Author

Jing Xu

Date

December 2006

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Environmental and Biomolecular Systems

Institution

Oregon Health & Science University

Abstract

Luteinizing hormone (LH) is essential for the development/function of the primate corpus luteum (CL) during the menstrual cycle. However, the cellular/molecular processes whereby LH controls luteal structure/function are poorly understood. Therefore, studies were initiated to investigate systematically the genes that are regulated by LH in the monkey CL during the menstrual cycle. Rhesus monkeys (Macaca mulatta) were either untreated or received the gonadotropin releasing hormone antagonist to inhibit pituitary LH secretion. RNA from the CL was analyzed using a human gene spotted microarray. A total of 206 cDNAs exhibited a ≥2-fold change after treatment. Sixty-four of them were up-regulated and 142 were down-regulated. Selected cDNAs were validated via semi-quantitative RT-PCR. Twenty-two of 25 cDNAs exhibiting a ≥6-fold change were validated as differentially expressed in the treatment group. Eleven of 25 changed correspondingly in CL collected in the late-to-very late luteal phase of the natural menstrual cycle, which is consistent with the concept that the proteins encoded by up-regulated genes following LH withdrawal may serve as initiators or mediators of luteal regression. Genes down-regulated following the loss of LH support may promote luteal activities. Microarray analysis provided unexpected evidence that the level of mRNA specifying CRH-binding protein (CRHBP) increased significantly in the primate CL following LH withdrawal. Therefore, further experiments were designed to determine if other components of the CRH/urocortin (UCN)-receptor (R)-BP system are expressed in the macaque CL and regulated by LH. Components were quantitated for mRNA by real-time PCR and for protein by western blotting. The mRNA and protein was localized by in situ hybridization and immunohistochemistry. All genes encoding the CRH/UCN-R-BP components, except UCN3, were expressed in the monkey CL. Suppressing LH secretion reduced (P < 0.05) UCN2 and increased (P < 0.05) CRHBP mRNA levels. The mRNA and protein levels of UCNs, CRHR1, and CRHR2 were maximal (P < 0.05) in CL at early-to-mid luteal phase, whereas CRHBP peaked (P < 0.05) in the late luteal phase. The mRNAs for CRHRs and CRHBP, but not the ligands, were detected in the granulosa and theca cells of the preovulatory follicle. However, intense staining for the ligands CRH, UCN, and UCN2, as well as for CRHRs and CRHBP, was evident in the CL, notably in the steroidogenic luteal cells. Ligand and receptor proteins were localized to the granulosa-lutein cells of the CL; whereas CRHBP was limited to the theca and thecalutein cells of the preovulatory follicle and CL. The role of the CRH/UCN-R-BP system was investigated by injecting a CRHR antagonist directly into the preovulatory follicle of monkeys. CRHR antagonist treatment had no effect on ovulation. However, estradiol and progesterone levels were significantly less (P < 0.05) in the treatment group during the following luteal phase, compared to controls. Histologic evaluation provided preliminary evidence that CRHR antagonist treatment also suppressed luteal structure, as judged by a luteal cavity and TUNEL-labeled cells. Thus, a local CRH/UCN-R-BP system exists in a cell-specific manner in the primate CL that is dynamically expressed and LH-regulated during the luteal phase of the menstrual cycle. Initial evidence is consistent with the concept that ligand-receptor activation during the early-to-mid luteal phase promotes luteal development and/or structure/function, whereas its loss in the late luteal phase contributes to luteal regression.

Identifier

doi:10.6083/M4B56GPP

School

OGI School of Science and Engineering

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