A Recurrent Missense Mutation In ZP3 Causes Empty Follicle Syndrome And Female Infertility

Chapter I Identification of the causative mutation of empty follicle syndromeObjective:Empty follicle syndrome(EFS)is defined as the failure to obtain oocytes from mature ovarian follicles despite normal estrogen levels and normal follicular development under ultrasound.It happens both in natural cycles and during in vitro fertilization(IVF).For individuals with recurrent EFS,there are no effective treatment options besides oocyte donation.Except for some cases caused by pharmacological or iatrogenic problems,the etiology of EFS remains enigmatic.Some have believed that early oocyte atresia,oocyte aging and poor ovary reserve were related with EFS.As for genetic mechanisms of EFS,previous studies included pericentric inversion of chromosome 2 and LHCGR mutations.However,these studies were all about simplex cases or small pedigrees and there have been no studies of large EFS pedigrees till now.In the present study,we describe a large family(family A)with a dominant inheritance pattern of female infertility characterized by recurrent EFS.The proband and her elder sister both showed recurrent EFS in multiple IVF cycles.Two paternal aunts and two cousins of the proband also suffered unexplained primary infertility.In Chapter I of this study,we planned to use genetic approaches to map the causative mutation.Method:In order to identify disease-associated regions,a genome-wide linkage scan was performed on 17 family members.To further determine the causative mutation,whole-exome sequencing(WES)was carried out in 3 affected females,2 potential male carriers as well as 2 unaffected females.The identified variants were then verified and filtered by public databases and women controls with normal fertility.Subsequently,the selected variant was sequenced in another large EFS family(family B)and a cohort of 21 EFS cases.For EFS cases with the mutation,their pedigrees were traced.Finally,based on these results together,the causative mutation would be confirmed.Result:The genome-wide linkage analysis yielded a maximum LOD score of 2.35 in chromosome 7 and the common minimal region was chromosome 7p12.3-q21.13.Exome sequencing revealed a heterozygous missense variant(c.400G>A)[p.Ala134Thr]in exon 2 of ZP3 as the potential pathogenic variant,segregating perfectly with the disease status in affected and unaffected female individuals of the family.It is noteworthy that this variant was found on chromosome 7q 11.23,which localized to the previously identified disease-associated region by linkage analysis.In addition,c.400G>A was found to co-segregate with disease status in family B as well.Haplotype analysis revealed that the disease allele of family A and family B originated independently.Furthermore,in a cohort of 21 individuals who exhibited EFS during IVF attempts,the same heterozygous mutation of ZP3 c.400G>A was identified in 2 individuals(SC-1 and SC-2),who were both simplex cases.The parents of SC-1 were both wild-type at this allele,suggesting the mutation of SC-1 was de novo.Taken together,we concluded that ZP3 c.400G>A(p.Ala134Thr)is the mutation responsible for EFS.Conclusion:ZP3 c.400 G>A(p.Ala134Thr)was responsible for EFS.Chapter Ⅱ In vitro functional study of the causative mutationObjective:ZP3 glycoprotein encoded by ZP3 is a major component of zona pellucida(ZP).ZP is a structure surrounding the oocyte,which plays an important role in oocyte development,fertilization and early embryo development.The mutation of p.Ala134Thr locates in the ZP domain of ZP3.ZP domain is crucial for the assembly of zona pellucida.In Chapter II of this study,we planned to uncover the function of the mutation via in vitro experiments.Method:The pathological changes of the proband’s oocyte and ovary was explored by immunofluorescence and staining of ovarian sections.To elucidate the molecular mechanism of the mutation,expression vectors of both wildtype and mutated protein was constructed and transfected into CHO-K1 cells.Protein was extracted from cell lysates and co-immunoprecipitation(Co-IP)analysis was performed to explore whether the mutation affected protein interactions.Result:In the immunofluorescence assay,ZP3 was barely detectable in the proband’s oocyte and the nucleus was totally disassembled.In HE stain and immunohistochemistry of ovary section,the proband’s oocyte was found to have neither an obvious ZP3 signal nor zona pellucida(ZP).In the Co-IP assay,when mutant ZP3 was co-expressed with wildtype ZP3,the interaction between wildtype ZP3 and ZP2 was markedly decreased due to the binding of wildtype ZP3 and mutant ZP3,via dominant negative inhibition.As a result,the assembly of ZP was impeded and the communication between cumulus cells and the oocyte was prevented,resulting in oocyte degeneration.Conclusion:The mutation of p.Ala134Thr could destroy the assembly of the ZP and led to oocyte degeneration,via dominant negative inhibition.Chapter Ⅲ Mouse model study of the causative mutationObjective:The structure of zona pellucida is highly conserved in mammalian animals.The mouse zona pellucida is composed of Zpl,Zp2 and Zp3,in which Zp2 and Zp3 consist of filaments crosslinked by Zp1.In previous studies,the Zp3-/-mouse was infertile.Very few oocytes could be recovered from oviducts of super ovulated Zp3-/-mouse,any oocytes that were retrieved from cumulus masses lacked a ZP,which was similar as the previously described EFS induvial.As the amino acid sequenced at this point was not conserved between human and mouse,in which it was alanine in human while valine in mice,the mouse model of p.Val134Thr was generated.In Chapter III of this study,we planned to explore the phenotype of p.Val34Thr knock-in mice.Method:The mouse model of p.Val134Thr mutation was generated via CRISPR-Cas9-mediated gene-modified semi-cloned mice from androgenetic haploid embryonic stem cells.The fertility was observed by mating with wild type male mice.Superovulation was then conducted and the number of retrieved oocytes was counted.HE staining,immunohistochemistry(IHC)of ZP3 and Periodic Acid-Schiff stain(PAS)was performed in the ovary sections to investigate the morphology of the oocytes.Result:Mice with homozygous mutation was infertile.In the superovulation experiment,most of the retrieved COCs were empty in homozygous mice,with only few of them contained a degenerated oocyte without ZP.In heterozygous mice,the retrieved oocytes were less than wildtype with a thinner ZP.The HE,IHC and PAS staining of ovary sections showed the oocyte of homozygous mice lacked ZP,which was consistent with EFS individuals.Conclusion:Mice with homozygous p.Val134Thr was completely infertile,most of the retrieved COCs in superovulation were empty,any oocytes that were retrieved were degenerated and lacked a ZP.This phenotype is similar as EFS individuals.