A Study On The Function OfAcrosomal Protein IQCF1in The Male Fertility

There are a group of various transcripts in mature mammalian spermatozoa withfunction unknown. The reserved mechanism and the potential function ofspermatozoal transcripts are still unclear. Using serial analysis of gene expression(SAGE) strategy, we constructed the SAGE library of human mature spermatozoa andanalyzed the spermatozoal transcripts. In the SAGE library of human spermatozoa,there were some tags that could not be matched to the tag database. Based on theimproved two-step analysis of the unknown SAGE tags (TSAT-PCR), we identifiedmore transcripts in spermatozoa, including IQCF1(IQ motif containing F1).Considering that many reports have shown that transcripts in spermatozoa played acritical role in spermatogenesis, fertilization and zygotic development, in thisdissertation, using mouse as the model, through kinds of biochemical and molecularbiological techniques, we investigated the function of IQCF1in male fertility and themechanism of Iqcf1transcript reserved in spermatozoa.First of all, the open reading fragment of mouse Iqcf1was cloned and expressedin E. coli, followed by purification of the fused IQCF1in vitro. The purified IQCF1fused protein was used to immunize the rabbit to produce anti-IQCF1serum, whichwere purified through affinity purification for the subsequent experiments. What’smore, the location and the potential motifs of IQCF1were predicted with the help ofbioinformatics.Then, through Western blotting and reverse transcription-PCR, the expressionprofile of IQCF1in mouse tissues and organs was detected. The location of IQCF1intestes and sperm of mouse was determined using IHC, IF, and immune electronmicroscopy. Besides, the expression of IQCF1in different cells of testes were detected for speculating the transcription and translation process of IQCF1in testes,and the mechanism of Iqcf1reserved transcript in spermatozoa. The fused protein ofIQCF1and GFP was using to locate IQCF1in the eukaryotic cell cultured in vitro.The mouse testes of different ages were collected and detected for determining theprecise time of IQCF1’s expression in testes, and investigating the relationshipbetween IQCF1expression level and mouse spermatogenesis capacity. The transcriptand protein of IQCF1in the oocyte and zygote of mouse were detected to see ifIQCF1functioned in zygotic development.Finally, through transcription activator-like effector nucleases (TALEN) strategy,the Iqcf1-knockout mice were produced for analyzing the phenotypes in male fertilityof Iqcf1-knockout mice, including reproductive capacity, sperm count and motility,and acrosomal reaction ratio, to investigate the functions of IQCF1in male fertility.The yeast two-hybridization assay, Co-IP, and co-location using IF were used to verifythe interaction of IQCF1and calmodulin in vitro and in vivo. According to thefunctions of calmodulin in sperm capacitation and acrosomal reaction, the molecularmechanism of IQCF1in male fertility was investigated.The analyses results of IQCF1using bioinformatics showed that there were threeIQ motifs in IQCF1, as a potential calmodulin-binding region, and the location ofIQCF1in cell was predicted to be nuclear with probability more than60%. The purityof the fused IQCF1protein met the requirements to produce and purify anti-IQCF1polyclonal antibody, which was demonstrated to be with high specificity forsubsequent analyses.The expression profile of IQCF1in mouse tissues and organs showed that thetranscript and protein of IQCF1existed only in the testes and sperm. In testes, Iqcf1was transcribed in spermatogonia and spermatocyte, and translated in roundspermatids, of which IQCF1was located in the acrosome; in mature spermatozoa,IQCF1was predominantly located in the acrosome and lost after acrosomal reaction.Corresponding with the prediction of bioinformatics, IQCF1was located in thenuclear of the eukaryotic cells cultured in vitro, but could not be expressed steadily.Even in the testes of new-born mice, the Iqcf1transcript was detectable, though extremely low, and then the expression level increased along with the sex maturationand got steady35days post-partum (dpp) till adulthood. Whereas the IQCF1proteinwas detected for the first time at19dpp and increased along with sex maturation tilladulthood, similarly to the transcript level. In the testes of old mice (0.454±0.0890)and experimental cryptorchidism mice (0.256±0.021), the IQCF1expression levelwas significantly reduced compared to the young adult mice (1.52±0.144, P<0.001),indicating that the IQCF1expression level was positively correlated withspermatogenesis capacity. The RT-PCR and Western blotting results showed that thetranscript and protein of IQCF1were undetectable in the oocyte, indicating thatIQCF1was probably not involved in the zygotic early development.The sequencing results of the genomic DNA of Iqcf1-knockout mice showed that8nucleotides were lost in the third exon, causing frame shift and prematuretranslation termination. In the mutant mice, only80amino acids residues were coded,with all the three IQ motifs destroyed. Compared to the wild-type male mice, thereproductive capacity (Iqcf1/:9.92±4.97; Iqcf1+/+:13.43±3.31, P<0.01) of theIqcf1-knockout mice was significantly reduced, which was caused by the reducedsperm motility (Iqcf1/:53.00±2.65%; Iqcf1+/+:76.67±8.33%, P<0.01) andacrosomal reaction ratio (Iqcf1/:52.53±4.46%; Iqcf1+/+:61.93±6.69%, P<0.01). Allthe three method demonstrated that IQCF1interacted with calmodulin in sperm as aprotein complex. In the Iqcf1/sperm, the expression level of calmodulin (Iqcf1/:0.834±0.047; Iqcf1+/+:1.201±0.075, P<0.01) and the calmodulin closely-relatedtyrosine phosphorylation level (Iqcf1/:2.47±0.275; Iqcf1+/+:6.54±0.885, P<0.01) ofcapacitated sperm proteins, were reduce compared to the wild type.In conclusion, IQCF1was expressed specially in testes and spermatozoa, as akind of acrosomal protein. In testes, Iqcf1was transcribed in spermatogonia andspermatocyte, and translated in spermatids, with residual transcript in the maturespermatozoa; in sperm, IQCF1was involved in the tyrosine phosphorylation of spermproteins during capacitation and acrosomal reaction, as a calmodulin-interactingprotein. The reproductive capacity of Iqcf1-knockout male mice was significantlyreduced because of the reduced sperm motility and acrosomal reaction ratio, indicating that IQCF1played an important role in male fertility functioning probablyin tyrosine phosphorylation with molecular mechanism to be explored.