Progesterone stimulates expression of follistatin splice variants Fst288 and Fst315 in the mouse uterus  R.G. Craythorn, W.R. Winnall, F. Lederman, E.J. Gold, A.E. O’Connor, D.M. de Kretser, M.P. Hedger, P.A.W. Rogers, J.E. Girling  Reproductive BioMedicine Online  Volume 24, Issue 3, Pages 364-374 (March 2012) DOI: 10.1016/j.rbmo.2011.12.004 Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Follistatin splice variant mRNA expression increases on day 3 of pregnancy in the mouse uterus. Expression of (A) total follistatin, (B) Fst288, (C) Fst315, (D) Inhba and (E) Inha mRNA in the mouse uterus during the first 5days of pregnancy. Values are fold induction from the average of all samples from day 1: note the different scales in the different parts. Sample size: days 1–3, n=10 mice; day 4, n=11; day 5, n=12. Columns represent back-transformed logarithmic means and 95% confidence internals. Columns with different letters are statistically different (P<0.05). Reproductive BioMedicine Online 2012 24, 364-374DOI: (10.1016/j.rbmo.2011.12.004) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Inhba, but not Fst288 or Fst315, mRNA expression increases in response to 17β-oestradiol treatment. Expression of (A) total follistatin, (B) Fst288, (C) Fst315, (D) Inhba and (E) Inha mRNA in the mouse uterus following treatment with either a single injection of 17β-oestradiol or vehicle and dissection 24h after the injection. Values are fold induction from the average of all vehicle-treated samples: note the different scales in the different parts. Sample size: n=6 per treatment group. Columns represent back-transformed logarithmic means and 95% confidence internals. Columns with different letters are statistically different (P<0.05). Reproductive BioMedicine Online 2012 24, 364-374DOI: (10.1016/j.rbmo.2011.12.004) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Follistatin splice variant expression increases in response to progesterone. Expression of (A) total follistatin, (B) Fst288, (C) Fst315, (D) Inhba and (E) Inha mRNA in the uteri of ovariectomized mice treated with vehicle, 17β-oestradiol only (E only), progesterone-only (P only) and progesterone following 17β-oestradiol priming (E+P). Values are fold induction from the average of all vehicle-treated samples: note the different scales in the different parts. Sample sizes: vehicle, oestradiol only and P-only, n=7; E+P, n=8. Columns represent back-transformed logarithmic means and 95% confidence internals. Columns with different letters are statistically different (P<0.05). Reproductive BioMedicine Online 2012 24, 364-374DOI: (10.1016/j.rbmo.2011.12.004) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Activin βA immunostaining in the mouse uterus. (A–C) Representative micrographs of activin βA immunostaining in the mouse uterus on (A) day 2 and (B,C) day 5 of pregnancy: (C) provides a higher magnification image of the area indicated in (B) and illustrates the variation in stromal staining intensity. (D–F) Representative micrographs of activin βA immunostaining in ovariectomized mouse uteri treated with (D) vehicle, (E) 17β-oestradiol only or (F) progesterone only. g=glandular epithelium; l=lumen; m=myometrium; arrows indicate blood vessels. (G,H) Semi-quantitative grading of activin βA immunostaining in the (G) glandular and (H) luminal epithelium of the mouse uterus on days 1–5 of pregnancy. Graphs are representative of data from three animals: dots illustrate the grading from each animal; horizontal lines represent the median. Values with different letters are statistically different (P<0.05). Reproductive BioMedicine Online 2012 24, 364-374DOI: (10.1016/j.rbmo.2011.12.004) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions