The Role And Mechanism Of PIWIL2 Mutation In The Pathogenesis Of Sertoli Cell-only Syndrome

Part 1 Identification of causative variants of SCOS through whole exome sequencing [Purpose] A large portion of SCOS patients remain unexplained,but the cause is highly suspected to be genetic abnotmalities.The exploration of genetic causes of SCOS is an important apect of improving SCOS diagnosis system.In this study,whole exome sequencing(WES)was used to screen genetic causes of a SCOS patient with unkown cause.[Methods] An infertile man with consanguineous parents was included in the study and diagnosed by laboratory examination and testicular biopsy.Genomic DNA of the included patient was extracted from peripheral blood and then used for WES analysis.Homozygous variants with MAF?5%,which also may change protein structure and have deleterious effects,were further screened for causative variants.Genomic DNA of patient's immediate relatives was collected to perform Sanger sequencing to identify the variants on the same mutation sites,and the inheritance pattern of the causative variant was determined according to the Sanger sequencing results.The testicular expression of pathogenic gene of the patient and a fertile man was determined by immunohistochemistry.The mutation of the pathogenic gene in 757 cases of non-obstructive azoospermia(NOA)and 709 fertile men was analyzed.[Results] The infertile man included in the study has normal karyotype and no microdeletions on the Y chromosome.He was diagnosed as NOA(SCOS pattern),considering no sperm was observed through three unrelated semen analyses,negative seminal DDX4 mRNA and no observed spermatogenic cells in testicular biopsy.After analyzing WES data,ten homozygous variants that met the screening criteria were identified,of which c.731?732del AT in PIWIL2 gene was considered as the causative variant because PIWIL2 is involved in spermatogenesis.The loss-of-function variant of PIWIL2 led to a truncated PIWIL2 protein which lacks functional domains.Immunohistochemistry showed no signal of intact PIWIL2 protein in patient's testes and PIWIL2 located in spermatogonia,spermatocytes and spermatids of the fertile man.Patient's father and brother carried the same heterozygous variant of PIWIL2,indicating that the inheritance pattern of PIWIL2 pathogenic variant was autosome recessive.Additionally,one NOA patient with unknown testicular histology(undone testicular biopsy)and cause carried a homozygous PIWIL2 missense variant g.22145115C>T,and the patient had normal karyotype and no microdeletions on the Y chromosome.[Conclusions] c.731?732del AT in PIWIL2 gene resulted in the traucated PIWIL2 protein which lacks important functions.PIWIL2 was highly suspected to be the genetic cause of SCOS because of its crucial role in spermatogenesis.The inheritance pattern of the PIWIL2 mutation is autosome recessive.It is the first report of the loss-of-function in PIWIL2 in SCOS.Part 2 Generation of an iPSC line from the SCOS patient [Purpose] The location of PIWIL2 in germ cells is different in human and mouse indicating that the exact functions of PIWIL2 may be different in human and mouse.The role of PIWIL2 mutation in SCOS can not be determined only by PIWIL2(Mili)knockout mouse model.In oreder to vadiate the effects of PIWIL2 mutation on human germ cells through in vitro differentiation of iPSC into germ cells,we generated an induced pluripotent stem cell(iPSC)line from the SCOS patient and validated the pluripotency of the established iPSC line.[Methods] After implementing the informed consent of the patient,skin samples were taken from the incision of micro-dissection of testicular sperm extraction to isolate skin fibroblasts.Once stably cultured,fibroblasts were infected with lentivirus containing OCT4,SOX2,KLF4 and C-MYC to be reprogrammed into iPSCs.The pluripotency of the established iPSC line(passage 10)was confirmed by staining alkaline phosphatase,determining the expression of pluripotent genes and in vitro embryoid body formation followed by immunostaining of germ layer genes.The karyotype of the iPSC line(passage 10)was also analyzed.Sanger sequencing was used to detect the c.731?732del AT in PIWIL2 gene.[Results] Fibroblasts were isolated from patient's skin samples and successfully reprogrammed into iPSCs.The established iPSC line showed a morphology of human embryonic stem cells and was alkaline phosphatase positive.RT-PCR and immunofluorescence showed the iPSC line expressing OCT4,SOX2,NANOG and TRA 1-60.iPSCs were able to form embryoid bodies in vitro which expressed the markers of three germ layers,the ectodermal marker MAP2,the mesodermal marker SMA and the endodermal marker SOX17.The iPSC line has normal karyotype and c.731?732del AT in PIWIL2 gene.[Conclusions] The iPSC line carrying c.731?732del AT in PIWIL2 gene was successfully established from fibroblasts of the SCOS patient.After several passages,the iPSC line had normal karyotype and was able to differentiate into different types of cells.The iPSC line generated from the SCOS patient could be used to in vitro differentiate into germ cells.Part 3 Effect of PIWIL2 deletion on the differentiation of iPSCs into germ cells[Purpose] It is very likely that SCOS characterized by no spermatogenic cells in testes is caused by abnormalities in primordial germ cells(PGCs)and spermatogonial stem cells (SSCs).We investigated the effect of PIWIL2 deletion on in vitro differentiation of iPSC into germ cells(PGC-like cells and SSC-like cells)to facilitate the understanding of the role of loss-of-function mutation in PIWIL2 gene in SCOS.[Methods] PIWIL2 was deleted in the iPSC line derived from a normal fertile man(control iPSC)by CRISPR/Cas9 and the PIWIL2 knockout iPSC line was regard as the positive control of the patient iPSC line.The iPSC lines with homozygous deletion or heterozygous deletion of PIWIL2(PIWIL2-/-iPSC and PIWIL2^+/-iPSC,respectively)were retained for further studies,and PIWIL2^+/-iPSC was the control of PIWIL2-/-iPSC.The pluripotent genes and transposon expression,cell proliferation and cell cycle of all established iPSC lines were determined.All the iPSC lines were differentiated into PGC-like cells(PGCLCs).The induction rate was determined at 2,4,6 days of differentiation by flow cytometry.RT-qPCR was used to investigate the expression of PGC genes and pluripotent genes at 4 days of differentiation.The migration of PGCLCs was observed through immunofluorescence staining CD38.Valproic acid(VPA)was introduced into SSC-like cell(SSCLC)induction system to improve the induction rate of SSCLC and the dynamics of SSCLC induction rate,the cell apoptosis and the expression of SSC genes were determined during SSCLC induction.The feasibility of developed SSCLC induction system was assessed by several stem cell lines.All the iPSC lines were differentiated into SSCLC using the developed SSCLC induction system.The induction rate was measured at 8,10,12,14 days of differentiation by flow cytometry.RT-qPCR and immunofluorescence were used to investigate the expression of SSC genes and pluripotent genes at 12 days of differentiation.[Results] The expression of pluripotent genes and transposon LINE-1,cell proliferation and cell cycle of all four iPSC lines were comparable.Deletion of PIWIL2 did not alter the features of iPSCs.No significant difference of induction rate among four groups was observed at 2 days of PGCLC differentiation.The PGCLC induction rate of PIWIL2-/-,PIWIL2^+/-and patient groups was reduced compared with the control group at 4 and 6 days of induction,but the induction rate was not significantly different between PIWIL2-/-and PIWIL2^+/-groups.RT-qPCR results showed that the expression of PGC genes was decreased in PIWIL2-/-and patient groups compared with the control group,but the difference was not significant between PIWIL2-/-and PIWIL2^+/-groups.Additionally,PGCLCs of all the groups showed the ability to migrate.Deletion of PIWIL2 was unlikely to influence the PGCLC specification and migration.VPA could raise the induction rate of SSCLC,which was more effective than vitamin C(VC)of the reference article.Combination of relatively low concentrations of VC and VPA led to the lower apoptosis rate during induction and the highest induction rate of SSCLC at 12 days of differentiation.This induction system could be used to induce SSCLCs from different NOA iPSC lines,and SSCLCs derived through the developed induction system expressed several SSC genes.The elevated induction rate may because of changed histone modification induced by the developed induction system.The four iPSC lines were differentiated into SSCLCs using the developed induction system.The induction rate of PIWIL2-/-and patient groups was decreased compared with control and PIWIL2^+/-group at 8,10,12 and 14 days of differentiation,and the SSCLC rate in these two groups could not be maintained at a certain level from 10 to 14 days of differentiation.The results of RT-qPCR and immunofluorescence also showed that the expression of SSC genes of PIWIL2-/-and patient groups was decreased at 12 days of differentiation.Deletion of PIWIL2 negtively affected the formation and maintenance of SSCLCs.[Conclusions] Combination of relatively low concentrations of VC and VPA led to the highest induction rate of SSCLC,and the developed induction system was able to differentiate several stem cell lines into SSCLCs with high efficacy and stability.This SSCLC induction system could be used as a cell model to study the formation and development of human SSCs in vitro and assess the associaton of newly identified pathogenic genes and NOA.Deletion of PIWIL2 in iPSCs was unlikely to affect PGCLC specification and migration,but negatively affected the formation and maintenance of SSCLC,which was different from the Mili(PIWIL2)knockout mice with abnormalities in SSC maintenance and differentiation.SCOS patient with the loss-of-function mutation in PIWIL2 gene was likely to have abnormalities in SSC formation and maintenance.SSCs have a critical role in ensuring normal spermatogenesis after birth,the gradual loss of SSCs will lead to the complate failure of spermatogenesis and cause SCOS.This study further comfirmed that PIWIL2 is a pathogenic gene of SCOS.Part 4 The mechanism of impaired establishment and maintenance of spermatogonial stem cell-like cells caused by PIWIL2 deletion [Purpose] Deletion of PIWIL2 negatively affected the formation and maintenance of spermatogonial stem cell-like cells(SSCLCs).In this part,we investigated the mechanism of impaired formation and maintenance of SSCLC caused by PIWIL2 deletion to provide explanations of the mechanism of loss-of-function mutation in PIWIL2 gene in SCOS.[Methods] At 12 days of differentiation,the rate of apoptosis cells of the control,PIWIL2-/-,PIWIL2^+/-and patient groups was detected by flow cytometry and RT-qPCR was used to determine LINE-1 expression of four groups.An EGFP reporter plasmid containing PLZF mRNA 3'UTR was constructed and transfected into control and PIWIL2-/-iPSCs to explore whether PIWIL2 deletion could directly affect the stability of PLZF(SSC gene)mRNA.EGFP was observed at 24 h and 48 h after transfection.To explore whether PLZF expression was affected by epigenetics,immunofluorescence was used to detect PLZF and histone modification(H3K9ac and H3K27me3).RNA-seq was performed to compare the transcriptome between control and PIWIL2-/-groups(iPSC lines and cells at 8 and 12 days of SSCLC differentiation),and differently expressed genes were analysed by GO and KEGG.The differently expressed genes were verified in the control,PIWIL2-/-,PIWIL2^+/-and patient groups at 8 and 12 days of SSCLC differentiation by RT-qPCR.[Results] At 12 days of differentiation,the rate of apoptosis cells and the expression of LINE-1 were significantly increased in PIWIL2-/-and patient groups compared with the control group,indicating that deletion of PIWIL2 induced apoptosis and derepressed LINE-1 during SSCLC induction.After transfected with UTR-EGFP at 24 h and 48 h,no difference of EGFP was observed between control and PIWIL2-/-groups,indicating that deletion of PIWIL2 did not directly affect the stability of PLZF mRNA.The co-location immunostaining of PLZF and H3K9 ac was weak in PIWIL2-/-and patient groups,and the immunostaining of H3K27me3 in these two groups was strong,indicating that the expression of PLZF was affected by histone modification.RNA-seq analysis showed the activation of WNT signaling pathway and SSC genes and the repression of pluripotent genes through the process of iPSCs differentiating into SSCLCs,which simulated the in vivo formation and development of human SSCs to some extent.The decreased expression of WNT signaling pathway genes and derepressed expression of pluripotent genes in PIWIL2-/-group may lead to reduced SSCLC induction rate and increased apoptosis,and the same results existed in the patient group validated by determined the expression of WNT signaling genes and pluripotent genes at 8 and 12 days of SSCLC differentiation by RT-qPCR.Loss-of-funtion mutation in PIWIL2 gene in SCOS might attenuate WNT signaling pathway and derepress pluripotent genes.[Conclusions] During iPSCs differenating into SSCLCs,WNT sianaling pathway was activiated and pluripotent genes were repressed and SSC genes were gradually upregulated,which simulated the in vivo formation and development of human SSCs to some extent.PIWIL2 deletion resulted in derepression of transposon and affected histone modification.Furthermore,PIWIL2 deletion affected the activation of WNT signaling pathway and the repression of pluripotent genes expression and induced apoptosis,leading to the impairment of SSC formation and maintenance.PIWIL2 might activate WNT signaling and repress pluripotent genes to facilitate the formation and maintenance of human SSCs,but the specific regulatory mechanism of PIWIL2 needed to be further studied.