Local adrenomedullin gene delivery inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo.

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3ß-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.

Studies on steroid monooxygenase from Cylindrocarpon radicicola ATCC 11011. Oxygenative lactonization of androstenedione to testololactone.

A steroid monooxygenase of Cylindrocarpon radicicola was found to catalyze oxygenative lactonization of 17-ketosteroid, androstenedione, to yield D-homo-17 alpha-oxasteroid, testololactone, i.e., the androstenedione monooxygenase reaction, in addition to catalyzing the progesterone monooxygenase reaction. The reaction product was identified by TLC, GLC, and mass spectrometry. The oxygenation proceeded with unitary stoichiometry for 17-ketosteroid, NADPH, and molecular oxygen, indicating that it is a typical monooxygenase reaction of the external electron donor type. The enzyme catalyzed successively the side chain cleavage reaction of 17 alpha-hydroxy-20-ketosteroid to produce its 17-keto derivative and the lactonization of the product. The effects of pH and of the concentration of substrate steroids on the androstenedione monooxygenase reaction were different from those on the progesterone monooxygenase reaction. Progesterone is a strong and competitive inhibitor of the lactonization of 17-ketosteroids. The steroid monooxygenase is concluded to have the activities of both oxygenative esterification of 20-ketosteroids and oxygenative lactonization of 17-ketosteroids.