| Summary: | 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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