Ryun Sup Ahn

Effect of ascorbic acid supplementation on testicular steroidogenesis and germ cell death in cadmium-treated male rats.

Cadmium (Cd) is one of the environmental pollutants affecting various tissues and organs including testis. Harmful effect of Cd in testis is known to be germ cell degeneration and impairment of testicular steroidogenesis. Animals treated with high doses of Cd (0.2 and 0.3 mg/100g BW) showed a significant decrease in serum testosterone (T) level, but a significant induction of testicular lipid peroxidation levels. TUNEL assay showed that low doses of Cd (0.13 and 0.15 mg/100g BW) exhibited typical characteristics of apoptosis while high doses of Cd caused more necrosis than apoptosis. In contrast, supplementation with ascorbic acid reduced testicular lipid peroxidation levels. Ascorbic acid supplementation restored testicular 3beta-hydroxysteroiddehydrogenase (HSD) and 17beta-HSD enzyme activities, 3beta-HSD and cytochrome P450 side chain cleavage (P450(scc)) mRNA levels and serum T concentration to normal in Cd-administered rats. Moreover, administration of ascorbic acid prevented germ cell apoptosis as demonstrated by the reduced number of TUNEL-positive cells in germinal epithelium and inhibited Cd-induced necrosis. These results indicate that ascorbic acid have protective roles in vivo on the Cd-induced overall testicular damage including impaired steroidogenesis and germ cell death possibly through scavenging the reactive oxygen species generated by Cd administration.

Differential G protein coupling preference of mammalian and nonmammalian gonadotropin-releasing hormone receptors.

Recently, we have identified three distinct types of gonadotropin-releasing hormone receptor (GnRHR) in the bullfrog (designated bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we compared G protein coupling preference of mammalian and nonmammalian GnRHRs. In a transient expression system, stimulation of either bfGnRHRs or rat GnRHR by GnRH significantly increased both inositol phosphates (IP) and cAMP productions, but ratios of IP to cAMP induction levels were quite different among the receptors, indicating differential G protein coupling preference. Using cAMP-dependent protein kinase A (PKA)-specific (CRE-luc) or protein kinase C (PKC)-specific reporter (c-fos-luc) systems, we further examined G(s) and G(q/11) coupling preference of these GnRHRs. Since activities of CRE-luc and c-fos-luc were highly dependent on cell types, GnRH-induced CRE-luc or c-fos-luc activity was normalized by forskolin-induced CRE-luc or 12-O-tetradecanoylphenol-13-acetate (TPA)-induced c-fos-luc activity, respectively. This normalized result indicated that bfGnRHR-2 couples to G(s) more actively than G(q/11), while bfGnRHR-1 and -3 couple to G(s) and G(q/11) with similar strength. However, the rat GnRHR appeared to couple to G(q/11) more efficiently than G(s). This study was further confirmed by an experiment in which GnRH augmented CRE-driven luciferase activity in alphaT3-1 cells when CRE-luc was cotransfected with bfGnRHRs but not with vehicle or rat GnRHR. Collectively, these results indicate that mammalian and nonmammalian GnRHRs may induce diverse cellular and physiological responses through differential activation of PKA and PKC signaling pathways.

Phosphoenolpyruvate Carboxykinase and Glucose-6-phosphatase Are Required for Steroidogenesis in Testicular Leydig Cells

Background: PEPCK is expressed in Leydig cells of testes, but its role has not been studied. Results: Knockdown and inhibition of PEPCK and Glc-6-Pase inhibited steroidogenesis via activation of AMPK. Conclusion: PEPCK and Glc-6-Pase are required for steroidogenesis in testicular Leydig cells. Significance: Male fertility may be adversely affected by conditions and factors that inhibit PEPCK and Glc-6-Pase expression in Leydig cells. Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.

ERα/E2 signaling suppresses the expression of steroidogenic enzyme genes via cross-talk with orphan nuclear receptor Nur77 in the testes

Estrogen receptor alpha (ERα) has been reported to affect steroidogenesis in testicular Leydig cells, but its molecular mechanism remains unclear. Here, we investigate the effect of estrogen and ERα on Nur77, a major transcription factor that regulates the expression of steroidogenic enzyme genes. In MA-10 Leydig cells, estradiol (E2) treatment, and interestingly ERα overexpression, suppressed the cAMP-induced and Nur77-activated promoter activity of steroidogenic enzyme genes via the suppression of Nur77 transactivation. ERα physically interacted with Nur77 and inhibited its DNA binding activity. In addition, ERα/E2 signaling decreased Nur77 protein levels. Consistent with the above results, the testicular testosterone level was higher in Leydig cell-specific ERα knock-out mice (ERα(flox/flox)Cyp17iCre) than in wild-type mice (ERα(flox/flox)). Taken together, these results suggest that ERα/E2 signaling controls the Nur77-mediated expression of steroidogenic enzyme genes in Leydig cells. These findings may provide a mechanistic explanation for the local regulation of testicular steroidogenesis by estrogenic compounds and ERα.

Reduced testicular steroidogenesis in tumor necrosis factor-α knockout mice

We previously demonstrated that the expression of Mullerian inhibiting substance (MIS) in Sertoli cells is downregulated by tumor necrosis factor alpha (TNF-alpha), which is secreted by meiotic germ cells, in mouse testes. Several studies have reported that MIS that is secreted by Sertoli cells inhibits steroidogenesis and, thus, the synthesis of testosterone in testicular Leydig cells. Here, we demonstrate that in TNF-alpha knockout testes, which show high levels of MIS, steroidogenesis is decreased compared to that in wild-type testes. The levels of testosterone and the mRNA levels of steroidogenesis-related genes were significantly lower after puberty in TNF-alpha knockout testes than in wild-type testes. Furthermore, the number of sperm was reduced in TNF-alpha knockout mice. Histological analysis revealed that spermatogenesis is also delayed in TNF-alpha knockout testes. In conclusion, TNF-alpha knockout mice show reduced testicular steroidogenesis, which is likely due to the high level of testicular MIS compared to that seen in wild-type mice.

Effects of butyltin compounds on follicular steroidogenesis in the bullfrog(Rana catesbeiana)

The effects of butyltin compounds on follicular steroidogenesis in amphibians were examined using ovarian follicles of Rana catesbeiana. Isolated follicles were cultured for 18h in the presence and absence of frog pituitary homogenate (FPH) or various steroid precursors, and the steroid levels in the follicles or culture media were measured by radioimmunoassay (RIA). Among the butyltin compounds, tributyltin (TBT) strongly inhibited the FPH-induced synthesis of pregnenolone (P(5)), progesterone (P(4)) and testosterone (T). It also inhibited the conversion of P(5)-P(4) and T to estradiol-17β(E(2)) and it partially suppressed the conversion of androstenedione (AD) to T, but not P(4) to 17α-hydroxyprogesterone (17α-OHP(4)). A high concentration of dibutyltin (DBT) also inhibited steroidogenesis by the follicles while monobutyltin (MBT) and tetrabutylin (TeBT) had no effect. These results suggest that the initial step of steroidogenesis (P(5) synthesis) and enzymes such as 3β-HSD, 17β-HSD and aromatase are inhibited by TBT or DBT. However, 17α-hydroxylase was not suppressed by TBT or the other butyltin compounds.

Evidence for two-cell model of steroidogenesis in four species of amphibian

Previously, based on studies conducted using Rana nigromaculata, a two-cell model involving the theca and granulosa cells was proposed to account for the steroidogenic activity of amphibian ovarian follicles. Experiments were carried out to ascertain whether the model was applicable to four other frog species with different reproductive cycles (R. dybowskii, R. rugosa, R. catesbeiana, and Bombina orientalis). Ovarian follicles were collected from each species and manually microdissected to obtain various follicular components: theca-epithelium (THEP) and granulosa cell-enclosed oocyte (GCEO). Subsequent to collection, equal numbers of intact follicles and various follicular components were cultured for 6 hr in the presence of known inducers of steroidogenesis (frog pituitary homogenate [FPH] or 3-iso-butyl-1-methylxanthine [IBMX] + forskolin) or various steroids that serve as substrates for specific steroidogenic enzymes. Following incubation, culture medium was collected and analyzed by radioimmunoassay (RIA). Both FPH and IBMX + forskolin consistently stimulated secretion of androstenedione (AD), testosterone (T), and estradiol (E2) from intact follicles obtained from all four frog species. Additionally, in R. dybowskii, these treatments stimulated secretion of progesterone (P4) and 17α-hydroxyprogesterone (17α-OHP4) into the culture medium. Intact follicles obtained from all species readily converted pregnenolone (P5), P4, and 17α-OHP4 to AD, T, and E2. In contrast GCEO converted P5, P4, and 17α-OHP4 to AD and E2, but not to T. However, AD, but not P5, P4, or 17α-OHP4, was converted to T when cultured in the presence of isolated THEP. The microdissection procedure was also modified to isolate THEP without contaminating granulosa cells. The steroidogenic capacities of “impure” THEP and “pure” theca-epithelium (P-THEP) were then compared. Basal amounts of P4 were produced when P5 was added to P-THEP, whereas significantly higher amounts were produced in the presence of impure THEP. No significant conversion of P5 or P4 to 17α-OHP4 occurred following culture with pure or impure THEP layer. Results suggest that the enzyme activity necessary to metabolize AD T is localized in the THEP, whereas the metabolic capacities to convert P5 AD and T E2 are present in the granulosa cell. Furthermore, the data show that the two-cell model is applicable to other frog species. J. Exp. Zool. 284:91–99, 1999.

Transforming growth factor-β1 signaling represses testicular steroidogenesis through cross-talk with orphan nuclear receptor Nur77.

Transforming growth factor- β1 (TGF-β1) has been reported to inhibit luteinizing hormone (LH) mediated-steroidogenesis in testicular Leydig cells. However, the mechanism by which TGF-β1 controls the steroidogenesis in Leydig cells is not well understood. Here, we investigated the possibility that TGF-β1 represses steroidogenesis through cross-talk with the orphan nuclear receptor Nur77. Nur77, which is induced by LH/cAMP signaling, is one of major transcription factors that regulate the expression of steroidogenic genes in Leydig cells. TGF-β1 signaling inhibited cAMP-induced testosterone production and the expression of steroidogenic genes such as P450c17, StAR and 3β-HSD in mouse Leydig cells. Further, TGF-β1/ALK5 signaling repressed cAMP-induced and Nur77-activated promoter activity of steroidogenic genes. In addition, TGF-β1/ALK5-activated Smad3 repressed Nur77 transactivation of steroidogenic gene promoters by interfering with Nur77 binding to DNA. In primary Leydig cells isolated from Tgfbr2flox/flox Cyp17iCre mice, TGF-β1-mediated repression of cAMP-induced steroidogenic gene expression was significantly less than that in primary Leydig cells from Tgfbr2flox/flox mice. Taken together, these results suggest that TGF-β1/ALK5/Smad3 signaling represses the expression of steroidogenic genes via the suppression of Nur77 transactivation in testicular Leydig cells. These findings may provide a molecular mechanism involved in the TGF-β1-mediated repression of testicular steroidogenesis.

Differences in Cardiovascular and Hypothalamic-Pituitary-Adrenal Axis Functions between High-Altitude Visitors and Natives during a Trek on the Annapurna Circuit

Objective: Differences in the cardiovascular and hypothalamic-pituitary-adrenal (HPA) axis functions at high altitudes (HAs) between visitors to and natives of HA were examined. Methods: The cardiovascular functions and peripheral oxygen saturation (SPO2) were monitored, and the cortisol awakening response (CAR) and nighttime cortisol concentration (NCC), as indices of the HPA axis function, were determined in 25 trekkers and 21 Sherpas during an Annapurna circuit trek. Results: SPO2 decreased less in the Sherpas than in the trekkers at HAs (3,540, 3,800, and 4,800 m). Blood pressure and heart rate in the Sherpas changed concurrently during the trek; however, a tachycardic response occurred without changes in blood pressure in the trekkers at HAs. The CAR and NCC at HAs in the trekkers differed from those observed at 1,100 m and those observed at HAs in the Sherpas. The trekkers exhibited an elevated morning cortisol level at 3,540 and 3,800 m, a heightened CAR at 4,800 m, and an elevated NCC at 3,800 m. Alteration of the CAR resulted in an increase in the integrated volume of cortisol released within the first hour after awakening (CARauc) in the trekkers. The changes in SPO2 occurred concurrently with the changes in the CARauc and the heart rate in the trekkers. Conclusions: The alterations of CAR occurred at HAs where blood pressure levels reached a peak plateau, which is associated with an increase in heart rate at HAs in the trekkers. The CAR was unaltered in the Sherpas during the trek.

Puberty-related changes in cortisol, dehydroepiandrosterone, and estradiol-17β secretions within the first hour after waking in premenarcheal girls.

Objective: The onset of menstruation is the hallmark of female pubertal development. The present study determined whether pubertal girls experience adrenocortical and ovarian steroid secretions within their first waking hour before getting their period, similar to those observed in adult females with regular cycles. Methods: Cortisol, dehydroepiandrosterone (DHEA), and estradiol-17β concentrations were measured in saliva samples collected after awakening (0, 30, and 60 min after awakening) from 158 normal premenarcheal pubertal girls and 69 adult females with regular menstrual cycles. The girls were subgrouped according to self-reported Tanner breast (B) and pubic hair (PH) stages (B1PH1, B2PH1, B2PH2, B3PH1, and B3PH2). Results: All the subgroups showed a similar pattern of cortisol secretion. However, cortisol levels were higher in girls at B3PH1 and at B3PH2 than other subgroups. DHEA secretion showed a similar pattern across the groups examined. The largest increase in DHEA levels occurred between B1PH1 and B2PH1 stages, and further increased with pubertal progression. DHEA levels in girls at B3PH2 were approximately one half of the adult value. Estradiol-17β profiles in girls at B3PH1 and B3PH2 differed from those of other subgroups of girl. A sharp increase in estradiol-17β levels after awakening which observed in adult females emerged in girls at B3PH1 and B3PH2. However, the estradiol-17β levels did not reach adult values until B3PH2 stage. Conclusions: The progression of female puberty includes an increase in the levels of adrenocortical and ovarian steroid secretions and a gain of adult female-like patterns of estradiol-17β secretion within their first waking hour.