Gene Targeting of Cul4b in Mice as a Model for Spermatogenesis and Placentation Study

• Main Authors: Chien-Yu Lin, 林建宇
• Other Authors: Shu-Wha Lin
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/68538521997777264977

博士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 102 === In mammals, Cullin genes constitute a family of eight proteins (CUL1, 2, 3, 4A, 4B, 5, 7, 9). Cullin 4B (CUL4B), a member of the Cullin protein family, serves as the structural scaffolds of the CUL4B-RING ligase complex, which recognizes and ubiquitinates selective substrates for protein degradation via the ubiquitin-proteasome system. Mutation of CUL4B in human results in X-linked intellectual disability (XLID) associated with impaired behavior and hypogonadism. However, the pathogenic role of CUL4B mutation in neuronal or other developmental defects is not understood and a mouse model for targeted Cul4b has not been described. Part I: To investigate the biological function of CUL4B, we here report the generation of Cul4b genetically engineered Cul4b mutant mice, in which exons 4 to 5 were deleted by gene targeting approach using Cre/loxP recombination system. We generated Cul4b mutant mice by crossing females carrying Cul4b floxed alleles with Sox2-Cre transgenic males, in which the deletion of Cul4b takes place specifically in embryo proper during embryogenesis. Firstly, Cul4b mutant mice were analyzed by behavior and neurological manners and we found mutant mice had abnormal spatial learning and memory ability and fewer parvalbumin-positive hippocampal neurons. Moreover, Cul4b mutant hippocampal neurons exhibited reduced dendritic complexity and spine density compared with control neurons. These data indicated the genetically engineered Cul4b mutant mouse as a potential model for human X-linked intellectual disability. In addition, CUL4B is strongly expressed in testes, suggesting that CUL4B- dependent protein degradation is involved in the control of the precisely timed and highly organized process of spermatogenesis. We found that Cul4b mutant male mice were infertile and displayed a progressive loss of germ cells from an initially normal germ epithelium of the tubules leading to oligoasthenospermia. Adult Cul4b mutant epididymides contained very low number of mature spermatozoa with pronounced morphological abnormalities. Mitosis of spermatogonial stem cells and meiosis of spermatocytes appeared unaffected. However, the loss-of-function allele affected the post-meiotic haploid spermatids during spermiogenesis. Decreased spermatids and an increased number of apoptotic germ cells were observed in Cul4b mutant testes. Because the most prominent defects were found during haploid differentiation, CUL4B was demonstrated to be critical for acrosome formation, chromatin remodeling and nuclear condensation which controls the cell death and sperm head shaping. In Cul4b mutant testes, spermatids with normal Golgi phase acrosome could be detected. However there were a variety of acrosome abnormalities including overly extended acrosomes and acrosomes encircled the nuclei in acrosome phase spermatids. Analysis of the first wave of spermatogenesis in Cul4b mutant mice also showed degeneration of round spermatids, amorphous acrosomes and disintegrated nuclei by day 27 and this phenomenon was consistent with adult spermatogenic cycle. We further isolated total protein from control and mutant testes at P20 and P27 to proceed with multidimensional liquid chromatography and analyzed by mass spectrometry. Quantification of identified proteins and relative expression changes were compared using an ANOVA statistical measurement to present the proteomic dataset. To obtain a global view of the molecular pathways and process networks, differential proteins were determined by MetaCore database. The pathways and networks with the higher representation were related with cytoskeleton rearrangement, cell adhesion, apoptosis, ubiquitin-proteasomal proteolysis and protein folding. Taken together, these collective data indicated that perturbed CUL4B function, as evidenced in the Cul4b mutant mice, results in disrupted haploid spermatid differentiation and male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility.Part II:In part I, mutation of Cul4b gene in mice causes abnormal spatial learning ability and male infertility. However, the epiblast-specific Cul4b knockout mice could not present the early embryogenesis and placentation. We here report the generation of Cul4b knockout mice, in which exons 4 to 5 were deleted by gene targeting approach using Cre/loxP recombination system. Cul4b conditional knockout mice were mated with Prm1-cre and Zp3-cre transgenic mice as deletion of Cul4b was exclusively occurred in spermatid and oocyte. We found that Cul4b null embryos exhibit arrested development and lethality around embryonic day 7.5. (E7.5). Cul4b heterozygotes had different phenotypes due to parent-of-origin mutant allele. Cul4b+/Δ heterozygotes were viable, fertile, normal in size and did not display any gross physical abnormalities. However, Cul4bΔ/+ exhibited a severe developmental delay from E11.5 and mostly suffered prenatal death due to the paternal X chromosome is preferentially inactivated in the placenta and resulted in Cul4b null placentas in Cul4bΔ/+ heterozygotes. Cul4bΔ/+ placentas exhibited deficiency of lower count of trophoblast giant cells at E8.5, decreased size in spongiotrophoblast layer from E11.5, disorganized labyrinth layer and impaired vascularization during E11.5-E18.5. The blood spaces within the labyrinthine layer were disrupted and the fetal blood vessels and the maternal sinusoids were considerably larger, leading to a reduction in the surface area available for nutrient and gas exchange. Although Cul4b null embryos exhibited more pronounced phenotypes than Cul4bΔ/+ heterozygotes, the lethality could be rescued by epiblast-specific deletion (Sox2-cre) of Cul4b and gave rise to viable Cul4b null mice and Cul4bΔ/+ heterozygotes. Together, our results showed that CUL4B is required in extra-embryonic tissues for placental development but indispensable for embryonic development in the mouse.