The Effects Of Folate Deficiency On Spermatogenesis And Their Underlying Mechanisms

Part I The correlationship between seminal plasma folate and semen parametersObjective: Semen parameters are good for predicting male fertility.However,since 1930 s,human semen quality,especially sperm density,has been declining gradually,but the reasons are unclear.Folic acid,an important trace element,is involved in the synthesis of DNA,RNA and protein,as well as methylation reactions,which is closely related to spermatogenesis.Previous studies have reported that seminal plasma folate concentration represents well folate nutritional status for human.Therefore,this study aimed to explore the correlationship between seminal plasma folate level and semen parameters(including sperm DNA fragmentation index,DFI),and the relationship between folate level in seminal plasma and male infertility.Methods: Participants were recruited from the reproductive medicine center of Tongji Medical College,Huazhong University of Science and Technology and the Hubei sperm bank.After screening,their semen and blood samples were collected.First of all,computer assisted sperm analysis system(CASA)and sperm chromatin structure analysis(SCSA)were used to analyze human semen and sperm integrity,respectively.After centrifugation,plasma and serum were obtained,and then automatic biochemical analyzer was used to detect folic acid and homocysteine(Hcy)level.The SPSS 17 software was used to compare and analyse the correlation between biochemical indexes.Results: After screening,419 subjects were included in the study;including 313 infertile male patients(71 non-obstructive azoospermia,12 severe oligospermia,45 oligospermia and 185 with normal sperm density)and 106 healthy adult volunteers(19 oligospermia and with 87 normal sperm density).All subjects aged 22 ~ 51 years old(31.4±5.3);body mass index(BMI)17.1 ~ 31.1 kg/m2(22.6 ± 4.5)kg/m2.There was no significant difference in age and BMI between the infertility and the fertility group.All subjects have underwent detection for seminal plasma folate,its concentration 11.27 ~ 82.67 nmol/L,the median value of 26.57 nmol/L;among infertility group,seminal plasma folate concentration [ 25.12(11.27-69.64)nmol/L ] was significantly lower than that of fertile group [27.32(13.65-82.67)nmol/L](P(27)0.01).According to sperm density,azoospermia [23.14(11.27-47.56)nmol/L] and severe oligozoospermia [23.16(13.32-49.65)nmol/L] were significantly lower than the participants with normal sperm density [28.12(14.75-82.67)nmol/L](P(27)0.01).While compared to participants with normal sperm density,oligozoospermia [26.83(13.65-69.64)nmol/L] was lower,but the difference was not statistically significant(P(29)0.05).105 infertile patients(20 azoospermia,25 oligozoospermia and 60 normal sperm density)completed the detection for folate and Hcy in seminal plasma and serum,the distributions have no significant difference.After excluding azoospermia,the determination of the sperm integrity of 85 infertile patients with DFI,oligospermia’s DFI(20.92±13.67)% was lower than that of patients with normal sperm density(26.61±13.67)%,but the difference was not statistically significant(P=0.08).After adjustment for age and BMI,folate levels in seminal plasma folate and in serum was correlated(r=0.62,P(27)0.01);and sperm density(r=0.24,P(27)0.01);DFI(r=-0.41,P(27)0.05).Conclusion: seminal plasma folate concentration may be a good indicator of the nutritional status of folic acid.Low seminal plasma folate level destroys sperm’s genome integrity,which probably inhibits spermatogenesis and causes to decrease of sperm density.Therefore,low folate level in seminal plasma may be an independent risk factor for male infertility.Part II The correlation between seminal plasma folate levels and sperm DNA methylationObjective: Folate is the main donor of methyl in body,and involved in methylation reaction through one-carbon pathway,so the seminal plasma folate may affect sperm DNA methylation.Because of the compact structure of the sperm genome,it is difficult to obtain high quality sperm DNA.Therefore,the purposes of the current study were to optimize and compare the sperm genome extraction methods,and to determine relationship between seminal plasma folate levels and sperm DNA methylation.Methods: Improved guanidine thiocyanate method,conventional phenol chloroform method and Tiangen kit method were used to extract human sperm DNA,and common PCR and Methy Light method are used to compare the three extraction methods.Samples with low and normal folate content were selected for sequencing analysis by Reduced Representation Bisulfite Sequencing(RRBS),and the results were verified by Bisulfite Sequencing PCR (BSP).The effects of the level of folic acid on the m RNA expression of key gene ESR1,CAV1 and ELAVL1 in spermatogenesis were evaluated by fluorescence quantitative PCR.Results: compared with the traditional phenol chloroform method and Tiangen kit,modified guanidine thiocyanate can obtained great DNA yield and high purity with good integrity,which is suitable for methylation analysis.Compared to the patients with normal seminal plasma folate,1287 defferent methylation region(DMRs)were found in the low folate group,including 464 up and 823 down.Bioinformatics analysis showed that DMRs related genes were concentrated on cellular metabolism,biological regulation,immune response,cell development,substance metabolism,damage repair,apoptosis and signaling pathways.BSP failed to find significant differences in the level of methylation in the ESR1,CAV1 and ELAVL1 promoter regions between two groups.Low human seminal plasma folate level down-regulate ESR1,CAV1 and ELAVL1 m RNA.Conclusion: the modified guanidine thiocyanate method is able to extract high quality sperm DNA,and is suitable for methylation analysis.Folate level in seminal plasma changes the methylation pattern of sperm genome and interferes with the expression of important genes related to spermatogenesis,such as ESR1,CAV1 and ELAVL1,which may be the potential cause of male infertility.Part III Folate deficiency affects spermatogenesis via Cav1/Perk/Eif2a/Atf4/Chop signaling pathway in vivoObjective: Study reported that folate deficiency induced apoptosis of germ cells through endoplasmic reticulum stress(ER stress).PERK(Per K/Eif2a/Atf4/Chop)signaling pathway is one of the most important three signaling pathways among ER stress.According to our sequencing data and publised studies,the purpose of this study is to clarify the effect of folate deficiency on spermatogenesis and to preliminarily study the PERK(Per K/Eif2a/Atf4/Chop)signaling pathway among ER stress.Methods: the folate deficiency animal model was established with the ordered feed.The effects of folic acid deficiency on testicular tissue were observed by epididymal sperm count,HE staining,TUNEL and testicular tissue immunofluorescence.The effects of folate deficiency on the key genes of Cav1,Esr1 and Elavl1,as well as the expression of Per K/Eif2a/Atf4/Chop signaling pathway were detected by RT-PCR and Western blot.Results: compared with control group,folate deficiency reduced the sperm density and increased tissue injury,apoptosis and DNA double strand break rate in testes;Meanwhile,folate deficiency reduced Cav1,Esr1 and Elavl1 m RNA and protein expression and increased expression of Per K/Eif2a/Atf4/Chop signaling pathway,the differences were statistically significant(P(27)0.05).Conclusion: Folate deficiency animal models confirmed that low folate levels can damage the testicular germ cell genome,increase the apoptosis of testicular tissue,decrease sperm density,inhibit gene expression(Cav1,Esr1 and Elavl1)related to spermatogenesis,and may inhibit spermatogenesisi by down-regulating the key gene expression(Cav1),which may activate the PERK(Perk/Eif2a/Atf4/Chop)signaling pathway.Part IV Folate deficiency induce GC-2 apoptosis via Cav1/Perk/Eif2a/Atf4/Chop signaling pathway in vitroObjective: As described in the second and third parts of this article,folate deficiency reduces the expression of Cav1 and activates the PERK pathway,and Cav1 is closely related to the PERK pathway.In order to further explore the mechanism of increased apoptosis induced by folate deficiency,we used RNAi technology to silence Cav1,in order to verify whether folate deficiency can increase cell apoptosis through Cav1/PERK/Eif2a/Atf4/Chop signaling pathway.Methods: GC-2,GC-1,TM3 and TM4 cells were treated with no folic acid medium.Cell viability was analyzed by using Cell Counting Kit-8,and cell cycle and apoptosis rate were detected by flow cytometry.Si RNA silenced Cav1 of GC-2 cells and then we detected the effects of folate deficiency on the expression of Cav1 and Per K/Eif2a/Atf4/Chop signaling pathways with RT-PCR and Western blot.Results: compared with control group with normal folate,folate deficiency inhibited cell viability and cell proliferation;increased cell apoptosis;we found that folate deficiency reduce the expression of Cav1,increase the expression of Per K/Eif2a/Atf4/Chop signaling pathway,and the differences were statistically significant(P(27)0.05).Silencing of Cav1 also increased the expression of Per K/Eif2a/Atf4/Chop signaling pathway,the differences were statistically significant(P(27)0.05).Conclusion: Folate deficiency increases the apoptosis rate of mouse testicular cell lines,GC-2,with the most sensitive,suggesting that spermatogonial cells are more vulnerable and sensitive to the external environment.No folic acid treatment combined with in vitro transient silencing Cav1 assay suggests that folate deficiency may promote the apoptosis of spermatogonial cells by activating Cav1/Per K/ Eif2a/Atf4/Chop signaling pathway.