JBRA Assist. Reprod. 2025;29(Suppl 1):36-36
POSTER PRESENTATION
doi: 10.5935/1518-0557.20250090
1Instituto de Biología y Medicina Experimental (IBYME; CONICET) (Buenos Aires, Argentina)
2Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral (Santa Fe, Argentina)
Objective: Preventing fertilization failure requires a better understanding of sperm-egg interactions and predictive testing. Fertilization involves binding and fusion of the spermatozoon and the oocyte. Using biochemical and cell biology approaches, IZUMO1 was identified as the first sperm protein essential for fertilization. Its receptor, oocyte’s JUNO/IZUMO1R, was later found essential for gamete interaction. Their significance in this event led to further molecular characterization and identification of amino acids involved in the IZUMO1-JUNO binding: amino acids Trp148, His157 and Arg160 in IZUMO, and Lys81 and Trp62 in JUNO. In addition, Trp88 and Trp113 were found needed for IZUMO fusion. In recent years, other proteins present in both sperm and oocytes have been found to play a role in gamete interactions, thus increasing the process complexity. Our laboratory identified IZUMO4, another IZUMO family member, involvement in sperm-oocyte binding/fusion. A deep and thorough molecular and genomic characterization of these genes using bioinformatics and computational biology groundbreaking tools will provide essential knowledge to identify key players and better understand sperm-oocyte recognition. Moreover, they will lead to improved infertility diagnosis and treatment with cutting-edge innovative and precise molecular approaches. General
Objective: To explore innovative molecular approaches to further characterize fertilization-related events and provide novel approaches for future infertility diagnosis. Specific
Objectives: To evaluate 1) IZUMO1 and IZUMO4 RNA expression in healthy and pathological samples; 2) To model IZUMO-JUNO protein interaction and assess the amino acids involved, and 3) To explore and identify protein functional hotspots (amino acids predicted crucial for protein function) and genetic variants (changes in DNA sequence).
Methods: Bioinformatics and computational biology tools were used, including the Human Protein Atlas (HPA) and Gene Expression Omnibus (GEO), AlphaFold2, gnomAD, and AlphaMissense.
Results: RNA expression analysis in tissues and sperm: Both IZUMO1 and IZUMO4 RNAs expression was found enriched in the testis (nTPM = 18.4 and nTPM = 430.8, respectively) in meiotic and postmeiotic germ cells (HPA database). IZUMO4 but not IZUMO1 RNAs were less abundant in patients with teratozoospermia compared to normozoospermic individuals (GEO; GSE6969, Mann-Whitney test, p<0.0001). Protein interaction modeling: IZUMO4-JUNO interaction was analyzed using AlphaFold2 modeling, resulting in a reliable model (ranking confidence=0,67, pTM=0,73, ipTM=0,65). Amino acids relevant for their interaction were identified. The IZUMO1-JUNO interaction model was re-assessed considering IZUMO4 participation.Protein functional amino acids: AlphaMissense was used to identify functional hotspots in IZUMO1 and IZUMO4, by calculating the average pathogenicity score for each residue, considering all possible substitutions with high confidence. For IZUMO1, Trp148 and Trp113, previously shown to be critical for JUNO binding and fusion, respectively, along with 31 other amino acids were identified as hotspots. For IZUMO4, 11 amino acids were identified as functional hotspots.Genetic variation and hotspots: Heterozygous carriers of missense variants affecting key amino acids were identified in both genes. IZUMO1: gnomAD metrics revealed low tolerance to missense mutations. Carriers for variants affecting Trp88 (essential for fusion), Trp148, His157 and Arg160 (essential for binding) were found. Carriers of variants affecting 22 out of 33 functional hotspots were identified (including Trp148). IZUMO4: carriers of variants affecting amino acids involved in interaction with JUNO were identified, as well as carriers of variants in 10 of the 11 functional hotspots.
Conclusion: Bioinformatics and computational biology tools here presented have enhanced our understanding on essential proteins in successful fertilization. Alongside RNA expression and protein modeling, genetic analysis has identified heterozygous carriers of pathogenic variants in the general population, underscoring the importance of genetic studies as part of infertility diagnostics. These findings will help redefine the molecular diagnosis of male infertility, reduce the risk of fertilization failure, and guide effective therapeutic strategies in the evolving field of medically assisted reproduction.