Ovarian failure in LH/hCG receptor knockout animals

LH/hCG 受体敲除动物的卵巢衰竭

• 批准号: 6637891
• 负责人: Ch V RAO
• 金额: $ 29.6万
• 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/6637891
• 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和我们的小组最近都成功地通过胚胎干细胞的基因靶向产生LH受体敲除小鼠。 虽然这种基因敲除不致命，但它使动物不育。 野生型和杂合子动物的卵巢含有LH受体，而同窝出生的纯合子动物的卵巢不含LH受体。 此外，虽然野生型和杂合子动物的卵巢大小正常，含有排卵前卵泡和黄体，但同窝纯合子动物的卵巢小而苍白，在窦期卵泡生长停滞。 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，LH水平显著升高，FSH水平中度升高，雌二醇和孕酮水平降低，但未完全抑制。敲除动物在回答LH生物学中的一些未知问题方面非常有用。 例如，我们可以了解：1）LH作用是否是原始卵泡、初级卵泡、腔前卵泡和腔卵泡正常数量存在所必需的; 2）在完全没有LH作用的情况下，FSH是否可以诱导卵泡生长和排卵; 3）LH信号在出生后一周至一岁的卵巢发育和功能中起什么作用; 4）LH的哪些卵巢作用是由雌二醇、孕酮和睾酮介导的; 5）鉴定和表征先前未鉴定的由雌二醇、孕酮、睾酮、LH或其组合调节的卵巢基因;以及6）是否使用基因治疗将LH受体引入无效动物的卵巢中使它们周期性排卵，但仍然不能怀孕，因为它们在子宫中没有LH受体。 这些只是使用无效动物如何促进我们目前对LH在不同卵巢功能中作用的理解的几个例子。 我们在本申请中提出了三个具体目的：1）研究7天、25天、60天和1岁大的无效小鼠的卵巢中的结构和功能缺陷，以与它们的年龄匹配的野生型和杂合同胞进行比较。2)研究雌二醇、孕酮和睾酮替代疗法是否可以纠正LH受体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LH受体基因转移是否可以纠正无效动物卵巢中的结构和功能缺陷，使其成为周期性和排卵，即使由于子宫中缺乏LH受体而可能不会怀孕。 这一提议有几个优点。 首先是使用基因敲除技术研究LH生物学。 其次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一种强有力的技术。 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。