Dehydroepiandrosterone Antagonizes Pain Stress-Induced Suppression of Testosterone Production in Male Rats

Abstract

Background: Leydig cells secrete the steroid hormone, testosterone, which is essential for male fertility and reproductive health. Stress increases the secretion of glucocorticoid [corticosterone, (CORT) in rats] that decreases circulating testosterone levels in part through a direct action on its receptors in Leydig cells. Intratesticular CORT level is dependent on oxidative inactivation of CORT by 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) in rat Leydig cells. Pain may cause the stress, thus affecting testosterone production in Leydig cells. Methods: Adult male Sprague-Dawley rats orally received vehicle control or 5 or 10 mg/kg dehydroepiandrosterone (DHEA) 0.5 h before being subjected to pain stimulation for 1, 3, and 6 h. In the present study, we investigated the time-course changes of steroidogenic gene expression levels after acute pain-induced stress in rats and the possible mechanism of DHEA that prevented it. Plasma CORT, luteinizing hormone (LH), and testosterone (T) levels were measured, and Leydig cell gene expression levels were determined. The direct regulation of HSD11B1 catalytic direction by DHEA was detected in purified rat Leydig, liver, and rat Hsd11b1-transfected COS1 cells. Results: Plasma CORT levels were significantly increased at hour 1, 3, and 6 during the pain stimulation, while plasma T levels were significantly decreased starting at hour 3 and 6. Pain-induced stress also decreased Star, Hsd3b1, and Cyp17a1 expression levels at hour 3. When 5 and 10 mg/kg DHEA were orally administered to rats 0.5 h before starting pain stimulation, DHEA prevented pain-mediated decrease in plasma T levels and the expression of Star, Hsd3b1, and Cyp17a1 without affecting plasma CORT levels. DHEA was found to modulate HSD11B1 activities by increasing its oxidative activity and decreasing its reductive activity, thus decreasing the intracellular CORT levels in Leydig cells. Conclusion: Stress induced by acute pain can inhibit Leydig cell T production by upregulation of corticosterone. DHEA can prevent the negative effects of excessive corticosterone by modulating HSD11B1 activity.

Keywords: 11β-hydroxysteroid dehydrogenase 1; DHEA; Leydig cell; acute stress; corticosterone; pain; steroidogenic enzymes.

FIGURE 1

Plasma corticosterone (CORT), luteinizing hormone (LH), testosterone (T) concentrations, and T production of rat Leydig cells during the course of pain. Serum CORT, LH, and T levels were measured at each time point and Leydig cells isolated at hour 3 after pain were stimulated for 3 h without (basal) or with 100 ng/ml LH (LH) or 20 μM 22R-hydroxycholesterol (22OHC). (A) Plasma CORT; (B) plasma LH; (C) plasma T levels; (D) basal; (E) LH-stimulated; (F) 22OHC-mediated T production. Mean values ± SEM, n = 6. Asterisks “^∗∗∗” indicate significant difference when compared to the control (without pain) at each time-point at P < 0.001.

FIGURE 2

Messenger RNA levels in testis and pituitary during the course of pain. Black circle, control; Black square, pain. Testis genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp11a1, Cyp17a1, Hsd17b3, Hsd11b1, Nr5a1, Nr3c1, Sgk1, and Nos2; Pituitary genes: Lhb, Gnrhr, Esr1, and Nr3c1. Mean values ± SEM, n = 6. ^∗, ^∗∗, and ^∗∗∗ indicate significant differences when compared to the control (without pain) at each time point at P < 0.05, 0.01, 0.001, respectively.

FIGURE 3

The expression levels of STAR and LHCGR, as well as steroidogenic enzyme activities in the testes from control (CON) and pain-stimulated (PAIN) rats. Protein levels were adjusted by β-actin (ACTB). (A) STAR; (B) LHCGR; (C) CYP11A1; (D) HSD3B1; (E) CYP17A1; and (F) HSD17B3. Mean values ± SEM, n = 4–6. Asterisks ^∗, ^∗∗, and ^∗∗∗ indicate significant differences when compared to the control (without pain) at each time point at P < 0.05, 0.01, and 0.001, respectively.

FIGURE 4

Interstitial fluid T levels of control (CON) and pain (PAIN) rats without or with RU486 (+RU486) treatment at hour 3 during the course of pain stimulation. Rats were assigned to control (No pain), no pain + RU486 (+RU486) treatment, pain (Pain), and pain + RU486 treatment groups. Mean ± SEM, n = 6. Identical letters indicate no significant difference between two groups at P < 0.05.

FIGURE 5

Effects of DHEA on the HSD11B oxidase and reductase activity in Leydig, liver, and COS1-Hsd11b1 cells. (A,D) Leydig cells; (B,E) liver cells; (C,F) COS1-Hsd11b1 cells. (A–C) HSD11B oxidase; (D–F) HSD11B reductase. Mean values ± SEM, n = 4.

FIGURE 6

Mode of action of DHEA on HSD11B1 reductase and molecular docking simulation analysis of its interaction with human enzyme. (A) Lineweaver–Burk plot of rat 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) reductase versus DHC in the presence of DHEA; (B) docking of DHEA on human HSD11B1 which had two openings: one for cofactor NADP⁺/NADPH (white arrow) and the other for steroid substrate or DHEA (black arrow); (C) DHEA (blue color) matched HSD11B1 substrate cortisone (pink color) and was interacting with NADP⁺ (blue color).

FIGURE 7

Plasma corticosterone (CORT), luteinizing hormone (LH), and testosterone (T) concentrations at hour 3 of pain stress and the effects of DHEA. Plasma CORT, LH, and T levels were measured at hour 3 after pain were stimulated. (A) Plasma CORT; (B) plasma LH; (C) plasma T levels. Mean values ± SEM, n = 6. Identical letters indicate no significant difference between two groups at P < 0.05.

FIGURE 8

Messenger RNA levels in testis and pituitary in the pain-stressed rats after DHEA treatment. Testis genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp11a1, Cyp17a1, Hsd17b3, Hsd11b1, Nr5a1, Nr3c1, Sgk1, and Nos2; Pituitary genes: Lhb, Gnrhr, Esr1, and Nr3c1. Mean values ± SEM, n = 6. Identical letters indicate no significant difference between two groups at P < 0.05.