Differential regulation of gonadotropin-releasing hormone (GnRH)I and GnRHII messenger ribonucleic acid by gonadal steroids in human granulosa luteal cells

Abstract

In humans, reproduction was generally believed to be controlled by only one form of GnRH (called mammalian GnRH or 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. Because gonadal sex steroids are one of the main regulators of reproduction, we investigated, in the present study, the regulation of GnRHI and GnRHII mRNA expression by 17beta-estradiol (E2) and RU486 (a progesterone antagonist) in human granulosa luteal cells (hGLCs). The levels of the mRNA transcripts encoding the two GnRH forms were examined using semiquantitative RT-PCR followed by Southern blot analysis. With time in culture, GnRHI and GnRHII mRNA levels significantly increased, by 120% and 210%, at d 8 and d 1, respectively. The levels remained elevated until the termination of these experiments at d 10. A 24-h treatment of hGLCs with E2 (10(-9) to 10(-7) M) resulted in a dose-dependent decrease and increase in mRNA expression of GnRHI and GnRHII, respectively. E2 (10(-9) M) significantly decreased GnRHI mRNA levels (by 55%) and increased GnRHII mRNA levels (by 294%). Time-course studies demonstrated that E2 (10(-9) M) significantly decreased GnRHI mRNA levels in a time-dependent manner, with maximal inhibition of 77% at 48 h. In contrast, GnRHII mRNA levels significantly increased in a time-dependent fashion, reaching a maximum level of 280% at 24 h. Cotreatment of hGLCs with E2 and tamoxifen (an E2 antagonist) reversed the inhibitory and stimulatory effects of E2 on the mRNA expression of GnRHI and GnRHII, respectively. 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 24 h (with 10(-5)M RU486). In summary, the present study demonstrated that the expression of GnRHI and GnRHII at the transcriptional level is differently regulated by E2 and P4 in hGLCs.