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.