Effects of oestrogen on prolactin transcription patterns in living pituitary tissue Amanda Louise Patist PhD in the Faculty of Medical and Human Sciences, September 2013, The University of ManchesterOestrogen is a well-known modulator of the transcription and secretion of prolactin as well as having a role in the physiological proliferation and possibly also in pathological hyperplasia of lactotrophs. Our group has studied prolactin gene promoter regulation in single living pituitary cells in Fischer 344 (d2eGFP-hPRL 455) rats, that express a destabilised eGFP reporter gene under the control of a human prolactin genomic fragment, and identified prolactin transcription cycles that occur in a non-circadian and non-synchronised fashion. Pulsatile transcription has been identified in fetal tissue, stabilising during development. Here we assess the effects of physiological and supraphysiological oestrogen exposure in vivo on prolactin transcription in the adult rat. We have established and validated models of both states by evaluating the expression of the hPRL-d2eGFP transgene during the oestrous cycle and in males with long-term oestradiol-releasing implants, respectively.The oestrous cycles of adult female rats were synchronised by IP LHRH injection. A 1.8-fold increase in the number of cells expressing the prolactin transgene at oestrus (n=7) as compared to diestrus (n=5) and a 10.6 fold increase in mean fluorescence per cell was identified by flow cytometry. In males, chronic oestrogen stimulation induced a 2.5-fold increase in pituitary weight, a 5.2-fold increase in number of cells expressing the transgene and 4.4-fold increase in fluorescence per cell, as indicated by flow cytometry (n=3). Immunofluorescence, qPCR and serum analysis confirmed the high-production state of lactotroph cells in both female and male models. 250µm pituitary slices were imaged for 48 hours using time-lapse confocal microscopy and pulsatile fluorescent reporter activity was evident in both female and male models. Interestingly, no significant difference was seen between transcription cycle patterns (timing or amplitude of transcriptional pulses) in individual cells between high and low oestrogen states. Using a novel mathematical model, that calculates transcription rate from fluorescence data, we have been able to study the transcription rates displayed by single cells and the estimated points at which a cell switches from one rate to another. Patterns in switches in transcription rates were similar between high and low oestrogen states, suggesting that individual pituitary cells within the context of tissue, continue to display cyclical patterns of gene expression, in states of both high and low prolactin production. This implies that cyclical transcription is a fundamental property of prolactin gene expression that persists in different physiological states, and that modulation of cycle characteristics is not the mechanism for increased prolactin synthesis in these circumstances.