Sexual differences of the effects of prenatal stress on the expression of extracellular signal-regulated kinaseas in the hippocampus of offspring rats

BACKGROUND: Prenatal stress has been shown to inhibit cell proliferation in the dentate gyrus and hippocampus, reduce hippocampal volume, and cause neuronal loss and oxidative damage in the hippocampus of offspring rats, but the sexual difference of the effects on offsprings is seldom referred to. OBJECTIVE: To observe the effect of prenatal stress to adult pregnant rats on expression of extracellular signal-regulated kinases (ERK) in hippocampus of the offspring rats of different genders. DESIGN: A randomized and control animal experiment. SETTING: Department of Physiology and Pathophysiology, School of Medicine, Xi’an Jiaotong University. MATERIALS: The experiments were carried out in the Key Laboratory of Environment and Gene Related Diseases (Xi’an Jiaotong University), Ministry of Education between October 2005 and March 2006. Fifteen female and five male adult Sprague-Dawley rats were adopted. Female rats weighing 230-250 g and male rats weighing 280-350 g were used. METHODS: The virgin female rats were placed overnight with adult male rats (3:1) for mating. A total of twelve pregnant rats were randomly assigned to prenatal stress group (PNS group, n=6) and control group (n=6). The pregnant rats of the PNS group were exposed to restraint stress on days 14-20 of pregnancy three times a day, 45 minutes for each time[9,13]. The restraint device was a transparent plastic tube (6.8 cm in diameter) with air holes for breathing and closed end. The length could be adjusted to accommodate the size of the animals. To prevent habituation of animals to the daily procedure, restraint periods were randomly shifted within certain time periods (8:00-11:00, 11:00-14:00, and 16:00-19:00). After birth, offsprings of all groups were culled to 8-10 litters in each group and housed in the same animal room, and kept together with their biologic mothers. The pregnant rats of the control group were left undisturbed. On day 21, after all the offspring were weaned, male and female pups were separated and housed four in each cage respectively until test at 30 days of age. At the end of postnatal day 30, one male and female offspring rats from the same dam were selected with a random choice and a total of 24 animals from 12 different dams were used. The experimental rats were sacrificed by decapitation under anesthesia. Bilateral hippocampal tissues were isolated and homogenized in cold condition. Alkaline carbonate buffer (BCA) method was used to detect the concentration of extracellular signal-regulated kinases (ERK), then mixed with loading buffer, the constant voltage was 100 V. Finally, BCIP/NBT staining and electrophoresis were performed, the absorbance (A) value for the bands was detected with the Bandscan analytical software, and the expression of ERK in hippocampus of offspring rats of different genders in each group was quantitatively analyzed. MAIN OUTCOME MEASURES: The level of ERK expression in hippocampus of offspring rats of different genders in each group was observed. RESULTS: All the 24 offspring rats were involved in the analysis of results. ① The staining results of ERP activity in the extract of brain tissue detected with Western blotting technique and specific antibody analysis showed that the ERP in hippocampus of offspring rats had two subtypes of ERK-1 and ERK-2, and the latter was the main type. ② Standardized by the average A value in the control group, the quantitative data of the general A value of total ERK showed that the expression of ERK-2 in hippocampus of female offspring rats was obviously higher in the PNS group than in the control group (A value: 126±6.76,100±4.89,P < 0.01). ③ The expression of ERK-2 had no obvious difference between the female and male offspring rats in the control group. ④ The expression of ERK-2 in hippocampus of male offspring rats was a little higher in the PNS group than in the control group (A value: 104±6.27,102±5.48,P > 0.05). CONCLUSION: PNS significantly affects the increase of ERK expression in hippocampus of female offspring rats, but has no obvious influence on that of male ones.