The Establishment Of Mice Model Of Fscb Gene Knockout And Its Effects On The Reproductive Function

BackgroundThe incidence of male infertility is increasing with the influence of environmental pollution, genetic or lifestyle changes in recent years[1].Studies have found that the fibrous sheath of spermatozoon is dysplasia in the severe asthenospermia or the patients whose sperm do not have the capability of movement[5].In the process of spermitogenesis and spermiogenensis, the specific genes in testis can express the coresponding proteins timely and orderly, and then the organelles in sperm could synthesis and assembly coordinately.The transportation and intergration of the related proteins in the scaffolding structure of sperm orderly is the fundamental basis for the normal morphology and function of sperm [6].Fibrous Sheath CABYR-binding protein(FSCB), firstly discovered and identified by Prof. Li Yanfeng, is a new type of protein specifically expressed in spermiogenesis with the final localization at the surface of both the longitudinal columns and ribs of fibrous sheath of the sperm flagellum[7]. This protein is testis/sperm-specific, and is only expressed in the later phase of spermiogenesis. Previous researches [14] showed that proteins such as FSCB and CABYR can combine and assembly on scaffold protein AKAPs of fibrous sheath through direct or indirect interactions, form giant molecular protein complexes and play the physiological functions of fibrous sheath together. FSCB protein includes the conservative PXXP gene sequence, proline-rich region and extensin-like domain, and has calcium-binding capacity. Its serine/threonine can be phosphorylated by protein kinase A(PKA) in vivo and in vitro. Therefore, it is deduced that this protein may be an important kind of protein that participates in the flagellar movement of the sperm, and is related to the sperm capacitation and sperm hyperactivity[7].The comparative analysis on the time phase and localization features of FSCB protein expression and other proteins in the spermiogenesis, showed that the expression of thisprotein has high similarity with the expression of AKAP4, the major structural protein of fibrous sheath. The abundant expression of AKAP4 is localized in the fibrous sheath during sperm development, and it can provide scaffold for various kinases and proteases. Studies in gene knockout mice proved that Akap4–/– homozygous male mice produced normal amount of sperms with the fibrous sheath formation defects such as shortened longitudinal columns and thinned circular ribs; meanwhile, the sperm progressive motility is damaged,which leads to infertility [13]. To further investigate the functions of FSCB protein in spermiogenesis and sperm motility, this study built the Fscb gene targeting vector in mouse with CRISPR/Cas9 system, knocked out the full-length Fscb gene, and established the Fscb-gene knockout animal model. The phenotypic traits of Fscb-gene knockout mouse were tested and determined by the analysis of sperm quality and motility, analysis of in vivo and in vitro fertilization, Western blot and immunohistochemistry(ICH), etc. so as to study the influence of the deletion of Fscb gene on the fertility of male mice.Part I: The construction of targeting vectors using CRISPR/Cas9 system and the generation of Fscb knockout mice.Objective To build the targeting vectors for the deletion of mice Fscb gene by using the CRISPR/Cas9 system, and to generate Fscb gene knockout mice for the studying of its function in the sperm activation and capacitation.Methods The targeting sites were designed according to the full length sequence of Fscb gene and three pairs of single-guide RNA( sg RNA) were chemically synthesized, and inserted into the linearized plasmid pUC57-T7-GDNA. The Cas9 RNA and sgRNAs were transcribed by T7 RNA polymerase in vitro.Cas9 mRNA and sgRNA mixtures were microinjected into fertilized eggs to generate mice with targeted mutation. The genotypes of homozygous were identified by PCR, DNA sequencing and western blot.Results The vector of expressing sg RNA was successfully built, and the sg RNA and Cas9 mRNA were transcripted in vitro. The active sgRNA and Cas9 RNA were then injected into the fertilized eggs of mice, and 39 positive mice as the F0 generation were generated. Three select male mice with clear deletion of the Fscb gene fragment and with obvious frame shifting breed with wild-type female mice, and 5 positive F1 mice(two males and three females) were achieved. The heterozygous males mated with females to generate the F2 homozygous mice. Two different genotypes of homozygous were generated.Western blot analysis confirmed that the expression of FSCB protein in the testis and sperm of the homozygous male mice were disappeared.Conclusion The Fscb gene knockout model was successfully built by using the CRISPR/Cas9 system, which offered a tool for the studying of the function of FSCB protein.Part 2:The influence of Fscb gene knockout on the reproduction of male mice Objective To investigate the influence of the deletion of Fscb gene on the fertility of male mice in Fscb-gene knockout mice.Methods The expression of FSCB protein in the testicular tissue and the sperm of Fscb-gene knockout mice was observed by immunohistochemistry; The sperm motility,sperm viability, sperm motility parameters, and single sperm motility traits before and after capacitation in vitro were analyzed by computer-aided sperm analysis; the capacity in vitro fertilization(IVF) of Fscb-gene knockout mice sperm was detected under the culture conditions of in vitro capacitation; The homozygous of Fscb-gene knockout(KO), the heterozygote(Het), and the wild type(WT) male mice were mated with wild type C57BL/6J adult female mice, and the pregnancy rate of female mice, the number of the embryos, and the morphological differences of the gonads in filial generation mice were observed.Results Observations with immunofluorescence showed that there was no expression of FSCB protein in the testicular tissue and in the sperm in KO mice, and the expression of FSCB protein in the testicular tissue or the sperm in Het was significantly lower than that in the WT mice, which was consistent with expected results. No statistically significant differences of the sperm motility and motility parameters were found among KO, Het and WT mice.(P>0.05); and also, no obvious differences of the sperm morphology and single sperm motility traits were found among the three groups; the results of the IVF experiment showed that no significant differences of the average numbers of sperm-ZP(zona pellucida)binding, the fertilization rate of egg cells with intact zona pellucid, and the average numbers of sperm binding with zone-free egg cells were showed(all P>0.05). The result of fertilization capacity analysis indicated that no statistically significant differences of the pregnancy rate, the number of the embryos and the natural litter size of the female mice that mated with KO, Het and WT mice(P>0.05) were indicated, as well as that no significantdifferences in the gonadogenesis(the development of the gonads) in filial generation of the adult male mice was demonstrated.Conclusion As a spermiogenesis-related gene, FSCB is specifically expressed in spermiogenesis with the final localization on the fibrous sheath of sperm flagellum. No significant difference of the sperm motility and the capacity of in vivo and in vitro fertilization in the Fscb gene knockout male mice, indicates that the functions of the FSCB protein may be unnecessary or replaceable for the maintenance of sperm motility and fertilization capacity.