| Autism Spectrum Disorder(ASD)is a common neurodevelopmental disorder with high heterogeneity,resulting from the combined effects of environmental and genetic factors.Severe exposure to high levels of androgens during pregnancy can significantly affect the neurodevelopment of offspring and increase the risk of developing ASD.The hippocampus is an important brain region closely related to the occurrence and development of ASD,affecting learning,memory and emotional regulation,with high plasticity.The subgranular zone of the hippocampal dentate gyrus is a region where neurogenesis persists throughout life,and neural stem cells in this layer can continuously produce new neurons,which integrate into neural circuits and participate in higher brain functions with stable synaptic connections with pyramidal neurons.The development of dendritic spines,which play important roles in learning,memory and motor coordination by determining synaptic strength and stability,is a key factor affecting the function of neurons.Impairment of dendritic spine morphology and function can affect higher brain functions.Currently,little is known about the mechanisms underlying the adverse effects of prenatal androgen exposure on offspring neurodevelopment and ASD-like behavior.In this study,we established a model of prenatal androgen exposure using dihydrotestosterone(DHT),and observed the development of dendritic spines and neurobehavioral changes in offspring.Additionally,we used m RNA transcriptome sequencing technology to identify key molecules involved in the effects of prenatal androgen exposure on offspring neurodevelopment and verified their role.Objective:This study used C57BL/6J mice as the research subjects to investigate the effects of prenatal androgen exposure on hippocampal neurodevelopment and ASD-like behavior in offspring,and identified key genes responsible for abnormal phenotypes using m RNA transcriptomics and bioinformatics.Our findings provide a theoretical basis for exploring the role of these genes in Autism Spectrum Disorder.Methods:The effects of prenatal exposure to androgens on hippocampal neurodevelopment and ASD-like behavior in mouse offspring.1.The pregnant mice were randomly divided into two groups:the Control group and the DHT group,each with 10 mice.On gestational days16～18(G16～18),the DHT group mice were subcutaneously injected on the neck and back with 10 mg/kg of dihydrotestosterone solvent,while the Control group was injected with an equal amount of solvent.On G19,serum androgen levels were measured in both groups using enzyme-linked immunosorbent assay(ELISA).After the birth of the offspring,7-day-old(Postnatal,P7)pups were intraperitoneally injected with a solution containing50 mg/kg of Brd U.Two hours after injection,brain tissues were collected and analyzed by immunofluorescence microscopy to examine the number of Brd U-positive cells in the hippocampal dentate gyrus,reflecting cell proliferation.P7 pups were also intraperitoneally injected with a solution containing 100 mg/kg of Brd U,and at P28,brain tissues were collected and analyzed by immunofluorescence microscopy to observe the number of Brd U~+/Neu N~+,Brd U~+/GFAP~+,and Brd U~+/Iba1~+positive cells in the hippocampal dentate gyrus,reflecting the differentiation of neural stem cells and the situation of new-born astrocytes and microglia,respectively,thereby evaluating early hippocampal neurogenesis in the offspring.At 2 months of age,10 mice were randomly selected from each of the control and DHT groups for behavioral experiments,including the open field test to assess anxiety status,the buried marble test to detect repetitive and stereotyped behavior,the three-chamber social test to evaluate social ability and preference,the novel object recognition test and Morris water maze test to evaluate learning and memory,and the forced swimming test to assess depression status.After behavioral experiments,the mice were perfused and their brain tissues were subjected to Golgi-Cox staining to observe the dendritic spine density and morphology in the hippocampal CA1 region.2.Regulation of gene expression in prenatal hyperandrogen exposed mice.There were differences in gene expression patterns between hippocampal tissues of hyperandrogen-exposed rats during pregnancy and control groups.A total of 736 DEGs were detected by m RNA transcriptome sequencing technology,of which 373 gene expression was upregulated and 363 gene expression was down-regulated.Through the KEGG pathway enrichment analysis of DEGs,the results show that compared with the Control group,a total of 33 signaling pathways with significant differences were enriched in the DHT group,of which 12 were up-regulated and 21 were down-regulated.GO enrichment results showed that in the biological process,144 were upregulated and 229 were down-regulated;Among the cellular components,23 were up-regulated and 60 down-regulated;In molecular function,32 are up-regulated and 36 are down-regulated.The key autism risk gene Nr4a2 was further screened in biological processes related to neurodevelopment and synaptic function.The expression of Nr4a2 was detected at the transcription and translation levels,and the results of q RT-PCR and Western Blot showed that compared with the Control group,the expression of Nr4a2 m RNA and protein in the DHT group was significantly reduced,and the results were consistent with the sequencing results.3.Effects of Nr4a2 on hippocampal dendritic spine development and autistic like behavior in prenatal hyperandrogen exposed mice.1)Effects of Nr4a2 overexpression on hippocampal dendritic spine development and autistic like behavior in prenatal hyperandrogen exposed ratsNr4a2 overexpressed adeno-associated virus was constructed,and GFP adeno-associated virus control vector(AAV+Control)and overexpression Nr4a2 adeno-associated virus vector(DHT+AAV+OE-Nr4a2)were injected into the mouse hippocampal CA1 region using brain stereopocampal localization technology.The experiment was divided into three groups:AAV+Control group,DHT+AAV+Control group,and DHT+AAV+OE-Nr4a2group.The expression levels of Nr4a2 m RNA and protein in the hippocampal tissues of each group were detected by q RT-PCR and Western Blot,and the effect of overexpression of Nr4a2 on the neurobehavior of sub-mice was detected by behavioral experiments.Then,the mice were perfused and fixed,and the brain tissues were removed for Golgi-Cox staining to observe the density and morphology of CA1 dendritic spines in the hippocampus.2)Effects of amodiaquine on hippocampal neurodevelopment and autism-like behavior in hyperandrogen-exposed mice during pregnancyThe mice were randomly divided into three groups:Saline group,DHT+Saline group,and DHT+AQ group.20 mg/kg of amodiaquine solution was injected intraperitoneally,the control group was injected with the same amount of normal saline,and the material was taken two weeks after the last injection,and the expression levels of Nr4a2 m RNA and protein in the hippocampal tissues of each group were detected by q RT-PCR and Western Blot,and then neurobehavioral experiments were performed to detect behavioral changes.After the end,the group of rats was perfused and fixed,and the density and morphology of CA1 dendritic spines in the hippocampus were observed by Golgi-Cox.Results:1.Effects of prenatal high androgen exposure on hippocampal neurodevelopment and autistic like behavior in offspring mice.The results of ELISA experiments showed that compared with the pregnant mice in the Control group,the serum content of the pregnant mice in the DHT group was significantly increased,indicating the success of modeling.The results of immunofluorescence experiments showed that compared with the Control group at P7,the number of mice hippocampal gyrus Brd U~+cells in the DHT group was significantly reduced,and compared with the Control group at P28,the number of rat hippocampal gyrus Brd U~+/Neu N~+co-labeled cells in the DHT group was significantly reduced,the number of Brd U~+/GFAP~+co-labeled cells was significantly changed,and the number of Brd U~+/Iba1~+co-labeled cells was significantly increased,indicating that hyperandrogen exposure during pregnancy damaged the neurogenesis of offsprings.The results of the open field experiment showed that compared with the control group,the time and distance percentage of the DHT group were significantly reduced in the central area.The results of marble burying test showed that the number of buried marble in the DHT group increased compared with the control group of mice.The results of the social chamber test showed that the DHT group sniffed Stranger mice and Novel mice for less time than the control group mice.The results of the new object recognition test showed that compared with the control group,the time for sub-mice in the DHT group to recognize new objects was significantly reduced.The results of the Morris water maze test showed that compared with the Control group,the sub-rats in the DHT group had a significant increase in the evasion latency period on day 5,and the percentage of time spent in the target quadrant and the number of crossing the platform were significantly reduced.The results of forced swimming experiments showed that compared with the control group,the cumulative immobility time of mice in the DHT group was significantly increased.Suggests that high androgen exposure during pregnancy led to autism-like behavior in mice.Compared with the Control group,the density of dendritic spines in the DHT group was significantly reduced,the percentage of mature mushroom and slender dendritic spines was significantly reduced,and the percentage of immature coarse and short dendritic spines and filamentous pseudopodia increased,indicating that hyperandrogen exposure during pregnancy reduced the total density and maturity of dendritic spines in the CA1 region of the hippocampus.2.Regulation of gene expression in prenatal hyperandrogen exposed mice.There were differences in gene expression patterns between hippocampal tissues of hyperandrogen-exposed mice during pregnancy and control groups.A total of 736 DEGs were detected by m RNA transcriptome sequencing technology,of which 373 gene expression was upregulated and 363 gene expression was down-regulated.Through the KEGG pathway enrichment analysis of DEGs,the results show that compared with the Control group,a total of 33 signaling pathways with significant differences were enriched in the DHT group,of which 12 were up-regulated and 21 were down-regulated.GO enrichment results showed that in the biological process,144 were upregulated and 229 were down-regulated;Among the cellular components,23 were up-regulated and 60 down-regulated;In molecular function,32 are up-regulated and 36 are down-regulated.The key autism risk gene Nr4a2 was further screened in biological processes related to neurodevelopment and synaptic function.The expression of Nr4a2 was detected at the transcription and translation levels,and the results of q RT-PCR and Western Blot showed that compared with the Control group,the expression of Nr4a2 m RNA and protein in the DHT group was significantly reduced,and the results were consistent with the sequencing results.3.Effects of Nr4a2 on hippocampal dendritic spine development and autistic like behavior in prenatal hyperandrogen exposed mice.The qRT-PCR experiment revealed that,compared to the AAV+Control group,the expression of Nr4a2 m RNA in the hippocampus of the DHT+AAV+Control group was significantly reduced,while the expression of Nr4a2 m RNA in the DHT+AAV+OE-Nr4a2 group was significantly increased.Compared to the DHT+AAV+Control group,the expression of Nr4a2 m RNA in the DHT+AAV+OE-Nr4a2 group was significantly increased.The Western blot experiment showed that,compared to the AAV+Control group,the expression of Nr4a2 protein in the hippocampus of the DHT+AAV+Control group was significantly reduced,while the expression of Nr4a2 protein in the DHT+AAV+OE-Nr4a2 group was significantly increased.Compared to the DHT+AAV+Control group,the expression of Nr4a2 protein in the DHT+AAV+OE-Nr4a2 group was significantly increased.In the open field experiment,the percentage of activity distance and time in the central zone of the DHT+AAV+Control group were significantly lower than those in the AAV+Control group,while the percentage of activity distance and time in the central zone of the DHT+AAV+OE-Nr4a2 group were significantly higher than those in the DHT+AAV+Control group.In the buried bead experiment,compared to the AAV+Control group,the number of buried beads in the DHT+AAV+Control group was significantly increased,while the number of buried beads in the DHT+AAV+OE-Nr4a2 group was significantly decreased compared to the DHT+AAV+Control group.In the three-chamber social experiment,compared to the AAV+Control group,the time coefficient for sniffing the empty cage of the DHT+AAV+Control group was increased,and the time coefficient for sniffing the unfamiliar mouse was decreased.Compared to the DHT+AAV+Control group,the time coefficient for sniffing the empty cage of the DHT+AAV+OE-Nr4a2 group was decreased,and the time coefficient for sniffing the unfamiliar mouse was increased.In the novel object recognition experiment,compared to the AAV+Control group,the time for sniffing the novel object in the DHT+AAV+Control group was decreased,while the time for sniffing the novel object in the DHT+AAV+OE-Nr4a2group was increased.The Morris water maze experiment showed that,compared to the AAV+Control group,the escape latency on the fifth day was significantly increased in the DHT+AAV+Control group,and the percentage of time spent in the target quadrant and the number of platform crossings were significantly reduced.Compared to the DHT+AAV+Control group,the escape latency on the fifth day was significantly reduced in the DHT+AAV+OE-Nr4a2 group,and the percentage of time spent in the target quadrant and the number of platform crossings were significantly increased.The forced swimming experiment showed that,compared to the AAV+Control group,the immobility time of the DHT+AAV+Control group was significantly increased,while the immobility time of the DHT+AAV+OE-Nr4a2 group was significantly decreased.These results suggest that overexpression of Nr4a2can rescue abnormal behavioral phenotypes of mice exposed to high levels of pregnancy androgen.The Golgi-Cox staining experiment showed that,compared to the AAV+Control group,the dendritic spine density in the hippocampal CA1 area of the DHT+AAV+Control group was significantly reduced,and the percentage of mature mushroom-shaped and slender dendritic spines was significantly decreased,while the percentage of immature stubby dendritic spines and filopodia was increased.Compared to the DHT+AAV+Control group,there was no significant change in dendritic spine density in the DHT+AAV+OE-Nr4a2 group,and the percentage of mature mushroom-shaped dendritic spines was significantly increased,while the percentage of immature stubby dendritic spines and filopodia was decreased.These results suggest that overexpression of Nr4a2 can promote dendritic spine maturation in the hippocampal CA1 area of mice exposed to high levels of pregnancy androgen.The q RT-PCR experiment showed that compared to the Saline group,the expression of Nr4a2 m RNA in the hippocampus of the DHT+Saline group was significantly reduced,while the expression of Nr4a2 m RNA in the hippocampus of the DHT+AQ group was significantly increased.Compared to the DHT+Saline group,the expression of Nr4a2 m RNA in the DHT+AQ group was significantly increased.The Western Blot experiment showed that compared to the Saline group,the expression of Nr4a2 protein in the hippocampus of the DHT+Saline group was significantly reduced,while there was no difference in the expression of Nr4a2 in the hippocampus of the DHT+AQ group.Compared to the DHT+Saline group,the expression of Nr4a2 in the DHT+AQ group was significantly increased.The open field test showed that compared to the Saline group,the percentage of center activity distance and activity time in the DHT+Saline group was significantly reduced.Compared to the DHT+Saline group,the percentage of center activity distance and activity time in the DHT+AQ group was significantly increased.The buried-bead test results showed that compared to the Saline group,the number of buried beads in the DHT+Saline group was significantly increased.Compared to the DHT+Saline group,the number of buried beads in the DHT+AQ group was significantly decreased.The three-chamber social interaction test results showed that compared to the Saline group,the time coefficient of sniffing the empty cage was increased,while the time coefficient of sniffing the unfamiliar mouse was reduced in the DHT+Saline group.Compared to the DHT+Saline group,the time coefficient of sniffing the empty cage was reduced,while the time coefficient of sniffing the unfamiliar mouse was increased in the DHT+AQ group.The novel object recognition test results showed that compared to the Saline group,the time spent sniffing the novel object was reduced in the DHT+Saline group.Compared to the DHT+Saline group,the time spent sniffing the novel object was increased in the DHT+AQ group.The Morris water maze experiment results showed that compared to the Saline group,the escape latency on the fifth day was significantly increased,and the percentage of time spent in the target quadrant as well as the number of platform crossings were significantly reduced in the DHT+Saline group.Compared to the DHT+Saline group,the escape latency on the fifth day was significantly reduced,and the percentage of time spent in the target quadrant as well as the number of platform crossings were significantly increased in the DHT+AQ group.The forced swimming test results showed that compared to the Saline group,the cumulative immobility time in the DHT+Saline group was significantly increased.Compared to the DHT+Saline group,the cumulative immobility time in the DHT+AQ group was significantly decreased.These findings indicate that Amiodarone quinoline can effectively rescue abnormal behavioral phenotypes in offspring mice exposed to high levels of gestational androgen.The Golgi-Cox staining experiment results showed that compared to the Saline group,the dendritic spine density in the hippocampal CA1 region of the DHT+Saline group was significantly reduced,the percentage of mature mushroom-shaped and thin spines was significantly reduced,and the percentage of immature stubby-shaped spines and filopodia-like protrusions was significantly increased.Compared to the DHT+Saline group,the dendritic spine density in the DHT+AQ group showed no significant change,while the percentage of mature mushroom-shaped spines was significantly increased,and the percentage of immature stubby-shaped spines and filopodia-like protrusions was significantly decreased.These results suggest that Amiodarone quinoline can promote dendritic spine maturation in the hippocampal CA1 region of offspring mice exposed to high levels of gestational androgen.Conclusions:1.High androgen exposure during pregnancy leads to autistic behavior in offspring mice,which damages the development of hippocampal dendritic spines.2.Nr4a2 may be a key differential gene for abnormal neurodevelopment and autism-like behavior in hyperandrogen exposed mice during pregnancy.3.Increased Nr4a2 expression improved autistic behavior and promoted the development of hippocampal dendritic spines in rats exposed to hyperandrogen during pregnancy. |
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