| The rate of older-age fertility increases year by year,which has become a global trend.In China,the second-child policy has been implemented on January 1,2016.Therefore,the issue about delayed motherhood arose more attention.There are many negative effects of delayed motherhood,including the higher incidence of Down syndrome and mental disorders in offspring.Epidemiological studies based on large samples demonstrated that delayed motherhood have a negative impact on the mental development of offspring.In our lab,it has been proved that the learning and memory ability decreased in offspring born to old female SD rats through Morris water maze.However,there is no relevant data in mice model.The development of intelligence is related to hippocampus which locates between brain thalamus and medial temporal lobe.In hippocampus,the learning and memory functions are mainly performed by neurons and glial cells.Neurons,which are marked by MAP2,exchange information by receiving,integrating,transmitting,and exporting information.Astrocytes marked by GFAP,which are the main components of glial cells,play a crucial role in maintaining the growth,development,and survival of neurons.Therefore,abnormal development or function of the hippocampus will cause changes in learning and memory ability.Heat shock proteins(Hsps)are a group of highly conserved proteins that play an important role in the correct folding of nascent polypeptides,the formation and stabilization of protein complexes.Heat shock cognate protein 70(HSC70)is a major structural active member of the Hsps family.Unlike Hsp70 which is highly expressed when cells are in a stress environment to exert stress protection,HSC70 is fundamentally expressed in normal cells to maintain the basic shape and function of cells.HSC70 is abundantly expressed in the nervous system and is regulated temporally and spatially with the development of the nervous system,suggesting its involvement in neurodevelopment.This study was to investigate the effect of older-age fertility in C57BL/6 female mice on learning and memory of the offspring,and to detect the expression characteristics of HSC70 in the hippocampus of offspring during the process of neural development,and to provide new ideas for medical research and fertility guidance related to older-age fertilityMethods 1.Experimental animals and groups: Clean grade C57BL/6 mice.Older female mice(11 months of age)and young female mice(3 months of age)were individually caged with 3 month old male mice.After the female is pregnant,the male and female mice are divided into cages.After the females gave birth,the offspring of the older females were recorded as the old mother group,and the offspring of the young females were recorded as young mother group,and the experimental mice were weighed.2.Morris water maze test mice learning and memory ability: Postnatal 28 days(P28)is brain maturity and postnatal 60 days(P60)is adulthood.The mice were divided into two groups,the first group was raised to postnatal 28 days for water maze test,the second group was raised to postnatal 60 day for water maze test,and record their pre-experimental body weight and related indicators in the place navigation test and spatial probe test.3.Immunohistochemistry staining was used to detect the expression of HSC70 protein in hippocampus: Whole brain tissue of the P28 young mother group and old mother group were sacrificed and immunohistochemical staining was performed to detect the expression of HSC70,MAP2 and GFAP in the hippocampus.The fluorescence intensity was measured by the confocal microscope and Image-Pro Plus software.4.Western blot detection of HSC70 protein expression in hippocampus during development: hippocampus was taken after 1,3,7,14,21,28,and 60 days after birth in the young mother group and old mother groups,Western blotting was used to dynamically detect the expression of HSC70 protein in mouse hippocampus during development.5.Western blot detection of HSC70 and HSP70 protein expression in hippocampus before and after water maze test: to detect the changes of HSC70 and HSP70 expression in hippocampus before and after the mouse water maze in young mother group at P28.6.Statistical analysis: The data statistics involved in this article were analyzed by SPSS 17.0 software.The place navigation test uses multi-factor analysis of variance of repeated measurement data.When two sets of samples are compared,two independent sample t-tests are used if they have a normal distribution and the variance is equal;a normal t-test is used when the distribution is normal but the variance is not correct;the rank sum test is used when the normal distribution is not satisfied.The test level is ? = 0.05.Results 1.Body weight of mice: At the P28 young mother and old mother groups,the mean body weights of mice were 16.98 ± 1.11 g and 16.05 ± 1.15 g;at the P60 young mother and old mother groups,the mean body weight was 27.21 ± 1.64 g and 26.37 ± 1.74 g.No statistical significance(P> 0.05).2.Morris water maze results: With the extension of training time from day 1 to day 6 in the place navigation test,the incubation period of the old mother group and young mother group(P28 and P60)reached the platform was significantly reduced(P < 0.05).On the day 6 of training,at P28 the incubation period was significantly longer in the old mother group than in the young mother group(P < 0.05),and there was no significant difference in the incubation period between the old mother group and the young mother group at P60(P > 0.05);there was no significant difference in swimming distance in the old mother group compared with the young mother group at P28 and P60(P > 0.05).On the day 7 of spatial probe test,the percent time in the target quadrant in the old mother group was significantly lower than in the young mother group at P28 and P60(P < 0.05);the target crossing in old mother group was significantly lower than in young mother group at P28 and P60(P < 0.05).3.Immunohistochemical fluorescence results: HSC70 was widely expressed in mice brain,including cortex,hippocampus and other brain regions.HSC70 was expressed on neurons(MAP2 positive staining)and astrocytes(GFAP positive staining),and it was mainly expressed in the cytoplasm;in the old mother group,the expression of HSC70 protein was lower than in the young group in the hippocampal CA3 area.4.Western blot results: The expression of HSC70 protein in hippocampus was detected at 1,3,7,14,21,28,60 days after birth in young and old mother groups.The expression of HSC70 protein in hippocampus increased gradually with age,and peaked at 28 days and declined on the 60 days;The expression of HSC70 protein in the hippocampus of old mother group was significantly lower than that of the young group at P28(P < 0.001),there was no significant difference in the expression of the HSC70 protein between young mother group and the old mother group(P > 0.05);There was no significant difference in the expression of HSC70 protein in the hippocampus before and after the water maze test in the young group at P28(P > 0.05),the expression of Hsp70 protein in hippocampus after water maze test was significantly higher than that before water maze(P < 0.05).Conclusion 1.The offspring's learning and memory ability declines in older female mice.2.With the development of nerves,the expression of HSC70 in the hippocampus gradually increased,reached the highest point in the brain maturation period.3.The expression of HSC70 in the hippocampus of offspring mice born in older female mice was lower than that in young female mice at the maturity of brain development,and there was no difference in adulthood. |
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