Investigating The Molecular Genetic Mechanisms Of Impaired Spermatogenesis Caused By Rare AKAP4 Gene Variants

Background:Infertility has been identified as a new type of disease that affects human life and health,with a global incidence rate of approximately 8-15%,of which male factors account for around 50%.Male infertility is typically caused by multiple factors or a combination of various diseases.The phenotypes of male infertility exhibit significant heterogeneity,with non-obstructive azoospermia being a severe sterility condition.The genetic origin has been recognized according to the accumulating knowledges of male germ cell biology and the complexity of sperm development processes involving numerous testis-specific genes.Objectives:1.To determine highly candidate pathogenic variants for specific phenotypes through clinical genetic analysis based on male infertility pedigrees.2.To construct humanized mouse models with candidate gene knock out or knock-in to evaluate the effects of candidate genes on male mouse fertility and the detrimental effects of candidate gene variants on male reproductive abilities and explore the molecular mechanisms underlying male infertility.3.To explore the molecular mechanisms of impaired fertility caused by candidate gene mutations through in-depth analysis of single-cell transcriptomic data from mouse testes on public data platforms.4.To observe the impact of candidate variations through constructing candidate gene expression vectors on protein and cell functions through in vitro experiments.Methods:1.A family which the two probands were diagnosed as non-obstructive azoospermia was recruited in the Andrology clinic.Candidate genes and variants were identified through whole-exome sequencing and a stringent filtering process.Further filtering of candidate genes was performed using protein databases and mouse phenotype databases to obtain prioritized candidate variants.Sanger sequencing validation and co-segregation analysis within the pedigree were conducted.2.CRISPR/Cas9 technology was applied to generate humanized mutation mice.Fertility testing experiments,litter size analysis,epididymal sperm counting,testicular histopathology,transmission electron microscope,and western blotting experiments were performed to determine the reproductive phenotypes,ultrastructural changes in sperm flagellum,and potential molecular mechanisms of gene-edited mice.3.Deep exploration of single-cell transcriptomic data of Akap4 knock out mouse testes from public databases.Differential expression analysis,RNA velocity analysis,and pseudotime analysis were conducted to evaluate the effects of Akap4 deletion on spermatogenic cell development.Gene ontology（GO）,Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis,and protein-protein interaction（PPI）analysis were performed to investigate the molecular mechanisms underlying Akap4-mediated impairment of spermatogenesis.4.Overexpression plasmid for AKAP4-WT and AKAP4-MUT were constructed to observe the effects of amino acid changes on protein stability,as well as cellular functions such as proliferation and apoptosis.Results:1.We identified a hemizygous variant（NM＿003886.2:c.1286G>A;p.Arg429His）in AKAP4 as candidate variants for non-obstructive azoospermia in infertile males through pedigree-based studies and another compound heterozygous variants（NM＿003575.2:c.904G>A;p.Gly302Ser/c.224C>T;p.Pro75Leu）in ZNF282 could not be precluded.2.We successfully generated Zfp282 knock out mice,the homologous gene of ZNF282,exhibited normal fertility and spermatogenesis and determined that ZNF282 variants did not affect male fertility.3.Female mice housed with Akap4^R428H males exhibited a decreased pregnancy rate（33.33% vs 96.00%）and litter size（8.46 vs 4.63）.4.Akap4^R428H male mice displayed normal spermatogenesis in testes,whatever,exhibited a reduced number in epididymal sperm count,and mutated sperm showed abnormal features such as short tails,coiled tails,and bent tails.Transmission electron microscopy revealed the poor development of the fibrous sheath in the principal piece of sperm from Aksp4^R428H male mice.5.Akap4^R428H male miceshowed a significant decrease in Akap4 protein levels in the testes.Additionally,the levels of Akap3 and Qrich2 proteins were reduced in the testes of Akap4^R428H male mice.6.Akap4 knock out mouse testicular single cell transcriptomic data mining revealed widespread transcriptional changes in testicular cells due to Akap4 deficiency.Akap4 deficiency leads to a decrease in RNA synthesis rate in germ cells,mainly manifested in haploid sperm cells.Pseudotime analysis revealed changes in the developmental state of round spermatids.Enrichment analysis of elongating spermatids differentially expression genes show the significant enrich effect in spermatogenisis pathway.PPI analysis constructs a spermatozoa dysfunction protein interaction network with Akap4 and Hlfnt as central components.7.Transfection of 293T cells revealed that AKAP4 p.R429H leads to a decrease in protein stability,and this mutation leads to a decrease in 293T cell proliferation activity and an increase in apoptosis level.Conclusion:1.A Pathogenic missense mutations in X-Iinked AKAP4 gene was identified（NM＿003886.2:c.1286G>A;p.R429H）to be the genetic cause in two non-obstructive azoospermia patients.2.The mice with homozygous point mutations in Akap4 exhibited reduced fertility and impaired development of fibrous sheath which confirmed the loss of function mechanism of the missense mutation.3.Akap4 deficiency leaded to widespread transcriptional changes in testicular cells.4.AKAP4 p.R429H leaded to a decrease in protein stability,a decrease proliferation activity,as well as an increased apoptosis level in 293T cell.