Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells(SSCs).However,such therapy for infertility has n...Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells(SSCs).However,such therapy for infertility has not been experimentally investigated yet.In this study,a mouse model with an X-linked testis-expressed 11(TEX11)mutation(Tex11PM/Y)identified in azoospermia patients exhibited meiotic arrest due to aberrant chromosome segregation.Tex11PM/Y SSCs could be isolated and expanded in vitro normally,and the mutation was corrected by clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated endonuclease 9(Cas9),leading to the generation of repaired SSC lines.Whole-genome sequencing demonstrated that the mutation rate in repaired SSCs is comparable with that of autonomous mutation in untreated Tex11PM/Y SSCs,and no predicted off-target sites are modified.Repaired SSCs could restore spermatogenesis in infertile males and give rise to fertile offspring at a high efficiency.In summary,our study establishes a paradigm for the treatment of male azoospermia by combining in vitro expansion of SSCs and gene therapy.展开更多
There are many unknown genetic factors that lead to infertility in nonobstructive azoospermia men.Here,we performed whole-exome sequencing in blood samples obtained from 40 azoospermia patients with meiotic arrest and...There are many unknown genetic factors that lead to infertility in nonobstructive azoospermia men.Here,we performed whole-exome sequencing in blood samples obtained from 40 azoospermia patients with meiotic arrest and found a novel c.151_154del(p.D51fs)frame-shift mutation in exon 3 of the testis expressed 11(TEX11)gene in one patient.Sanger sequencing analysis of the patient and 288 fertile men was performed to validate the mutation.Immunohistochemical analysis showed TEX11 expression in late-pachytene spermatocytes and in round spermatids in fertile human testes.In contrast,testes of the patient with TEX11 mutation underwent meiotic arrest and lacked TEX11 expression.Western blotting of human embryonic kidney(HEK293)cells transfected with a vector for the p.D51fs TEX11 variant detected no TEX11 expression.In conclusion,we identified a novel frame-shift mutation in the TEX11 gene in an azoospermia patient,emphasizing that this gene should be included in genetic screening panels for the clinical evaluation of azoospermia patients.展开更多
Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks(DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. Howeve...Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks(DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction.Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I.Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.展开更多
基金This study was supported by Genome Tagging Project and grants from the Chinese Academy of Sciences,the National Key Research and Development Program of China,Shanghai Municipal Commission for Science and Technology,and the National Natural Science Foundation of China(XDB19010204,2019YFA0109900,OYZDJ-SSW-SMC023Facility-based Open Research Program,19411951800,17JC1420102,31821004,32030029,31730062,31530048,and 81672117)The research is partly supported by the Fountain-Valley Life Sciences Fund of University of Chinese Academy of Sciences Education Foundation。
文摘Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells(SSCs).However,such therapy for infertility has not been experimentally investigated yet.In this study,a mouse model with an X-linked testis-expressed 11(TEX11)mutation(Tex11PM/Y)identified in azoospermia patients exhibited meiotic arrest due to aberrant chromosome segregation.Tex11PM/Y SSCs could be isolated and expanded in vitro normally,and the mutation was corrected by clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated endonuclease 9(Cas9),leading to the generation of repaired SSC lines.Whole-genome sequencing demonstrated that the mutation rate in repaired SSCs is comparable with that of autonomous mutation in untreated Tex11PM/Y SSCs,and no predicted off-target sites are modified.Repaired SSCs could restore spermatogenesis in infertile males and give rise to fertile offspring at a high efficiency.In summary,our study establishes a paradigm for the treatment of male azoospermia by combining in vitro expansion of SSCs and gene therapy.
基金the Key Research and Development Program of Shandong Province(2019GSF108237)the Young Scholars Program of Shandong University(2016WLJH50)the Natural Science Foundation of Shandong Province(ZR2017MH049).
文摘There are many unknown genetic factors that lead to infertility in nonobstructive azoospermia men.Here,we performed whole-exome sequencing in blood samples obtained from 40 azoospermia patients with meiotic arrest and found a novel c.151_154del(p.D51fs)frame-shift mutation in exon 3 of the testis expressed 11(TEX11)gene in one patient.Sanger sequencing analysis of the patient and 288 fertile men was performed to validate the mutation.Immunohistochemical analysis showed TEX11 expression in late-pachytene spermatocytes and in round spermatids in fertile human testes.In contrast,testes of the patient with TEX11 mutation underwent meiotic arrest and lacked TEX11 expression.Western blotting of human embryonic kidney(HEK293)cells transfected with a vector for the p.D51fs TEX11 variant detected no TEX11 expression.In conclusion,we identified a novel frame-shift mutation in the TEX11 gene in an azoospermia patient,emphasizing that this gene should be included in genetic screening panels for the clinical evaluation of azoospermia patients.
基金supported by the National Key Research and Developmental Program of China (2018YFC1003700, 2016YFC1000600, 2018YFC1003400 and 2018YFC1004700)the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000)the National Natural Science Foundation of China (31890780, 31630050, 31871514 and 31771668)。
文摘Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks(DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction.Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I.Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.
