Pregnant female rats of the Sprague Dawley strain aged between 10–12 weeks (bred in the Animalario General USC; Santiago de Compostela, Spain), were housed in a temperature-regulated room with a 12 h light/12 h dark cycle, with tap water and standard rat chow ad libitum or a food-restricted diet (see below). At the end of the study period, animals were sacrificed, hypothalami and placentas were removed from each mother, and snap-frozen in liquid nitrogen for RNA extraction and real time semi-quantitative RT-PCR. All experiments and procedures in this study were carried out according to a protocol approved by the Ethics Committee of the University of Santiago de Compostela in accordance with the European Union normative for the care and use of experimental animals.

A maternal chronic food-restriction model was used throughout pregnancy to study the effect of long-term undernutrition on placental neuropeptide mRNA expression as previously described 17. Briefly, virgin rats were mated on the day of proestrus and the day on which spermatozoa were present in vaginal smear, was designated gestational day 1. The pregnant rats were divided into two dietary groups: pregnant rats fed ad libitum and food-restricted group of pregnant rats fed 30% of ad libitum intake. Food-restricted animals were fed every day at 18:00 hours. Rats were sacrificed at gestational days 12, 16 and 21. We used 9 hypothalami and placentas per experimental group, extracted from 9 different mothers. All the samples were analyzed individually and samples were not pooled.

Placental neuropeptide gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR) as previously validated 181920. Total RNA was isolated using Trizol according to the manufacturer's protocol (Invitrogen, CA). First-strand cDNA was synthesized from 2 μg of total RNA by random primer reverse transcription. A negative control without MMLV reverse transcriptase was used to ensure specificity of the PCR amplification. The resulting cDNA was subjected to PCR amplification using sense and antisense primers specific for rat AgRP, CART, NPY and POMC mRNAs (Table 1). Primer pairs were designed overlapping on different exons to prevent amplification from any contaminated genomic DNA (data not shown). To verify the identity of amplified cDNAs, PCR products were electrophoresed on a 1.5% agarose gel, which yielded DNA fragments of the expected length for all mRNAs and were confirmed by sequencing (data not shown). Rat hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as a control housekeeping gene. PCR was performed by real-time in LightCycler real-time PCR machine 2 (Roche Diagnostics, Germany) and the software version 3.5, according to the method described elsewhere 192021. Briefly, the PCR protocol consisted of initial denaturation at 96°C for 5 min, followed by 35 cycles of denaturation (96°C for 2 s), annealing (62°C for 15 s) and elongation (72°C for 15 s). This was followed by melting curve analysis consisting of 1 cycle at 95°C for 30 s, 60°C for 15 s and a temperature rise to 85°C at a slope of 0.2°C/s with continuous measurement of fluorescence. The primers specify was also assured by the melting curve of each gene. All samples were standardized against HPRT by using the 2ΔΔct method 1922.

Firstly, we studied the hypothalamic mRNA levels of NPY during pregnancy and after food restriction. Our data showed that the transcriptional expression of NPY is increased at the latest pregnancy stage (21 days; P < 0.001) with food restriction inducing a further increase at both 16 and 21 days of pregnancy (P < 0.001 and P < 0.05, respectively) (Figure 1A). Next, we evaluated the transcriptional expression of NPY in rat placenta; our results showed that placental mRNA levels of NPY were significantly higher during the initial period of gestation studied (12 days), and decreased significantly (P < 0.001) with gestational age (Figure 1B). We also studied the effect of food restriction on placental mRNA expression of NPY and these data showed that the expression profile of NPY was decreased by food restriction only at day 12 (P < 0.01), and remain unchanged between 16 to 21 days, compared to the ad libitum group (Figure 1B).

Throughout pregnancy, hypothalamic mRNA expression levels of AgRP were decreased (P < 0.01) at 16 and 21 days of gestation compared to 12 days and these levels were increased by food restriction (P < 0.05 and P < 0.01) at all days of gestation studied compared to the ad libitum (Figure 2A). In rat placenta, throughout the same experimental period, the expression mRNA profile of AgRP was opposite to NPY (Figure 2B). Placental AgRP expression remained unchanged between 12 and 16 days of gestation reaching the highest levels at the end of the gestation period (P < 0.001)(Figure 2B). Food restriction induced a significant and progressive increase (P < 0.05) in placenta AgRP mRNA expression at all studied time points.

Hypothalamic POMC mRNA expression was increased during pregnancy (P < 0.05) and decreased by food restriction (P < 0.01) at 12, 16 and 21 days of gestation (Figure 3A). Placental POMC mRNA expression showed a significantly (P < 0.001) increasing trend of expression from 12 to 21 days of gestation in rats fed ad libitum (Figure 3B). Placental POMC expression was significantly (P < 0.01) lower in fed ad libitum rats than in food-restricted group at 12 days. Conversely, significantly (P < 0.01) lower levels of POMC expression were detected in the food-restricted group at compared to fed ad libitum experimental group from 16 to 21 days of gestation.

Hypothalamic CART mRNA expression did not show any change during the different stages of pregnancy and was decreased by food restriction (P < 0.01) at 21 days of gestation (Figure 4A). Placental CART mRNA expression did not change significantly from 12 to 16 days, showing the highest levels (P < 0.001) at 21 days of gestational age. In addition, CART mRNA expression remained unchanged throughout pregnancy from 12 to 16 days of pregnancy in food-restricted rats. Conversely, at 21 days of gestational age mRNA levels of CART were significantly lower in food-restricted rats than in fed ad libitum rats (P < 0.01) (Figure 4B).