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Regulation of GnRHI and GnRHII mRNA in the Human Ovary and the Role of these Two GnRH Forms in Apoptosis Khosravi, Shahram

Abstract

In humans, reproduction was generally believed to be controlled by only one form of GnRH called GnRHI. However, recently a second form of GnRH, analogous to chicken GnRHII, was discovered in several tissues including the human ovary. The regulation and function of GnRHI in the hypothalamus has been well studied. However, the function and regulation of GnRHI and particularly GnRHII in the ovary is less well understood. Since gonadal sex steroids are one of the main regulators of reproduction, in the present study we investigated the regulation of GnRHI and GnRHII mRNA expression by 17p-estradiol (E2) and progesterone (P4) in human granulosa luteal cells (hGLCs). Additionally, GnRHI and GnRHII have been shown to directly induce apoptosis in the ovaries of some vertebrates, and according to several studies, luteolysis might happen via apoptosis. Consequently, in the present study we also examined the ability of the two GnRH forms to induce apoptosis in hGLCs, in an attempt to define the putative roles of GnRHI and GnRHII in luteolysis. The levels of the mRNA transcripts encoding the two GnRH forms were examined using semi-quantitative RT-PCR followed by southern blot analysis. DNA fragmentation was measured using cell death detection ELISA kit. With time in culture, GnRHI and GnRHII mRNA levels significantly increased by 120% and 210% at day 8 compared to day 1, respectively. The levels remained elevated until the termination of these experiments at day 10. A 24h dose dependent treatment of hGLCs with E2 (1-100 nM) resulted in a significant decrease and increase in mRNA expression of GnRHI and GnRHII, respectively. One nM of E2 decreased GnRHI mRNA levels by 55% and increased GnRHII mRNA levels by 294%. Time dependent treatment studies demonstrated that E2 (InM) significantly decreased GnRHI mRNA levels in a time dependent manner with maximal inhibition of 77% at 48h. In contrast, GnRHII mRNA levels significantly increased in a time dependent fashion, reaching a maximum level of 280% at 24h. Co-treatment of hGLCs with E2 and tamoxifen reversed the regulatory effects of E2 on the mRNA expression of GnRHI and GnRHII. Time and dose dependent treatment with RU486 did not affect GnRHI mRNA levels in hGLCs. In contrast, RU486 treatment significantly increased GnRHII mRNA levels in hGLCs in a time and dose dependent fashion, with a maximum increase being observed at 24h with a lOOOOnM of RU486. With time in culture, a significant increase (51%) was observed in the percentage of cells undergoing apoptosis at day 8 compared to day 1. The level of apoptosis in the cells remained elevated until the end of these studies at day 10. The present study also demonstrated that 10 nM of GnRHI or GnRHII were capable of enhancing apoptosis in hGLCs after 12h. In conclusion, the present study demonstrated that the expression of GnRHI and GnRHII at the transcriptional level is differently regulated by E2 and P4 in hGLCs, and the effect of E2 on mRNA expression of the two GnRH forms is exerted through the conventional estrogen receptors (ERs). GnRHI and GnRHII were also shown to be capable of inducing apoptosis in hGLCs. Therefore, the dynamic balance between E2 and P4 and the subsequent increase or decrease in GnRHI or GnRHII, may play a role in regulating the fate of the corpus luteum.

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