Mutant ZP1 in familial infertility

Abstract

The human zona pellucida is composed of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) and has an important role in reproduction. Here we describe a form of infertility with an autosomal recessive mode of inheritance, characterized by abnormal eggs that lack a zona pellucida. We identified a homozygous frameshift mutation in ZP1 in six family members. In vitro studies showed that defective ZP1 proteins and normal ZP3 proteins colocalized throughout the cells and were not expressed at the cell surface, suggesting that the aberrant ZP1 results in the sequestration of ZP3 in the cytoplasm, thereby preventing the formation of the zona pellucida around the oocyte.

Figure 1. Phenotypic Features of the Patients

Panel A shows a normal oocyte with surrounding insoluble zona pellucida, and Panel B shows a structure diagram of the human zona pellucida. Panel C shows the pedigree of the family. Squares denote male family members, circles female family members, and solid symbols affected members; slashes denote deceased family members, equal signs infertility, and the double line a consanguineous marriage. The arrow indicates the index patient (family member IV-3). The zona pellucida 1 (ZP1) genotype for each person is indicated, with NM indicating no mutation in the normal allele and a dash (–) indicating the homozygous deletion of the eight nucleotides in ZP1 that are responsible for infertility. Panel D shows six oocytes from the index patient, which were cultured for 6 hours after follicular aspiration, separated from granulosa cells, and then imaged immediately. Panel E shows four oocytes from the fifth sister (IV-5). Oocytes 1, 2, 3, and were successfully isolated; oocyte 4 was closely surrounded by granular cells.

Figure 2. (facing page). Immunofluorescence in Eggs and Pathogenic Model

Panel A depicts the process through which the zona pellucida (ZP) proteins form a normal zona pellucida (upper diagram) and the hypothetical pathogenic mechanism that prevents the formation of the zona pellucida (lower diagram); ZP1, ZP2, and ZP3 are vital normal ZP proteins. Panel B shows oocytes imaged with the use of confocal laser scanning microscopy and visible inverted microscopy. The fluorescent signals of ZP proteins 1 (green) and 3 (red) were imaged individually and merged (orange). The signal pattern of normal oocytes is different from that of the oocytes from the family member IV-5.

Figure 3. Genetic and Bioinformatic Analysis of ZP1

Panel A shows the results of polyacrylamide-gel electrophoresis (PAGE) of the products of polymerase-chain-reaction amplification of a region of exon 7. The results are consistent with six members of the family (IV-1, IV-3, IV-4, IV-5, IV-6, and IV-7) having a homozygous deletion and other family members (II-1, II-2, III-2, and IV-2) having a heterozygous mutation. Panel B shows a normal sequencing profile and profiles for the homozygous and heterozygous deletion of TTTTCCCA between codons 1169 and 1176 in ZP1. Six family members had a homozygous deletion, and four carried a heterozygous deletion, which is consistent with the results of PAGE. The mutation leads to a frameshift and the formation of a premature stop codon, I390fs404X. Panel C shows the domain organization (top) and the crystallographic models (bottom) of nonmutant (left) and mutant ZP1 (right). The schematic diagram shows the deleted portions of ZP1 (e.g., the transmembrane domain, part of the zona pellucida domain, and other C-terminal domains).