The regulatory effect of sex steroids on the RhoA/ROCK pathway in the rat distal vagina.

BACKGROUND: Sex steroids have been demonstrated as important modulators of vaginal function. The RhoA/ROCK calcium-sensitizing pathway plays a role in genital smooth muscle contractile mechanism, but its regulation has never been elucidated. AIM: This study investigated the sex steroid regulation of the vaginal smooth muscle RhoA/ROCK pathway using a validated animal model. METHODS: Ovariectomized (OVX) Sprague-Dawley rats were treated with 17ß-estradiol (E2), testosterone (T), and T with letrozole (T + L) and compared with intact animals. Contractility studies were performed to test the effect of the ROCK inhibitor Y-27632 and the nitric oxide (NO) synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated; mRNA expression was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction; and RhoA membrane translocation was evaluated by Western blot. Finally, rat vaginal smooth muscle cells (rvSMCs) were isolated from the distal vagina of intact and OVX animals, and quantification of the RhoA inhibitory protein RhoGDI was performed after stimulation with NO donor sodium nitroprusside, with or without administration of the soluble guanylate cyclase inhibitor ODQ or PRKG1 inhibitor KT5823. OUTCOMES: Androgens are critical in inhibiting the RhoA/ROCK pathway of the smooth muscle compartment in the distal vagina. RESULTS: ROCK1 was immunolocalized in the smooth muscle bundles and blood vessel wall of the vagina, with weak positivity detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline precontracted vaginal strips, decreased by OVX and restored by E2, while T and T + L decreased it below the OVX level. In Western blot analysis, when compared with control, OVX significantly induced RhoA activation, as revealed by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing NO formation via L-NAME increased Y-27632 responsiveness in the OVX + T group; L-NAME had partial effects in controls while not modulating Y-27632 responsiveness in the OVX and OVX + E2 groups. Finally, stimulation of rvSMCs from control animals with sodium nitroprusside significantly increased RhoGDI protein expression, counteracted by ODQ and partially by KT5823 incubation; no effect was observed in rvSMCs from OVX rats. CLINICAL IMPLICATIONS: Androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring sexual intercourse. STRENGTHS AND LIMITATIONS: This study describes the role of androgens in maintaining vaginal well-being. The absence of a sham-operated animal group and the use of the only intact animal as control represented a limitation to the study.

Effects of Benzo[a]pyrene on Human Sperm Functions: An In Vitro Study.

Benzo(a)pyrene (BaP) is considered one of the most dangerous air pollutants for adverse health effects, including reproductive toxicity. It is found both in male and female reproductive fluids likely affecting spermatozoa after the selection process through cervical mucus, a process mimicked in vitro with the swim-up procedure. In vitro effects of BaP (1, 5, 10 µM) were evaluated both in unselected and swim-up selected spermatozoa after 3 and 24 h of incubation. BaP reduced total, progressive and hyperactivated motility and migration in a viscous medium both in swim-up selected and unselected spermatozoa. Viability was not significantly affected in swim-up selected but was reduced in unselected spermatozoa. In swim-up selected spermatozoa, increases in the percentage of spontaneous acrosome reaction and DNA fragmentation were observed after 24 h of incubation, whereas no differences between the control and BaP-treated samples were observed in caspase-3 and -7 activity, indicating no effects on apoptotic pathways. ROS species, evaluated by staining with CellROX® Orange and Dihydroethidium, did not differ in viable spermatozoa after BaP treatment. Conversely, the percentage of unviable ROS-positive spermatozoa increased. Our study suggests that BaP present in male and female genital fluids may heavily affect reproductive functions of human spermatozoa.

Acetylcholine modulates K+ and Na+ currents in human basal forebrain cholinergic neuroblasts through an autocrine/paracrine mechanism.

The Nucleus Basalis of Meynert (NBM) is the main source of cholinergic neurons in the basal forebrain to be crucially involved in cognitive functions and whose degeneration correlates with cognitive decline in major degenerative pathologies as Alzheimer's and Parkinson's diseases. However, knowledge concerning NBM neurons derived from human brain is very limited to date. We recently characterized a primary culture of proliferating neuroblasts isolated from the human fetal NBM (hfNBM) as immature cholinergic neurons expressing the machinery to synthetize and release acetylcholine. Here we studied in detail electrophysiological features and cholinergic effects in this cell culture by patch-clamp recordings. Our data demonstrate that atropine-blocked muscarinic receptor activation by acetylcholine or carbachol enhanced IK and reduced INa currents by stimulating Gi -coupled M2 or phospholipase C-coupled M3 receptors, respectively. Inhibition of acetylcholine esterase activity by neostigmine unveiled a spontaneous acetylcholine release from hfNBM neuroblasts that might account for an autocrine/paracrine signaling during human brain development. Present data provide the first description of cholinergic effects in human NBM neurons and point to a role of acetylcholine as an autocrine/paracrine modulator of voltage-dependent channels. Our research could be of relevance in understanding the mechanisms of cholinergic system development and functions in the human brain, either in health or disease.

Neuroprotective Effects of Testosterone in the Hypothalamus of an Animal Model of Metabolic Syndrome.

Metabolic syndrome (MetS) is known to be associated to inflammation and alteration in the hypothalamus, a brain region implicated in the control of several physiological functions, including energy homeostasis and reproduction. Previous studies demonstrated the beneficial effects of testosterone treatment (TTh) in counteracting some MetS symptoms in both animal models and clinical studies. This study investigated the effect of TTh (30 mg/kg/week for 12 weeks) on the hypothalamus in a high-fat diet (HFD)-induced animal model of MetS, utilizing quantitative RT-PCR and immunohistochemical analyses. The animal model recapitulates the human MetS features, including low testosterone/gonadotropin plasma levels. TTh significantly improved MetS-induced hypertension, visceral adipose tissue accumulation, and glucose homeostasis derangements. Within hypothalamus, TTh significantly counteracted HFD-induced inflammation, as detected in terms of expression of inflammatory markers and microglial activation. Moreover, TTh remarkably reverted the HFD-associated alterations in the expression of important regulators of energy status and reproduction, such as the melanocortin and the GnRH-controlling network. Our results suggest that TTh may exert neuroprotective effects on the HFD-related hypothalamic alterations, with positive outcomes on the circuits implicated in the control of energy metabolism and reproductive tasks, thus supporting a possible role of TTh in the clinical management of MetS.

Tumor Necrosis Factor α Influences Phenotypic Plasticity and Promotes Epigenetic Changes in Human Basal Forebrain Cholinergic Neuroblasts.

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and ß-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.

Physical activity counteracts metabolic syndrome-induced hypogonadotropic hypogonadism and erectile dysfunction in the rabbit.

Metabolic syndrome (MetS) clusters cardiovascular and metabolic risk factors along with hypogonadism and erectile dysfunction. Lifestyle modifications including physical exercise (PhyEx) are well-known treatments for this condition. In this study, we analyzed the effect of PhyEx on hypothalamic-pituitary-testis axis and erectile function by use of an animal MetS model, previously established in rabbits fed a high-fat diet (HFD). Rabbits fed a regular diet (RD) were used as controls. A subset of both groups was trained on a treadmill. HFD rabbits showed typical MetS features, including HG (reduced T and LH) and impairment of erectile function. PhyEx in HFD rabbits completely restored plasma T and LH and the penile alterations. At testicular and hypothalamic levels, an HFD-induced inflammatory status was accompanied by reduced T synthesis and gonadotropin-releasing hormone (GnRH) immunopositivity, respectively. In the testis, PhyEx normalized HFD-related macrophage infiltration and increased the expression of steroidogenic enzymes and T synthesis. In the hypothalamus, PhyEx normalized HFD-induced gene expression changes related to inflammation and glucose metabolism, restored GnRH expression, particularly doubling mRNA levels, and regulated expression of molecules related to GnRH release (kisspeptin, dynorphin). Concerning MetS components, PhyEx significantly reduced circulating cholesterol and visceral fat. In multivariate analyses, cholesterol levels resulted as the main factor associated with MetS-related alterations in penile, testicular, and hypothalamic districts. In conclusion, our results show that PhyEx may rescue erectile function, exert anti-inflammatory effects on hypothalamus and testis, and increase LH levels and T production, thus supporting a primary role for lifestyle modification to combat MetS-associated hypogonadism and erectile dysfunction.

An electrophysiological study on the effects of BDNF and FGF2 on voltage dependent Ca(2+) currents in developing human striatal primordium.

Over the past decades, studies in both Huntington's disease animal models and pilot clinical trials have demonstrated that replacement of degenerated striatum and repair of circuitries by grafting fetal striatal primordium is feasible, safe and may counteract disease progression. However, a better comprehension of striatal ontogenesis is required to assess the fetal graft regenerative potential. During neuronal development, neurotrophins exert pleiotropic actions in regulating cell fate and synaptic plasticity. In this regard, brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF2) are crucially implicated in the control of fate choice of striatal progenitor cells. In this study, we intended to refine the functional features of human striatal precursor (HSP) cells isolated from ganglionic eminence of 9-12week old human fetuses, by studying with electrophysiological methods the effect of BDNF and FGF2 on the membrane biophysical properties and the voltage-dependent Ca(2+) currents. These features are particularly relevant to evaluate neuronal cell functioning and can be considered reliable markers of the developmental phenotype of human striatal primordium. Our results have demonstrated that BDNF and FGF2 induced membrane hyperpolarization, increased the membrane capacitance and reduced the resting total and specific conductance values, suggesting a more efficient control of resting ionic fluxes. Moreover, the treatment with both neurotrophins enhanced N-type Ca(2+) current amplitude and reduced L- and T-type ones. Overall, our data indicate that BDNF and FGF2 may help HSP cells to attain a more functionally mature phenotype.

Differential Effects of Testosterone and Estradiol on Clitoral Function: An Experimental Study in Rats.

INTRODUCTION: Female sexual response is a complex phenomenon in which psychological, neurologic, and vascular mechanisms and hormonal factors interact. During the arousal phase, they cooperate to increase genital blood flow, thus inducing engorgement of the clitoris and lubrication of the vagina. Regulation of vascular and non-vascular smooth muscle tone is the crucial event in the erectile process. Preclinical studies have suggested that nitric oxide (NO) is the main vasodilator neurotransmitter modulating, through the second messenger cyclic guanosine monophosphate (cGMP), clitoral flow vessels. AIM: To investigate the effects of sexual steroid hormones on pro-erectile and relaxant (mediated by NO and cGMP) and anti-erectile and contractile (mediated by ras homolog gene family member A [RhoA] and Rho-associated protein kinase [ROCK]) mechanisms in the clitoris using a validated animal model of female ovariectomized Sprague-Dawley rats. METHODS: Subgroups of ovariectomized rats were treated with 17ß-estradiol, progesterone, testosterone, or testosterone and letrozole for 6 weeks. The experimental groups were compared with a control group of intact rats. MAIN OUTCOME MEASURES: Sex steroids plasma levels were assessed and in vitro contractility studies were carried out in order to investigate the effect of ovariectomy and in vivo treatments on clitoris smooth muscle activity. Smooth muscle cells (SMCs) from rat clitoral biopsies were isolated and characterized. RhoA activity was determined in SMCs cell cultures. RNA from tissues and cells was analyzed by quantitative real-time RT-PCR. RESULTS: Using real-time polymerase chain reaction, testosterone treatment upregulated the expression of NO-mediated pathway genes (endothelial and neuronal NO synthase, guanylate cyclase soluble subunit-α3, guanylate cyclase soluble subunit-ß3, cGMP-dependent protein kinase 1, and phosphodiesterase type 5). Conversely, estrogen replacement upregulated the expression of calcium-sensitizing RhoA-ROCK pathway genes. In vitro contractility studies were performed on phenylephrine pre-contracted clitoris strips. Ovariectomy resulted in a decreased responsiveness to Y-27632, a ROCK inhibitor, which was fully restored by 17ß-estradiol supplementation. To further examine the effect of 17ß-estradiol on the RhoA-ROCK pathway, smooth muscle cells were isolated from rat clitoris and their migration capacity was evaluated. CONCLUSION: Collectively, these data demonstrate that testosterone improves the relaxation of vascular smooth muscle cells through the NO-cGMP pathway, and that testosterone and 17ß-estradiol are necessary to maintain a functional contractile and relaxant machinery in the clitoris. This new concept might provide support for the concomitant use of estrogen and testosterone during the treatment of sexual arousal disorders related to hormonal imbalance or insufficiency.

Estrogen mediates metabolic syndrome-induced erectile dysfunction: a study in the rabbit.

INTRODUCTION: Estrogen receptor (ER) α is critical in mediating the harmful effects of hyperestrogenism in fetal or neonatal life on the developing penis. In contrast, little is known on the impact of an excess of estrogens on penile function in adulthood. AIM: To investigate the effect of estrogens on metabolic syndrome (MetS)-associated erectile dysfunction (ED). METHODS: We employed a recently established animal model of high fat diet (HFD)-induced MetS. Subgroups of MetS rabbits were dosed with either testosterone (T) or tamoxifen. We evaluated penile responsiveness to acetylcholine (Ach) as well as the expression of genes related to penile smooth muscle relaxation and contractility. MAIN OUTCOME MEASURE: Associations between MetS-induced penile alterations and sex steroids were investigated in an animal model of HFD-induced MetS. To understand the role of either androgen deficiency or estrogen excess on ED, we treated subgroups of MetS rabbits with either T or tamoxifen, a classical ER antagonist. RESULTS: Feeding an HFD-induced MetS was associated to elevated estradiol (E2) and low T levels. E2, but not T, was independently and negatively associated with genes able to affect penile erection. Smooth muscle-related markers decreased as a function of E2 and were positively associated with all the variables investigated. Increasing concentrations of circulating E2 were negatively associated with Ach-induced relaxation. In HFD rabbits, in vivo T dosing significantly improved MetS and completely normalized circulating E2. Conversely, in vivo tamoxifen dosing reduced visceral adiposity and partially restored T level. Ach-induced relaxation was severely impaired by HFD and significantly restored, up to the control level, by both tamoxifen and T dosing. In rabbit smooth muscle cells cultures 17ß-E2 (1 nM) significantly reduced the expression of α-smooth muscle actin, transgelin, and phosphodiesterase type 5. The effects of 17ß-E2 were completely reverted by tamoxifen (100 nM). CONCLUSIONS: This study demonstrates, for the first time, that HFD-induced ED is more associated with a high E2, rather than to a low T, milieu. HFD-induced ED is partially restored by in vivo treatment not only with T but also with the nonsteroidal ER antagonist, tamoxifen.

Tadalafil effect on metabolic syndrome-associated bladder alterations: an experimental study in a rabbit model.

INTRODUCTION: Metabolic syndrome (MetS) and lower urinary tract symptoms (LUTS) are often associated. Bladder detrusor hyper-contractility-a major LUTS determinant-is characterized by increased Ras homolog gene family, member A/Rho-associated protein kinase (RhoA/ROCK) signaling, which is often upregulated in MetS. AIM: This study investigated the effects of tadalafil dosing on RhoA/ROCK signaling in bladder, in a rabbit model of high-fat diet (HFD)-induced MetS. METHODS: Adult male rabbits feeding a HFD for 12 weeks. A subset of HFD animals was treated with tadalafil (2 mg/kg/day, 1 week: the last of the 12 weeks) and compared with HFD and control (feeding a regular diet) rabbits. MAIN OUTCOME MEASURES: In vitro contractility studies to evaluate the relaxant effect of the selective ROCK inhibitor, Y-27632, in carbachol precontracted bladder strips. Evaluation of RhoA activation by its membrane translocation. Immunohistochemistry for ROCK expression has been performed to evaluate ROCK expression in bladder from the different experimental groups. mRNA expression of inflammation, pro-fibrotic markers by quantitative RT-PCR has been performed to evaluate the effect of tadalafil on MetS-induced inflammation and fibrosis within the bladder. The in vitro effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cells was evaluated by using chemotaxis assay. RESULTS: Bladder strips from HFD rabbits showed hyper-responsiveness to Y-27632, indicating RhoA/ROCK overactivity in HFD bladder compared with matched controls. Accordingly, the fraction of activated (translocated to the membrane) RhoA as well as ROCK expression are increased in HFD bladder. Tadalafil dosing normalized HFD-induced bladder hypersensitivity to Y-27632, by reducing RhoA membrane translocation and ROCK overexpression. Tadalafil dosing reduced mRNA expression of inflammatory, pro-fibrotic, and hypoxia markers. A direct inhibitory effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cell was demonstrated by using chemotaxis assay. Pre-treatment with tadalafil inhibited both basal and PDGF-induced migration of bladder smooth muscle cells. CONCLUSIONS: Tadalafil dosing reduced RhoA/ROCK signaling and smooth muscle overactivity in an animal model of MetS-associated bladder alterations. Our findings suggest a novel mechanism of action of tadalafil in alleviating LUTS in MetS patients.

Metformin in vitro and in vivo increases adenosine signaling in rabbit corpora cavernosa.

INTRODUCTION: In subjects with erectile dysfunction responding poorly to sildenafil, metformin was reported to improve erections. AIMS: The aim of this study is to investigate metformin's mechanism of action on erectile function, particularly focusing on adenosine (ADO) and nitric oxide (NO) signaling in an animal model of high-fat diet (HFD)-induced metabolic syndrome. METHODS: In vitro contractility studies of penile strips. Penile expression of genes related to ADO or NO signaling was also evaluated. MAIN OUTCOME MEASURE: In vitro contractility studies were used to investigate the effect of in vivo and ex vivo metformin administration on ADO- or acetylcholine (Ach)-induced relaxation of penile strips from HFD as compared with animals fed a regular diet (RD). RESULTS: Expression of ADO receptor type 3 (A3 R), ADO deaminase (ADA), AMP deaminase type 1 (AMPD1), and 2 (AMPD2) was decreased in HFD as compared with RD. Accordingly, in HFD the ADO relaxant effect was potentiated as compared with RD (P < 0.02). In vivo metformin treatment in both RD and HFD significantly increased the ADO relaxing effect (P < 0.0001 and P < 0.01, respectively, vs. relative untreated groups) although to a different extent. In fact, the half-maximal inhibitory concentration (IC50 )/IC50 ratio in RD increased fourfold vs. HFD (RD IC50 ratio = 13.75 ± 2.96; HFD IC50 ratio = 2.85 ± 0.52). In corpora cavernosa (CC) from HFD, in vivo metformin (i) normalized A3 R, ADA, and AMPD1; (ii) further decreased AMPD2; (iii) increased dimethylarginine dimethylamino-hydrolase; and (iv) partially restored impaired Ach-induced relaxation. Ex vivo metformin time and dose dependently increased the relaxant effect of ADO in RD. The potentiating effect of metformin on ADO-induced relaxation was significantly reduced by preincubation with NO synthase inhibitor N(ω) -Nitro-L-arginine methyl ester hydrochloride (L-NAME). Interestingly, in vivo testosterone supplementation in HFD rabbits (i) increased penile expression of endothelial NO synthase and AMPD2 and (ii) restored metformin's potentiating effect on ADO-induced relaxation up to RD level. CONCLUSION: Metformin in vivo and ex vivo increases ADO signaling in CC, most probably interfering with NO formation and ADO breakdown.

Sciatic Nerve and Its Anatomical Variations: In-Depth Understanding Acquired During Dissection Classes.

Knowledge of anatomical variability is extremely important in order to better understand the etiology of pain, if present, or to avoid iatrogenic consequences. Sometimes the anatomical "anomalies" have the same anamnesis but different causes. For example, sciatic neuralgia may be caused by a herniated disc or it may have a different origin. The sciatic nerve (SN), also known as the ischial nerve, is the widest in the human body. This huge peripheral nerve originates from the roots of the lumbosacral plexus (L4-S3) and passes through the great sciatic foramen, under the piriformis muscle (PM). However, there is much variability in the pattern of SNs about the muscle, which has been known since the first half of the 20th century. In the present study, we describe six different case reports of anatomical variations of the SN and its interplay with the PM. The observations were made during dissection classes at the ICLO Teaching and Research Centre (Verona, Italy), on both male and female cadavers aged between 58 and 84 years. The SN was reported as a single and divided nerve into the tibial nerve (TN) and the common peroneal nerve (CPN), passing alone above, below, or between the PM. However, the two parts of the SN may also interact with the PM in different ways, adding to the anatomical variability. A thorough knowledge of the anatomical variations in any part of the human body is extremely important. The various techniques used, from imaging to autopsy or surgery, are also useful in the SN pathway. Thus, the anatomical features and the understanding of each variation are useful for a correct approach that can lead to an effective and correct treatment with a favorable outcome.

Mechanism of action of phosphodiesterase type 5 inhibition in metabolic syndrome-associated prostate alterations: an experimental study in the rabbit.

BACKGROUND: Phosphodiesterase type 5 (PDE5) inhibitors improve benign prostatic hyperplasia (BPH)-related lower urinary tract symptoms (LUTS), often associated with metabolic syndrome (MetS). This study investigated the effects of PDE5 inhibition in the prostate of rabbits fed a high fat diet (HFD) for 12 weeks. HFD-rabbits develop the most important features of human MetS (glucose intolerance, dyslipidemia, increased abdominal adiposity, and hypertension), along with hypogonadism and LUT abnormalities (prostate and bladder inflammation/tissue remodeling). METHODS: Gene expression was evaluated by quantitative RT-PCR. Prostate morphological changes and oxygenation were evaluated by immunohistochemistry. RESULTS: HFD prostates showed increased PDE5 expression, suggesting a peculiar sensitivity of prostate to the action of PDE5 inhibitors during MetS. Accordingly, prostate PDE5 mRNA was negatively associated to plasma testosterone/estradiol ratio, whose reduction characterizes MetS, and positively with the expression in prostate of several genes exploring pathogenetic processes for BPH/LUTS, such as inflammation, leukocyte infiltration, and fibrosis/myofibroblast activation. Most of these genes was up-regulated by HFD, and significantly reduced by PDE5 inhibition, through either chronic (12 weeks) or, at a lower extent, acute (1-week) tadalafil dosing. Tadalafil was also able to reduce blood pressure and visceral fat in HFD rabbits, without changing any other MetS parameter. Interestingly, 1-week tadalafil administration to HFD rabbits, significantly blunted prostate inflammation (increased CD45 immunopositivity), fibrosis (reduced muscle/fiber ratio) and hypo-oxygenation, thus suggesting a potential curative effect of PDE5 inhibition on MetS-related prostate alterations. CONCLUSIONS: Our data provide the experimental evidences to support the multiple potentiality of PDE5 inhibitors as a useful therapeutic tool in LUTS.

Fat boosts, while androgen receptor activation counteracts, BPH-associated prostate inflammation.

BACKGROUND: Metabolic syndrome (MetS) and benign prostate hyperplasia (BPH) are often comorbid. Chronic inflammation, a determinant pathogenic factor for BPH, is a putative link between the two conditions. METHODS: In a multi-center cohort of BPH patients (n = 244) who underwent prostatectomy, we evaluated whether MetS is associated with prostatic inflammation in BPH specimens. In addition, we investigated the in vitro inflammatory effects of metabolic insults on human prostatic myofibroblastic cells (hBPH). RESULTS: Inflammatory infiltrates score (IS) in prostatectomy specimens showed a step-wise association with the number of MetS factors present (P = 0.001). After adjusting for age, reduced HDL cholesterol, and elevated triglycerides were the only factors significantly associated with IS. Increased IS was also significantly associated with hypogonadism. In an age- and testosterone (T)-adjusted model, dyslipidemia was still associated with IS. To investigate whether metabolic factors could directly trigger prostate inflammation, we performed preliminary studies in myofibroblastic hBPH. Among the different factors, oxidized low-density lipoprotein (oxLDL) showed the highest secretion of IL-8 (>10-fold)-a surrogate marker of prostate inflammation--as well as IL-6, and bFGF. Co-treatment with DHT significantly inhibited oxLDL-induced secretion of IL-8, whilst an AR-antagonist, bicalutamide, reversed DHT effects. DHT suppresses oxLDL receptor (LOX-1) expression. CONCLUSIONS: Our data suggest that fats and insulin could have a detrimental effect on prostate health, boosting inflammation, a key pathogenic factor in BPH. Conversely, beneficial effects of DHT in counteracting lipid- and insulin-induced prostatic alterations, suggest that T-via its conversion into DHT-may have unexpected beneficial effects on prostate health.

PDE5 inhibitors blunt inflammation in human BPH: a potential mechanism of action for PDE5 inhibitors in LUTS.

BACKGROUND: Metabolic syndrome (MetS) and benign prostate hyperplasia (BPH)/low urinary tract symptoms (LUTS) are often comorbid. Chronic inflammation is one of the putative links between these diseases. Phosphodiesterase type 5 inhibitors (PDE5i) are recognized as an effective treatment of BPH-related LUTS. One proposed mechanism of action of PDE5 is the inhibition of intraprostatic inflammation. In this study we investigate whether PDE5i could blunt inflammation in the human prostate. METHODS: Evaluation of the effect of tadalafil and vardenafil on secretion of interleukin 8 (IL-8, a surrogate marker of prostate inflammation) by human myofibroblast prostatic cells (hBPH) exposed to different inflammatory stimuli. We preliminary evaluate histological features of prostatic inflammatory infiltrates in BPH patients enrolled in a randomized, double bind, placebo controlled study aimed at investigating the efficacy of vardenafil (10 mg/day, for 12 weeks) on BPH/LUTS. RESULTS: In vitro treatment with tadalafil or vardenafil on hBPH reduced IL-8 secretion induced by either TNFα or metabolic factors, including oxidized low-density lipoprotein, oxLDL, to the same extent as a PDE5-insensitive PKG agonist Sp-8-Br-PET-cGMP. These effects were reverted by the PKG inhibitor KT5823, suggesting a cGMP/PKG-dependency. Treatment with tadalafil or vardenafil significantly suppressed oxLDL receptor (LOX-1) expression. Histological evaluation of anti-CD45 staining (CD45 score) in prostatectomy specimens of BPH patients showed a positive association with MetS severity. Reduced HDL-cholesterol and elevated triglycerides were the only MetS factors significantly associated with CD45 score. In the MetS cohort there was a significant lower CD45 score in the vardenafil-arm versus the placebo-one.

Effects of benzo[a]pyrene on the reproductive axis: Impairment of kisspeptin signaling in human gonadotropin-releasing hormone primary neurons.

The neuroendocrine control of reproduction is strictly coordinated at the central level by the pulsatile release of gonadotropin-releasing hormone (GnRH) by the hypothalamic GnRH neurons. Alterations of the GnRH-network, especially during development, lead to long-term reproductive and systemic consequences, also causing infertility. Recent evidence shows that benzo[a]pyrene (BaP), a diffuse pollutant that can play a role as an endocrine disruptor, affects gonadal function and gamete maturation, whereas data demonstrating its impact at hypothalamic level are very scarce. This study investigated the effects of BaP (10 µM) in a primary cell culture isolated from the human fetal hypothalamus (hfHypo) and exhibiting a clear GnRH neuron phenotype. BaP significantly decreased gene and protein expression of both GnRH and kisspeptin receptor (KISS1R), the master regulator of GnRH neuron function. Moreover, BaP exposure increased phospho-ERK1/2 signaling, a well-known mechanism associated with KISS1R activation. Interestingly, BaP altered the electrophysiological membrane properties leading to a significant depolarizing effect and it also significantly increased GnRH release, with both effects being not affected by kisspeptin addition. In conclusion, our findings demonstrate that BaP may alter GnRH neuron phenotype and function, mainly interfering with KISS1R signaling and GnRH secretion and therefore with crucial mechanisms implicated in the central neuroendocrine control of reproduction.

Defective jagged-1 signaling affects GnRH development and contributes to congenital hypogonadotropic hypogonadism.

In vertebrate species, fertility is controlled by gonadotropin-releasing hormone (GnRH) neurons. GnRH cells arise outside the central nervous system, in the developing olfactory pit, and migrate along olfactory/vomeronasal/terminal nerve axons into the forebrain during embryonic development. Congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome are rare genetic disorders characterized by infertility, and they are associated with defects in GnRH neuron migration and/or altered GnRH secretion and signaling. Here, we documented the expression of the jagged-1/Notch signaling pathway in GnRH neurons and along the GnRH neuron migratory route both in zebrafish embryos and in human fetuses. Genetic knockdown of the zebrafish ortholog of JAG1 (jag1b) resulted in altered GnRH migration and olfactory axonal projections to the olfactory bulbs. Next-generation sequencing was performed in 467 CHH unrelated probands, leading to the identification of heterozygous rare variants in JAG1. Functional in vitro validation of JAG1 mutants revealed that 7 out of the 9 studied variants exhibited reduced protein levels and altered subcellular localization. Together our data provide compelling evidence that Jag1/Notch signaling plays a prominent role in the development of GnRH neurons, and we propose that JAG1 insufficiency may contribute to the pathogenesis of CHH in humans.

Long-COVID cognitive impairments and reproductive hormone deficits in men may stem from GnRH neuronal death.

BACKGROUND: We have recently demonstrated a causal link between loss of gonadotropin-releasing hormone (GnRH), the master molecule regulating reproduction, and cognitive deficits during pathological aging, including Down syndrome and Alzheimer's disease. Olfactory and cognitive alterations, which persist in some COVID-19 patients, and long-term hypotestosteronaemia in SARS-CoV-2-infected men are also reminiscent of the consequences of deficient GnRH, suggesting that GnRH system neuroinvasion could underlie certain post-COVID symptoms and thus lead to accelerated or exacerbated cognitive decline. METHODS: We explored the hormonal profile of COVID-19 patients and targets of SARS-CoV-2 infection in post-mortem patient brains and human fetal tissue. FINDINGS: We found that persistent hypotestosteronaemia in some men could indeed be of hypothalamic origin, favouring post-COVID cognitive or neurological symptoms, and that changes in testosterone levels and body weight over time were inversely correlated. Infection of olfactory sensory neurons and multifunctional hypothalamic glia called tanycytes highlighted at least two viable neuroinvasion routes. Furthermore, GnRH neurons themselves were dying in all patient brains studied, dramatically reducing GnRH expression. Human fetal olfactory and vomeronasal epithelia, from which GnRH neurons arise, and fetal GnRH neurons also appeared susceptible to infection. INTERPRETATION: Putative GnRH neuron and tanycyte dysfunction following SARS-CoV-2 neuroinvasion could be responsible for serious reproductive, metabolic, and mental health consequences in long-COVID and lead to an increased risk of neurodevelopmental and neurodegenerative pathologies over time in all age groups. FUNDING: European Research Council (ERC) grant agreements No 810331, No 725149, No 804236, the European Union Horizon 2020 research and innovation program No 847941, the Fondation pour la Recherche Médicale (FRM) and the Agence Nationale de la Recherche en Santé (ANRS) No ECTZ200878 Long Covid 2021 ANRS0167 SIGNAL, Agence Nationale de la recherche (ANR) grant agreements No ANR-19-CE16-0021-02, No ANR-11-LABEX-0009, No. ANR-10-LABEX-0046, No. ANR-16-IDEX-0004, Inserm Cross-Cutting Scientific Program HuDeCA, the CHU Lille Bonus H, the UK Medical Research Council (MRC) and National Institute of Health and care Research (NIHR).

Farnesoid X receptor activation improves erectile dysfunction in models of metabolic syndrome and diabetes.

The metabolic syndrome (MetS) is an insulin-resistant state characterized by a cluster of cardiovascular risk factors, including abdominal obesity, hyperglycemia, elevated blood pressure and combined dyslipidemia. In this review, we discuss the potential of farnesoid X receptor (FXR) agonists in the treatment of erectile dysfunction (ED), a multifactorial disorder often comorbid with MetS. FXR not only regulates lipid and glucose homeostasis but also influences endothelial function and atherosclerosis, suggesting a regulatory role for this hormone nuclear receptor in the cardiovascular complications associated with the MetS, including ED. MetS induces ED via several mechanisms, and in particular through endothelial dysfunction in penile vessels. In a high-fat diet rabbit model of MetS, a 3-month treatment with the potent and selective FXR agonist INT-747 restores endothelium-dependent relaxation in isolated cavernous tissue, normalizing responsiveness to acetylcholine and to electrical field stimulation. Accordingly, eNOS expression in the penis is greatly up-regulated by INT-747 treatment. Experiments in a rat model of chemically-induced type 1 diabetes further demonstrate that INT-747 treatment preserves erectile function induced by electrical stimulation of the cavernous nerve. These results add a new facet to the pleiotropic activities mediated by FXR, and reveal novel beneficial effects of FXR activation with potential clinical relevance. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

Testosterone/estradiol ratio regulates NO-induced bladder relaxation and responsiveness to PDE5 inhibitors.

INTRODUCTION: The efficacy of phosphodiesterase type 5 inhibitors (PDE5i) in treating lower urinary tract symptoms is supported by the extremely high expression and activity of PDE5 in male bladder. Although bladder function regulation is similar among genders, no data are available on PDE5 expression and activity in female bladder. AIM: To investigate sex differences in PDE5 expression and biological activity in female bladder, as opposed to the male counterpart. MAIN OUTCOME MEASURE: Gene and protein expression and enzymatic activity of PDE5. METHODS: We studied gene and protein expression, and enzymatic activity of PDE5 in bladder of male and female rats. A subgroup of female rats was ovariectomized and alternatively replaced with estradiol (E2), progesterone, and testosterone (T) alone or in combination with letrozole to completely abrogate T-induced E formation. As a readout of PDE5 activity, we studied vardenafil efficacy in potentiating sodium nitroprusside (SNP)-induced relaxation in bladder of the different experimental groups. RESULTS: SNP was three-log unit less potent in relaxing the male bladder than the female one. On the contrary, the PDE5-resistant cyclic guanosine monophosphate (cGMP) analog (Bromo-ß-phenyl-1, N(2) -ethenoguanosine-3', 5'-cyclic monophosphorothioate, Sp-isomer [SP-8-Br-PET-cGMPS]) was equipotent in relaxing male and female bladder. Vardenafil was more effective in potentiating SNP-induced bladder relaxation in male than in female. Accordingly, the cGMP-hydrolyzing activity of PDE5 was higher in male vs. female homogenates. In ovariectomized female rats, with or without sex-steroid replacement, vardenafil activity in potentiating SNP-induced bladder relaxation was associated with an increased T/E2 ratio. In particular, masculinization of ovariectomized rats--by the administration of T + letrozole--dramatically increased vardenafil capacity to potentiate SNP-induced relaxation. CONCLUSION: In this study, we demonstrated that PDE5 activity is more pronounced in male as compared with female bladder and that T/E ratio positively regulates responsiveness to PDE5i, thus suggesting that male bladder is a more suitable target for PDE5i than the female counterpart.