Testosterone, not 5alpha-dihydrotestosterone Stimulates Liver Receptor homolog-1 Expression by Activating the Androgen Receptor in Primary Rat Granulosa Cells.

Abstract

Androgens are crucial for normal folliculogenesis and female fertility. Using primary rat granulosa cells, we recently demonstrated that in the absence of gonadotropins, testosterone increases aromatase (Cyp19) and P450 side change cleavage (P450scc) expression. Interestingly, 5alpha-dihydrotestosterone (DHT), a more potent activator of the AR, does not stimulate the expression of Cyp19 and P450scc to the same extent as testosterone. An explanation for this unique effect of testosterone remains elusive. Here, we have explored the nature of the mechanisms involved in the stimulation of gene expression by testosterone in granulosa cells (GCs), and the differential response of these cells to testosterone and DHT. Using primary GCs, we showed that testosterone, but not DHT, stimulates liver receptor homolog-1 (LRH-1) mRNA expression. LRH-1 is a transcription factor known to be involved in the up-regulation of Cyp19 expression. The effects of testosterone on LRH-1 expression are mediated by an increase in the transcription of this gene since testosterone also increased LRH-1 promoter activity. In agreement with the lack of stimulation of LRH-1 mRNA expression by DHT, this androgen did not increase the activity of the LRH-1 promoter in GCs. Based on these results; we reasoned that if DHT and T occupy the same androgen receptor (AR), then treatment with increasing concentrations of DHT might inhibit the ability of testosterone to stimulate LRH-1. This presupposes that the DHT pretreatment saturates and substantially reduces the number of ARs available to testosterone. The results of this experiment demonstrated that the stimulation of LRH-1 expression by testosterone could be out competed in a concentration-dependent manner not only by DHT but also by R1881, a potent AR agonist. This result suggests that the effects of testosterone are indeed mediated by the activation of the AR. Interestingly, both testosterone and DHT stimulated the activity of a reporter construct containing three consensus AR response elements. Moreover, in contrast to the selective response of the LRH-1 promoter for testosterone observed in ovarian GCs, both testosterone and DHT stimulated the activity of the LRH-1 promoter in human embryonic kidney cells (293T cells). These results indicate that only granulosa cells provide the appropriated milieu of coregulators needed for the differential stimulation of LRH-1 expression by testosterone and DHT. To begin to understand the mechanisms by which testosterone regulates LRH-1 expression in GCs, studies using progressive mutant deletions of the LRH-1 promoter were performed. These experiments identified the region between -348bp and -266bp of the LRH-1 promoter as the target for the testosterone-AR complex. Within this region a selective AR response element was found. These results suggest strongly that in ovarian granulosa cells, pathways activated primarily by testosterone may control the expression of LRH-1. Supported by NIH R01HD057110 and R21HD066233. (poster)