Pineau C, McCool S, Glucksman M J, Jégou B, Pierotti A R
GERM-INSERM U.435, Campus de Beaulieu, Université de Rennes I, Bretagne, France.
J Cell Sci. 1999 Oct;112 ( Pt 20):3455-62. doi: 10.1242/jcs.112.20.3455.
Thimet oligopeptidase (TOP:E.C. 3.4.24.15) is a thiol sensitive metalloendopeptidase which is widely distributed and active in most tissues including testis, brain and pituitary. In the median eminence it is postulated to play a role in the degradation of GnRH released from the hypothalamus and thus to modulate LH levels. In the rat and human, the testis is the richest source of TOP activity with levels 3- to 5-fold higher than that of the brain. In order to define the exact localisation of this enzyme within the rat and human testis, the distribution of TOP in the developing and adult gonad was examined in situ and in isolated cells by immunohistochemistry, western blotting and northern blotting analysis. Ontogeny studies have demonstrated that TOP is detectable by western blotting from 9 days with levels of expression increasing with the age of the animal. Immunolocalisation of the protein in the interstitium was positive from 9 days onwards but was negative within the seminiferous tubules before 35 days of age, whereas TOP mRNA was not detected within the testis until 35 days of age with subsequent stable expression levels up to 90 days. In the adult rat testis, a strong TOP immunoreactivity was observed within seminiferous tubules, in elongating and elongated spermatids and residual bodies. In the interstitial compartment, immunoreactivity was also observed in Leydig cells and throughout the interstitial space. Western blot analyses confirmed the distribution of expression observed using immunochemistry, however Leydig cells display a lower signal than expected from the immunohistochemical data. Northern hybridization showed that the transcript is present in pachytene spermatocytes, early spermatids, and residual bodies, whereas its presence was not observed in Leydig cells probably due to very low levels of expression of the message. Analyses of various human tissue extracts showed that the testis displays the highest levels of TOP mRNA, with immunohistochemical experiments revealing that, as in the rat, the protein is principally expressed in elongated spermatids/residual bodies, and in Leydig cells. It is concluded that in the human and rat testes, TOP is highly expressed, in particular in post-meiotic germ cells and Leydig cells. The possible involvement of TOP in proteolytic events associated with the process of spermiogenesis and Leydig cell function is currently under investigation.
硫醇寡肽酶（TOP：E.C. 3.4.24.15）是一种对硫醇敏感的金属内肽酶，广泛分布于包括睾丸、脑和垂体在内的大多数组织中且具有活性。据推测，在正中隆起中，它在下丘脑释放的促性腺激素释放激素（GnRH）的降解过程中发挥作用，从而调节促黄体生成素（LH）水平。在大鼠和人类中，睾丸是TOP活性最丰富的来源，其水平比脑高3至5倍。为了明确该酶在大鼠和人类睾丸中的具体定位，通过免疫组织化学、蛋白质免疫印迹法和Northern印迹分析，在原位和分离细胞中检测了发育中和成年性腺中TOP的分布。个体发育研究表明，从第9天开始通过蛋白质免疫印迹法可检测到TOP，其表达水平随动物年龄增长而增加。该蛋白在间质中的免疫定位从第9天开始呈阳性，但在35日龄前在生精小管内呈阴性，而直到35日龄才在睾丸中检测到TOP mRNA，随后表达水平稳定直至90日龄。在成年大鼠睾丸中，在生精小管、伸长和已伸长的精子细胞及残余小体中观察到强烈的TOP免疫反应性。在间质区室中，在睾丸间质细胞和整个间质空间也观察到免疫反应性。蛋白质免疫印迹分析证实了免疫化学观察到的表达分布，然而睾丸间质细胞显示的信号低于免疫组织化学数据预期的信号。Northern杂交表明，该转录本存在于粗线期精母细胞、早期精子细胞和残余小体中，而在睾丸间质细胞中未观察到其存在，可能是由于该信息的表达水平非常低。对各种人类组织提取物的分析表明，睾丸中TOP mRNA水平最高，免疫组织化学实验显示，与大鼠一样，该蛋白主要在已伸长的精子细胞/残余小体和睾丸间质细胞中表达。得出的结论是，在人类和大鼠睾丸中，TOP高度表达，特别是在减数分裂后的生殖细胞和睾丸间质细胞中。目前正在研究TOP可能参与与精子发生过程和睾丸间质细胞功能相关的蛋白水解事件。
