Lephart E D, Doody K J, McPhaul M J, Simpson E R
Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas 75235.
J Steroid Biochem Mol Biol. 1992 Jun;42(5):439-47. doi: 10.1016/0960-0760(92)90255-h.
In the present study, we examined the changes in enzyme activity and mRNA levels of aromatase cytochrome P450 (P450AROM) and 5 alpha-reductase in ovarian tissue from adult cyclic rats. For each stage of the estrous cycle, the enzymatic activities were quantified by means of the 3H2O-release assay in the case of P450AROM and thin-layer chromatography in the case of 5 alpha-reductase. Levels of mRNA encoding P450AROM and 5 alpha-reductase in the ovary were determined by Northern blot analysis utilizing 32P-labeled rat cDNAs as probes. Serum LH levels were determined by RIA. Three P450AROM mRNA species were detected (at 1.7, 2.2 and 2.7 kb) in ovarian tissue from cyclic rats. All three P450AROM transcripts were expressed in a co-ordinated fashion throughout the cycle. The P450AROM levels were highest during diestrus and proestrus, decreased during estrus while at metestrus the levels were nearly nondetectable. Conversely, one 5 alpha-reductase mRNA species at 2.5 kb was detected in ovarian tissue from cyclic animals. Ovarian 5 alpha-reductase mRNA levels were lowest during diestrus and proestrus, increased at estrus and were most abundant during metestrus; a pattern opposite to that of P450AROM. The pattern of change in P450AROM and 5 alpha-reductase activities paralleled that of the respective mRNA profiles but lagged behind the mRNA profiles by about 24 h, or one stage of the estrous cycle. Aromatase activity was 1.5 pmol/h/mg protein during diestrus, increased over 3-fold at proestrus (approximately 5.5 pmol/h/mg protein), decreased at estrus and declined to the lowest values at metestrus (approximately 1.0 pmol/h/mg protein). In contrast, the 5 alpha-reductase activity pattern was essentially the mirror image of the P450AROM activity pattern during the estrous cycle. 5 alpha-Reductase levels were lowest during proestrus (approximately 5 pmol/h/mg protein) and estrus (approximately 8 pmol/h/mg protein), increased over 3-fold during metestrus, while the highest activity levels occurred during diestrus (approximately 36 pmol/h/mg protein). The normalization of the P450AROM and 5 alpha-reductase mRNA levels and their respective enzyme activities revealed a correspondence between mRNA abundance and subsequent increases (24 h later) in enzyme activity levels during the estrous cycle. These findings suggest that: (a) a temporal relationship exists between the profiles of the enzymatic activities that follows the changes in the levels of their respective mRNAs and (b) an inverse pattern exists between P450AROM and 5 alpha-reductase in terms of both enzymatic activity and mRNA expression during the estrous cycle in rat.
在本研究中，我们检测了成年周期性发情大鼠卵巢组织中芳香化酶细胞色素P450（P450AROM）和5α-还原酶的酶活性及mRNA水平的变化。对于发情周期的每个阶段，P450AROM的酶活性通过3H2O释放测定法进行定量，5α-还原酶的酶活性则通过薄层色谱法进行定量。利用32P标记的大鼠cDNA作为探针，通过Northern印迹分析确定卵巢中编码P450AROM和5α-还原酶的mRNA水平。血清促黄体生成素（LH）水平通过放射免疫分析（RIA）测定。在周期性发情大鼠的卵巢组织中检测到三种P450AROM mRNA种类（分别为1.7、2.2和2.7 kb）。在整个发情周期中，所有三种P450AROM转录本均以协调的方式表达。P450AROM水平在间情期和发情前期最高，在发情期降低，而在动情后期几乎检测不到。相反，在周期性发情动物的卵巢组织中检测到一种2.5 kb的5α-还原酶mRNA种类。卵巢5α-还原酶mRNA水平在间情期和发情前期最低，在发情期升高，在动情后期最为丰富；这与P450AROM的模式相反。P450AROM和5α-还原酶活性的变化模式与各自的mRNA谱相似，但比mRNA谱滞后约24小时，即发情周期的一个阶段。间情期的芳香化酶活性为1.5 pmol/h/mg蛋白质，发情前期增加超过3倍（约5.5 pmol/h/mg蛋白质），发情期降低，在动情后期降至最低值（约1.0 pmol/h/mg蛋白质）。相比之下，5α-还原酶活性模式在发情周期中基本上是P450AROM活性模式的镜像。5α-还原酶水平在发情前期（约5 pmol/h/mg蛋白质）和发情期（约8 pmol/h/mg蛋白质）最低，在动情后期增加超过3倍，而最高活性水平出现在间情期（约36 pmol/h/mg蛋白质）。P450AROM和�α-还原酶mRNA水平及其各自酶活性的标准化显示，在发情周期中，mRNA丰度与随后（24小时后）酶活性水平的增加之间存在对应关系。这些发现表明：（a）酶活性谱与各自mRNA水平变化之间存在时间关系；（b）在大鼠发情周期中，P450AROM和5α-还原酶在酶活性和mRNA表达方面均存在相反模式。
