Kwon Jungkee, Wang Yu-Lai, Setsuie Rieko, Sekiguchi Satoshi, Sakurai Mikako, Sato Yae, Lee Won-Woo, Ishii Yoshiyuki, Kyuwa Shigeru, Noda Mami, Wada Keiji, Yoshikawa Yasuhiro
Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Biol Reprod. 2004 Aug;71(2):515-21. doi: 10.1095/biolreprod.104.027565. Epub 2004 Apr 14.
The ubiquitin pathway functions in the process of protein turnover in eukaryotic cells. This pathway comprises the enzymes that ubiquitinate/deubiquitinate target proteins and the proteasome that degrades ubiquitin-conjugated proteins. Ubiquitin C-terminal hydrolases (UCHs) are thought to be essential for maintaining ubiquitination activity by releasing ubiquitin (Ub) from its substrates. Mammalian UCH-L1 and UCH-L3 are small proteins that share considerable homology at the amino acid level. Both of these UCHs are highly expressed in the testis/ ovary and neuronal cells. Our previous work demonstrated that UCH-L1-deficient gracile axonal dystrophy (gad) mice exhibit progressively decreasing spermatogonial stem cell proliferation, suggesting that UCH isozymes in the testis function during spermatogenesis. To analyze the expression patterns of UCH isozymes during spermatogenesis, we isolated nearly homogeneous populations of spermatogonia, spermatocytes, spermatids, and Sertoli cells from mouse testes. Western blot analysis detected UCH-L1 in spermatogonia and Sertoli cells, whereas UCH-L3 was detected in spermatocytes and spermatids. Moreover, reverse transcription-polymerase chain reaction analysis of UCH isozymes showed that UCH-L1 and UCH-L4 mRNAs are expressed in spermatogonia, whereas UCH-L3 and UCH-L5 mRNAs are expressed mainly in spermatocytes and spermatids. These results suggest that UCH-L1 and UCH-L3 have distinct functions during spermatogenesis, namely, that UCH-L1 may act during mitotic proliferation of spermatogonial stem cells whereas UCH-L3 may function in the meiotic differentiation of spermatocytes into spermatids.
泛素途径在真核细胞的蛋白质周转过程中发挥作用。该途径包括使靶蛋白泛素化/去泛素化的酶以及降解泛素结合蛋白的蛋白酶体。泛素C末端水解酶（UCHs）被认为通过从其底物中释放泛素（Ub）来维持泛素化活性至关重要。哺乳动物的UCH-L1和UCH-L3是小蛋白，在氨基酸水平上具有相当高的同源性。这两种UCHs在睾丸/卵巢和神经元细胞中均高表达。我们之前的研究表明，UCH-L1缺陷型薄束轴索性营养不良（gad）小鼠的精原干细胞增殖逐渐减少，这表明睾丸中的UCH同工酶在精子发生过程中发挥作用。为了分析精子发生过程中UCH同工酶的表达模式，我们从小鼠睾丸中分离出了几乎均一的精原细胞、精母细胞、精子细胞和支持细胞群体。蛋白质印迹分析在精原细胞和支持细胞中检测到了UCH-L1，而在精母细胞和精子细胞中检测到了UCH-L3。此外，UCH同工酶的逆转录-聚合酶链反应分析表明，UCH-L1和UCH-L4 mRNA在精原细胞中表达，而UCH-L3和UCH-L5 mRNA主要在精母细胞和精子细胞中表达。这些结果表明，UCH-L1和UCH-L3在精子发生过程中具有不同的功能，即UCH-L1可能在精原干细胞的有丝分裂增殖过程中起作用，而UCH-L3可能在精母细胞向精子细胞的减数分裂分化过程中发挥作用。
