Profiling transcript levels for steroidogenic enzymes in fetal tissues

Cytochrome P450 (CYP) and hydroxysteroid dehydrogenase enzymes are involved in the conversion of cholesterol to steroid hormones. These enzymes are primarily expressed in the placenta, adrenal and gonads. Interestingly, some of these enzyme activities have been demonstrated in non-endocrine tissues, where they may be involved in important paracrine and autocrine actions. This is particularly the case in the human fetus where steroid precursors circulate at high levels and could be metabolized within tissues to produce active steroid hormones. Herein, we tested the hypothesis that transcripts for steroidogenic enzymes are expressed in fetal tissues other than the classical steroidogenic organs. To test this hypothesis, real-time reverse transcription polymerase chain reaction (RT-RTPCR) assays were developed that quantify mRNA levels for steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A), 3β-hydroxysteroid dehydrogenase types 1 and 2 (HSD3B1 and HSD3B2), 17α-hydroxylase (CYP17), 21-hydroxylase (CYP21), 11β-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2) and aromatase (CYP19). The use of RT-RTPCR allows the specific detection of these transcripts at levels that would not be detectable using northern analysis. In addition, this method can detect levels of transcript that would not lead to sufficient protein for detection of enzymatic activity of protein by western analysis. Thus, this methodology can detect low levels of expression that could play a role in regulating intra-tissue concentrations of steroid hormone. Total RNAs used for RT-RTPCR analysis were isolated from several human fetal tissues, including adrenal, testis, ovary, placenta, aorta, brain, liver, kidney, heart, lung, pancreas, prostate, stomach, and thymus. Our findings suggest that RT-RTPCR is a powerful tool for the examination of steroidogenic enzyme mRNA expressions. Using this approach, we have identified and quantified transcript levels of StAR and steroidogenic enzymes in several endocrine and non-endocrine fetal tissues. Even though some of the mRNA levels measured in these peripheral tissues are extremely lower in respect to the steroidogenic tissues, they could be sufficient to produce local (i.e. autocrine and paracrine) effects because produced steroids are not diluted into the entire circulation. These findings open new perspectives on the role of steroid hormones synthesized locally as probable regulatory factors of the development of several organ systems.