Identification of Amnionless Gene as an Estrogen/ER Alpha-Regulated Gene in the Mouse Pituitary.

Abstract

Estrogen plays a key role in regulating gonadotropin secretion from the pituitary through both negative and positive feedback pathways. However, the direct mechanisms of this regulation have yet to be defined. We hypothesize that the positive feedback effect of estrogen is achieved by ERα-mediated transcriptional regulation of cellular components needed for LH vesicle transport and secretion of its components into the bloodstream, thus equipping cells with the secretory machinery required for the LH surge. To identify these ERα-regulated components of LH secretion, total RNA was extracted from the pituitaries of cycling mice in metestrus or proestrus (low and high levels of circulating estrogen, respectively), ovariectomized (OVX) WT mice treated with vehicle or estrogen, and OVX ERαKO mice treated with vehicle or estrogen, and subjected to microarray analysis. Overall, 183 genes were found to be differentially expressed in an estrogen/ERα-dependent manner. In this study, we characterized the expression pattern of a gene (amn) that codes for Amnionless (Amn), a type I transmembrane protein known to be involved in vitamin B12 absorption by distal intestinal epithelia and proximal tubules of the kidney. The amn gene was selected as one gene of interest due to significant effect of estrogen treatment on transcription levels and the novelty of its characterization in the pituitary. The microarray analysis revealed a 30-fold increase of amn transcription in proestrus compared to metestrus, and a fold change of 25 in OVX WT mice with estrogen compared to vehicle treatment. ERαKO mice showed no increase in transcription of amn with estrogen treatment. This pattern of amn expression was confirmed using RT-PCR, showing a 9 fold increase in transcription levels in proestrus versus metestrus, and 16-fold increase in the OVX WT estrogen treated compared to vehicle treated groups. Immunohistochemistry confirmed that protein levels increased concurrently with mRNA levels in proestrus versus metestrus, but not in ERαKO mice. In silico analysis of the amn proximal promoter reveals 8 putative EREs within 2 kilo bases upstream of the start codon, evidence of a possible direct regulation of amn expression by the classical ERα pathway, which will be determined by ChIP assay. Currently, we are determining the functional significance of ERα-regulated amn expression in LH secretion using in vitro models. Both primary pituitary cells and isolated gonadotroph cells are treated with anti-Amnionless antibody, in order to block the functional capacity of the protein, followed by GnRH treatment. This experiment in the primary pituitary cells will allow us to determine if Amn in the pituitary plays a critical role in facilitating LH secretion, and the experiment in isolated gonadotroph cells will clarify if the effect on LH secretion is due to Amn in gonadotrophs themselves, or secondarily due to Amn expression in other cell types of the pituitary. For isolation of gonadotroph cells, we have generated ROSA-GFP CgaCre mice, who express Cre, and thus GFP, specifically in gonadotroph. Using flow cytometry, live gonadotrophs can be segregated from other cell types of the pituitary. Future directions will be aimed at elucidation of the role of Amn in preparing the gonadotroph for LH secretion in relation to the well-known function of Amn in facilitating vitamin B12 absorption. In summary, the gene encoding the Amnionless protein has been identified as an estrogen/ERα-regulated gene in the pituitary. The findings we may make from the study of Amn function in the pituitary will add to the understanding of the molecular mechanisms of estrogen priming of the pituitary in preparation for the LH surge. Supported by a COBRE grant from NIH NCRR: P20 RR15592. (poster)