Novel PLCZ1 mutation caused polyspermy during in vitro fertilization

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.

Defects in phospholipase C zeta cause polyspermy and low fertilization after conventional IVF:not just ICSI failure

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.

Male-Female Crosstalk during Pollen Germination, Tube Growth and Guidance, and Double Fertilization

Sperm cells of flowering plants are non-motile and thus require transportation to the egg apparatus via the pollen tube to execute double fertilization. During its journey, the pollen tube interacts with various sporophytic cell types that support its growth and guide it towards the surface of the ovule. The final steps of tube guidance and sperm delivery are controlled by the cells of the female gametophyte. During fertilization, cell-cell communication events take place to achieve and maximize reproductive success. Additional layers of crosstalk exist, including self-recognition and specialized processes to prevent self-fertilization and consequent inbreeding. In this review, we focus on intercellular communication between the pollen grain/pollen tube including the sperm cells with the various sporophytic maternal tissues and the cells of the female gametophyte. Polymorphic-secreted peptides and small proteins, especially those belonging to various subclasses of small cysteine-rich proteins （CRPs）, reactive oxygen species （ROS）/NO signaling, and the second messenger Ca2＋, play center stage in most of these processes.

“Love Is Strong, and You＇re so Sweet”： JAGGER Is Essential for Persistent Synergid Degeneration and Polytubey Block in Arabidopsis thaliana

Successful double fertilization and subsequent seed development in flowering plants requires the delivery of two sperm cells, transported by a pollen tube, into the embryo sac of an ovule. The embryo sac cells tightly control synergid cell death, and as a result the polyspermy block. Arabinogalactan proteins are highly glycosylated proteins thought to be involved in several steps of the reproductive process. We show that JAGGER, Arabinogalactan Protein 4, is an important molecule necessary to prevent the growth of multiple pollen tubes into one embryo sac in Arabidopsis thaliana. In jagger, an AGP4 knockout mutant, the pistils show impaired pollen tube blockage as a consequence of the survival of the persistent synergid. JAGGER seems to be involved in the signaling pathway that leads to a blockage of pollen tube attraction. Our results shed light on the mechanism responsible for preventing polyspermy in Arabidopsis and for safe- guarding successful fertilization of all ovules in one pistil, ensuring seed set and the next generation.