Failure of oocyte activation,including polyspermy and defects in pronuclear(PN)formation,triggers early embryonic developmental arrest.Many studies have shown that phospholipase C zeta 1(PLCZ1)mutations cause failure ...Failure of oocyte activation,including polyspermy and defects in pronuclear(PN)formation,triggers early embryonic developmental arrest.Many studies have shown that phospholipase C zeta 1(PLCZ1)mutations cause failure of PN formation following intracytoplasmic sperm injection(ICSI);however,whether PLCZ1 mutation is associated with polyspermy during in vitro fertilization(IVF)remains unknown.Whole-exome sequencing(WES)was performed to identify candidate mutations in couples with primary infertility.Sanger sequencing was used to validate the mutations.Multiple PLCZ1-mutated sperm were injected into human and mouse oocytes to explore whether PN formation was induced.Assisted oocyte activation(AOA)after ICSI was performed to overcome the failure of oocyte activation.We identified three PLCZ1 mutations in three patients who experienced polyspermy during IVF cycles,including a novel missense mutation c.1154C>T,p.R385Q.PN formation failure was observed during the ICSI cycle.However,injection of multiple PLCZ1-mutated sperm induced PN formation,suggesting that the Ca2+oscillations induced by the sperm exceeded the necessary threshold for PN formation.AOA after ICSI enabled normal fertilization,and all patients achieved successful pregnancies.These findings expand the mutational spectrum of PLCZ1 and suggest an important role for PLCZ1 in terms of blocking polyspermy.Furthermore,this study may benefit genetic diagnoses in cases of abnormal fertilization and provide potential appropriate therapeutic measures for these patients with sperm-derived polyspermy.展开更多
Phospholipase C zeta(PLC)is a key sperm-borne oocyte-activating factor that triggers Ca^(2+)oscillations and the subsequent block to polyspermy following gamete fusion.Mutations in PLCZ1,the gene encoding PLCζ,cause ...Phospholipase C zeta(PLC)is a key sperm-borne oocyte-activating factor that triggers Ca^(2+)oscillations and the subsequent block to polyspermy following gamete fusion.Mutations in PLCZ1,the gene encoding PLCζ,cause male infertility and intracytoplasmic sperm injection(ICSI)fertilization failure;and PLCζ expression and localization patterns are significantly correlated with ICSI fertilization rate(FR).However,in conventional in vitro fertilization(cIVF),whether and how sperm PLCζ affects fertilization remain unclear.Herein,we identified one previously reported and two novel PLCZ1 mutations associated with polyspermy in vitro that are characterized by excessive sperm-zona binding and a delay in pronuclei(PN)formation.Immunofluorescence staining and oocyte activation testing revealed that virtually all spermatozoa from patients lacked functional PLCζ and were thus unable to evoke Ca^(2+) oscillations.ICSI with an artificial oocyte activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth.Furthermore,we analyzed PLCζ in an additional 58 males after cIVF treatment in the Reproductive and Genetic Hospital of CiTiC-Xiangya(Changsha,China)between February 2019 and January 2022.We found that the proportion of spermatozoa that expressed PLCζ was positively correlated with both 2PN rate and total FR.The optimal cutoff value below which males were likely to experience low FR(total FR≤30%)after clVF was 56.7%for the proportion of spermatozoa expressing PLC5.Our study expands the mutation and the phenotypic spectrum of PLCZ1 and further suggests that PLCζ constitutes a promising biomarker for identifying low FRs cases in cIVF due to sperm-related oocyte activation deficiency and that sperm PLCζ analysis may benefit the widermale population and not onlymen with IcsI failure.展开更多
基金the General Project of Chongqing Natural Science foundation of China(cstc2021jcyj-msxmX0877)the Chongqing Medical Scientific Research Project(Joint Project of Chongqing Health Commission and Science and Technology Bureau,2023MSXM054)the General Project of Chongqing Health Center for Women and Children(2020YJMS01 and 2021YJMS05).
文摘Failure of oocyte activation,including polyspermy and defects in pronuclear(PN)formation,triggers early embryonic developmental arrest.Many studies have shown that phospholipase C zeta 1(PLCZ1)mutations cause failure of PN formation following intracytoplasmic sperm injection(ICSI);however,whether PLCZ1 mutation is associated with polyspermy during in vitro fertilization(IVF)remains unknown.Whole-exome sequencing(WES)was performed to identify candidate mutations in couples with primary infertility.Sanger sequencing was used to validate the mutations.Multiple PLCZ1-mutated sperm were injected into human and mouse oocytes to explore whether PN formation was induced.Assisted oocyte activation(AOA)after ICSI was performed to overcome the failure of oocyte activation.We identified three PLCZ1 mutations in three patients who experienced polyspermy during IVF cycles,including a novel missense mutation c.1154C>T,p.R385Q.PN formation failure was observed during the ICSI cycle.However,injection of multiple PLCZ1-mutated sperm induced PN formation,suggesting that the Ca2+oscillations induced by the sperm exceeded the necessary threshold for PN formation.AOA after ICSI enabled normal fertilization,and all patients achieved successful pregnancies.These findings expand the mutational spectrum of PLCZ1 and suggest an important role for PLCZ1 in terms of blocking polyspermy.Furthermore,this study may benefit genetic diagnoses in cases of abnormal fertilization and provide potential appropriate therapeutic measures for these patients with sperm-derived polyspermy.
基金supported by grants from the National Natural Science Foundation of China(No.81901553 to CD)the Natural Science Foundation of Hunan Province,China(No.2021JJ30461 to CD).
文摘Phospholipase C zeta(PLC)is a key sperm-borne oocyte-activating factor that triggers Ca^(2+)oscillations and the subsequent block to polyspermy following gamete fusion.Mutations in PLCZ1,the gene encoding PLCζ,cause male infertility and intracytoplasmic sperm injection(ICSI)fertilization failure;and PLCζ expression and localization patterns are significantly correlated with ICSI fertilization rate(FR).However,in conventional in vitro fertilization(cIVF),whether and how sperm PLCζ affects fertilization remain unclear.Herein,we identified one previously reported and two novel PLCZ1 mutations associated with polyspermy in vitro that are characterized by excessive sperm-zona binding and a delay in pronuclei(PN)formation.Immunofluorescence staining and oocyte activation testing revealed that virtually all spermatozoa from patients lacked functional PLCζ and were thus unable to evoke Ca^(2+) oscillations.ICSI with an artificial oocyte activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth.Furthermore,we analyzed PLCζ in an additional 58 males after cIVF treatment in the Reproductive and Genetic Hospital of CiTiC-Xiangya(Changsha,China)between February 2019 and January 2022.We found that the proportion of spermatozoa that expressed PLCζ was positively correlated with both 2PN rate and total FR.The optimal cutoff value below which males were likely to experience low FR(total FR≤30%)after clVF was 56.7%for the proportion of spermatozoa expressing PLC5.Our study expands the mutation and the phenotypic spectrum of PLCZ1 and further suggests that PLCζ constitutes a promising biomarker for identifying low FRs cases in cIVF due to sperm-related oocyte activation deficiency and that sperm PLCζ analysis may benefit the widermale population and not onlymen with IcsI failure.
