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 and supraphysiological prolactin upregulation in the adult, we have evaluated the expression of the hPRL-d2eGFP transgene during the oestrous cycle and in males with long-term oestradiol releasing implants, respectively.
Rats, injected with LHRH to synchronise oestrous cycles, were culled and pituitary glands harvested at proestrus, oestrus and diestrus. Flow cytometry indicated a 1.8-fold increase in the number of cells expressing detectable levels of the d2EGFP reporter at oestrus (n=7) as opposed to diestrus (n=5). Mean fluorescence per cell increased by 10.6-fold. Validation by qPCR, confirmed a 4.1-fold increase in the expression of d2EGFP mRNA and a 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 the high level secretory output during proestrus and oestrus. Supraphysiological oestrogen stimulation in males caused a 2.5-fold increase in pituitary weight and transgene expression was shown to be upregulated by flow cytometry and qPCR.
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 how they are modified by relative cell positions in the tissue.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This work was supported, however funding details are unavailable.
05 - 09 May 2012
European Society of Endocrinology