Mechanisms involved in the homologous down-regulation of transcription of the follicle-stimulating hormone receptor gene in Sertoli cells

Abstract

The action of follicle-stimulating hormone (FSH) in spermatogenesis is regulated at a fundamental level by controlling the number of competent receptors present at the surface of Sertoli cells. By controlling the number of receptors, the cell is able to modulate the timing and magnitude of subsequent signal transduction in response to FSH. One mechanism of control is the down-regulation of the steady state levels of the FSH receptor gene after exposure to FSH or agents that stimulate or prolong the cAMP signal transduction cascade (homologous down-regulation) in Sertoli cells. The goals of this study were to examine possible mechanisms involved in the down-regulation of mRNA levels of this gene. Analysis of transcription and processing by a PCR-based assay showed that treatment of Sertoli cells with FSH caused at least a 50% reduction of hnRNA for the FSH receptor gene. Reporter genes controlled by 5′ flanking sequences of the FSH receptor gene that were transiently transfected into Sertoli cells were not down-regulated. In electrophoretic mobility shift assays (EMSA), cAMP-inducible nuclear protein complex containing c-Fos formed on the activator protein-1/cAMP responsive element-like site located at −216 to −210 in the promoter of the rat FSH receptor gene. We concluded from this study that there was no evidence for the putative role of ICER in the down-regulation of the FSH receptor promoter. In addition, the FSH-induced down-regulation of the transcription of the FSH receptor gene in Sertoli cells was prevented by the treatment of Sertoli cells with trichostatin A prior to the addition of FSH. This experiment coupled with other observations suggested that the down-regulation may be mediated by changes in chromatin structure.

Introduction

One of the most important biochemical signaling networks involved in controlling normal spermatogenesis in mammals exists between the anterior lobe of the pituitary gland and Sertoli cells in the testis (Griswold et al., 1975, Griswold et al., 1976, Griswold et al., 1977, Orth, 1984, Singh and Handelsman, 1996, Kumar et al., 1997, Tapanainen et al., 1997, Dierich et al., 1998). The biological events controlled by this endocrine mechanism result from the interaction of follicle-stimulating hormone (FSH) with the FSH receptor and the subsequent transduction of molecular information across the membrane leading to the production of second messengers such as cAMP (Means et al., 1980) and Ca²⁺ (Chaudhary et al., 1996, Lalevee et al., 1999) and changes in gene expression.

Continuous stimulation of Sertoli cells with FSH leads to a desensitization of the cells to FSH (Gnanaprakasam et al., 1979). Desensitization of the FSH response in Sertoli cells involves multiple identified steps in the FSH/cAMP signal transduction pathway including the following: (1) the rapid internalization and sequestration of the ligand-bound receptor (Fletcher and Reichert, 1984, Saez and Jaillard, 1986, Shimizu et al., 1987); (2) post-translational modification of the receptor (Quintana et al., 1994, Hipkin et al., 1995); (3) reduction in adenylate cyclase activity (Le Gac et al., 1985); (4) increased phosphodiesterase activity (Conti et al., 1983, Conti et al., 1986); (5) direct inhibition of protein kinase A by protein kinase A inhibitor (Tash et al., 1979, Tash et al., 1981); and (6) the down-regulation of the transcription of the FSH receptor gene (Themmen et al., 1991, Monaco et al., 1995, Maguire et al., 1997). Given that the concentration of FSH in the serum of non-seasonal-breeding males is relatively constant, the regulation of the number of FSH receptors and their competency to bind FSH and transduce signal may be an important level of control on the action of FSH in males (McGuiness and Griswold, 1995).

Homologous down-regulation of transcription of the FSH receptor gene in cultured rat Sertoli cells was first reported by Themmen et al. (1991). The loss of approximately 90% of the steady-state level of FSH receptor mRNA and a parallel loss of ¹²⁵I FSH binding was measured 4 h after the addition of 500 ng/ml Ovine FSH (oFSH) to Sertoli cells in primary culture (Themmen et al., 1991). Themmen and coworkers suggested that down-regulation was stimulated by (Bu)₂cAMP, did not require de novo transcription or translation and concluded that RNA degradation was likely responsible for the homologous down-regulation of FSH receptor mRNA in Sertoli cells. Maguire and coworkers measured the relative contribution of FSH receptor mRNA decay to the process of homologous down-regulation by uncoupling transcription and mRNA decay with the transcription inhibitor actinomycin D (Maguire et al., 1997). Maguire and coworkers also demonstrated homologous down-regulation of the FSH receptor gene in vivo. The results of their work did not support the hypothesis that homologous down-regulation occurred via a cAMP-inducible mRNA decay pathway but rather suggested a role for transcription in homologous down-regulation is likely.

A model for the mechanism of homologous down-regulation of the FSH receptor in Sertoli cells that involved the inputs of de novo transcription and translation was presented by Monaco et al. (1995). They presented evidence that the inducible cAMP early repressor (ICER) bound to the rat FSH receptor promoter at a presumptive CRE-like site in vitro and repressed expression of a FSH promoter driven reporter gene. This was observed when ICER was overexpressed from the SV40 promoter on a co-transfected template in transient transfection assays (Monaco et al., 1995). In this study we have re-examined this putative CRE-like site and demonstrate that it functions as an AP-1 site. The transcription factor, c-Fos, induced by cAMP or FSH, can bind to the AP1 site in the promoter region of the FSH receptor gene in vitro and in vivo.

The restricted pattern of expression of the FSH receptor gene to Sertoli cells in males and homologous down-regulation of the gene are relaxed when non-chromatin templates are used to study the regulation of this important gene (Linder et al., 1994, Monaco et al., 1995). We were able to demonstrate that treatment of immature rat Sertoli cells in primary culture with trichostatin A, which inhibits histone deacetylase activity completely prevents the homologous down-regulation of the transcription of the FSH receptor gene.