| Research Backgroud: Human spermatogenesis is a set of continuous processes consisting of the proliferation and differentiation of spermatogonia,meiosis of spermatocytes and spermiogenesis,which are precisely controlled in human testes.Disturbances on any of these processes will lead to spermatogenesis abnormality,which consequently lead to male infertility caused by oligospermia or azoospermia.It has been proved that genetic effect;hormone level and the environments we live are the critical factors for normal spermatogenesis.Homologous pairing,synapsis,recombination and crossover formation during meiosis prophase I in spermatocytes are the key processes of spermatogenesis.During meiosis,any disturbance on these processes may lead to the generation of abnormal gametes.Non-obstructive azoospermia is a sort of disease with the most complex etiology and difficulty on therapy in male infertility patients.The pathogenesis and mechanism of some Non-obstructive azoospermia patients are still unknown,which bring challenge to the therapy of this disease.Thus,getting a growing understanding of the genetic causes,the pathogeny and the mechanisms for the development of non-obstructive azoospermia,as well as the researches on abnormal gamete formation are of important significance for therapy.Purpose and significance: To study the synapsis and recombination processes in meiosis prophase I on spermatocytes of non-obstructive azoospermia patients by immunofluorescence staining.To explore the mechanism of spermatogenesis defect formation by studying the pathogeny of representative patients,and provide therapeutic cornerstones for the clinical diagnosis,therapy and genetic counseling.Methods: Carry out spermatocyte spreading and immunohistochemical staining on testicular tissues from 6 non-obstructive azoospermia patients and 5 fertile men.Use SCP3 antibody to stain the synaptonemal complex,use recombinase MLH1 antibody to mark the recombination sites,and use anti-Crest antibody to mark the centromeres to study the homolog pairing,synapsis and recombination of meiosis in non-obstructive azoospermia patients.By analysing the pattern of SCP3 staining,meiosis prophase I is divided into substages including leptotene,zygotene,pachytene and diplotene,which helps us to understand the meiosis progression.Analyse the expression of ?-H2 AX and BRCA1 and the expression of genes on translocated chromosomes by RT-PCR.To test the gene mutation on AR in complete androgen insensitivity syndrome(CAIS)patient by exon sequencing.And study the pathology characteristics of the testes of patients by H&E staining and IHC.Results: We identified the substages of meiosis prophase I in the spermatocytes of 6non-obstructive azoospermia patients by immunohistochemical staining.The ratio of leptotene spermatocytes in P1 and zygotene spermatocytes in P1,P2,P3 and P4 is significantly increased in control group.The ratio of pachytene spermatocytes in P1,P2 and P4 in control decreased significantly(P<0.05).No spermatocyte was found in P6 patient.We further analysed 326 pachytene spermatocytes in the 5 non-obstructive azoospermia patients with spermatocytes.The average number of MLH1 sites were42.2±12.6,48.1±6.5,49.5±5.0,44.0±4.8 and 45.1±7.0 in P1 to P5 respectively.Among which P1 and P4 beared significant reduce on the average MLH1 site per-cell.P1 is a patient with reciprocal translocations with a karyotype of 46,X,t(Y;1)(p11.3;p31).H&E and IHC staining with H1T2 antibody showed that the spermatogenesis of the patient arrested before late pacytene stage.Surface spread of the spermatocytes of the patients showed that the translocated chromosomes,together with the X chromosome,synapsed with each other to form tetravalent structure in some cells.Further analysis showed that the synapsis,DSB repair and crossover formation were significantly affected even on autosomes without translocations.We also observed significant downregulation on the expression of the genes around the breakpoint of translocated chromosomes,which can be caused by MSUC of the unsynapsed region around the breakpoint.The possibility of meiotic silence of genes was essential for meiosis progression and triggering of synapsis checkpoint by the unpaired regions,which may explain the spermatogenesis defect of the patient.In addition,MLH1 staining showed that X and Y chromosome recombination rate in patient with PAR area decreased significantly,which may make chromosomes not properly to arrange to the equatorial plate and activate the spindle checkpoint.It may be the main reason that leads to spermatogenesis processes arrested for the patient.P6 is a patient with normal karyotype of 46,XY.By phenotype analysis,we suspected the patient is a complete androgen insensitivity syndrome(CAIS)patient.DNA sequencing of the AR gene detected a missense mutation C.1715A>G(p.Y572C)in exon 2.H&E staining showed that most seminiferous tubules of the patient have only sertoli cells,while only a few tubules contained spermatogonia.Tubules were then stained with SOX9 and AMH,which revealed Setoli cell maturation arrest.Thus,we suggested that loss of the function of AR by mutation promoted the expression of AMH,which consequently lead to maturation failure of the Sertoli cells.Conclusion: The meiosis progression is delayed in non-obstructive azoospermia patients,with a possible reduction on the recombination rate in pachytene spermatocytes,which might be partially responsible for the defect on spermatogenesis.The chromosomal translocation patient showed meiosis progression arrest,reduction on recombination rate,gene silence on unsynapsed region,which subsequently lead to spermatogenesis defect and infertility in the patient.Mutation on AR may cause spermatogenesis defect by affecting the maturation of sertoli cells in testes.Our reseaches proved that spermatogenesis is not only affected by the chromosome structure and gene expressions,but also need the participation of sertoli cells and external hormones.Disturbance on any of these factors may lead to spermatogenetic defects. |
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