Ovarian failure in LH/hCG receptor knockout animals

IIpo Huhtaniemi和我们的团队最近都成功地通过胚胎干细胞基因靶向产生了LH受体敲除小鼠。虽然这种基因敲除不是致命的，但它使动物不育。野生型和杂合子的子房含有LH受体，而纯合子的子房不含LH受体。此外，野生型和杂合子的卵巢大小正常，含有排卵期前卵泡和黄体，而纯合子的子房则小而苍白，卵泡生长在中期停止。初步研究表明，这种停滞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水平明显升高，FSH水平中度升高，雌二醇和黄体酮水平下降但未完全抑制。敲除动物在回答LH生物学中的一些未知方面非常有用。例如，我们可以了解：1)原始卵泡、原发卵泡、窦前卵泡和窦前卵泡的正常数量是否需要LH作用；2)在完全没有LH作用的情况下，FSH是否能诱导卵泡生长和排卵；3)从出生后1周到1岁，黄体生成素信号在卵巢发育和功能中的作用；4)黄体酮、黄体酮、睾酮对卵巢LH有哪些调节作用；5)鉴定和描述以前未被发现的受雌二醇、黄体酮、睾酮、LH或它们的联合调控的卵巢基因；6)是否使用基因疗法将黄体生成素受体引入无体动物的卵巢，使它们有周期和排卵，但仍不能怀孕，因为它们子宫中没有黄体生成素受体。这些仅仅是使用零动物如何推进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目的：1)研究7天、25天、60天和1岁小鼠卵巢的结构和功能缺陷，并与它们的年龄匹配、野生型和杂合的兄弟姐妹进行比较。2)探讨雌二醇、黄体酮和睾酮替代治疗是否能纠正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null动物卵巢的结构和功能缺陷，使其即使由于子宫中缺乏LH受体而无法怀孕也能恢复周期和排卵。这个提议有几个优点。最重要的是利用基因敲除技术研究LH生物学。第二种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，这是一种基因表达分析的强大技术。拟议研究中使用的所有技术都已确定，以便获得提案中提出的初步数据。

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.