The Function Of The ZP4 Studied Based On CRISPR-Cas9 Editing Technique

Background:Abnormal development of the ovum is a major cause of infertility.The zona pellucida（ZP）is a glycoprotein layer surrounding the ovum that is synthesized and secreted by oocytes during the egg growth phase and transported to the outside of the cell.The ZP plays a vital role in oogenesis,fertilization,and preimplantation embryonic development.Various morphological changes in the ZP,such as deletion,deformation,thinning,and fissure,are associated with infertility or reduced fertility.The ZP of mammalian eggs consists of 3-6 zona pellucida glycoproteins（ZPGs）,with humans and rats having four ZPGs（h ZP1-4 and r ZP1-4）,while the mouse ZP has only three ZPGs（m ZP1-3）.Investigation of the functions of ZP1,ZP2,and ZP3 in mice through gene knockout technology showed that ZP1-3-knockout mice have deletion or thinning of the ZP surrounding the oocytes together with reduced fertility or infertility.ZP1-3 plays a key role in several important biological processes,including maintenance of ZP integrity and participation in fertilization（including sperm-egg-specific recognition,triggering the sperm acrosome reaction,and prevention of polyspermic fertilization）.At the time of the inception of the present study,there was no research on the function of ZP4.Objective:CRISPR-Cas9 technology was used to construct ZP4^-/-rats and the effects of the knockout on the morphology and function of the ZP as well as on the fertility of the rats were investigated.The ZP1^WT/MTZP4^-/-,ZP2^WT/MTZP4^-/-,ZP3^+/-ZP4^-/-double-knockout rat models were constructed by hybridization to study the association between ZP4 and ZP1-3.Method:Part 1.The ZP4^-/-rat model1.Construction and identification of the ZP4^-/-rat model:CRISPR-Cas9 was used to construct the ZP4^-/-rat model.Identification was at the g DNA,m RNA,and protein levels,and off-target detection was also performed.2.Assessment of growth and fertility in female ZP4^-/-rats:（1）The growth and development of the rats were evaluated by comparing the ovarian and body weights;（2）The natural fertility of the ZP4^-/-and ZP4^+/+female rats was compared by calculating the number of births and the number of offspring born per litter;（3）Eggs were obtained through super-ovulation and were fertilized in vitro.The numbers of eggs and the fertilization rates were compared.3.Evaluation of ZP morphology in eggs:（1）Morphological differences in the ZPs of ZP4^-/-and ZP4^+/+female rats were evaluated by light microscopy.（2）The growing follicles of the ZP4^-/-and ZP4^+/+rats were observed by Periodic-Acid Schiff（PAS）staining.4.Evaluation of the structural properties of the ZP in eggs:（1）The permeability of the ZP in ZP4^-/-and ZP4^+/+rats was compared by fluorescent nanosphere penetration.（2）Scanning electron microscopy was used to compare the mesh structures of the ZPs.（3）The numbers of sperm binding to the ova were compared between ZP4^-/-and ZP4^+/+rats.5.ZP1-3 m RNA expression levels in ZP4^+/+and ZP4^-/-rats:Total RNA was extracted from the ovary tissue and the differences in ZP1-3expression between the ZP4^-/-and ZP4^+/+rats were assessed at the m RNA level.6.Comparison of ZP4 between humans,rats,and rabbits:Bioinformatic analyses were used to compare ZP4 differences in terms of nucleic acid sequences,amino acid sequences,amino acid compositions,hydrophobicity,secondary structures,transmembrane domains,and signal peptides.Part 2.Effects of ZP4 knockout on ZP1-31.Female rats with ZP1-3&ZP4:ZP1^WT/MT,ZP2^WT/MTand ZP3^+/-genotypes were naturally caged with ZP4^-/-males to obtain ZP1^WT/MTZP4^-/-,ZP2^WT/MTZP4^-/-,and ZP3^+/-ZP4^-/-female rats,respectively,and the genotypes were confirmed.2.Evaluation of growth,development,and fertility of ZP1-3&ZP4female rats with the above genotypes:（1）The growth and development of the female rats were monitored by weekly weighing;（2）The average numbers of pregnancies and litter sizes were calculated after caging with ZP4^+/+male rats;（3）The total numbers of eggs and the average numbers of eggs were compared.3.Evaluation of ZP morphology and structure in ZP1-3&ZP4 female rats with the above genotypes:（1）ZP morphology and thickness were observed under light microscopy;（2）The morphology of the eggs and ZP were assessed by PAS staining;（3）ZP permeability was measured by fluorescence nanoparticle.ResultsPart 1.The ZP4^-/-rat model1.Construction and identification of ZP4^-/-rats:（1）ZP4^-/-rats were successfully constructed.（2）Measurement of g DNA suggested that ZP4 in the ZP4^-/-rats lacked 9751 bp.（3）m RNA measurements indicated that the coding sequence（CDS）of the coding region in the ZP4^-/-rats was reduced by 1618 bp.（4）ZP4 protein was not detected in ZP4^-/-rats.（5）No abnormal mutation was detected in 20 off target sites.2.Assessment of growth and fertility in female ZP4^-/-rats:（1）There were no significant differences in growth and development between ZP4^-/-and ZP4^+/+female rats（P>0.05）.（2）No significant difference in the litter size was observed between ZP4^-/-and ZP4^+/+female rats（P>0.05）.（3）There were no significant differences in the number of eggs retrieved and fertilization rate between ZP4^-/-and ZP4^+/+female rats（P>0.05）.3.Evaluation of ZP morphology in eggs:The morphology and thickness of the ZP between ZP4^-/-and ZP4^+/+female rats did not differ significantly,nor did the densities or constituent ratios of the growing follicles（P>0.05）.4.Evaluation of ZP structural features in eggs:（1）There was no significant change in the permeability of the ZP between ZP4^-/-and ZP4^+/+female rats（P>0.05）.（2）Scanning electron microscopy showed no significant differences in the ZP mesh structure between ZP4^-/-and ZP4^+/+female rats（P>0.05）.（3）No significant differences in the numbers of sperm binding between the ZP4^-/-and ZP4^+/+female rats were seen under light microscopy.5.Comparison of the m RNA levels of ZP1-3 in ZP4^+/+and ZP4^-/-rats:There was no significant difference in the m RNA levels of ZP1-3 between ZP4^-/-and ZP4^+/+female rats（P>0.05）.6.Differences between human,rat,and rabbit ZP4 structures.Compared with rats,humans and rabbits showed more similarity in both the nucleic acid and amino acid sequences of ZP4.The isoelectric points of ZP4 protein differed significantly among the three species.Differences in hydrophilicity were seen in two places,while the differences in the secondary structure were not significant.Part 2.Effects of ZP4 knockout on ZP1-31.The growth,development,and fertility of ZP1-3&ZP4 female rats were as follows:（1）No significant differences were seen between ZP1^WT/MTZP4^+/-,ZP1^WT/MTZP4^-/-,ZP2^WT/MTZP4^+/-,ZP2^WT/MTZP4^-/-,ZP3^+/-ZP4^+/-,ZP3^+/-ZP4^-/-,and wild-type female rats（P>0.05）;（2）The average litter size of the ZP1^WT/MTZP4^-/-rats did not differ significantly from the ZP1^WT/MTZP4^+/-rats（P>0.05）;The average litter size of ZP2^WT/MTZP4^-/-females was significantly lower than that of ZP2^WT/MTZP4^+/-rats（P<0.05）;The average litter size of ZP3^+/-ZP4^-/-females was significantly lower than that of ZP3^+/-ZP4^+/-females（P<0.05）;（3）The superovulation results showed that both the total and average egg numbers in ZP1^WT/MTZP4^-/-rats were lower than in ZP1^WT/MTZP4^+/-rats although the differences were not statistically significant（P>0.05）;Both the total and average egg numbers in the ZP2^WT/MTZP4^-/-rats were significantly lower than in ZP2^WT/MTZP4^+/-rats（P<0.05）;The total and average egg numbers in the ZP3^+/-ZP4^-/-females were significantly lower than in ZP3^+/-ZP4^+/-females（P<0.05）.2.The morphology of the ZP in ZP1-3&ZP4 female rats:（1）Although the ZP in the ova of ZP1^WT/MTZP4^-/-rats was complete,the average thickness was significantly less than that in ZP1^WT/MTZP4^+/-rats（P<0.05）;The permeability of the ZP1^WT/MTZP4^-/-ZP to fluorescent antiparticles did not differ significantly from that in ZP1^WT/MTZP4^-/-females;（2）The abnormal morphology of ZP2^WT/MTZP4^-/-oocytes increased,but with essentially complete ZP;The ZP thickness in the ZP2^WT/MTZP4^-/-ovum did not differ significantly from that of the ZP2^WT/MTZP4^+/-ova（P>0.05）;The permeability of the ZP to fluorescent antiparticles was not significantly different between ZP2^WT/MTZP4^-/-and ZP2^WT/MTZP4^+/-females;（3）Cytoplasmic shrinkage was observed in the ZP3^+/-ZP4^-/-rat oocytes,with significant widening of the periegg space,while the ZP was complete;The ZP in the ZP3^+/-ZP4^-/-and ZP3^+/-ZP4^+/-rats were significantly thinner than those in the wild-type（P<0.05）.Conclusion:1.CRISPR-Cas9 gene editing was used to investigate the function of ZP4 in the rat.The results showed that complete knockout of ZP4 did not affect the formation of the ZP nor the reproductive processes of female rats,suggesting that ZP4 may not be required for the maintenance of normal ZP function.2.The lack of phenotypic effects in the full ZP4 knockout,together with the observation of abnormal ZP morphology and impaired fertility in the rats with homozygous deletion of ZP4 and heterozygous mutation of ZP1-3 suggests that ZP4 is a modifier gene of ZP1-3,with potentially complementary compensatory effects.