The opposite roles of glucocorticoid and α1-adrenergic receptors in stress triggered apoptosis of rat Leydig cells

Abstract

The stress-induced initiation of proapoptotic signaling in Leydig cells is relatively well defined, but the duration of this signaling and the mechanism(s) involved in opposing the stress responses have not been addressed. In this study, immobilization stress (IMO) was applied for 2 h daily, and animals were euthanized immediately after the first (IMO1), second (IMO2), and 10th (IMO10) sessions. In IMO1 and IMO2 rats, serum corticosterone and adrenaline were elevated, whereas serum androgens and mRNA transcription of insulin-like factor-3 in Leydig cells were inhibited. Reduced oxygen consumption and the mitochondrial membrane potential coupled with a leak of cytochrome c from mitochondria and increased caspase-9 expression, caspase-3 activity, and number of apoptotic Leydig cells was also observed. Corticosterone and adrenaline were also elevated in IMO10 rats but were accompanied with a partial recovery of androgen secretion and normalization of insulin-like factor-3 transcription coupled with increased cytochrome c expression, abolition of proapoptotic signaling, and normalization of the apoptotic events. Blockade of intratesticular glucocorticoid receptors diminished proapoptotic effects without affecting antiapoptotic effects, whereas blockade of intratesticular α(1)-adrenergic receptors diminished the antiapoptotic effects without affecting proapoptotic effects. These results confirmed a critical role of glucocorticoids in mitochondria-dependent apoptosis and showed for the first time the relevance of stress-induced upregulation of α(1)-adrenergic receptor expression in cell apoptotic resistance to repetitive IMOs. The opposite role of two hormones in control of the apoptotic rate in Leydig cells also provides a rationale for a partial recovery of androgen production in chronically stressed animals.

Fig. 1.

Effects of stress on circulating levels of stress hormones, LH, androgens, transcription of the Insl3 gene, and apoptosis of Leydig cells. A–D: stress-induced increase in circulating corticosterone (CORT; A) and adrenaline (B) levels was accompanied with a decrease in LH (C) and androgen [testosterone + dehydrotestosterone (T + DHT)] levels (D). Note that there is a difference in LH and androgen levels after immobilization stress (IMO)1 and IMO10 (1st and 10th sessions of IMO, respectively). E and F: IMO affected Insl3 transcription (E), and changes in apoptotic rates (F) mirrored androgen and Insl3 profiles. In this and the following figures, rats were subjected to IMO1, IMO2, or IMO10 or left undisturbed (controls; 0), trunk blood was collected for hormonal analysis, and Leydig cells were isolated (for details, see materials and methods). Data bars are means ± SE of 3–5 independent in vivo experiments. Statistical significance was at level P < 0.05: *vs. control group; ^vs. IMO1 group.

Fig. 2.

Stress disturbed mitochondrial function and distribution of cytochrome c (CYTC) in Leydig cells. A: IMO1 and IMO2 transiently inhibited oxygen consumption by Leydig cells (top) and the mitochondrial membrane potential (bottom). B: distribution of CYTC in Leydig cell mitochondrial fraction (MF), postmitochondrial fraction (PMF), and whole cell lysate (WCL) was also affected by IMO stress. Representative blots of CYTC and β-actin (ACTB; used as an internal control) are shown at top, and pooled data from scanning densitometry normalized on ACTB values are shown at bottom as means ± SE from 3 independent experiments. Statistical significance at level P < 0.05: *vs. control group.

Fig. 3.

Stress triggered changes in caspase-9 (CASP9) protein transcription, caspase-3 (CASP3) activity, and phospho (p)-Akt/Akt distribution in Leydig cells. A: IMO-induced changes in the expression of CASP9 protein. Representative blots are shown at top, and pooled data from scanning densitometry normalized on ACTB values are shown at bottom as means ± SE from 3 independent experiments. B: IMO-induced changes in CASP3 activity. C and D: distribution of p-Akt/Akt in Leydig cells in WCL, MF, and PMF fractions. Top: representative blots. Bottom: pooled data from scanning densitometry normalized on Akt and ACTB values. Normalized data shown are means ± SE from triplicate determination. Statistical significance at level P < 0.05: *vs. control group; ^vs. IMO1 group.

Fig. 4.

Time course effects of RU-486, a glucocorticoid receptor antagonist, on hormonal profile and apoptotic signaling in unstressed and stressed animals. RU-486 (20 μg in 20 μl/testis) or vehicle (20 μl/testis) was injected intratesticularly to unstressed and stressed rats 12 h before the IMO session; animals were euthanized at the end of the IMO session, blood was collected, and Leydig cells were prepared (for details, see materials and methods). A–D, left: in unstressed animals, RU-486 alone did not affect serum LH (A), but it decreased serum androgens (B), the mitochondrial membrane potential (C), and the rate of Leydig cell apoptosis (D). A–D, right: in stressed animals, RU-486 attenuated IMO-induced inhibition of serum androgens (B) without affecting serum LH levels (A). It also inhibited IMO-induced decrease in mitochondrial membrane potential (C) and reduced the percentage of apoptotic cells (D). Data shown are means ± SE with 4 animals/group. Statistical significance at level P < 0.05: *vs. untreated group; ^vs. IMO1 group; #vs. corresponding IMO group. E: RU-486 inhibited the IMO-induced caspase-9 expression. Bottom: representative blots for caspase-9 and ACTB. Top: mean ± SE values from 3 experiments. Statistical significance at level P < 0.05: *vs. vehicle. F: RU-486 abolished the IMO10-induced expression of Hsd11b2 gene in Leydig cells. Bars illustrate mean ± SE values from 3 experiments. *P < 0.05 vs. vehicle.

Fig. 5.

Time-dependent effects of prazosin (Pra), an α₁-adrenergic receptor antagonist, on hormonal profile and apoptotic signaling in unstressed and stressed animals. Pra (7.5 μg in 20 μl/testis) or vehicle (20 μl/testis) was injected intratesticularly to unstressed and stressed rats 30 min before the stress session; animals were euthanized at the end of the stress session, blood was collected, and Leydig cells were prepared (for details, see materials and methods). A–D, left: in unstressed animals, Pra decreased serum androgen levels (B) without affecting serum LH levels (A). It also increased the percentage of apoptotic cells during repetitive application (D) without affecting mitochondrial membrane potential (C). A–D, right: in stressed animals, Pra did not affect LH levels (A) or the IMO1- and IMO2-induced drop in androgenesis, but it protected the partial recovery of androgenesis in IMO10 rats (B). It also protected recovery of mitochondrial membrane potential (C) and sustained apoptosis (D) in IMO10 rats. E: Pra alone stimulated CASP9 expression and enhanced IMO10-induced expression of this enzyme. Bottom: representative blots for CASP9 and ACTB. Top: mean ± SE values from 3 experiments. *P < 0.05 vs. vehicle; #P < 0.05 vs. corresponding IMO group. F: Pra did not affect the expression of the Hsd11b2 gene in Leydig cells. G and H: the level of Akt (G) and p-ERK/ERK ratio (H) in Leydig cells. Representative blots are shown at bottom, and pooled data from scanning densitometry normalized on ACTB or ERK values are shown as means ± SE for 3 independent experiments. Data bars are means ± SE values of 3 independent experiments. Statistical significance at level P < 0.05: *vs. control group. TMRE, tetramethylrhodamine ethylester.