Gene Expression of Basic Helix-Loop-Helix Transcription Factor, SHARP-2, Is Regulated by Gonadotropins in the Rat Ovary and MA-10 Cells1

Abstract

Basic helix-loop-helix (bHLH) proteins regulate transcription from the E box sequence (5'-CANNTG-3') located in the regulatory region of most gene promoters. The rat enhancer of split- and hairy-related protein 2 (SHARP-2) is a member of the bHLH protein family. To analyze the possible role of SHARP-2 in the rat ovary, the regulation of the expression of the SHARP-2 gene was examined, and the SHARP-2 protein was characterized. Northern blot analysis revealed that the level of SHARP-2 mRNA abruptly and temporarily increases as the result of the action of LH, i.e., eCG or hCG treatment alone or hCG after eCG treatment, in the rat ovary, as indicated by the treatment of primary cultured rat granulosa cells with hCG after FSH treatment or of mouse Leydig MA-10 cells with hCG or 8-bromoadenosine 3',5'-cyclic monophosphate. An in situ hybridization analysis showed that eCG treatment increases the level of the SHARP-2 transcript in theca interna cells and that hCG treatment, after the administration of eCG, increases the level of the SHARP-2 transcript in granulosa cells. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into primary cultured granulosa cells and MA-10 cells revealed that the entire coding sequence of SHARP-2 fused to the GFP is localized in the nucleus. The transcriptional activity of SHARP-2 also was examined using transient DNA transfection experiments. When an expression vector encoding the full length of SHARP-2 was cotransfected with thymidine kinase promoter-luciferase reporter plasmids, with or without E box sequences, into MA-10 cells, the luciferase activity was decreased in an E box-dependent manner. We conclude that the level of SHARP-2 mRNA is regulated by gonadotropins and that SHARP-2 functions as a transcriptional repressor localized in the nucleus.

Reproductive biology of female Bmal1 null mice

“…During the 24 h of diestrus, the amplitude of the clock gene rhythms in wild-type ovaries appeared to be reduced compared to estrus. This unexpected result may be due to the influence of gonadotropins altering the rhythm amplitude (Yamada et al 2004). Nevertheless, Bmal1, as well as Dbp and Nr1d1, were rhythmically expressed, with Dbp and Nr1d1 in antiphase to Bmal1, confirming that the endogenous clock system was functional in wild-type mice.…”

“…One of the genes implicated in the accessory feedback loop underpinning cellular rhythmicity, Bhlhe40 (also known as Dec1 and Sharp2), is expressed in the rat ovary, and is induced temporarily by equine and hCGs in both theca and granulosa cells (Yamada et al 2004). In the ovary, BHLHE40 appears to act as a repressor (Yamada et al 2004), and either directly or indirectly alters the expression of FSH receptor, prostaglandin endoperoxidase synthase 2 mRNA, and other E-box-dependent genes in a gonadotropin-dependent manner. Given the observed circadian rhythm of Bhlhe40 expression in the ovary, it is interesting to speculate that there may be circadian gating of cellular processes at the ovarian level as well as within the hypothalamus at the time of ovulation.…”

Circadian rhythms and reproduction

“…DEC1 and DEC2 proteins may block circadian gene expression, in part by the formation of a non-functional heterodimer with BMAL1 (and hence inhibiting the expression of all genes dependent on an E-box), as well as playing a role in light induction of genes in the SCN (Honma et al 2002). Gonadotrophins and insulin are known to induce dec1 gene expression and this may provide an avenue for the reproductive system and pancreas to influence circadian timing both centrally and in other peripheral tissues (Yamada et al 2003(Yamada et al , 2004.…”

Association, mutual stabilization, and transcriptional activity of the STRA13 and MSP58 proteins

“…Its broad-range expression in embryonic and adult tissues combined with responsiveness to various stimuli, including hypoxia [2,3], cytokines [4,5], retinoic acid [6], cAMP [7], serum deprivation, a histone deacetylase inhibitor trichostatin A [8], gonadotropins [9], and insulin [10] imply important roles for STRA13 in multiple signal transduction pathways and various biological processes from regulation of cell metabolism to morphogenesis. Indeed, this transcription factor has been implicated in neurogenesis [6], adipogenesis [11], chondrogenesis [12], and regulation of mammalian circadian rhythms [13,14].…”