Summary of time line for mouse model of menstruation and regeneration.

<p>Colour coding pink = ‘proliferative phase’, blue = ‘secretory phase’, red = ‘menstrual phase’. Ovex; ovariectomy, E; β-oestradiol, P; progesterone. β-oestradiol concentrations in brackets (ng/100 µl), P4 pellet (1 mg/ml). One uterine horn was stimulated on day 15 via oil injection into the luminal cavity; “menses” was induced by P4 pellet removal on day 19. BrdU was injected 90 minutes prior to tissue recovery at 4, 8, 12 and 24 hours after the removal of the P4 secreting pellet.</p

Proliferation of uterine cells between 4 and 24 hours after P4 withdrawal.

<p>To identify proliferating cells, animals were injected with BrdU 90 minutes prior to tissue recovery. A; Proliferating luminal epithelial cells detected in tissues 4 hours after progesterone withdrawal. B; In the same tissue, stromal cells in the basal layer are positive for BrdU (arrows). C; At 12 hours, luminal epithelial cells were positive for BrdU, no BrdU positive cells were identified in the shed cell mass. D; In the same tissue, stromal cells close to the luminal edge were positive for BrdU (arrowheads), new epithelial cells were identified lining the lumen in an area of tissue where the decidualised tissue had shed (arrows). E; At 24 hours after withdrawal, endothelial cells were positive for BrdU (arrowheads), the intact luminal epithelium was also positive for BrdU. F; In another sample at 24 hours, the stromal compartment was exposed to the lumen (arrowheads); note stromal cells positive for BrdU are interspersed throughout the basal layer and evidence of glandular epithelial cell proliferation was also detected (arrows). BL; Basal layer, LE; luminal epithelium, DS; decidualised stromal cells, M; myometrium, SC; shed cells. Scale bars are equal to 100 µm or 50 µm where indicated.</p

Endometrial remodelling was associated with dynamic changes in concentrations of mRNAs expressed in stromal and epithelial cell types as well as those encoded by genes implicated in MET.

<p>mRNA concentrations for candidate genes involved in mesenchymal to epithelial transition and tissue remodelling; <i>Snai1</i> (Snail), <i>Snai2</i> (Slug), <i>Snai3</i> (Smuc), <i>Wt1</i>, <i>Twist</i> and <i>Mmp3</i> following progesterone withdrawal. mRNA expression for the decidualised horn (black bars) was normalised against the control 0 hour horn. Statistical analysis was performed by Student t test, comparing each time-point to the decidualised 0 hour time-point, *p<0.05, **p<0.01, ***p<0.001.</p

Immunostaining for WT1 illustrates dynamic changes in cellular localisation during breakdown and repair.

<p>A; Immunopoistive staining for WT1 was detected in decidualised stromal cells, stromal cells of the basal layer and the luminal epithelium at the time of progesterone withdrawal. B; At 4 hours, strong immunostaining was maintained in the basal and decidual layers of the tissue. C; By 8 hours, strong immunopositive staining was localised to areas close to the luminal epithelium. D; By 12 hours, fewer immunopositive cells were observed, these were limited to the stroma, no immunostaining for WT1 was detected in the luminal epithelium. LE; luminal epithelium, G: glandular epithelium, SC; shed cells, DS; decidualised stroma, M; myometrium. Inset; negative control. Scale bars are equal to 100 µm where indicated.</p

TGFβ1 inhibited expression of decidualization markers when ESC were decidualized in the presence or absence of TGFβ1 for up to 72 h.

<p>A: TGFβ1 reduced expression of mRNA IGFBP-1 in a time dependent manner, 48 h, p<0.01, 72 h, p<0.001. 1.0 =  absolute value of ΔCT  = 12. B: TGFβ1 inhibited release of IGFBP-1 protein release after only 2 h of treatment (p<0.001) and this continued to decline in a time-dependent manner (all time points p<0.001). C: TGFβ1 inhibited expression of PRL mRNA in a time dependent manner, 48 h (p<0.01), 72 h (p<0.001). 1.0 =  absolute value of ΔCT  = 11. D: TGFβ1 inhibited PRL protein release after only 2 h of treatment (p<0.05) and this was sustained for up to 72 h (12 h, p<0.01, 24 h, p<0.05, 48 h and 72 h, p<0.01). E: TGFβ1 inhibited expression of TF mRNA in a time dependent manner; 24 h (p<0.05), 48 h (p<0.001), 72 h (p<0.001). 1.0 =  absolute value of ΔCT  = 14. Data are mean ± S.E.M; * p<0.05, ** p<0.01, *** p<0.001 vs. control. n = 8 endometrial samples (in triplicate).</p

Number of gene changes in MET PCR array.

<p>Number of gene changes in MET PCR array.</p

Dynamic changes in concentration of mRNAs specific to stromal and epithelial cell compartments.

<p>Comparison between concentrations of mRNAs encoded by genes typically expressed in stromal (<i>Cdh2, Wnt4</i>, vimentin) and epithelial (<i>Cdh1, Wnt7a, Krt8</i>) cells at 0 hours (full decidualisation) and following P4 withdrawal 4, 8, 12 and 24 hours prior to tissue recovery. Statistical analysis was performed by Student t test, comparing each time-point to the 0 hour time-point: *p<0.05, **p<0.01, ***p<0.001.</p

Metacore analysis of genes detected in mouse uterus that were associated with regulation of E cadherin and N cadherin.

<p>To filter data, the full gene array list was input into Metacore™ software and any gene that was found to have no known interaction with E and N cadherin was excluded. Arrows indicate direct effects on other genes in the pathway. Green arrows indicate activation, whereas red arrows show inhibitory action.</p

TGFβ1 suppresses expression and release of markers of decidualization by cells from 1^st trimester decidua.

<p>A: TGFβ1 reduced expression of IGFBP-1 mRNA after 72 h treatment. (p<0.05) 1.0 =  absolute value of ΔCT  = 12.2. B: TGFβ1 was without significant effect on protein release of IGFBP-1. C: TGFβ1 reduced expression of PRL mRNA after 72 h treatment (p<0.001). 1.0 =  absolute value of ΔCT  = 11.5. D: TGFβ1 was without significant effect on PRL protein release, although did display a trend toward inhibition. E: TGFβ1 reduced expression of TF mRNA after 72 h treatment (p<0.05). 1.0 =  absolute value of ΔCT  = 13.75. Data are mean ± S.E.M; * p<0.05, *** p<0.001 vs. no TGFβ1 treatment. n = 7 decidual samples (in triplicate).</p

Gross morphology, bleeding and progesterone concentrations.

<p>A, Mouse at 12 hours after P4 withdrawal showing blood in vagina; B, The non decidualised control (left) and the decidualised horn (right) upon dissection (12 hours after withdrawal); C, Blood cells are detectable in lumen of the non decidualised horn following vaginal lavage (24 hours after withdrawal); D, Shed tissue expelled from cervix (24 hours after withdrawal), decidualised horn shows regression; E, Serum concentrations of progesterone (ng/ml). Statistical analysis was carried out by Student t test, comparing each time-point to the 0 hour time-point *p<0.05 **p<0.01 and ***p<0.001; F. Percentage of mice bleeding between 4 and 24 hours. This was calculated as a percentage of mice that were identified to be bleeding at each time-point of the total number of mice examined at each time-point.</p