Effect Of Maternal Dietary N-3 Polyunsaturated Fatty Acids During Pregnancy And Lactation On Neurogenesis And Apoptosis In Adult Brain Of The Offspring

Background and aims: N-3 polyunsaturated fatty acids(n-3 PUFAs), especially docosahexaenoic acid(DHA, C22:6 n-3 PUFAs) plays crucial roles in the development and function of brain. DHA accretion in neural tissue peaks in the last trimester of pregnancy and 1~2 years after birth. Nowdays, maternal diets in many countries are lack of n-3 PUFAs when compared with the recommended amount by the WHO(at least 200 mg DHA/day). Inadequate intake of n-3 PUFAs can influence brain plasticity, and lead to abnormal learning ability and congnitive behaviour. Moreover, these effects may persist into adulthood and increase the occurrence of neuropsychiatric disorders, such as depression, anxiety and autism. However, it is not entirely clear about the related mechanism of n-3 PUFAs lasting impact on brain function. In our previous study, while feeding maternal mice on diets with variable n-3 PUFAs(ratios of n-6/n-3 PUFAs intended to 15.7:1, 6.3:1, 1.6:1 to 1:5.7)and n-3 PUFAs deficiency during pregnancy and lactation, we found n-3 PUFA containing diets, particularly those with n-6/n-3 PUFA ratios of 6.3:1 and 1.6:1, significantly accumulate brain development. And on this basis we utilized n-3 PUFAs deficient and n-3 PUFAs adequate diets(n-6/n-3 PUFA ratios of 6.3:1), supplied during maternal pregnancy and lactation, to asses the histological changes of hippocampal neurogenesis and apoptosis, bdnf transcripts(II, IV, VI and IX) levels in adult brain of the offsprings, as well as the epigenetics mechanism of bdnf transcripts expression modulated by n-3 PUFAs in early life.Methods: Twenty 3-4 weeks old C57BL/6J female mice were randomly assigned to 2 dietary groups: n-3 PUFA deficient diet(n-3 def) vs n-3 PUFA adequate diet(n-3 adq). Followed by 8 weeks’ feeding, the mice were mated for breeding pups. At 21 d postnatally, equal male pups from each of the n-3 def diet and n-3 adq diet groups were selected, and then crossed over the opposite diet. The rest male pups were fed with the same diets as their mothers. Thus, four experimental groups were generated, i.e., the continued feeding with the n-3 def diet group during the whole life period(n-3 def); feeding with the n-3 adq diet to pups from the maternal n-3 def diet group(def-adq); the continued feeding with the n-3 adq diet group during the whole life period(n-3 adq); feeding with the n-3 def diet to pups from the maternal n-3 adq diet group(adq-Def). At 3 months of age, the offspring mice were sacrificed and the brain was selected. Seven to nine pups were chosen for each group, Fatty acids in the brain were analyzed by gas chromatography and expressed as percent(%) of the total fatty acids. Expressions of the Calretinin(CR), B-cell lymphoma-2(Bcl-2) and Bcl-2 associated X protein(Bax) in the hippocampus were measured by immunohistochemistry. The mRNA expressions of BDNF transcripts in the brain cortex were measured by real-time RT-PCR. Bisulfite sequencing-PCR(BSP) was used to determine the methylation status of the bdnf promoter.Results:1. Effect of maternal n-3 PUFAs nutrition during pregnancy and lactation on fatty acids profiles in adult brains of the offspringsWhen compared to the n-3 def group, the def-adq group significantly increased concentrations of epicosapentaenoic acid(EPA), DHA and total n-3 PUFAs, and decreased concentrations of arachidonic acid(AA) and total n-6 PUFAs. Moreover, in these parameters, brain DHA, total n-6 and n-3 PUFAs levels in adulthood reached to levels of the n-3 adq group. Meanwhile, compared to the n-3 adq group, the adq-def group decreased the levels of DHA and total n-3 PUFAs, all of which were still higher than those of the n-3 def group.2. Effect of maternal n-3 PUFAs nutrition during pregnancy and lactation on histological changes in hippocampus CA3 of adult offspringsImmunohistochemical analyses with antibodies specific for brain calretinin demonstrated changes in the expression level of this marker among four experimental groups. In general, compared to the n-3 def group, the average optical density(OD) and average numerical density on area(NA) of calretinin positive cells in hippocampus CA3 showed no changes in the def-adq group(P>0.05), but were still lower than that in the n-3 adq group(P<0.05). Compared to the n-3 adq group, the adq-def group did not reduce the expression of calretinin, which was still higher than that in the n-3 def group(P<0.05).Overall, compared to the pups in the n-3 def group, the OD and NA on area of the Bcl-2 did not show any differences in the def-adq group(P>0.05), but were still lower than that in the n-3 adq group(P<0.05). Compared to the n-3 adq group, no changes occurred in Bcl-2 expression in the adq-def group, which were still higher than that in the n-3 def group(P<0.05). There was no difference in the expression of Bax among these four groups.3. Effect of maternal n-3 PUFAs nutrition during pregnancy and lactation on expression levels of brain BDNF transcripts in adult offspringsThe mRNA levels of four BDNF transcripts(II, IV, VI and IX) in brain cortex were detected. For BDNF transcript II and VI, the mRNA expressions were shown no difference among the n-3 def, def-adq, n-3 adq and adq-def groups. For the mRNA expression of BDNF transcript IV, no significant differences were found in the def group and the def-adq group, which were lower than that of the n-3 adq group. Meanwhile, no changes also occurred in the expression of BDNF transcript IV between the n-3 adq and adq-def groups.For BDNF transcript IX, the mRNA expression in the def-adq group increased when compared to the n-3 def group, but was still lower than that in the n-3 adq group. No differences were shown in the expression of BDNF transcript IX between the n-3 adq and adq-def groups.4. Effect of maternal n-3 PUFAs nutrition during pregnancy and lactation on methylation levels of brain BDNF transcripts of adult offspringsIn the subsequent experiment, we detected the changes of DNA methylation at BDNF transcript IV and IX, which showed mRNA expression changes with supplementation of n-3 PUFA in early life. Compared to the n-3 def group, the methylation levels of CG4, CG5, CG7 and CG9 at BDNF transcript IV, and CG1, CG4 at BDNF transcript IX decreased significantly in n-3 adq group. But no changes occurred in the methylation levels of most CG sites at BDNF transcript IV(except CG9) and IX between the n-3 def and def-adq groups. Similarly, there was no difference in the methylation levels of the n-3 adq and adq-def groups.Conclusions:The appropriate n-3 PUFA intake during maternal pregnancy and lactation may be beneficial for neurogenesis and anti-apoptosis of offspring in adulthood. N-3 PUFAs in early life may be beneficial for maintaining the transcription of brain bdnf in adults, and DNA methylation may be one of the mechanisms that contribute to it.