1 Introduction [1–3] [4,5] [6] [7] [8] [9] [8] [10] [11] In this study we determined the degree of oxidative stress present in the murine placenta at different gestational ages in normal pregnancies. We also tested whether there was a temporal and spatial association between oxidative stress and two markers of placental function, one physiological and one pathological. These were the induction of cyclooxygenase (COX) enzymes and apoptosis respectively. [12–16] [17] [18] [19] [20] 2 Materials and methods 2.1 Tissue collection Placentas were collected from C57BL/6J inbred mice, and all experiments were carried out in accordance with the UK Government Home Office licensing procedures. Stages E14, E16, E18, and E19 were studied, where E1 of gestation was the morning when a copulation plug was found. 2.2 Western blotting Table 1 2.3 Immunohistochemistry [21] Table 1 In order to localise expression of the antigens to specific cell types staining intensity was graded visually from 0–3, where 0 represented the negative control. Three people scored the sections independently, blinded to the gestational age or experimental protocol, and the mean value was taken. 2.4 Immunofluorescent dual-labelling Dual immunofluorescent labelling was performed in order to test for co-localisation of markers of oxidative stress and COX expression in individual cells. Sections were dewaxed, permeabilised in TBS containing Triton X-100 (0.1%) and Tween 20 (0.1%) (TBS-TT) for 30–60 min, and blocked in 5% bovine serum albumin for 20 min at room temperature. Primary antibodies diluted in TBS-TT were applied overnight at 4 °C. Negative control sections had primary antibodies omitted. After three 10-min washes in TBS-TT, sections were incubated for 1 h at room temperature with a mixture of fluorescent secondary antibodies, containing anti-goat Alexa 488 and anti-rabbit Alexa 568 (both used 1/100; from Molecular Probes) in TBS-TT. Sections were washed in TBS-TT as before and then twice in distilled water for 5 min and subsequently mounted in Vectashield mounting medium containing DAPI (Vector, UK). Images were captured using a Leica confocal microscope (LeicaTCS-NT, Leica Instruments GmbH). Co-localisation was identified in overlay images by a yellow signal caused by the combination of the green signal (COX-1/COX-2) and the red signal (HNE). 2.5 TUNEL assay Apoptotic Index  ( AI : % ) = Number of TUNEL stained nuclei / Number of DAPI stained nuclei × 100 . 2.6 Dual-fluorescence TUNEL assay & Active Caspase-3, and HNE co-localisation Staining was performed using In-Situ Cell Death Detection kit-Texas Red (Roche Applied Science) according to the manufacturer's instructions, and using an Anti-Active Caspase-3 antibody (Promega) at 1:50, and Anti-HNE as previously at 1:200. Samples were also DAPI stained to intercalate the DNA and fluoresce cell nuclei. For negative controls, omission of primary antibody and TdT enzyme were performed separately. For positive controls, post-lactational mouse mammary glands were used. For confocal microscopy, all samples were analysed during one session to avoid bias, and multiple (usually 5) fields of view per labyrinth were saved for further analysis. 2.7 Ex-vivo culture experiment [22] 3 2 [23] 2 2 2 2 2 2 2.8 Statistical analyses P 3 Results 3.1 Markers of oxidative stress P Fig. 1 Fig. 1 Fig. 1 Fig. 1 Nitrotyrosine (NT) staining indicates the formation of the prooxidant peroxynitrite, and of an imbalance in the production of the superoxide ions and nitric oxide. Immunostaining revealed a similar pattern to HNE, with almost undetectable staining in early samples and significant increases from day 16 onwards (data not shown). Therefore, oxidative stress increases during gestation in the murine placenta, specifically in syncytiotrophoblast and glycogen cells. 3.2 COX-1 P Fig. 1 Fig. 2 Fig. 2 3.3 COX-2 P Fig. 1 Fig. 3 Fig. 3 3.4 Detection of apoptotic changes [24] Fig. 4 Immunoreactivity for M30 also increased with gestational age, and was most abundant at E18, declining slightly at E19 (data not shown). P Fig. 4 Fig. 4 Fig. 4 3.5 Ex-vivo culture experiments [22] Fig. 5 Fig. 5 P Fig. 5 From these data it can be concluded that there is a significant increase in oxidative stress following H/R, and that this is associated with increased expression of COX-1 and COX-2 expression in the LZ and JZ, and with increased apoptosis in the labyrinth. 4 Discussion These results indicate that oxidative stress increases with gestational age in the murine placenta during normal pregnancies, and that it may play a significant physiological role by inducing higher concentrations of the COX-1 and COX-2 enzymes, and hence increasing prostaglandin synthesis. Our data also indicate a close temporal and spatial association between oxidative stress and trophoblast apoptosis within the labyrinth. Trophoblast apoptosis has been linked to the pathophysiology of preeclampsia, and the mouse might provide a useful genetic model in which to elucidate the mechanisms underlying the shedding of apoptotic debris. [3,25] [26–30] [31] [32] [10,11,33] [24] [34] [20] [35] [36] [37–39] [6]