All animal procedures were approved by the University of Cambridge Ethical Review Board and were performed in accordance with the UK Animals (Scientific Procedures) Act 1986. The animal experiments were conducted at the Barcroft Centre at the University of Cambridge.

At 100 ± 1 day (d) gestation (term, ∼145 days), 38 pregnant Welsh mountain ewes carrying singleton pregnancies underwent a sterile laparotomy under general anesthesia (1.5%–2.0% isofluorane in 60:40 O₂:N₂O) to determine fetal sex and catheterisation of the maternal femoral artery and vein, as previously described (Brain et al., 2015; Allison et al., 2016; Brain et al., 2019; Botting et al., 2020). To control for sex differences, only female fetuses were included in this postnatal study. Male fetuses were assigned to fetal studies [Brain et al., 2015; McGillick et al., 2017; McGillick et al., 2022; McGillick et al., 2025].

At 103 d gestation, when the fetal lung is in the canalicular phase of development, ewes were randomly assigned to one of two experimental groups: Normoxic (N; n = 20) or Hypoxic (H; n = 18). Ewes allocated to the Normoxic group remained housed in individual floor pens for the duration of the experimental protocol. Pregnant ewes assigned to the Hypoxic group were housed in bespoke isobaric hypoxic chambers (Telstar Ace, Dewsbury, UK) and exposed to ∼10% O₂ from 105 d by altering the incoming inspirate mixture as previously described (Brain et al., 2015). While the Control ewes were not housed in the same chambers as the hypoxic group, we controlled for as many factors as possible, including the pen floor surface area, humidity, temperature, noise levels and the feeding regimen (Brain et al., 2015). Critically, animals in both groups could always see other sheep, thereby minimizing stress. Measurements of stress hormones in maternal plasma during chronic hypoxia confirmed a lack of any change from baseline (Brain et al., 2015). This model of maternal chronic hypoxia also does not affect maternal food intake (Brain et al., 2015).

At 105 d gestation, Normoxic and Hypoxic ewes were randomly divided into 2 groups receiving either a daily bolus (between 09:00-10:00) of intravenous Saline (0.6 mL/kg; Normoxic + Saline, N = 11; Hypoxic + Saline, H = 8) or Vitamin C (200 mg/kg i. v. daily Ascorbate; A-5960; Sigma Chemicals, UK; 1.14 mmol/kg/day dissolved in 0.6 mL/kg saline; Normoxic + Vitamin C, NVC = 9; Hypoxic + Vitamin C, HVC = 10) until 135 d gestation. Vitamin C was chosen for administration in this study due to it being a commonly used water-soluble antioxidant supplement that can cross the placenta (Sales et al., 2019) and shows powerful antioxidant protection in the offspring (Thaket al. 2010; Giussani et al., 2012; Richter et al., 2012; Brain et al., 2019). While Vitamin C can be easily administered orally daily in humans, the maternal i. v. route was chosen in this study to ensure complete and controlled delivery into the maternal circulation. The effect of maternal chronic hypoxia and antenatal Vitamin C administration on maternal and fetal blood gas status, cortisol and Vitamin C concentrations have been previously reported (Brain et al., 2015; McGillick et al., 2017; McGillick et al., 2022).

At 138 d gestation, pregnant ewes were transferred from the hypoxic chambers to individual pens in a barn (same as Control ewes) with a 12:12-h light–dark cycle, where they were returned to normoxic conditions. Ewes were allowed to deliver naturally, lambs were weighed within 12 h of birth, and they remained with their mothers until weaning at 3 months. Following weaning, lambs were maintained in a farm grazing on grass with free access to water in paddocks that belong to the Barcroft Centre at the University of Cambridge.

At 9 months after birth, past sexual maturity and into early adulthood, lambs were euthanised by an overdose of sodium pentobarbitone (0.4 mL/kg, intravenous administration, Pentoject; Animal Ltd., York, UK). Body and organ weights were recorded. A piece of left lung tissue was snap-frozen in liquid nitrogen and stored at −80°C for measuring hormone concentrations, and gene and protein expression analysis. A section of right lung tissue was immersion fixed in 4% paraformaldehyde and processed to paraffin for immunohistochemical analysis.

The tissues generated in this study were part of a programme of work designed with the primary objective of investigating cardiovascular physiology (Brain et al., 2019). This study used the tissues generated in a subset of the cohort to address additional scientific questions retrospectively. This scientific approach is strongly promoted by the UK Home Office 3R principle of Replacement, Reduction and Refinement, thereby making best use of the valuable experimental material (Russell and Burch, 1959). Consequently, no prospective pulmonary functional outcomes were performed, and the lung tissue collected was immersion fixed rather than perfusion fixed.

Lamb lung hormone concentrations (N = 11, NVC = 9, H = 8, NVC = 10) were determined by liquid chromatography (LC; Shimadzu Nexera XR, Shimadzu, Japan) coupled to a SCIEX 6500 Triple-Quad tandem mass spectrometry system (MS/MS; SCIEX, US) using an adapted protocol (McBride et al., 2020; Dimasi et al., 2023). Lung tissue hormones measured were cortisol, cortisone, triiodothyronine (T3) and thyroxine (T4). Initially, lung tissue was homogenised in 500 μL 0.9% NaCl at 50 h/z for 2 min and then centrifuged at 16,000 g for 10 min at 4°C. 100 μL of supernatant was added to 300 μL acetonitrile containing 50 ng/mL internal standard (cortisol-9,11,12,12-d4; Toronto Research Chemicals, Toronto, Canada), vortexed for 1 min and then centrifuged at 12,000 g for 10 min. Supernatant was transferred to a fresh Eppendorf tube and the remaining pellet was resuspended in 300 μL ethyl acetate, vortexed for 1 min and then centrifuged at 12,000 g for 10 min. Supernatant was added to the acetonitrile, mixed by inversion, and then evaporated to dryness using the GeneVac EZ-2 Evaporating System (GeneVac, UK). Dried samples were reconstituted in 50% methanol and then injected onto an ACQUITY UPLC BEH C18 Column 130Å, 1.7 µm, 2.1 mm × 100 mm (Waters Corp, US). Mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Flow rate was 0.3 mL/min and mobile phase B was initially 10% and increased linearly to 90% over 10 min and then held at 90% for 2 min, after which it returned to 10% over 30°and then held for 3 min prior to injection of the next sample. Hormone concentrations were calculated via integration with a standard curve. Data are presented indexed to lung tissue weight. Data is presented as individual hormone concentrations as well as ratios of cortisol/cortisone and T4/T3.

RNA was extracted and cDNA synthesized from lamb lung tissue samples (∼50 mg; NS = 11; NVC = 9; HS = 8; HVC = 10) using QIAzol Lysis Reagent Solution and Qiagen miRNeasy purification columns (Qiagen, Victoria, Australia) and cDNA was synthesised using Superscript III First Strand Synthesis System (Invitrogen, Carlsbad, CA, United States) as previously described (Soo et al., 2012; McGillick et al., 2013; Lie et al., 2014; McGillick et al., 2022). The expression of target genes regulating oxidative stress, antioxidant defences, hypoxia signalling, glucocorticoid signalling, surfactant maturation (protein and lipid synthesis), inflammation and airway remodelling were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR; Table 1), as previously described (Soo et al., 2012; McGillick et al., 2013; Lie et al., 2014; McGillick et al., 2022). Gene expression was measured by qRT-PCR using Fast SYBRR Green Master Mix (Applied Biosystems, Foster City, CA, United States) in a final volume of 6 μL on a ViiA7 Fast Real-time PCR system (Applied Biosystems) as described previously (McGillick et al., 2013; McGillick et al., 2015; McGillick et al., 2016b). The abundance of each transcript relative to the abundance of stable reference genes (beta-actin, peptidylprolyl isomerase, tyrosine 3-monooxygenase) was calculated using DataAssist 3.0 analysis software and is expressed as mRNA mean normalized expression (MNE) ± SEM (Soo et al., 2012; McGillick et al., 2013; Lie et al., 2014; McGillick et al., 2022).

Protein was extracted by sonication of lamb lung tissue (∼100 mg, NS n = 6, NVC n = 7, HS n = 7, NVC n = 7) and protein content determined by a MicroBCA Protein Assay Kit (PIERCE, Thermo Fisher Scientific Inc., Rockford, Illinois) as previously described (Darby et al., 2019; McGillick et al., 2022). Extracted protein samples (75 μg) were subject to sodium dodecyl surface (SDS) page and stained with Coomassie blue to determine equal protein loading. Protein samples were transferred onto a 0.45 µm nitrocellulose membrane (Hybond ECL, GE Healthcare, NSW, Australia), and then stained with Ponceau S (0.5% Ponceau in 1% acetic acid) in order to determine the efficacy of the transfer. The membranes were briefly washed with 7% acetic acid followed by a reverse osmosis water rinse, and then imaged for Ponceau S using ImageQuant LAS4000 (GE Healthcare, Victoria, Australia). Following imaging, membranes were washed 3 × 5 min in tris-buffered saline (TBS). The membranes were blocked in 5% bovine serum albumin (BSA) in TBS with 1% Tween-20 (TBS-T) for 1 h at room temperature. The membranes underwent 3 × 5 min washes in TBS-T and were incubated with the primary antibody overnight at 4°C.

Primary antibodies of interest were Mitobiogenesis Western Blot Cocktail (MITOBIO: 1:250, in 5% BSA in TBST, #ab123545, Abcam; 70, 42 (β-actin internal loading control), 35 kDa), OxPhos Rodent Western blot Antibody Cocktail (1:500, in 5% BSA in TBST, #ab110413, Abcam, 20, 30, 40, 48, 55 kDa band), Catalase (1:1,000, in 5% BSA in TBS-T, #14097S, Cell Signalling Technology; 60 kDa band), PHD-1 (1:1,000, in 5% BSA in TBS-T, #ab108980, Abcam; 44 kDa band), 11βHSD-1 (1:1,000, in 5% BSA in TBS-T, #ab39364, abcam; 40 kDa band), ABCA3 (1:500, in 5% BSA in TBS-T, #WRAB-ABCA3, Seven Hills Bioreagents; 130 kDa band) and Elastin (1:1,000, in 5% BSA in TBS-T, #ab213720, Abcam; 68 kDa band). All antibodies were validated by testing multiple dilutions to identify proteins of interest at their respective kDa bands without non-specific staining by our group in sheep tissue. Protein expression determined using the MITOBIO, OxPhos, PHD-1 and 11βHSD-1 antibodies have been published previously by our group (Orgeig et al., 2015; Darby et al., 2019; Dimasi et al., 2023). The commercially available ABCA3 and Catalase antibodies were acquired specifically for this study and the kDa bands analyzed were cross-checked with the expected kDa from the antibody supplier and previous studies using the same antibody in a similar context. Apart from mitochondrial function antibodies, the remaining primary antibodies were chosen based on genes that changed in response to chronic fetal hypoxia and/or antenatal Vitamin C (McGillick et al., 2017; McGillick et al., 2022) to determine if the transcriptional changes observed translated into protein abundance differences. Following incubation with the primary antibody, the blots were washed and incubated with the relevant species of Horse Radish Peroxidase labelled secondary IgG antibody for 1 h at room temperature. Enhanced chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Australia) was used to detect the blots. The Western blot was imaged using ImageQuant LAS4000 and the protein abundance was quantified by densitometry using Image quant software (GE Healthcare, Victoria, Australia). Total target protein abundance was then normalized to Ponceau S (total protein stain) or reference protein Vinculin (1:2000, in 5% BSA IN TBST, XP HRP conjugate, #18799S, Cell Signalling Technology; 140 kDa band). Mitochondrial abundance was determined via a ratio of mitochondria DNA-encoded COX-I to nuclear DNA-encoded COXII as previously described (Dimasi et al., 2023).

To determine the effect of chronic fetal hypoxia and antenatal Vitamin C administration on the surfactant producing capacity of the postnatal lung at the structural level, immunohistochemistry was performed (NS = 5; NVC = 6; HS = 5; HVC = 6) using a rabbit anti-human mature surfactant protein B (SP-B) antibody (1:500, WRAB-48604, Seven Hills Bioreagents, Ohio), as previously described (McGillick et al., 2017; McGillick et al., 2022). In short, lung tissue slides (5 µm thickness) were baked at 60°C for 1 h followed by deparaffinization and rehydration. After rehydration, endogenous peroxide solution activity was blocked with 3% hydrogen peroxide (Sigma-Aldrich; St. Louis, MO), followed by heat-induced antigen retrieval in citrate buffer (pH 6.0). Slides were incubated overnight with a primary antibody at 4°C following incubation with non-immune serum (serum blocking solution; Histostain-Plus Kit; Invitrogen, Carlsbad, CA) to prevent non-specific binding. Negative control slides with the primary antibody omitted were used to demonstrate no nonspecific binding of the secondary antibody or reagent contamination (Figure 4A). In addition, negative control slides where incubation with primary antibody was substituted for rabbit serum (Sigma-Aldrich) at the same protein concentration as the diluted primary antibody (Figure 4B) (1:500) was carried out to highlight primary antibody specificity in the positive control slides in the alveolar epithelium observed in this study. A Histostain-Plus kit (Invitrogen) was used with horseradish peroxidase and Histostain-Plus broad spectrum 3,3′-diaminobenzidine chromagen for visualization of the SFTP-B positive cells. All sections were counterstained with Mayer’s hematoxylin (Sigma-Aldrich).

Stained lung tissue sections were examined using Visiopharm new Computer Assisted Stereological Toolbox (NewCAST) software (Visiopharm, Hoersholm, Denmark). Positively staining cells were identified by their cuboidal shape and location in the alveolar epithelium (Figures 4C–F). Analysis was carried out by a trained individual who was blinded to treatment groups. Sixty counting frames (×600 magnification) of the alveolar epithelium were randomly selected per section. Point counting with an unbiased counting frame with an area of 20,000 μm² was used to determine the numerical density of SFTP-B positive cells present in the alveolar epithelium of lung tissue, as previously described (Lock et al., 2015; McGillick et al., 2017; McGillick et al., 2022).

All statistical analyses were performed using GraphPad Prism v8. All data were evaluated for outliers ± 2 SD from the mean for each treatment group. All data were tested for normality and transformed if required. Data were analysed using a Two-Way ANOVA for main effects of treatment (Normoxic vs. Hypoxic) and drug (Saline vs. Vitamin C) and an interaction between factors (treatment x drug). When a significant interaction was detected between the two factors (CAT and HIF-2a gene expression), data were split by treatment to determine the effect of drug administration (S vs. VC) in Normoxic or Hypoxic lambs using the Students’ unpaired t-test. All data are presented as mean ± SEM. For all comparisons, P < 0.05 was considered statistically significant.

There was a significant effect of treatment with lambs exposed to chronic fetal hypoxia for a month in late gestation having reduced body weight at birth compared to lambs from normoxic pregnancies (Table 2). Conversely, there was no significant effect of exposure to chronic fetal hypoxia or antenatal Vitamin C on body and organ measures at 9 months of age (Table 2).

Chronic fetal hypoxia significantly reduced HMOX-1 expression (Figure 1A), while it increased NOX-4 mRNA expression in the lung of young adult lambs (Figure 1B). Antenatal Vitamin C treatment in hypoxic pregnancy normalized these changes (Figures 1A, B). Antenatal Vitamin C treatment in normoxic pregnancy also increased HMOX-1 (Figure 1A) and reduced NOX-4 mRNA expression (Figure 1B). There was no effect of chronic fetal hypoxia or antenatal Vitamin C on SOD-1 mRNA expression in the lungs of young adult lambs (Table 3). There was a significant effect of antenatal Vitamin C in hypoxic pregnancy, increasing CAT mRNA expression in the lung of young adult lambs (Figure 1C), but no significant effect on CAT protein expression (Figure 1D).

There was no effect of chronic fetal hypoxia or antenatal Vitamin C on expression of HIF-1α, EGLN-1 or EGLN-3 gene expression in the lung of young adult lambs (Table 3). Antenatal Vitamin C exposure upregulated HIF-2α gene expression in the lung of lambs exposed to chronic fetal hypoxia (Figure 1E). Antenatal Vitamin C exposure downregulated EGLN-2 gene (Figure 1F) and PHD-1 protein expression in the lung of young adult lambs of normoxic and hypoxic pregnancy (Figure 1G). There was no significant effect of chronic fetal hypoxia or antenatal Vitamin C on downstream signalling through genes with hypoxia-responsive elements including VEGF, ADM and KDM3A (Table 3).

There was no effect of chronic fetal hypoxia or antenatal Vitamin C exposure on mitochondrial abundance protein expression (Figure 2A). There was a significant effect of antenatal Vitamin C exposure, reducing expression of oxidative phosphorylation (OXPHOS) complex 1, 2 and 3 (Figure 2B–D), but no effect on complexes 4 or 5 (Figures 2E, F) in the lung of young adult lambs.

Lung tissue corticosterone, 11-deoxycortisol and progesterone concentrations were below the limit of quantitation (LOQ; corticosterone LOQ = 0.1 ng/mL, 11deoxycortisol LOQ = 0.5 ng/mL, progesterone LOQ = 0.05 ng/mL). There was no significant effect of chronic fetal hypoxia or Vitamin C exposure on lung tissue cortisol, cortisone, ratio of cortisol/cortisone, thyroid hormone triiodothyronine (T3), thyroid hormone Thyroxine (T4) or ratio of T3/T4 concentrations (Table 2).

Exposure to antenatal Vitamin C increased glucocorticoid activating enzyme HSD11B-1 gene expression (Figure 3A) and decreased 11β HSD-1 protein expression in the lung of young adult lambs (Figure 3B). There was no effect of chronic fetal hypoxia or antenatal Vitamin C on mRNA expression of glucocorticoid de-activating enzyme HSD11B-2 or the glucocorticoid receptor NR3C1 (Table 3).

There was no significant effect of chronic fetal hypoxia or antenatal Vitamin C on mRNA expression of the surfactant proteins or lipid transporter PCYT1A in the lung of young adult lambs (Table 3). Exposure to chronic fetal hypoxia significantly upregulated surfactant lipid transporter ABCA3 mRNA expression (Figure 3C) but had no significant effect on protein expression in the lung of young adult lambs (Figure 3D).

There was a significant decrease in IL-1B mRNA expression in the lung of young adult lambs exposed to chronic fetal hypoxia, while exposure to antenatal Vitamin C increased its expression compared to saline lambs (Figure 3E). There was no effect of exposure to chronic fetal hypoxia or antenatal Vitamin C on the expression of TGFB1 (Table 3).

There was no significant effect of chronic fetal hypoxia or antenatal Vitamin C on the number of SP-B producing cells in the alveolar epithelium in the lung of young adult lambs (Figures 4A–G). Chronic fetal hypoxia increased gene expression of ELN, while antenatal Vitamin C normalised its expression (Figure 4H). There was no significant effect of chronic fetal hypoxia or antenatal Vitamin C on elastin protein expression (Figure 4I) or mRNA expression of COL1A1 or MMP-9 (Table 3) in the lung of young adult lambs.