什么作用；4)黄体生成素的哪些卵巢作用是由雌二醇、孕酮和睾酮调节的；5)鉴定和鉴定以前未知的受雌二醇、孕酮、睾酮、黄体生成素或它们的组合调控的卵巢基因；6)用基因疗法将促黄体激素受体导入空动物的卵巢，是否因为子宫内没有促黄体激素受体而使其周期排卵，但仍不能怀孕。这些只是使用空动物如何提高我们目前对黄体生成素在不同卵巢功能中的作用的理解的几个例子。我们提出了三个具体的应用目标：1)研究7日龄、25日龄、60日龄和1年龄空小鼠卵巢的结构和功能缺陷，并与其年龄匹配的、野生型和杂合子兄弟姐妹进行比较。2)探讨雌激素、孕酮和睾酮替代治疗能否纠正黄体生成素受体基因敲除动物卵巢的结构和功能缺陷。3)确定逆转录病毒介导的促黄体生成素受体基因转移是否能纠正空白动物卵巢的结构和功能缺陷，使其即使在子宫中没有促黄体激素受体而不能怀孕的情况下也能周期排卵。这项提议有几个优点。最重要的是利用基因敲除技术研究黄体生成素生物学。第二是使用类固醇激素替代和基因疗法。第三是使用基因表达分析中的强大技术--cdna表达阵列。拟议研究中使用的所有技术都已确定，以获得提案中提出的初步数据。