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Molecular characterization and regulation of gonadotropin-releasing hormone (GnRH) and its receptor mRNA in the human ovary

University of British Columbia

With the recent detection of GnRH and its receptor (GnRH-R) in numerous extrapituitary tissues, it is hypothesized that GnRH acts as an autocrine/paracrine regulator of ovarian function. The direct involvement of GnRH in follicular atresia and modulation of ovarian steroidogenesis, further substantiate the extrapituitary actions of this decapeptide. In contrast to the hypothalamus and pituitary, the factors that regulate ovarian GnRH and its receptor remain poorly characterized. As gonadal steroids are key regulators of ovarian functions, the present study investigated the role of 17β-estradiol (E2) and progesterone (P4) in regulating the intrinsic ovarian GnRH axis. Using reverse transcription polymerase chain reaction (RT-PCR), we have isolated the full-length GnRH-R coding region from human granulosa-luteal cells (hGLCs). Sequence analysis revealed that the ovarian GnRH-R cDNA is identical to its pituitary counterpart. Basal expression studies demonstrated that GnRH and GnRH-R mRNA levels significantly increased with time in culture, reaching levels of 160% and 170% on day 8 and 10 of culture compared to day 1 (p<0.05), respectively. On day 5 in culture, hGLCs were treated with E2 and RU486 (a progesterone receptor antagonist) in a dose- and time-dependent fashion. A dose-dependent decrease was observed in GnRH and GnRH-R rnRNA levels after a 24h treatment with E2 (1-100 nM). Time course studies demonstrated that E2 (1 nM) decreased GnRH mRNA levels in a time dependent manner, with a maximal inhibition of 40% at 48h (p<0.05). In contrast, GnRH-R expression exhibited a biphasic pattern with time. A 6h treatment with E2 (1 nM) resulted in a 20% increase in GnRH-R message (p<0.05), whereas a long-term treatment (48h) resulted in a 60% decrease in GnRH-R expression (p<0.05) in hGLCs. Tamoxifen treatment reversed the E2-induced inhibition of GnRH and GnRH-R mRNA expression, indicating that the E2 effect was mediated through its receptor. The progesterone receptor antagonist had no signficant effect on GnRH mRNA levels in hGLCs. However, RU486 induced a timeand dose-dependent decrease in GnRH-R mRNA expression, with a maximal inhibition of 50% at a dose of 10μM for 24h (p<0.05). Since GnRH actions have been associated with atresia and luteolysis, the dynamic balance between E2 and P4 may contribute to the maintenance of the corpus luteum. With the recent identification of a second form of GnRH in the human brain (GnRH-II), the present study examined the expression and function of GnRH-II in human ovarian cells. Using RT-PCR we demonstrated the expression of GnRH-II in hGLCs, an ovarian cancer cell line (OVCAR-3), primary ovarian cancer cells, and human ovarian surface epithelial cells. Functionally like GnRH-I, GnRH-II significantly decreased progesterone secretion in hGLCs (p<0.001). Furthermore, GnRH-II decreased progesterone secretion more than GnRH-I (50% versus 30% compared to control; p<0.05). The GnRH-induced inhibition of progesterone secretion was reversed by antide, a GnRH antagonist. In summary, our studies demonstrate that the ovary possesses an intrinsic GnRH axis that is dynamically regulated during spontaneous luteinization in vitro, and that gonadal steroids are capable of regulating GnRH and its receptor in the human ovary. Coupled with the expression and functional analysis of a second form of GnRH (GnRH-II) in the ovary, our studies strengthen the notion that GnRH acts as an autocrine/paracrine modulator of ovarian functions.

Thesis/Dissertation

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2009-07-07

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http://hdl.handle.net/2429/10325

Reproductive and Developmental Sciences

University of British Columbia

UBCV

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