利用原型小鼠及Cul4b基因剔除小鼠研究CUL4B在小鼠睪丸中的表現形式

細胞中的蛋白質降解必須受到嚴格的調控，其中一個重要的降解系統為泛素-蛋白酶體降解系統 (Ubiquitin-proteasome degradation system, UPS)。Cul4b屬於cullin家族的E3泛素連接酶 (E3 ubiquitin ligase)，主要負責使受質經由26S 蛋白酶質體所辨認而進行降解。 2007年Tarpet研究團隊篩選了250個與X染色體相關的智能遲緩家族，其中8個與CUL4B 缺失有關。CUL4B缺失的智能遲緩病人具有幾種表徵，其中一個為性腺低下包括小睪丸、睪丸沒有降入陰囊以及陰莖短小。為了探討CUL4B的功能，實驗室已成功產製出Cul4b剔除小鼠，並發現Cul4b剔除公鼠 (Cul4b∆/Y) 患有不孕症，造成不孕的原因可能是精子數量稀少。所以本篇論文研究內容是分析CUL4B在成鼠睪丸中表現的形式，希望可以藉由了解CUL4B在野生型成鼠的精子細胞中表現的時期及表現的細胞種類，為Cul4b∆/Y不孕症提供可能的原因。首先我利用西方墨點法觀察到睪丸具有最大量的CUL4B。接著我進一步利用免疫組織螢光染色法偵測到CUL4B表現於未分化精原細胞 (Undifferentiated spermatogonia)、賽特利氏細胞 (Sertoli cell) 及精子細胞 (Spermatid)。由於CUL4B表現在精子細胞中，所以我利用頂體的標記物區分出各分化步驟精子細胞。從實驗結果可見CUL4B表現於步驟4-14精子細胞。我也發現造成Cul4b∆/Y精子數量較少的原因可能是精子細胞數量下降及精子釋放步驟發生異常。為了探討CUL4B缺失造成影響，我以西方墨點法及免疫組織化學染色法偵測CUL4B受質在睪丸中的表現，但至目前為止，實驗結果並沒有發現在Cul4b∆/Y中，CUL4B受質並無不正常累積，降解或是改變其表現的細胞種類。所以需要更進一步的實驗去找出CUL4B缺失會影響那些受質。

關鍵字

Cul4b ；睪丸；不孕症；精子生成；精子細胞

並列摘要

Protein degradation is regulated strictly in cells. One of the most important degradation systems is ubiquitin-proteasome degradation system. Cul4b, a member of cullin family, belongs to the E3 ubiquitin ligase. The function of Cul4b-associated E3 ligase promote substrate degradation via 26S proteasome. In 2007, Tarpet et al. screened 250 X-linked mental retardation (XLMR) families, and 8 of them had CUL4B dysfunction. XLMR patients had many phenotypes including hypogonadism such as small testis, undescending testis and small penis. In order to investigate the functions of CUL4B, Our lab had generated Cul4b knockout mice and found that Cul4b∆/Y exhibit infertilities resulted from low levels of spermatozoa. In this thesis, I examined the expression pattern of CUL4B in testis provide background knowledge that might help identify the possible mechanism of Cul4b-related infertility. First, I detected that CUL4B is highly expressed in testis by Western blot. By immunofluorescence assay, I observed that CUL4B is expressed in undifferentiated spermatogonia, Sertoli cells and spermatids. Next I distinguished the steps of spermatid by acrosome marker, and found that CUL4B is expressed in step 4-14 spermatid. I also found the possible reasons to cause low level of spermatozoa were decreased spermatid number and spermiation failure. In order to investigate the mice consequence of Cul4b deficiency, I examined the expression levels of CUL4B potential substrates in testis by Western blot and immunohistochemistry. I didn’t find abnormal accumulation or degradation nor changes of cellular localization in Cul4b∆/Y. Further experiments will be essential to unveil the candidate substrates affected by deficiency of Cul4b in the testis.

並列關鍵字

Cul4b ； Testis ； Infertility ； Spermatogenesis ； Spermatid

參考文獻

1. Goldstein, G., et al., Isolation of a Polypeptide That Has Lymphocyte-Differentiating Properties and Is Probably Represented Universally in Living Cells. Proceedings of the National Academy of Sciences of the United States of America, 1975. 72(1): p. 11-15.

2. Schlesinger, D.H., G. Goldstein, and H.D. Niall, The complete amino acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells. Biochemistry, 1975. 14(10): p. 2214-8.

3. Hershko, A., A. Ciechanover, and I.A. Rose, Resolution of the ATP-dependent proteolytic system from reticulocytes: a component that interacts with ATP. Proc Natl Acad Sci U S A, 1979. 76(7): p. 3107-10.

4. Pickart, C.M., Mechanisms underlying ubiquitination. Annual Review of Biochemistry, 2001. 70: p. 503-533.

5. Jin, J., et al., Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature, 2007. 447(7148): p. 1135-8.

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利用原型小鼠及Cul4b基因剔除小鼠研究CUL4B在小鼠睪丸中的表現形式

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