利用Cul4b基因剔除小鼠動物模型研究其在精子生成與胎盤發育之角色

Abstract

蛋白質降解必須維持嚴密之調控，以確保細胞正常生理作用，在真核細胞中，蛋白質主要以被泛素標記的方式進行降解，稱為泛素蛋白酶體降解系統。E3連接酶複合體具有辨識特定蛋白受質並將之泛素化之功能，其中一群由Cullin蛋白家族為主體而組成。Cullin 4B為該家族成員之一，其基因在人類與小鼠都位於X染色體，而蛋白將成為CUL4B-RING-E3連接酶複合體之骨架結構。臨床報告指出，人類CUL4B基因突變後將導致性聯遺傳智能障礙與性腺功能低下等病徵，然而相關研究病例僅有外觀描述，對於病理分析與致病機制目前仍一無所悉，此外也欠缺Cul4b基因剔除小鼠疾病動物模式進行病理機制研究。為了探討Cul4b在生物體內所扮演的角色及參與之功能，本論文以條件式基因剔除策略建立了Cul4b基因標的小鼠，在將其第四與第五外顯子於早期胚胎特異性剔除後，成功得到Cul4b全身性基因剔除小鼠。第一部分結果:首先將小鼠進行動物行為學與神經學分析，發現Cul4b基因剔除小鼠呈現出與臨床病人類似的學習障礙行為與病徵，且海馬迴的神經元數量、樹突複雜性和棘密度與對照組相比均顯著性降低，證實Cul4b基因剔除小鼠可作為具有潛力的人類疾病動物模型。此外，本論文亦發現在小鼠所有器官中，睪丸具有最高濃度CUL4B表現量，同時也觀察到Cul4b基因剔除公鼠無法產生任何子代，顯示CUL4B可能參與精子生成作用。分析結果顯示，基因剔除公鼠副睪中所含有的成熟精子數量非常稀少，且碩果僅存的精子其結構呈現廣泛性頭部損傷，顯示大部分精子於精子生成過程中死亡，少部分精子雖存活但於精子形成過程中產生變異，最終導致雄性不孕症。雖然成鼠睪丸中精原幹細胞有絲分裂與精母細胞減數分裂過程均不受CUL4B缺失的影響，但減數分裂後的單倍體精細胞數量卻逐漸降低，同時伴隨著凋亡細胞的增加。由於單倍體精細胞最重要的機制為精子形成，經過頂體形成、染色質重組與核濃縮過程後，可將圓形精細胞轉變為延長形成熟精蟲，這些過程也是控制精細胞凋亡與頭部濃縮成形的關鍵作用。共軛焦與電子顯微鏡分析發現基因剔除鼠單倍體精細胞在頂體形成初期的高爾基氏期沒有受到影響，但是在頂體期即出現膨大或環繞的頂體結構與不規則核濃縮的缺陷精細胞。這些細胞依損傷程度不同，有些會死亡、萎縮而被賽氏細胞吸收，有些則順利釋出至副睪，但異常頭部結構仍然使這些精蟲無法與卵子接觸受精。分析出生後第一波精子生成過程，發現在出生後二十七天出現的圓形精細胞數量顯著降低，且凋亡細胞於此時期顯著增加。此外，超微結構觀察亦發現精細胞於頂體形成的頂體期產生死亡、萎縮或結構異常，此結果與成鼠的精子生成循環過程相符合，證實不論是Cul4b基因剔除成鼠或發育中仔鼠，精子生成主要於頂體形成的頂體期產生死亡或缺陷。進一步將正值第一波精子生成的睪丸組織進行蛋白質體分析，區分出野生型小鼠與基因剔除小鼠間的差異性蛋白質，再經由軟體分析，判斷差異性蛋白質所參與的分子機制或細胞功能，藉此尋找CUL4B專一性調控的受質蛋白，解開缺乏CUL4B後導致精子生成異常的調控機制。目前已知CUL4B主要影響到的訊息傳遞路徑為細胞骨架重整、細胞附著、細胞凋亡與存活、蛋白質降解與蛋白質折疊等，未來將經由蛋白質交互作用分析來驗證顯著變化的蛋白質是否為CUL4B之受質蛋白。本論文結果證實Cul4b基因剔除公鼠不孕的原因為單倍體精細胞形成過程中頂體形成與核濃縮產生缺陷，導致精子細胞大量死亡、精子頭部變形與頂體結構異常，最終精子數量減少、頭型結構異常、缺乏泳動力，使得基因剔除公鼠無法藉由正常精卵結合過程產生子代。第二部分結果:第一部分指出小鼠Cul4b基因突變後將導致學習障礙與雄性不孕症，但由於採用了E6.5胚胎特異性剔除方式，無法觀察到最早期胚胎發育與胎盤形成之影響。為研究Cul4b在小鼠體內早期胚胎發育與胎盤形成所扮演的角色及參與之功能，本論文第二部分以精卵基因剔除策略建立Cul4b基因缺乏小鼠，將其第四與第五外顯子敲除後，發現全身性Cul4b基因剔除胚胎將於胚胎期7.5天發育中止且死亡。至於Cul4b異合子基因剔除母鼠的表現型會因突變對偶基因來自父源或母源而有所差異。突變基因來自父源的Cul4b+/Δ小鼠可以正常生長及繁衍子代，外觀上和野生型小鼠無異。然而，突變基因來自母源的Cul4bΔ/+小鼠將於胚胎期11.5天後產生嚴重的發育遲緩現象，且大多於出生前死亡。根據X染色體失活的原理，在胚胎外組織內，來自父源的X染色體將優先被靜默化，使得Cul4bΔ/+小鼠的胎盤組織將完全沒有CUL4B表現。結果發現Cul4bΔ/+小鼠胎盤的滋養層巨細胞在胚胎期8.5天僅剩一半，海綿層滋養母細胞在胚胎期11.5天厚度嚴重不足，而迷宮層的血管組織從胚胎期11.5天開始呈現失序的結構。除此之外，由於迷宮層裡的物質交換空間被破壞，導致小鼠血管與母體血竇都顯著擴大，造成母體與胎兒血液氧氣與養分交換的表面積下降，胎兒因此營養不良導致死亡。若將胚胎期6.5天的小鼠於外胚層特異性剔除Cul4b，保留胎盤的CUL4B表現，此策略則可以使Cul4b基因剔除鼠與Cul4b異合子基因剔除鼠順利娩出。總結而言，我們的結果證明胚胎外組織的發育需要CUL4B參與，而我們也在第一部分提供了一個策略產生Cul4b基因剔除鼠，藉以模擬CUL4B突變患者，進行組織病理分析與分子機制探討。

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.