Miura Takeshi, Miura Chiemi, Konda Yasuko, Yamauchi Kohei
Marine Bioresources Research Group, Field Science Center for Northern Biosphere, Hokkaido University, Hakodate 041-8611, Japan.
Development. 2002 Jun;129(11):2689-97. doi: 10.1242/dev.129.11.2689.
Under fresh-water cultivation conditions, spermatogenesis in the Japanese eel is arrested at an immature stage before initiation of spermatogonial proliferation. A single injection of human chorionic gonadotropin can, however, induce complete spermatogenesis, which suggests that spermatogenesis-preventing substances may be present in eel testis. To determine whether such substances exist, we have applied a subtractive hybridisation method to identify genes whose expression is suppressed after human chorionic gonadotropin treatment in vivo. We found one previously unidentified cDNA clone that was downregulated by human chorionic gonadotropin, and named it 'eel spermatogenesis related substances 21' (eSRS21). A homology search showed that eSRS21 shares amino acid sequence similarity with mammalian and chicken Müllerian-inhibiting substance. eSRS21 was expressed in Sertoli cells of immature testes, but disappeared after human chorionic gonadotropin injection. Expression of eSRS21 mRNA was also suppressed in vitro by 11-ketotestosterone, a spermatogenesis-inducing steroid in eel. To examine the function of eSRS21 in spermatogenesis, recombinant eSRS21 produced by a CHO cell expression system was added to a testicular organ culture system. Spermtogonial proliferation induced by 11-ketotestosterone in vitro was suppressed by recombinant eSRS21. Furthermore, addition of a specific anti-eSRS21 antibody induced spermatogonial proliferation in a germ cell/somatic cell co-culture system. We conclude that eSRS21 prevents the initiation of spermatogenesis and, therefore, suppression of eSRS21 expression is necessary to initiate spermatogenesis. In other words, eSRS21 is a spermatogenesis-preventing substance.
在淡水养殖条件下，日本鳗鲡的精子发生在精原细胞增殖开始前的未成熟阶段就会停滞。然而，单次注射人绒毛膜促性腺激素可诱导完全精子发生，这表明鳗鲡睾丸中可能存在抑制精子发生的物质。为了确定此类物质是否存在，我们应用消减杂交方法来鉴定在体内经人绒毛膜促性腺激素处理后表达受到抑制的基因。我们发现了一个先前未鉴定的cDNA克隆，其表达被人绒毛膜促性腺激素下调，并将其命名为“鳗鲡精子发生相关物质21”（eSRS21）。同源性搜索显示，eSRS21与哺乳动物和鸡的苗勒氏管抑制物质具有氨基酸序列相似性。eSRS21在未成熟睾丸的支持细胞中表达，但在注射人绒毛膜促性腺激素后消失。鳗鲡体内一种诱导精子发生的类固醇——11-酮睾酮，在体外也可抑制eSRS21 mRNA的表达。为了研究eSRS21在精子发生中的功能，将CHO细胞表达系统产生的重组eSRS21添加到睾丸器官培养系统中。体外由11-酮睾酮诱导的精原细胞增殖被重组eSRS21抑制。此外，在生殖细胞/体细胞共培养系统中添加特异性抗eSRS21抗体可诱导精原细胞增殖。我们得出结论，eSRS21可阻止精子发生的起始，因此，抑制eSRS21的表达对于启动精子发生是必要的。换句话说，eSRS21是一种抑制精子发生的物质。
