Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
Activation of the luteinizing hormone receptor (LHR) by luteinizing hormone (LH) is essential for steroid hormone production, spermatogenesis, and ovulation. Several naturally occurring heterozygous activating mutations have been found in the LHR gene of boys with sporadic or familial male limited precocious puberty. The majority of the activating mutations are located within transmembrane helix six of the seven transmembrane G protein coupled receptor, the most prevalent being an aspartic acid to glycine mutation at position 578. Patients with this condition undergo puberty between 1- 4 years of age and are characterized by high levels of testsoterone with prepubertal gonadotropin levels. The constitutive activation of aspartic acid to glycine mutation at position 582in mouse LHR which corresponds to position 578 in humans, was determined by signaling studies performed in transiently transfected HEK293 cells. The study showed a 14-fold elevation in basal level cAMP production compared to the wild type (WT) LHR. To study the effects of premature and constitutive activation of LHR on reproductive development we have created knock-in mice (KiLHR) expressing the point mutation D582G in the LHR gene. There were many steps involved in the creation of the KiLHR mice. The first step was to generate a targeting vector containing the mLHR exon 11 D582G sequence flanked by two regions of homology (5' and 3') which were required for homologous recombination. The targeting construct was electroporated into ES cells and antibiotic resistance selected those cells that incorporated the targeting construct into their DNA. ES cell clone DNA was isolated and screened by Southern blot analysis to verify the occurrence of homologous recombination at both the 5' and 3' ends. RT-PCR analysis of RNA from the gonads of the KiLHR mice showed expression of the WT and mutant alleles confirming the presence of the heterozygous condition. In preliminary analysis of KiLHR female mice, LHR activation caused an increase in ovarian steroid hormone production and resulted in precocious puberty as seen by an early vaginal opening compared to the WT mice. By 5 weeks of age, these mice exhibited irregular estrus cycles with enlarged ovaries consisting primarily of hemorrhagic cysts and luteal tissue. Follicular development was only seen along the periphery of the ovary with none past the preantral stage and all of the KiLHR females were infertile. The uteri of the 5-week old KiLHR mice were enlarged due to a dilated lumen, increased myometrial layer and attenuated stromal layer compared to the WT mice. Serum levels of LH and follicle stimulating hormone (FSH) were significantly lower while androgen and estrogen levels from ovarian homogenates were increased in KiLHR compared to WT animals. RNA was isolated from the KiLHR and WT ovary and relative expression of genes in the steroidogenic pathway analyzed by RT-PCR showed an increase in the expression of many steroidogenic enzymes in KiLHR ovaries. These data suggest that the KiLHR mice are a useful model for further studies addressing the effects of premature LHR activation on reproductive development.
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