F W Anthony1, D D Mukhtar, M A Pickett, I T Cameron. 1. Maternal, Fetal and Neonatal Physiology Group, Fetal Origins of Adult Disease (FOAD) Division, University of Southampton, Princess Anne Hospital, Coxford Road, Southampton SO16 5YA, United Kingdom. fwa@soton.ac.uk
Abstract
OBJECTIVE: To determine the change in expression of the Wilms tumor suppressor gene product, WT1, by progesterone alone in endometrial stromal cell culture and to study its relationship with prolactin, a marker of decidualization. In addition, to examine the change in ratio of WT1 isoforms with and without exon 5 message. METHODS: Endometrial biopsies were taken from eight patients who had hysterectomy. Stromal cells were isolated and cultured in the presence of progesterone alone (12 days) or progesterone and 8-bromo-cyclic adenosine monophosphate (cAMP) (6 days). RNA was extracted from cells, and reverse transcription, real-time polymerase chain reaction (PCR), and conventional PCR were done to analyze WT1 mRNA expression. Immunocytochemistry was performed on equivalent cells to study WT1 protein expression. Decidualization was identified by increased prolactin concentrations in the media and immunocytochemical markers IGFBP-1 and collagen IV. RESULTS: Reverse transcription and real-time PCR revealed a significant increase in WT1 mRNA with increasing progesterone concentrations when decidualization was occurring (n = 6, P =.002). Increasing progesterone concentrations also increased the proportion of the WT1 transcript containing a 17-amino-acid insert (+ exon 5 expression); changes in WT1 exon 5 expression have been shown to be involved in control of proliferation and differentiation. Significant correlations between WT1 message and prolactin existed at physiologic progesterone concentrations (6.25, 12.5, 25, and 50 nM; P <.05) until prolactin concentrations reached a plateau at 100 nM. At concentrations of progesterone alone (> 25 nM) and progesterone with 8-bromo-cAMP, WT1 protein was localized to the nuclei of many of the decidualized stromal cells. CONCLUSION: The changing expression of WT1 isoforms in endometrial stromal cells caused by progesterone may be important for differentiation into the decidualized phenotype.
OBJECTIVE: To determine the change in expression of the Wilms tumor suppressor gene product, WT1, by progesterone alone in endometrial stromal cell culture and to study its relationship with prolactin, a marker of decidualization. In addition, to examine the change in ratio of WT1 isoforms with and without exon 5 message. METHODS: Endometrial biopsies were taken from eight patients who had hysterectomy. Stromal cells were isolated and cultured in the presence of progesterone alone (12 days) or progesterone and 8-bromo-cyclic adenosine monophosphate (cAMP) (6 days). RNA was extracted from cells, and reverse transcription, real-time polymerase chain reaction (PCR), and conventional PCR were done to analyze WT1 mRNA expression. Immunocytochemistry was performed on equivalent cells to study WT1 protein expression. Decidualization was identified by increased prolactin concentrations in the media and immunocytochemical markers IGFBP-1 and collagen IV. RESULTS: Reverse transcription and real-time PCR revealed a significant increase in WT1 mRNA with increasing progesterone concentrations when decidualization was occurring (n = 6, P =.002). Increasing progesterone concentrations also increased the proportion of the WT1 transcript containing a 17-amino-acid insert (+ exon 5 expression); changes in WT1 exon 5 expression have been shown to be involved in control of proliferation and differentiation. Significant correlations between WT1 message and prolactin existed at physiologic progesterone concentrations (6.25, 12.5, 25, and 50 nM; P <.05) until prolactin concentrations reached a plateau at 100 nM. At concentrations of progesterone alone (> 25 nM) and progesterone with 8-bromo-cAMP, WT1 protein was localized to the nuclei of many of the decidualized stromal cells. CONCLUSION: The changing expression of WT1 isoforms in endometrial stromal cells caused by progesterone may be important for differentiation into the decidualized phenotype.
Authors: Lea Beinder; Nina Faehrmann; Rainer Wachtveitl; Ilona Winterfeld; Andrea Hartner; Carlos Menendez-Castro; Manfred Rauh; Matthias Ruebner; Hanna Huebner; Stephanie C Noegel; Helmuth G Doerr; Wolfgang Rascher; Fabian B Fahlbusch Journal: PLoS One Date: 2014-06-23 Impact factor: 3.240