The role of progesterone in endometrial angiogenesis in pregnant and ovariectomised mice

Abstract

The role of progesterone (and oestrogen) in endometrial angiogenesis remains controversial. The aims of this study were to quantify endometrial angiogenesis in pregnant mice and to investigate the role of progesterone in promoting endothelial cell proliferation in ovariectomized mice. Uteri were collected on days 1 to 4 of pregnancy when circulating progesterone concentrations were increasing, prior to implantation. Before dissection, mice were injected with bromodeoxyuridine (BrdU) enabling proliferating endothelial cells to be quantified with CD31/BrdU double-immunohistochemistry. There was a significant increase in proliferating endothelial cells on day 3 of pregnancy when plasma progesterone also increased. To determine if this endothelial cell proliferation was due to progesterone, an experiment was performed on ovariectomised mice. One group was treated with a single oestradiol injection on day 8 after ovariectomy, followed by a no-treatment day and three consecutive daily injections of progesterone. Other groups were treated with either the vehicle, oestradiol or progesterone injections only; all were dissected on day 13 following ovariectomy. Unexpectedly, mice treated with progesterone-only had the highest amount of endothelial cell proliferation and oestrogen priming was found to significantly reduce this progesterone-induced endothelial cell proliferation. To determine if this proliferation is mediated by vascular endothelial growth factor (VEGF), a further experiment in which VEGF anti-serum was administered concurrently with the progesterone injections was performed. Endothelial cell proliferation was reduced but not abolished suggesting progesterone-induced endometrial angiogenesis is only partly mediated by VEGF. Results indicate that oestrogen priming is not required for progesterone to stimulate endometrial endothelial cell proliferation and that oestrogen inhibits progesterone-induced angiogenesis in ovariectomised mice. Reproduction (2005) 129 765-777

Vascular endothelial growth factor-A isoform and (co)receptor expression are differentially regulated by 17β-oestradiol in the ovariectomised mouse uterus

“…Few studies have quantified endothelial cell proliferation in mouse uterus; however, our data are consistent with those previously published that have used a similar methodology. The number of PEC observed at 24 h in the current study is within the range observed in our previous studies of mouse endometrium during early pregnancy (days 1 and 2: no PEC; day 3: 142.3 PEC/mm 2 (2.3-219.4); day 4: 129.0 PEC/mm 2 (34.0-272.6)), after 3 days of progesterone treatment (97.3 PEC/mm 2 (60.8-203.4)), or 24 h following E 2 treatment (179.2 PEC/mm 2 (10.5-307.8)) (Walter et al 2005). While these data demonstrate that endothelial cell proliferation occurs in the endometrium under different hormonal conditions, they do not illustrate whether a specific vessel population is affected.…”

“…A mouse model was adapted from previous studies (Heryanto & Rogers 2002, Heryanto et al 2003, Walter et al 2005 to investigate the effects of E 2 on endometrial angiogenesis and VEGFA isoform and receptor expression in the uterus. Mice were bilaterally ovariectomised following anaesthesia with avertin (i.p.…”

“…A key hormone in this cyclicity is the ovarian steroid 17b-oestradiol (E 2 ), which is responsible for the rapid growth and proliferation of endometrial tissue that occurs during the proliferative stage of the human menstrual cycle. Using a mouse model, we have previously shown that E 2 stimulates endometrial vascular endothelial cell, but not mural cell, proliferation within 24 h of treatment (Heryanto & Rogers 2002, Walter et al 2005, Girling et al 2007. We have also demonstrated that the endothelial cell proliferation in this model was mediated by the endothelial cell mitogen vascular endothelial growth factor-A (VEGFA; Heryanto et al 2003, Walter et al 2005, which signals via the two tyrosine kinase receptors VEGF receptor-1 (VEGFR1) and VEGFR2 and the semaphorin family receptors neuropilin-1 (NRP1) and NRP2 (Neufeld et al 2002, Ferrara et al 2003, Adams & Alitalo 2007.…”

“…Nrp1 and Nrp2 mRNA expression were also analysed in whole uterine tissues in response to E 2 . mRNA expression patterns were compared with VEGFA and VEGFR2 protein expression and evaluated alongside the endothelial cell proliferation that occurs in this model (Heryanto & Rogers 2002, Heryanto et al 2003, Walter et al 2005. We hypothesised that uterine Vegfa 164 mRNA expression, in particular, as well as Vegfr2 mRNA expression would increase in response to E 2 treatment, and that this increase would be restricted to the endometrial stroma where E 2 -induced endothelial cell proliferation occurs.…”

Follistatin is essential for normal postnatal development and function of mouse oviduct and uterus

“…Transgenic mice were slightly older because tghFST315 mice do not demonstrate mating behaviour until ,8 wks of age (Lin et al 2008). Following bilateral ovariectomy, mice were left for a minimum of 7 days to recover and to allow for endometrial regression, before one of two steroid hormone protocols commenced (Heryanto and Rogers 2002;Walter et al 2005Walter et al , 2010Girling et al 2007). Protocol 1 (short-term oestrogen, E): mice were given a single subcutaneous (s.c.) injection of oestradiol-17b (100 ng in 100 mL peanut oil; Sigma-Aldrich, St Louis, MO, USA) or vehicle (100 mL peanut oil) 8 days post-ovariectomy (n ¼ 7-9 per group).…”