Abstract
Gonadotropin-releasing hormone (GnRH), a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7) neurons, which show pulsatile GnRH release in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of GnRH mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat Gnrh1 enhancer-derived noncoding RNAs (GnRH-E1 RNAs) are expressed. We investigated the characteristics and function of the endogenous mouse GnRH-E1 RNA. Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed GnRH-E1 RNAs that are transcribed in the sense and antisense directions from distinct 5’ start sites, are 3’ polyadenylated, and are over 2 kb in length. These RNAs are localized in the nucleus and have a half-life of over 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the sense or antisense mouse GnRH-E1 RNA in immature, migratory GnRH (GN11) neurons, which do not express either GnRH-E1 RNA or GnRH mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA is an inducer of Gnrh1 gene expression. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons.
Highlights
Regulation of gonadotropin-releasing hormone (Gnrh1) gene expression and Gonadotropin-releasing hormone (GnRH) secretion are essential for normal reproductive maturation and fertility
Mouse Gnrh1 enhancers and promoter are located in the genomic region 5’ of Gnrh1, where Gnrh1 enhancers 2 and 3 (E2 and E3) are located in the region overlapping the 3’ UTR of Kctd9, and Gnrh1 enhancer 1 (E1) and the promoter are located in the intergenic region 3’ of Kctd9
In addition to the reads aligning to Gnrh1, GT1-7 cells showed dense and robust RNA reads that align to the intergenic region of Gnrh1 and Kctd9 genes (Fig 1B)
Summary
Regulation of gonadotropin-releasing hormone (Gnrh1) gene expression and GnRH secretion are essential for normal reproductive maturation and fertility. GnRH released from hypothalamic neurons stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from gonadotrope cells in the pituitary, and these glycoprotein hormones stimulate the gonads to produce sperm, oocytes, and steroid hormones. These precise mechanisms govern normal reproductive development and function, including puberty, menstrual cycle, pregnancy, and menopause. Failure of GnRH neuron migration and maturation and dysregulation of Gnrh gene expression have been implicated in the incorrect timing of puberty, reproductive deficiencies, and infertility. Understanding the mechanisms regulating Gnrh gene expression will provide important insights into the biology and pathophysiology of mammalian reproduction
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