Endocrine Abstracts (2012) 28 P240

Transcriptional regulation of prolactin in the rat oestrous cycle

Amanda Patist1, Karen Featherstone1, David Spiller2, Sabrina Semprini4, Judith McNeilly3, Alan McNeilly3, John Mullins4, Michael White2 & Julian Davis1

1Faculty of Medical and Human Sciences, University of Manchester, Manchester, United Kingdom; 2Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom; 3MRC Centre for Reproductive Health, University of Edinburgh, Edinburgh, United Kingdom; 4Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom.

Circulating levels of prolactin are subject to acute and long-term regulation by many factors including oestrogen and dopamine. We have studied the regulation of prolactin promoter activity in living pituitary cells using transgenic Fischer rats in which reporter gene expression is regulated by the human prolactin gene locus (hPRL-d2EGFP). We have previously identified pulsatile prolactin transcription patterns in living lactotroph cells in fetal tissue, that became stabilised during neonatal development. In order to assess how transcription patterns are affected during physiological prolactin upregulation in the adult, we have evaluated the expression of the hPRL-d2eGFP transgene during the oestrous cycle. Transgenic Fischer rats were injected with LHRH in order to synchronise oestrous cycles. Animals were culled and pituitary glands harvested at proestrus, oestrus and diestrus for analysis. Flow cytometry showed a 1.8-fold increase in the number of cells expressing detectable levels of the d2EGFP reporter in rats in oestrus (n=7) as opposed to diestrus (n=5). There was a mean 10.6-fold increase in fluorescence signal per cell. Validation by qPCR, confirmed a 4.1-fold increase in the expression of d2EGFP mRNA and a similar 3.7-fold increase in the endogenous rat PRL mRNA. Immunofluorescence confirmed induction of EGFP and prolactin protein expression in tissue sections. These data indicate a major increase in transcription rate in individual cells, which is likely to be necessary to sustain high level secretory output during proestrus and oestrus. The apparent recruitment of transcriptionally active cells probably represents activation of quiescent low-expressing lactotroph cells. Using live cell imaging of tissue slice preparations from oestrous animals, fluctuating gene expression was detectable in a proportion of cells. The spatio-temporal nature of these transcription patterns is currently being subjected to mathematical analysis to determine if they are modified by the relative cell position in the tissue.

Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.

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