Maja M. Bjelic

Pharmacological Doses of Testosterone Upregulated Androgen Receptor and 3-Beta-Hydroxysteroid Dehydrogenase/Delta-5-Delta-4 Isomerase and Impaired Leydig Cells Steroidogenesis in Adult Rats

Anabolic androgenic steroids (AAS) are testosterone derivatives originally designed to enhance muscular mass and used for the treatment of many clinical conditions as well as in contraception. Despite popular interest and abuse, we still lack a broad understanding of effects of AAS on synthesis of steroid hormones on the molecular level. This study was designed to systematically analyze the effects of pharmacological/high doses of testosterone on steroidogenic machinery in Leydig cells. Two different experimental approaches were used: (1) In vivo experiment on groups of adult male rats treated with testosterone for 1 day, 2 weeks, and 2 months; (2) Direct in vitro testosterone treatment of Leydig cells isolated from intact rats. Result showed that prolonged in vivo treatment with testosterone decreased the expression of Scarb1 (scavenger receptor class B type 1), Tspo (translocator protein), Star (steroidogenic acute regulatory protein), Cyp11a1 (cholesterol side-chain cleavage enzyme), and Cyp17a1 (17α-hydroxylase/17, 20 lyase) in Leydig cells. Oppositely, the expression of Hsd3b (3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase), Ar (androgen receptor), and Pde4a/b (cyclic adenosine monophosphate-dependent phosphodiesterases) was increased. Androgenization for 2 weeks inhibited Cyp19 (aromatase) transcription, whereas 2-month exposure caused the opposite effect. Direct in vitro testosterone treatment also decreased the expression of Cyp11a1, Cyp17a1, and Cyp19a1, whereas Hsd3b was upregulated. The results of expression analysis were supported by declined steroidogenic capacity and activity of Leydig cells, although conversion of pregnenolone to progesterone was stimulated. The upregulation of AR and 3βHSD in testosterone-impaired Leydig cells steroidogenesis could be the possible mechanism that maintain and prevent loss of steroidogenic function.

Transient Rise of Serum Testosterone Level after Single Sildenafil Treatment of Adult Male Rats

INTRODUCTION Phosphodiesterase type 5 (PDE5) inhibitors have been established in therapy for a variety of physiological disorders including erectile dysfunction. Despite its popularity and wide usage in erectile dysfunction treatment, the short-term effect of PDE5 inhibition on Leydig cell functionality and testosterone dynamics is missing. AIM This study was designed to assess the acute in vivo effects of sildenafil citrate (Viagra) treatment on testosterone production. METHODS Male adult rats were given sildenafil (1.25 mg/kg BW) per os, and testosterone production were analyzed 30, 60, 120, and 180 minutes after treatment. Additionally, in vitro effect of sildenafil extract on Leydig cell steroidogenesis was estimated. MAIN OUTCOME MEASURES The formation of testicular interstitial fluid (TIF), and testosterone, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP) content was followed. Occurrence and phosphorylation of mature steroidogenic acute regulatory protein (StAR) and interaction with protein kinase G 1 (PRKG1) were assessed by immunoprecipitation and Western blot. RESULTS Serum testosterone was increased 60 and 120 minutes after sildenafil treatment. In 60 minutes, TIF volume was doubled and stayed increased till the end of the experimental period. cGMP and testosterone content in TIF were increased 30 minutes after treatment, and cAMP decreased in 60 minutes. Further, sildenafil-induced stimulation of testosterone production was abolished by ex vivo addition of PRKG1 inhibitor but not by protein kinase A inhibitor. Sildenafil treatment increased the level of phosphorylated and total StAR protein. Moreover, co-immunoprecipitation of StAR and PRKG1 was increased following sildenafil treatment suggesting the active role of this kinase in initiation of testosterone synthesis. Additionally, sildenafil extract applied in vitro on primary Leydig cell culture increased cGMP accumulation and testosterone production in time- and dose-dependent manner without effect on cAMP level. CONCLUSION Acute sildenafil treatment enlarged TIF volume but also stimulated testosterone production which may be significant considering the positive testosterone effect in regulation of sexual activity.

Age related changes of cAMP and MAPK signaling in Leydig cells of Wistar rats

Here, we chronologically analyzed age-associated changes of cAMP- and MAPK-signaling in Leydig cells (LCs) in relation with decreased testosterone (T) production. In Wistar rats, decreased serum T observed in 12 to 24-month-old rats was not related to decreased serum LH concentration but to reduced luteinizing hormone receptor (Lhr/LHR) and time-coordinated reduction of steroidogenic gene expression (decreased Cyp11a1, Cyp17a1 in 12-month-old rats followed by decreased Star/StAR, Hsd3b/HSD3B, Hsd17b4, and increased Cyp19a1 later in life). The predecessors of age-related changes noted in LCs from 6 to 12-month-old rats were increased level of soluble adenylate cyclase (Adcy/AC) 10, increased JNK phosphorylation but suppressed P38 MAPK. At approximately the same time changed mRNA abundance for transcription factors important for steroidogenesis was detected (increased Nur77 and decreased Sf1, Dax1). Aging caused biphasic expression pattern of ERK1/2 and Nur77: increased in 12-month but decreased in LCs from 24-month-old rats. Further, decreased basal cAMP level observed from 12 to 24th month coincidence with increased expression of cAMP-specific phosphodiesterase (Pde)4a, Pde4b and regulatory subunit of protein kinase A (Prkar/PKAR). Exposing of senescent LCs to permeable cAMP-analog improved transcription of Sf1, Nur77, Star, Cyp11a1,Cyp17a1, but without effect on aging pattern of Dax1, Pde4a/b, Prkar2a, Lhr and MAPK genes. Collectively, results indicated that age-related LC dysfunction is accompanied with changes in MAPK and cAMP signaling and coordinated reduction in the expression of many of the genes that participate in T synthesis. The predecessors of aged-related changes are increased ratio of pJNK/JNK, AC10 and decreased P38 level in LCs from 6-month-old rats.

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

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.

Circadian rhythm of the Leydig cells endocrine function is attenuated during aging

Although age-related hypofunction of Leydig cells is well illustrated across species, its circadian nature has not been analyzed. Here we describe changes in circadian behavior in Leydig cells isolated from adult (3-month) and aged (18- and 24-month) rats. The results showed reduced circadian pattern of testosterone secretion in both groups of aged rats despite unchanged LH circadian secretion. Although arrhythmic, the expression of Insl3, another secretory product of Leydig cells, was decreased in both groups. Intracellular cAMP and most important steroidogenic genes (Star, Cyp11a1 and Cyp17a1), together with positive steroidogenic regulator (Nur77), showed preserved circadian rhythm in aging although rhythm robustness and expression level were attenuated in both aged groups. Aging compromised cholesterol mobilization and uptake by Leydig cells: the oscillatory transcription pattern of genes encoding HDL-receptor (Scarb1), hormone sensitive lipase (Lipe, enzyme that converts cholesterol esters from lipid droplets into free cholesterol) and protein responsible for forming the cholesterol esters (Soat2) were flattened in 24-month group. The majority of examined clock genes displayed circadian behavior in expression but only a few of them (Bmal1, Per1, Per2, Per3 and Rev-Erba) were reduced in 24-month-old group. Furthermore, aging reduced oscillatory expression pattern of Sirt1 and Nampt, genes encoding key enzymes that connect cellular metabolism and circadian network. Altogether circadian amplitude of Leydig cells endocrine function decreased during aging. The results suggest that clock genes are more resistant to aging than genes involved in steroidogenesis supporting the hypothesis about peripheral clock involvement in rhythm maintenance during aging.

The Opposing Roles of Nitric Oxide and cGMP in the Age-Associated Decline in Rat Testicular Steroidogenesis

The molecular mechanism of the aging-associated dysfunction of Leydig cells (LCs) is complex and poorly understood. In this study, we analyzed the contribution of nitric oxide (NO) and cGMP signaling to the age-dependent decline in LC function. Significant (>50%) decreases in serum, intratesticular, and LC androgens in aging rats (15-24 months) were accompanied by a proportional increase in NO production, an up-regulation of cGMP levels, and the expression of soluble guanylyl cyclase-1B and protein kinase G1 in LCs. In contrast, LC cAMP levels decreased with age, most likely reflecting the up-regulation of cAMP-specific phosphodiesterase expression. Moreover, the expression of genes encoding enzymes responsible for cholesterol transport and its conversion to T were reduced. Exposing LCs from aged animals to NO further increased cGMP levels and decreased cAMP and androgen production, whereas the addition of cell-permeable 8-bromoguanosine-cGMP alone had the opposite effect. In vivo inhibition of cGMP-specific phosphodiesterase-5 for 3 and 6 months in aged rats led to a partial restoration of androgens, NO, and cyclic nucleotide levels, as well as the expression of steroidogenic and NO/cGMP signaling genes. These results indicate that a progressive increase in NO production contributes to the age-dependent decrease in steroidogenesis in a cGMP-independent manner, whereas the sustained elevation in cGMP levels significantly slows the decline in LC function.

Prolonged in vivo administration of testosterone-enanthate, the widely used and abused anabolic androgenic steroid, disturbs prolactin and cAMP signaling in Leydig cells of adult rats.

This study was designed to systematically analyze and define the effects of 1-day, 2-weeks, 10-weeks intramuscular administration of testosterone-enanthate, widely used and abused anabolic androgenic steroid (AAS), on main regulators of steroidogenesis and steroidogenic genes expression in testosterone-producing Leydig cells of adult rats. The results showed that prolonged (10-weeks) intramuscular administration of testosterone-enanthate, in clinically relevant dose, significantly increased prolactin, but decreased Prlr2 and Gnrhr in pituitary of adult rat. The levels of testosterone, Insl3, cAMP and mitochondrial membrane potential of Leydig cells were significantly reduced. This was followed by decreased expression of some steroidogenic enzymes and regulatory proteins such as Lhcgr, Prlr1/2, Tspo, Star, Cyp11a1, Cyp17a1, Dax1. Oppositely, Hsd3b1/2, Hsd3b5, Hsd17b4, Ar, Arr19 increased. In the same cells, transcriptional milieu of cAMP signaling elements was disturbed with remarkable up-regulation of PRKA (the main regulator of steroidogenesis). Increased prolactin together with stimulated transcription of Jak2/Jak3 could account for increased Hsd3b1/2 and Hsd3b5 in Leydig cells following 10-weeks in vivo treatment with testosterone-enanthate. In vitro studies revealed that testosterone is capable to increase level of Prlr1, Prlr2, Hsd3b1/2, Hsd3b5 in Leydig cells. Accordingly, testosterone-induced changes in prolactin receptor signaling together with up-regulation of PRKA, Hsd3b1/2, Hsd3b5, Ar in Leydig cells, could be the possible mechanism that contribute to the establishment of a new adaptive response to maintain homeostasis and prevent loss of steroidogenic function. Presented data provide new molecular insights into the relationship between disturbed testosterone homeostasis and mammalian reproduction and are important in terms of wide use and abuse of AASs and human reproductive health.

Melatonin replacement restores the circadian behavior in adult rat Leydig cells after pinealectomy

Melatonin actions on oscillators in reproductive organs are poorly understood. Here we analyzed melatonin effects on rhythmic expression of clock and steroidogenesis-related genes in adult rat Leydig cells (LCs). The effect of melatonin was tested both in vivo using pinealectomized and melatonin-substituted rats and in vitro on isolated LCs. Data revealed 24-h-rhythmic expression of clock genes (Bmal1, Per1,2,3, Rev-erba,b, Rorb), steroidogenic genes (Star, Cyp11a1, Cyp17a1), and genes of steroidogenic regulators (positive-Nur77, negative-Arr19). Pinealectomy increased 24-h-oscillations of serum testosterone and LCs cAMP levels, expression of Insl3, Per1, Star/StAR, Hsd3b1/2, Nur77, decreased Arr19 and canceled Per2 oscillatory expression pattern. At hypothalamic-pituitary level, pinealectomy increased mesor of Gnrh, Lhb and rhythm robustness of Mntr1a expression. All parameters disturbed were restored by melatonin-replacement. In vitro studies did not confirm direct melatonin effects on neither clock nor steroidogenic genes. Accordingly, melatonin influence 24-h-rhythmic LC-function likely through hypothalamic-pituitary axis and consequently cAMP-signaling in LCs.

Molecular adaptations of testosterone-producing Leydig cells during systemic in vivo blockade of the androgen receptor

This study systematically evaluates the effects of androgen receptor (AR) blockade on molecular events in Leydig cells. Results showed that intramuscular administration of testosterone-enanthate, at clinically relevant dose, decreased testosterone in interstitial fluid and Leydig cells from adult rats. AR-blocker (Androcur) prevented this effect and testosterone-reduced Leydig cells steroidogenic capacity/activity. Testosterone-reduced expression of some steroidogenic enzymes/proteins (Tspo,StAR,Hsd3b1/2) and transcription factors (Nur77,Gata4,Dax1) was completely abrogated, while decreased expression of Star,Cyp11a1,Cyp17a1,Hsd17b4,Creb1a was partially prevented. In the same cells, increased expression of Hsd3b5/HSD3B and Ar/AR was abolished. Androcur-treatment abolished testosterone-reduced cAMP, coupled with a changed expressional milieu of cAMP signaling elements. Results from in vitro experiments suggest that some of these effects are testosterone-AR dependent, while others could be due to disturbed LH and/or other signals. Presented data provide new molecular insight into Leydig cells function and are important in terms of human reproductive health and the wide-spread use of Androcur as well as use/abuse of testosterone-enanthate.

Intratesticular alpha1-adrenergic receptors mediate stress-disturbed transcription of steroidogenic stimulator NUR77 as well as steroidogenic repressors DAX1 and ARR19 in Leydig cells of adult rats

The aim of the present study was to define the role of testicular α1-adrenergic receptors (α1-ADRs) in stress-triggered adaptation of testosterone-producing Leydig cells of adult rats. Results showed that in vivo blockade of testicular α1-ADRs prevented partial recovery of circulating androgen levels registered after 10× repeated immobilization stress (10 × IMO). Moreover, α1-ADR-blockade diminished 10 × IMO-triggered recovery of Leydig cell androgen production, and abolished mitochondrial membrane potential recovery. In the same cells, 10 × IMO-induced increase in Star transcript was abolished, Lhcgr transcript decreased, while transcription of other steroidogenic proteins was not changed. α1-ADR-blockade recovered stress-induced decrease of Nur77, one of the main steroidogenic stimulator, while significantly reduced 10 × IMO-increased in the transcription of the main steroidogenic repressors, Arr19 and Dax1. In vitro experiments revealed an adrenaline-induced α1-ADR-mediated decrease in Nur77 transcription in Leydig cells. Adrenaline-induced increase of repressor Dax1 also involves ADRs in Leydig cells. Accordingly, α1-ADRs participate in some of the stress-triggered effects on the steroidogenic machinery of Leydig cells.