The Impact Of Paternal Frequent And Mild Scrotal Heat Stress On Embryo Development And Metabolic And Neurobehavior Of Offspring Mice

Part 1 The establishment of frequent and mild scrotal heat stress mouse model[Purpose]The scrotal heat stress that often happens to males and causes infertility in clinics is frequent and mild local scrotal heat exposure,which usually leads to oligoasthenospermia.We aimed to establish a fm SHS mouse model in this part,by comparing the impact of scrotal heat stress at different temperature on testis,epididymis and sperm quality.[Methods]Male C57/BL6 mice of 10 weeks old were anaesthetized and subjected to a scrotal heat stress at either 37? or 39? or 41? or no heat stress for 30 min once weekly for five consecutive weeks.Testis,epididymis and sperm quality were assessed one week later after the last heat stress.For mice in 39? and control group,DNA integrity were assessed by sperm chromosome structure analysis,and male mice were co-caged with female mice,whose plug was examined the next morning and pregnancy of plug-positive mice were confirmed 20 days later[Results]Comparing with control,the testis and epididymis weight of mice in 39? and 41? stress groups were all reduced,the also decreased were sperm quality,including reduced concentration(74.9�6.8 vs 49.9�7.5,P<0.05;74.9�6.8 vs 30.6�0.9,P<0.001),lower motility(53.4�3.1 vs 32.4�2.7,P<0.001;53.40�3.1 vs 24.2�1.0,P<0.0001)and decreased normal morphology rate(65.3�0.9 vs 30.2�0.9,P<0.0001;65.3�0.9 vs 24.5�1.6,P<0.0001).The impairment in 41? group of mice were severer than those of 39?mice,while no difference was found on these assessments between 37? and control mice(all P>0.05).When repeated these assessments between 39? and control group with more mice,those differences still existed.Moreover,sperm chromatin structural analysis showed that sperm DNA fragmentation index in 39? mice was significantly higher than that of control mice(12.5�1.0%vs 9.0�0.9%,P<0.05).Since heat stress at 39? was milder than 41?,and also brought about an appearance of oligoasthenospermia in mice,which was similar to that of clinics.We then tested the natural fertility of those mice by co-caging them with normal female mice,the examination of female mice's plug showed no difference.But the pregnancy test of those plug-positive female mice showed that none of the 23 plug-positive female mice in 39? group get pregnant,while 12 of the 15 mice in control group achieved(80.0%vs 0.0%,?2=26.9,P<0.0001)[Conclusions]Frequent scrotal heat stress at 39? in mice leads to sperm count and motility decreasing,and infertility,which are similar with clinical oligoasthenospermia caused by scrotal heat stress.Thus,frequent scrotal heat stress at 39? can be used to establish fmSHS model in micePart 2 Impact of fmSHS on preimplantation embryo development,and offspring sex ratio in mice[Purpose]To study the impact of fmSHS on sperm fertility,preimplantation embryo development,structure and invasiveness,and implantation ability at two cell stage in in vitro fertilization and embryo transfer(IVF-ET)process,as well as offspring sex ratio in mice[Methods]Sperm of fmSHS and control mice were collected and used for IVF with super ovulated oocytes,then oocytes fertility rate was observed,on-time development of the resultant embryos were examined from day 1(D1)after fertilization to D5.Inner cell mass and trophectoderm cell lineage differentiation of blastocysts were tested by immunofluorescence.Blastocysts invasiveness was assessed by observing the outgrowth area of hatched blastocyst on fibronectin coated 96 wells.Embryo implantation ability was tested with 2-cell embryos transferring into pseudo-pregnant mice.The gestational age,birth weight and sex ratio of the first generation(F1)offspring mice were recorded on the birth day.The ratio of Y/X chromosome-bearing sperm in fmSHS and control mice was individually assayed by absolute quantitative PCR[Results]Comparing with control group,the sperm fertilization rate(29.0�3.6%vs 47.2�4.4%,P<0.01),the proportion of resultant embryos at? 8-cell on D3(79.1�4.6%vs 93.7�3.8%,P<0.05),blastuation rate on D4 and D5(53.4�10.9%vs 82.7�5.0%,P<0.05),and blastocyst hatching rate(17.8�9.5%vs 46.8�5.6%,P<0.05)in fmSHS group were all reduced.The total cell number of blastocyst,inner cell mass and trophectoderm cell in well-developed blastocyst had no difference between groups(P>0.05).The outgrowth area of well-developed blastocyst at different time points had no difference between groups(all P>0.05).Two-cell embryos from fmSHS mice have lower implantation rate(22.0 � 6.4%vs 47.4 � 5.5%,P<0.01).The gestational age(P>0.05)and birthweight(P>0.05)of fmSHS offspring had no difference between groups.The fmSHS mice have a higher ratio of male offspring than that of control(72.9�12.6%vs 44.3�3.1%,P<0.05),and a higher ratio of Y chromosome-bearing sperm(68.6�2.4 vs 43.7�3.7,P<0.0001)[Conclusions]fmSHS affects not only sperm fertilization rate but also the formation of?8-cell embryos and blastocysts.fmSHS reduced blastocyst hatching ability and 2-cell embryo implantation ability.fmSHS didn't affect gestational age and birth weight of the resultant offspring mice.fmSHS changed the offspring sex ratio by altering Y/X chromosome-bearing sperm of their fathersPart 3 Impact of paternal fmSHS on glucose metabolism of F1 and F2 offspring mice and the relative mechanism[Purpose]To explore the impact of paternal fmSHS on glucose metabolism of F1 and F2 offspring mice and the relative mechanism[Methods]After being exposed to fmSHS,we bred the first generation(F1)offspring mice by IVF-ET and the second(F2)by natural mating of male F1 mice with wild female mice.The offspring of control mice were obtained by the same approaches.Glucose and insulin tolerance tests were applied to assay glucose metabolism of offspring mice.ELISA was used to test the serum insulin level of offspring mice during glucose tolerance tests Islet constructure was evaluated by immunofluorescence in F1 mice.Quantitative real time PCR(qPCR)was applied to assay the mRNA expression level of glucose metabolism-related genes in the special tissues of offspring mice.Western blot was applied to assay the protein expression level of glucose metabolism-related genes in the special tissues of offspring mice.Whole genome bisulfite sequence(WGBS)was applied to detect DNA methylation level of sperm from fmSHS-exposed and control mice(F0),the differentially methylated genes were confirmed by pyrosequencing in F0 sperm and F1 liver[Results]Comparing with control group,the glucose levels of fmSHS F1 male mice were higher at the five time points in glucose tolerance test(GTT),among of them,the glucose levels between groups at 15,30,60 and 120 min after glucose injection were statistically different(22.1�1.5 vs 15.6�1.1,P<0.01;20.5�2.2 vs 11.4�1.7,P<0.01;20.2�1.0 vs 11.8�1.3,P<0.001;14.5�0.7 vs 8.6�1.0,P<0.001);fmSHS F1 female mice showed a trend of being higher than the control(P<0.05);in addition,the blood insulin level of fm SHS F1 male mice at 0 and 15 min after glucose injection were significantly higher than control(0.5�0.07 vs 0.2�0.02,P<0.01;0.5�0.06 vs 0.3�0.04,P<0.05);there is no difference on blood insulin of female offspring mice between groups(P>0.05).During insuline tolerance test(ITT),the glucose levels of fmSHS F1 male mice were generally higher at the five time points,among of them,the difference at 30,60 and 90 min after insulin injection was statistically significant(6.9�0.7 vs 4.6�0.6,P<0.05;6.8�0.6 vs 4.5�0.9,P<0.05;8.1�1.0 vs 5.2�0.8,P<0.05).Islets,after evaluation,remained unchanged(P>0.05).The qPCR results showed that,among the 41 glucose metabolism related genes,11 genes showed aberrant mRNA expression in the liver of fmSHS F1 mice,including genes in insulin and insulin receptor signaling pathway(Irs2,Pik3r1,Pik3cg,Akt2),glucose transportation and relative genes(Glut4,Glut1,Nr4a1),glycogen synthesis enzyme(Gsy2)The protein expression of some key genes in fmSHS F1 liver were also reduced significantly(PIK3CG,0.1�0.01 vs 0.3�0.1,P<0.05;GLUT4,0.5�0.1 vs 0.8�0.04,P<0.05;NR4A1,0.2�0.05 vs 0.6�0.08;P<0.01).Those abnormal glucose tolerance in GTT test and abnormal insulin sensitivity in ITT test was still observed in fmSHS F2 mice of 9 weeks old,for the GTT test,all difference was not significant,but for the ITT test,differences at five time points were all statistically significant(6.5�0.5 vs 9.3�0.8,P<0.05;4.1�0.2 vs 3.2�0.3,P<0.05;5.8�0.6 vs 2.2�0.3,P<0.05;7.5�0.9 vs 3.4�1.1,P<0.001;8.5�0.8 vs 3.3�0.9,P<0.05).Those glucose metabolism related genes aberrantly expressed in fmSHS F1 mice still showed abnormal mRNA expression in fmSHS F2 liver(Irs2,P>0.05;Pik3r1,P<0.05;Pik3cg,P<0.05;Akt2,P>0.05;Glut4,P>0.05;Glut1,P>0.05;Nr4a1,P>0.05;Gsy2,P<0.05).The protein expression level of key genes in fmSHS F2 liver was still lower than that of control(PIK3CG,0.09 � 0.02 vs 0.3�0.05,P<0.05;GLUT4,0.5�0.1 vs 0.8�0.01,P<0.05;NR4A1,0.6�0.04 vs 0.4�0.08,P>0.05).Differentially methylated regions were found between the sperm of fmSHS-exposed mice and control by WGBS,including Pik3cg.Pyrosequence confirmed that the methylation level of Pik3cg were higher in both F0 sperm(for all five CG sites,P>0.05)and F1 liver(for the first three CG sites,P>0.05,for the fourth and the five,88.4�5.6 vs 47.4�12.8,P<0.05;97.4�1.4 vs 52.8�13.6,P<0.05)of fmSHS group than that of control.Additionally,the methylati on level of Nr4a1 were also higher in both F0 sperm(for all eight CG sites,P>0.05)and F1 liver(for the total of first 5 consecutive CG sites,12.2�1.0 vs 5.2�1.7,P<0.05 and the remained CG sites,P>0.05)of fmSHS group than that of control[Conclusions]Paternal fm SHS induces abnormal glucose metabolism in offspring mice,possibly by epigenetically affecting sperm DNA methylati on which influence the expression of glucose metabolism related genes.This influence can be inherited to the F2 offspringPart 4 Impact of paternal fmSHS on neurobehaviors of F1 and F2 offspring mice and the relative mechanism[Purpose]To explore the impact of paternal fmSHS on neurobehavior of F1 and F2 offspring mice and the relative mechanism[Methods]After being exposed to fmSHS,we bred the first generation offspring mice by IVF-ET and the second by natural mating of male F1 mice with wild female mice.The offspring of control mice were obtained by the same approaches.Open field test,social interaction test,new objective recognition test and morris water maze test were applied to examine the anxiety,interacting ability,non-spatial memory and spatial memory of the F1 offspring mice,respectively.Morris water maze test was also applied to examine the F2 offspring mice.Nissl staining was used to assay the number of granulosa cells and its content of Nissl bodies in F1 offspring hippocampus.Western blot was applied to test the expression levels of N-methyl-D-aspartate receptors 1(NMDAR1)and NMDAR2? in F1 offspring hippocampus[Results]Comparing with control,the behavior of the F1 fmSHS mice in open field test and new objective test was not changed.In the first stage of social interaction test,the time for fmSHS F1 mice interacting with stranger 1 mouse was significantly reduced than that for control F1 mice(270.0�27.7 vs 353.3�4.8,P<0.05).In the second stage of social interaction test,the duration time for fmSHS F1 mice staying in the familiar chamber was significantly reduced than that for control F1 mice(235.5�18.2 vs 346.3�25.0,P<0.05),while the duration time for fmSHS F1 mice staying in the stranger chamber was significantly higher than that for control F1 mice(238.3�22.0 vs 167.0�8.3,P<0.05).In water maze test,during the navigation experiment on day2-4,the latency for fmSHS-F1 mice to find the platform was higher than that for control F1 mice(45.2�3.9 vs 25.9�2.3,P<0.001;37.8�4.1 vs 22.1�3.7,P<0.5;43.0�4.4 vs 21.6�4.0,P<0.01).During spacial exploration test on day5,the duration time(13.9�2.6 vs 22.1�2.7,P<0.05)and distance(271.6�40.2 vs 472.7�67.0,P<0.05)of fmSHS-F1 mice swimming in the platform quadrant were all significantly shorter than those of the control-F1 mice.Similar to the behavior of their fathers in water maze test,the latency of fmSHS F2 mice to find the platform during the navigation experiment was higher than that of control F2 mice,the duration time and distance during spacial exploration test was lower than that of control F2 mice,but all these differences were not statistically significant(all P>0.05).No difference was found on the number of granulosa cells and its content of Nissl bodies in the hippocampus between both groups of F1 mice.The total protein content of NMDAR1 and NMDAR2? in hippocampus showed no difference by Western blot(P>0.05)[Conclusions]Paternal fmSHS brings about social dysfunction and spatial memory impairment to its F1 offspring mice.The spatial memory impairment could be inherited to its F2 offspring.