Assessment of Testicular Corticosterone Biosynthesis in Adult Male Rats

<div><p>Corticosterone is synthesized in the adrenal glands and is circulated throughout the body to perform regulatory functions in various tissues. The testis is known to synthesize and secrete testosterone and other androgens. We developed an accurate method to measure steroid content using liquid chromatography-mass spectrometry analysis. In the present study, significant levels of the precursor compounds of testosterone and corticosterone synthesis could be detected in rat testis using this method. After adrenalectomy, corticosterone remained in the blood and testicular tissue at approximately 1% of the amount present in the control testis. When the excised testicular tissue was washed and incubated with NADH, NADPH and progesterone, not only testosterone and its precursors but also 11-deoxycorticosterone and corticosterone were produced; the levels of 11-deoxycorticosterone and corticosterone increased with incubation time. The production rate of 11-deoxycorticosterone from progesterone was estimated to be approximately 1/20 that of 17-hydroxyprogesterone, and the corticosterone level was approximately 1/10 that of testosterone. These ratios coincided with those in the testicular tissue of the adrenalectomized rats, indicating that corticosterone was synthesized in the testis and not in the blood. A primary finding of this study was that corticosterone and testosterone were synthesized in a 1/10-20 ratio in the testis. It is concluded that corticosterone, which has various functions, such as the regulation of glycolysis and mediating spermatogenesis, is produced locally in the testis and that this the local production is convenient and functional to respond to local needs.</p></div

Amounts of the products in the enzyme assay using rat testis.

<p>The steroid production levels after the one-hour enzymatic reaction described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117795#pone.0117795.g002" target="_blank">Fig. 2</a> are shown in the column figure. The data for each group represents the mean ± S.E.M. of three experiments.</p

Steroid hormones and precursors in the testis of adrenalectomized rats.

<p>The adult male rats were adrenalectomized (AL) or sham-operated (Sham) as described in Materials and Methods. Two weeks after breeding, the levels of steroid hormones and their precursors were determined via LC-MS analysis, as described in Materials and Methods. The productions of pregnenolone (A), progesterone (B), 17-hydroxyprogesterone (C), androstenedione (D), testosterone (E), dihydrotestosterone (F), 11-deoxycorticosterone (G), corticosterone (H) and dehydrocorticosterone (I) are shown. The data for each group represents the mean ± S.E.M. of three to five rats. * P<0.05 and **P<0.01 compared with the control rats.</p

Concentrations of testosterone, corticosterone and aldosterone in the blood and testes of adrenalectomized adult male rats.

<p>Adult male rats were adrenalectomized and bred for one week as described in the Materials and Methods section. Then, the testosterone, corticosterone and aldosterone concentrations in the blood and the testicular tissue were assayed, as described in the Materials and Methods section. ND; not detectable (Limit of detection; 5.0 fmoles/ml of blood or g of tissue). The data for each group represents the mean ± S.E.M of three to five rats.</p><p>**P<0.01 compared with the control rats.</p><p>Concentrations of testosterone, corticosterone and aldosterone in the blood and testes of adrenalectomized adult male rats.</p

Enzyme activities of the synthesis steroids in rat testis.

<p>One half of the rat testis was washed with phosphate buffered saline at least three times, and the excised tissue was incubated with 30 μM progesterone in the culture medium containing 4 mM MgCl₂, 20 mM Tris-HCl buffer (pH 7.4), 100 μM NADPH and NADH at 37°C in a test tube. The reaction products in the medium were determined using LC-MS analysis, as described in Materials and Methods. The contents of 17-hydroxyprogesterone (HPGS) (A), androstenedione (ADS) (B), testosterone (TS) (C), 11-deoxycorticosterone (DCC) (D), and corticosterone (CCS) (E) are shown.</p

Production of 11-deoxycorticosterone and 17-hydroxyprogesterone in the enzyme assay using rat testis.

<p>After one hour of the enzymatic reaction described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117795#pone.0117795.g002" target="_blank">Fig. 2</a>, the production of 17-hydroxyprogesterone and 11-deoxycorticosterone was analyzed using LC-MS. Then, the two precursors with the same molecular weight in the same chromatogram were separated with LC; the ratio of the two products was 1:20.</p