Endoplasmic reticulum stress mediating downregulated StAR and 3-beta-HSD and low plasma testosterone caused by hypoxia is attenuated by CPU86017-RS and nifedipine

Abstract

Background: Hypoxia exposure initiates low serum testosterone levels that could be attributed to downregulated androgen biosynthesizing genes such as StAR (steroidogenic acute regulatory protein) and 3-beta-HSD (3-beta-hydroxysteroid dehydrogenase) in the testis. It was hypothesized that these abnormalities in the testis by hypoxia are associated with oxidative stress and an increase in chaperones of endoplasmic reticulum stress (ER stress) and ER stress could be modulated by a reduction in calcium influx. Therefore, we verify that if an application of CPU86017-RS (simplified as RS, a derivative to berberine) could alleviate the ER stress and depressed gene expressions of StAR and 3-beta-HSD, and low plasma testosterone in hypoxic rats, these were compared with those of nifedipine.

Methods: Adult male Sprague-Dawley rats were randomly divided into control, hypoxia for 28 days, and hypoxia treated (mg/kg, p.o.) during the last 14 days with nifedipine (Nif, 10) and three doses of RS (20, 40, 80), and normal rats treated with RS isomer (80). Serum testosterone (T) and luteinizing hormone (LH) were measured. The testicular expressions of biomarkers including StAR, 3-beta-HSD, immunoglobulin heavy chain binding protein (Bip), double-strand RNA-activated protein kinase-like ER kinase (PERK) and pro-apoptotic transcription factor C/EBP homologous protein (CHOP) were measured.

Results: In hypoxic rats, serum testosterone levels decreased and mRNA and protein expressions of the testosterone biosynthesis related genes, StAR and 3-beta-HSD were downregulated. These changes were linked to an increase in oxidants and upregulated ER stress chaperones: Bip, PERK, CHOP and distorted histological structure of the seminiferous tubules in the testis. These abnormalities were attenuated significantly by CPU86017-RS and nifedipine.

Conclusion: Downregulated StAR and 3-beta-HSD significantly contribute to low testosterone in hypoxic rats and is associated with ER stress which mediates testis damage caused by oxygen deprivation. CPU86017-RS is potential in ameliorating hypoxia-induced testicular injuries, possibly by its calcium antagonist effects on the testis.

Figure 1

Serum testosterone and LH levels. A reduction in testosterone and an elevated LH in serum were found in rats explored to intermittent hypoxia for 4 weeks, and these changes were relieved by medication (mg/kg, po) with CPU86017-RS (20, 40, 80, termed as RSL, RSM, RSH) and Nif (10) in the last 2 weeks. A, Testosterone, and B, LH. Mean ± SD, n = 10. *P < 0.05, **P < 0.01, vs. Normal; ^#P < 0.05, ^{# #}P < 0.01, vs. Hypoxia, ^$P < 0.05, vs. H+RSL (hypoxia + RS low dose).

Figure 2

MDA, GSH-px and LDH assay. Production of MDA in serum and the testis was augmented and activities of GSH-px and testicular LDH were reduced by hypoxia exposure for 4 weeks in rats. These changes were attenuated by medicated (mg/kg, po) with CPU86017-RS (20, 40 and 80) and nif (10) in the last two weeks; A, Serum MDA; B. Testicular MDA; C, Serum GSH-px; D, Testicular LDH. Mean ± SD, n = 10. *P < 0.05, **P < 0.01, vs. Normal; ^#P < 0.05, ^{# #}P < 0.01, vs. Hypoxia, ^$P < 0.05, vs. H+RSL (hypoxia + RS low dose).

Figure 3

The morphology changes in the testis. Alterations in testis histopathology were significant following 4 weeks exposure to hypoxia and were relieved by medication with CPU86017-RS and nifedipine in the last two weeks (200×) in rats. A, Control; B, Hypoxia; C, H+Nif; D, H+RSL; E, H+RSM; F, H+RSH; G, N+RSH.

Figure 4

StAR and 3-beta-HSD expression. Downregulation of mRNA or protein expression of StAR and 3-beta-HSD was significant in rat testis suffering from hypoxia exposure for 4 weeks and was relieved by CPU86017-RS and Nif, medicated in the last 2 weeks. A, StAR mRNA; B, StAR protein; C, 3-beta-HSD mRNA; D, 3-beta-HSD protein. Mean ± SD, n = 10. *P < 0.05, **P < 0.01, vs. Normal; ^#P < 0.05, ^{# #}P < 0.01, vs. Hypoxia, ^$P < 0.05, vs. H+RSL (RS low dose).

Figure 5

The expressions of ER stress chaperones. Upregulation of mRNA and protein expression of Bip, PERK and CHOP were caused by hypoxia in the testes and were attenuated by CPU86017-RS and Nif. A, Bip mRNA; B, Bip protein; C, PERK mRNA; D, PERK protein; E, CHOP mRNA; F, CHOP protein. Mean ± SD, n = 10. *P < 0.05, **P < 0.01, vs. Normal; ^#P < 0.05, ^{# #}P < 0.01, vs. Hypoxia, ^$P < 0.05, vs. H+RSL (hypoxia + RS low dose).