Disruption of nongenomic testosterone signaling in a model of spinal and bulbar muscular atrophy.

As one of the nine hereditary neurodegenerative polyQ disorders, spinal and bulbar muscular atrophy (SBMA) results from a polyQ tract expansion in androgen receptor (AR). Although protein aggregates are the pathological hallmark of many neurodegenerative diseases, their direct role in the neurodegeneration is more and more questioned. To determine the early molecular mechanisms causing motor neuron degeneration in SBMA, we established an in vitro system based on the tetracycline-inducible expression of normal (AR20Q), the mutated, 51 glutamine-extended (AR51Q), or polyQ-deleted (AR0Q) AR in NSC34, a motor neuron-like cell line lacking endogenous AR. Although no intracellular aggregates were formed, the expression of the AR51Q leads to a loss of function characterized by reduced neurite outgrowth and to a toxic gain of function resulting in decreased cell viability. In this study, we show that both AR20Q and AR51Q are recruited to lipid rafts in response to testosterone stimulation. However, whereas testosterone induces the activation of the c-jun N-terminal kinase/c-jun pathway via membrane-associated AR20Q, it does not so in NSC34 expressing AR51Q. Phosphorylation of c-jun N-terminal kinase plays a crucial role in AR20Q-dependent survival and differentiation of NSC34. Moreover, c-jun protein levels decrease more slowly in AR20Q- than in AR51Q-expressing NSC34 cells. This is due to a rapid and transient inhibition of glycogen synthase kinase 3α occurring in a phosphatidylinositol 3-kinase-independent manner. Our results demonstrate that the deregulation of nongenomic AR signaling may be involved in SBMA establishment, opening new therapeutic perspectives.

1α,25(OH)2-Vitamin D3 stimulates rapid plasma membrane calcium influx via MAPK activation in immature rat Sertoli cells.

It was characterized that the rapid response to 1α,25(OH)(2)-vitamin D(3) (1,25D(3)) on (45)Ca(2+) influx in rat Sertoli cells was mediated by voltage-dependent Ca(2+) channels (VDCCs), PKC, ERK1/2 and p38 MAPK pathways. In primary culture of 10 day-old rat Sertoli cells as well as in the whole testis, the time-course of (45)Ca(2+) influx did not change significantly in basal conditions. However, 1,25D(3) showed stimulatory effect on (45)Ca(2+) influx from 10(-15) to 10(-8) M after 60 s of incubation. The maximum effect was around 140% at 10(-12) M on purified Sertoli cells showing a steady state on (45)Ca(2+) influx between 10(-11) and 10(-9) M. Under this experimental condition, 1,25D(3) stimulated (45)Ca(2+) influx from 73% to 106% and no effect was observed at 10(-16), 10(-8) and 10(-7) M in whole testis. VDCC activities are mandatory for a full and complete stimulatory effect of 1,25D(3) in these approaches. K(+) and Cl(-) channels also are strongly involved in this rapid response coordinated by 1,25D(3). The participation of some selected kinases, points to PKC and ERK1/2 upstream activity to p38 MAPK activation suggesting an intracellular cross-talk between rapid (45)Ca(2+) influx and nuclear events. In addition, the comparative effect of microtubule disassembles and ClC-3 channel blocker on (45)Ca(2+) influx provides evidence of secretory activity of Sertoli cells triggered by 1,25D(3). Our results suggest that 1,25D(3) activates p38 MAPK and reorganizes microtubules, involving Ca(2+), PKC and ERK1/2 as upstream regulators and that extracellular Ca(2+) have a central role to rapidly start hormone-induced gene transcription and/or the secretory activity of Sertoli cell.

Role of estrogens in spermatogenesis.

Aromatase converts irreversibly androgens into estrogens and is present in the endoplasmic reticulum of various cells of mammalian testes ; at least in rodents, all testicular cells except peritubular cells express aromatase. In testis, high affinity estrogen receptors, ERalpha and/or ERbeta, together with a membrane rapid effect recently described, mediate the effects of estrogens. From the experimental models, in vitro studies and data collected in patients, it is now demonstrated that estrogens play an important role in the testis of vertebrates. At least it is supported by the widespread distribution of estrogen receptors in the testicular cells and the simultaneous presence of a biologically active aromatase in all germ cells (especially in meiotic and post-meiotic stages). Thus the role of estrogens (intracrine, autocrine and / or paracrine) in spermatogenesis (proliferation, apoptosis, survival and maturation) and more generally, in male reproduction is now evidenced, and much more complex than previously predicted.

17ß-Estradiol regulates cyclin A1 and cyclin B1 gene expression in adult rat seminiferous tubules.

Spermatogenesis, which is the fundamental mechanism allowing male gamete production, is controlled by several factors, and among them, estrogens are likely concerned. In order to enlighten the potential role of estrogen in rat spermatogenesis, seminiferous tubules (ST) from two groups of seminiferous epithelium stages (II-VIII and IX-I) were treated with either 17ß-estradiol (E(2)) agonists or antagonists for estrogen receptors (ESRs). In this study, we show that cyclin A1 and cyclin B1 gene expression is controlled by E(2) at a concentration of 10(-9) M only in stages IX-I. This effect is mimicked by a treatment with the G-protein coupled estrogen receptor (GPER) agonist G1 and is abolished by treatment with the ESR antagonist ICI 182 780. Moreover, using letrozole, a drug that blocks estrogen synthesis, we demonstrate that these genes are under the control of E(2) within rat ST. Thus, germ cell differentiation may be regulated by E(2) which acts through ESRs and GPER, expressed in adult rat ST.

Estrogens: new players in spermatogenesis.

Aromatase that irreversibly transforms androgens into estrogens is present in the smooth endoplasmic reticulum of nearly all cell types in the mammalian testis. In rodents, all testicular cells except for myoid cells express aromatase activity. We have demonstrated the presence of the functional aromatase (transcript or protein, and biological activity) in adult rat germ cells including pachytene spermatocytes and round spermatids. We have also demonstrated estrogen output from these cells equivalent to that of Leydig cells. Unlike androgen receptors, which are localized mainly in testicular somatic cells, estrogen receptors are present in both somatic and germ cells in the testis. Moreover, we have recently described the rapid membrane effects of estrogens (via G protein-coupled receptor [GPER]) in purified rat germ cells. On the basis of various experimental models, in vitro studies and clinical data, it can be concluded that estrogens play an essential role in male reproduction, specifically in the development of spermatozoa.

1α,25-Dihydroxyvitamin D(3) signaling pathways on calcium uptake in 30-day-old rat Sertoli cells.

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.

Effect of 1α,25-dihydroxyvitamin D3 in plasma membrane targets in immature rat testis: ionic channels and gamma-glutamyl transpeptidase activity.

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is critical for the maintenance of normal reproduction since reduced fertility is observed in vitamin D-deficient male rats. The aim of this study was to investigate the effect of 1,25D(3) in 30-day-old rat testicular plasma membrane targets (calcium uptake and gamma-glutamyl transpeptidase (GGTP) activity), as well as to highlight the role of protein kinases in the mechanism of action of 1,25D(3). The results demonstrated that 1,25D(3) induced a fast increase in calcium uptake in rat testis through a nongenomic mechanism of action. This effect was dependent on PKA, PKC and MEK. Moreover, ionic channels, such as ATP- and Ca(2+)-dependent K(+) channels and Ca(2+)-dependent Cl(-) channels, are involved in the mechanism of action. The use of BAPTA-AM showed that [Ca(2+)](i) was also implicated, and the incubation with digoxin produced an increase in (45)Ca(2+) uptake indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, 1,25D(3) was able to increase the GGTP activity. Considered together, our results indicate a PKA/PKC/MEK-dependent 1,25D(3) pathway as well as ionic involvement leading to (45)Ca(2+) uptake in immature rat testis. These findings demonstrate that 1,25D(3) stimulates calcium uptake and increases GGTP activity which may be involved in male reproductive functions.

Regulation of aromatase expression by 1α,25(OH)2 vitamin D3 in rat testicular cells.

It is well known that the vitamin D endocrine system is involved in physiological and biochemical events in numerous tissues, especially gut, bone and kidney but also testis. Therefore, in this study the effect and mechanisms of action of 1α,25(OH)(2) vitamin D(3) (1,25D) on aromatase gene expression in immature rat Sertoli cells were evaluated. Vitamin D receptor transcripts were present in immature Sertoli cells as well as in adult testicular germ cells and somatic cells. The treatment of immature Sertoli cells with 100 nM 1,25D increased the amount of aromatase transcript, mainly in 30-day-old rats. The protein kinase A (PKA) blocker, H89, partially inhibited the 1,25D effect. The stimulation of aromatase gene expression in 30-day-old Sertoli cells by the agonist 1α,25(OH)(2) lumisterol(3), and the suppression of the 1,25D effect by the antagonists 1ß,25(OH)(2) vitamin D(3) and (23S)-25-dehydro-1α (OH)-vitamin D(3)-26,23-lactone suggested, besides a genomic effect of 1,25D, the existence of non-genomic activation of the membrane-bound vitamin D receptor involving the PKA pathway.

Nongenomic and genomic effects of 1α,25(OH)2 vitamin D3 in rat testis.

The steroid hormone 1α,25(OH)(2)-vitamin D(3) (1,25D(3)) regulates gene transcription through a nuclear receptor (VDRnuc) and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDRmem). It has been described that successful mating and fertility rates are significantly decreased in vitamin D deficient male rats and a VDR null mutant rodent has decreased sperm count and motility and expresses rare spermatogenesis. Although the Sertoli cells are pointed as the major target of 1,25D(3) in the testis the mechanism of 1,25D(3) action, particularly in Sertoli cells, remains unclear. Several studies undertaken in the testicular cells showed that 1,25D(3) can produce both genomic and nongenotropic actions in those cells. 1,25D(3) can modulate kinase activities and ionic fluxes (Ca(2+) and Cl(-)) at the plasma membrane resulting in the regulation of secretory processes in Sertoli cells. The enormous complexity of the nongenomic actions of 1,25D(3) implies that specific receptor or specific ligand-binding sites located on the plasma membrane or in the nucleus are believed to initiate specific cell responses. Apparently the choice of the signaling pathways to be activated after the interaction of the hormone with cell surface receptors is directly related with the physiological action to be better accomplished. The demonstration that 1,25D(3) can regulate both Sertoli cell and sperm function may be useful for the study and development of new therapeutic strategies to the treatment of male reproductive disorders. This review summarizes recent research on the rapid actions of 1,25D(3) and identifies questions that remain to be answered in this area.

Effects of the methanol extract of Basella alba L (Basellaceae) on steroid production in Leydig cells.

In this study, Leydig cells were purified from 70 day-old Sprague Dawley male rats and incubated with 10 and 100 µg/mL of methanol extract of Basella alba (MEBa) for 4 hours followed by the evaluation of cell viability, steroid (testosterone and estradiol) production, and the level of aromatase mRNA. Results showed that MEBa did not affect Leydig cell viability. At the concentration of 10 µg/mL, MEBa significantly stimulated testosterone and estradiol production (p < 0.01 and p < 0.03, respectively), and enhanced aromatase mRNA level (p < 0.04). These observations suggest that MEBa directly stimulated testosterone, estradiol and aromatase mRNA levels in isolated Leydig cells.

Therapeutic effects of soy isoflavones on α-amylase activity, insulin deficiency, liver-kidney function and metabolic disorders in diabetic rats.

Natural estrogens have demonstrated a wide variety of biological activities, which makes them a good candidate for the treatment of diabetes. In vitro, this study evidenced that isoflavones enhanced insulin secretion and inhibited α-amylase activity. In vivo, the findings indicated that soy isoflavones stimulated insulin secretion, increased the hepatic glycogen content and suppressed blood glucose level. The soy isoflavones were also protected hepatic-kidney functions showed by the significant increase in superoxide dismutase, catalase and glutathione peroxidase activities and the decrease in thiobarbituric acid reactive substances, total bilirubin, creatinine and transaminases content. Moreover, soy isoflavones induced a decrease in LDL-cholesterol and triglycerides and an increase in HDL-cholesterol in plasma and liver. Overall, the findings of the current study indicate that soy isoflavones exhibit attractive properties and can, therefore, be considered a promising candidate for future application as alternative therapeutic agents, particularly in the development of anti-diabetic and hypolipidaemic drugs.

Estrogens in male germ cells.

The biosynthesis of steroids and the production of spermatozoa are two major functions of the mammalian testis which are tightly controlled by gonadotropins and numerous locally produced factors. Among these are the estrogens that are produced within the seminiferous epithelium via the irreversible transformation of androgens (C19) into estrogens (C18) by aromatase. We have recently reported that male germ cells are the new source of estrogens in the testis. For instance, estrogen receptors (ER) are found mainly in spermatids that give rise to spermatozoa. Moreover, it is noteworthy that GPR 30 (a transmembrane ER) induces rapid responses after estradiol binding, which, in turn, modulates cyclins and proapoptotic factors (e.g., BAX) to affect germ cell cycle progression and apoptosis. In summary, at least in the animal species that were studied thus far, germ cells are the major source and the target of estrogens, affecting normal male gonadal development and spermatogenesis, in particular spermiogenesis. These findings have also shed new light on the possible adverse effects of endocrine disruptors having estrogenic activities that can cause abnormal development of the male genital tract.

Inhibitory effects of estrogens on digestive enzymes, insulin deficiency, and pancreas toxicity in diabetic rats.

Diabetes mellitus, with its attendant disorders and dysfunctional behaviors, constitutes a growing concern to the population of the world. With this concern in mind, the present study investigated the anti-diabetic and hypolipedimic potential of 17ß-estradiol (called E2), particularly in terms of its inhibitory effects on maltase, sucrase, lactase, and lipase activities in the intestine of surviving diabetic rats. The findings revealed that this supplement helped protect the ß cells of the rats from death and damage. Interestingly, E2 induced considerable decreases of 29%, 46%, 42%, and 84% in the activities of intestinal maltase, lactase, sucrase, and lipase, respectively. The E2 extract also decreased the glucose, triglyceride, and total cholesterol rates in the plasma of diabetic rats by 39%, 27%, and 53%, respectively, and increased the HDL-cholesterol level by 74%, which helped maintain the homeostasis of blood lipid. When compared to those of the untreated diabetic rats, the superoxide dismutase, catalase, and glutathione peroxidase levels in the pancreas of the rats treated with this supplement were also enhanced by 330%, 170%, and 301%, respectively. A significant decrease was also observed in the lipid peroxidation level and lactate dehydrogenase activity in the pancreas of diabetic rats after E2 administration. Overall, the findings presented in this study demonstrate that E2 has both a promising potential with regard to the inhibition of intestinal maltase, sucrase, lactase, and lipase activities, and a valuable hypoglycemic and hypolipidemic function, which make it a potential strong candidate for industrial application as apharmacological agent for the treatment and prevention of hyperlipidemia, obesity, and cardiovascular diseases.

Potential protective effect on key steroidogenesis and metabolic enzymes and sperm abnormalities by fenugreek steroids in testis and epididymis of surviving diabetic rats.

The current study showed that the daily oral treatment of fenugreek steroids, designated F(steroids), to diabetic rats during 30 days demonstrated a significant (p < 0.05) decrease of blood glucose level and a considerable increase of the area of insulin-immunoreactive beta-cells in diabetic rats. Interestingly, this study showed that F(steroids) potentially unregulated the key steroidogenesis enzymes such as 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), malic enzyme, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and glucose-6-phosphate dehydrogenase (G6P-DH) activities as cholesterol rate in testis, which considerably enhanced testosterone and estradiol levels in the plasma of surviving diabetic rats. More interestingly, F(steroids) obviously prevented the alteration of the key carbohydrate enzymes such as hexokinase and pyruvate kinase activities as well as testicular glycogen and seminal fructose contents in surviving diabetic rats. Furthermore, F(steroids) administration to surviving diabetic rats significantly decreased the sperm shape abnormality and improved the sperm count. Above all, the potential protective action of reproductive systems was approved by the histological study of testis and epididymis.

Oestrogens and spermatogenesis.

The role of oestrogens in male reproductive tract physiology has for a long time been a subject of debate. The testis produces significant amounts of oestrogenic hormones, via aromatase, and oestrogen receptors (ERs)alpha (ESR1) and ERbeta (ESR2) are selectively expressed in cells of the testis as well as the epididymal epithelium, depending upon species. This review summarizes the current knowledge concerning the presence and activity of aromatase and ERs in testis and sperm and the potential roles that oestrogens may have in mammalian spermatogenesis. Data show that physiology of the male gonad is in part under the control of a balance of androgens and oestrogens, with aromatase serving as a modulator.

Aromatase, oestrogens and human male reproduction.

In most mammalian species aromatase is encoded by a single gene (Cyp19), which contains 18 exons, nine of them being translated. In man, the presence of a biologically active aromatase and oestrogen receptors (ERalpha and ERbeta) has been reported in Leydig cells, and also in immature germ cells and ejaculated spermatozoa. Concerning aromatase, the amount of transcript and enzymatic activity are decreased in immotile compared with motile sperm. We have amplified aromatase mRNA by real-time polymerase chain reaction in spermatozoa from asthenospermic, teratospermic and asthenoteratospermic men and recorded, respectively, 44, 52 and 67 per cent decreases of the amount of transcripts compared with fertile donors. A high degree of correlation (r = -0.64) between the abnormal spermatozoa (especially microcephaly and acrosome malformations) and aromatase/GAPDH transcript ratio has been observed. Idiopathic infertility is a wide health problem and no treatment is currently available. In humans, even if the role of oestrogens in spermatogenesis is still a matter of debate, the observations of decreased sperm number and motility in men genetically deficient in aromatase, together with our data and those reported in the literature, may suggest a role for aromatase/oestrogens not only during the development and maintenance of spermatogenesis but also in the final maturation of spermatozoa.

Aromatase gene expression in immature rat Sertoli cells: age-related changes in the FSH signalling pathway.

Aromatase, the enzyme responsible for the transformation of androgens into oestrogens, is encoded by the cyp19 gene expressed in the testis. The aim of the present study was to analyse the evolution of aromatase gene expression under FSH control in rat Sertoli cells between 10 and 30 days post partum, corresponding to the end of the proliferative period of Sertoli cells, establishment of the blood-testis barrier and acquisition of the mature phenotype. The maximum stimulatory effect of FSH on aromatase gene expression was obtained in 20-day-old rat Sertoli cells, compared with cells from 10- and 30-day-old rats, in parallel with the differentiation of Sertoli cells. Using two effectors of the protein kinase A pathway (i.e. forskolin and dibutyryl-cAMP) revealed differential effects between cells from rats aged 20 and 30 days, implying the involvement of another signalling pathway. Experiments using the specific phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 revealed that PI3-K is strongly involved in FSH-induced aromatase expression in Sertoli cells from both 20- and 30-day-old rats. In vivo, this decrease could be explained by a negative effect exerted by germ cells because, in coculture, aromatase gene expression in 20-day-old Sertoli cells is greatly diminished.

17 beta-estradiol activates rapid signaling pathways involved in rat pachytene spermatocytes apoptosis through GPR30 and ER alpha.

Aim of the present study was to investigate whether estrogens were able to directly activate rapid signaling pathways controlling spermatogenesis in rat pachytene spermatocytes (PS). Classically, estrogens act by binding to estrogen receptors (ERs) alpha and beta. Recently, it has been demonstrated that rapid estrogen action can also be activated through the G-protein-coupled receptor (GPR)-30. Herein, we demonstrated that rat PS express ER alpha, ER beta and GPR30. Treatment of PS with estradiol (E2), the selective GPR30 agonist G1 and the selective ER alpha agonist PPT determined activation of ERK1/2 which are part of GPR30 signaling cascade. ERK1/2 activation in response to E2 and G1 was correlated to an increased phosphorylation of c-Jun. All treatments failed to induce these responses in the presence of EGFR inhibitor AG1478, ERK inhibitor PD98059 and ER inhibitor ICI182780. mRNA expression of cell cycle regulators cyclin A1 and B1 was downregulated by E2 and G1 while an up-regulation of proapoptotic factor Bax was observed in the same conditions. These data demonstrate that E2, working through both ER alpha and/or GPR30, activates in PS the rapid EGFR/ERK/c-Jun pathway, modulating the expression of genes involved in the balance between cellular proliferation and apoptosis.

Immunomodulatory, beta-cell, and neuroprotective actions of fenugreek oil from alloxan-induced diabetes.

Immunological disorders and nephropathy are among the most frequent and serious complications of diabetes mellitus. This shows that fewer infiltrated inflammatory cells evidenced by reverted back to near the normal value of white blood cell, mean corpuscular volume, and lymphocytes counts as interleukin-6 in pancreas. Also, fenugreek oil significantly improved blood glucose levels, glucose intolerance, and insulin sensitivity compared to the diabetic group. The pancreatic islet and less beta-cells damage were observed after the administration of fenugreek oil to diabetic rats. Moreover, diabetic rats showed low activities of superoxide dismutase, catalase, glutathione peroxidase, and reduced glutathione content in kidney, which were restored to near normal levels by treatment with fenugreek oil. The increased levels of lipid peroxidation, creatinine, albumin, and urea in diabetic rats decreased significantly in diabetic rats treated with fenugreek oil. Diabetic rats treated with fenugreek oil restored almost a normal architecture of pancreas and kidney. In conclusion, this study reveals the efficacy of fenugreek oil in the amelioration of diabetes, hematological status, and renal toxicity which may be attributed to its immunomodulatory activity and insulin stimulation action along with its antioxidant potential.