Carreau S, Levallet J
CNRS EP 9 Department of Biochemistry, University of Caen, France.
Folia Histochem Cytobiol. 1997;35(4):195-202.
The ability of the male gonad to convert androgens into estrogens is well known; the microsomal enzymatic complex involved in this transformation is named aromatase and is composed of a specific cytochrome P450 aromatase (P450arom) and an ubiquitous reductase. According to age, aromatase activity has been already measured in immature and mature rat Leydig cells as well as in Sertoli cells. Recently, in different studies, a cytochrome P450arom has even been immunolocalized not only in Leydig cells but also in germ cells of mouse, brown bear and rooster whereas in pig, ram and human the aromatase is mainly present in Leydig cells. Our purpose was to investigate the testicular cell distribution of cytochrome P450arom mRNA in adult rat using RT-PCR. With two highly specific primers located on exons 8 and 9, we have been able to amplify a 289 bp aromatase fragment not only in Leydig cells and Sertoli cells but also in highly-enriched preparations of pachytene spermatocytes, round spermatids and testicular spermatozoa. These amplified products showed 100% homology with the corresponding fragment of the rat ovary cDNA. In parallel, using an anti-human cytochrome P450arom antibody we have demonstrated the presence of a 55 kDa protein in seminiferous tubules and crude germ cells (pachytene spermatocytes and round spermatids) of the mature rat. After incubation with tritiated androstenedione, the aromatase activity in the microsomal fractions of purified testicular spermatozoa was 2.96 pmoles/mg/h and was found to be 5-fold higher when compared to that of either purified pachytene spermatocytes or round spermatids. Using a quantitative RT-PCR method with a standard cDNA 29 bp shorter, we have compared the amount of cytochrome P450arom mRNA in mature rat Leydig cells and Sertoli cells. In purified Leydig cells from mature rats the P450arom mRNA level was: 36 x 10(-3) amoles/microgram RNA whereas in Sertoli cells the mRNA level was 10 fold lower. In pachytene spermatocytes, round spermatids and testicular spermatozoa the P450arom mRNA levels were respectively 367, 117 and < 1 x 10(-3) amoles/microgram RNA. Therefore, we evidenced that not only the Leydig cells but also the Sertoli cells of the rat during the testicular maturation have the capacity to express the gene of the cytochrome P450 aromatase. More importantly a biologically active cytochrome P450 aromatase is also present in germ cells (pachytene spermatocytes, round spermatids and spermatozoa). The existence of an additional source of estrogens within the genital tract of the male is now well documented and that suggests a putative role for these hormones during the male germ cell development and maturation not only in the testis but also in the epididymis.
雄性性腺将雄激素转化为雌激素的能力是众所周知的；参与这种转化的微粒体酶复合物被称为芳香化酶，它由一种特定的细胞色素P450芳香化酶（P450arom）和一种普遍存在的还原酶组成。根据年龄，已在未成熟和成熟大鼠的睾丸间质细胞以及支持细胞中测量了芳香化酶活性。最近，在不同的研究中，细胞色素P450arom不仅在睾丸间质细胞中，而且在小鼠、棕熊和公鸡的生殖细胞中都被免疫定位，而在猪、公羊和人类中，芳香化酶主要存在于睾丸间质细胞中。我们的目的是使用逆转录聚合酶链反应（RT-PCR）研究成年大鼠睾丸细胞中细胞色素P450arom mRNA的分布。使用位于外显子8和9上的两个高度特异性引物，我们不仅能够在睾丸间质细胞和支持细胞中，而且能够在粗线期精母细胞、圆形精子细胞和睾丸精子的高度富集制剂中扩增出一个289 bp的芳香化酶片段。这些扩增产物与大鼠卵巢cDNA的相应片段显示出100%的同源性。同时，使用抗人细胞色素P450arom抗体，我们证明了在成熟大鼠的生精小管和粗生殖细胞（粗线期精母细胞和圆形精子细胞）中存在一种55 kDa的蛋白质。用氚标记的雄烯二酮孵育后，纯化的睾丸精子微粒体部分中的芳香化酶活性为2.96 pmoles/mg/h，与纯化的粗线期精母细胞或圆形精子细胞相比，发现其活性高5倍。使用一种比标准cDNA短29 bp的定量RT-PCR方法，我们比较了成熟大鼠睾丸间质细胞和支持细胞中细胞色素P450arom mRNA的量。在来自成熟大鼠的纯化睾丸间质细胞中，P450arom mRNA水平为：36×10^(-3) amoles/μg RNA，而在支持细胞中，mRNA水平低10倍。在粗线期精母细胞、圆形精子细胞和睾丸精子中，P450arom mRNA水平分别为367、117和<1×10^(-3) amoles/μg RNA。因此，我们证明不仅睾丸间质细胞，而且大鼠睾丸成熟过程中的支持细胞都有能力表达细胞色素P450芳香化酶基因。更重要的是，一种具有生物活性的细胞色素P450芳香化酶也存在于生殖细胞（粗线期精母细胞、圆形精子细胞和精子）中。现在有充分的文献证明男性生殖道内存在额外的雌激素来源，这表明这些激素在男性生殖细胞发育和成熟过程中不仅在睾丸而且在附睾中可能发挥作用。
