2.1 Animals and treatment
Specific pathogen-free adult Wistar female (mean body weight: 209 ± 12 g) and male (mean body weight: 258 ± 17 g) rats (certification No. 42000600002258, license No. SCXK (E) 2012-2014) were provided by the Experimental Centre of the Hubei Medical Scientific Academy (Wuhan, China). Animal experiments were performed in the Center for Animal Experiment of Wuhan University (Wuhan, China), which has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The Committee on the Ethics of Animal Experiments of the Wuhan University School of Medicine approved the protocol (permit number: 14016). All animal experimental procedures were performed following the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee.
Animals were housed in metal cages in an air-conditioned room under standard conditions (environmental temperature: 18–22°C; humidity: 40–60%; light cycle: 12h light-dark cycle; 10–15 air changes per hour) and allowed free access to rat chow and tap water. All rats were acclimated one week before experimentation, and two female rats were placed together with one male rat overnight in a cage for mating. Mating was confirmed by the presence of sperm in the morning vaginal smear, recorded as the gestational day (GD) 0. Pregnant rats were transferred to individual cages and then randomly divided into the control group and PDE group. From GD 9 to GD 20, the PDE group was subcutaneously administrated with 0.2 mg/kg·d dexamethasone (Shuanghe, Pharmaceutical Co., Wuhan, China), while the control group was administered an equal volume of saline every morning (between 08:00-09:00 a.m.).
On GD 20, a subgroup of pregnant rats was anesthetized by isoflurane (Baxter Healthcare Co., Deerfield, IL, USA; 10019-360-40) inhalation, and the fetuses were removed by cesarean section. Each fetoplacental unit was quickly removed from the uterus, and the fetuses were weighed after being dried on filter papers. Pregnant rats with litter sizes of 12 to 14 were considered qualified. The IUGR diagnosis was based on two standard deviations of the fetal rat body weight lower than the mean weight of the control group. Fetal blood was pooled by female grouping per dam and was centrifuged for storage. The placental and fetal adrenal samples collected from littermates were pooled, immediately frozen in liquid nitrogen, and stored at −80°C for subsequent analyses. It should be noted that three whole fetal adrenals in three different fetal rats from each litter were randomly counted as one sample, and processed for gene analysis. The adrenals of five male fetuses in each group were randomly selected and fixed in 4% paraformaldehyde for 24 h. They were then dehydrated with alcohol and embedded in paraffin for further analysis.
The remaining pregnant rats (n=8 for each group) underwent normal delivery. On postnatal day (PD) 1, the numbers of pups were normalized to 8 pups per litter to assure adequate and standardized nutrition until weaning postnatal week (PW) 4. Three male offspring rats from each litter were selected and assigned to the studies at different postnatal time points or conditions (PW12, PW12 with chronic stress and PW28), respectively. The chronic stress test was achieved by forced 5-min ice-water swimming per day (5-7°C) from PW10 to PW12 [25]. At different postnatal time points, male rats were anesthetized with 2% isoflurane and euthanized. Then, the blood sample from the carotid artery was collected, the serum was isolated, and the adrenals and epididymis tissues were rapidly collected. All adrenals were dissected. The left partial adrenal was fixed in a 4% paraformaldehyde solution for histological examination, and the rest were separated and immediately frozen in liquid nitrogen, followed by storage at −80℃ for subsequent analyses. The schedule of animal treatment is shown in Fig S1.