The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo.

Introduction: Male reproduction is under the control of the hypothalamus-pituitary-gonadal (HPG) axis. The endocannabinoid system (ECS) and the kisspeptin system (KS) are two major signaling systems in the central and peripheral control of reproduction, but their possible interaction has been poorly investigated in mammals. This manuscript analyzes their possible reciprocal modulation in the control of the HPG axis. Materials and methods: Adolescent male rats were treated with kisspeptin-10 (Kp10) and endocannabinoid anandamide (AEA), the latter alone or in combination with the type 1 cannabinoid receptor (CB1) antagonist rimonabant (SR141716A). The hypothalamic KS system and GnRH expression, circulating sex steroids and kisspeptin (Kiss1) levels, and intratesticular KS and ECS were evaluated by immunohistochemical and molecular methods. Non-coding RNAs (i.e., miR145-5p, miR-132-3p, let7a-5p, let7b-5p) were also considered. Results: Circulating hormonal values were not significantly affected by Kp10 or AEA; in the hypothalamus, Kp10 significantly increased GnRH mRNA and aromatase Cyp19, Kiss1, and Kiss1 receptor (Kiss1R) proteins. By contrast, AEA treatment affected the hypothalamic KS at the protein levels, with opposite effects on the ligand and receptor, and SR141716A was capable of attenuating the AEA effects. Among the considered non-coding RNA, only the expression of miR145-5p was positively affected by AEA but not by Kp10 treatment. Localization of Kiss1+/Kiss1R+ neurons in the arcuate nucleus revealed an increase of Kiss1R-expressing neurons in Kp10- and AEA-treated animals associated with enlargement of the lateral ventricles in Kp10-treated animals. In the brain and testis, the selected non-coding RNA was differently modulated by Kp10 or AEA. Lastly, in the testis, AEA treatment affected the KS at the protein levels, whereas Kp10 affected the intragonadal levels of CB1 and FAAH, the main modulator of the AEA tone. Changes in pubertal transition-related miRNAs and the intratesticular distribution of Kiss1, Kiss1R, CB1, and CB2 following KP and AEA treatment corroborate the KS-ECS crosstalk also showing that the CB1 receptor is involved in this interplay. Conclusion: For the first time in mammals, we report the modulation of the KS in both the hypothalamus and testis by AEA and revealed the KP-dependent modulation of CB1 and FAAH in the testis. KP involvement in the progression of spermatogenesis is also suggested.

Central and Local Modulators of Reproduction and Fertility: An Update.

Infertility is currently one of the most important health troubles in industrialised countries after cardio-vascular diseases and cancer [...].

Differential Expression of Kisspeptin System and Kisspeptin Receptor Trafficking during Spermatozoa Transit in the Epididymis.

The hypothalamus-pituitary-testis axis controls the production of spermatozoa, and the kisspeptin system, comprising Kiss1 and Kiss1 receptor (Kiss1R), is the main central gatekeeper. The activity of the kisspeptin system also occurs in testis and spermatozoa, but currently the need of peripheral kisspeptin to produce gametes is not fully understood. Hence, we characterized kisspeptin system in rat spermatozoa and epididymis caput and cauda and analyzed the possible presence of Kiss1 in the epididymal fluid. The presence of Kiss1 and Kiss1R in spermatozoa collected from epididymis caput and cauda was evaluated by Western blot; significant high Kiss1 levels in the caput (p < 0.001 vs. cauda) and constant levels of Kiss1R proteins were observed. Immunofluorescence analysis revealed that the localization of Kiss1R in sperm head shifts from the posterior region in the epididymis caput to perforatorium in the epididymis cauda. In spermatozoa-free epididymis, Western blot revealed higher expression of Kiss1 and Kiss1R in caput (p < 0.05 vs. cauda). Moreover, immunohistochemistry revealed that Kiss1 and Kiss1R proteins were mainly localized in the secretory epithelial cell types and in contractile myoid cells, respectively. Finally, both dot blot and Elisa revealed the presence of Kiss1 in the epididymal fluid collected from epididymis cauda and caput, indicating that rat epididymis and spermatozoa possess a complete kisspeptin system. In conclusion, we reported for the first time in rodents Kiss1R trafficking in spermatozoa during the epididymis transit and Kiss1 measure in the epididymal fluid, thus suggesting a possible role for the system in spermatozoa maturation and storage within the epididymis.

Pleiotropic Outcomes of Glyphosate Exposure: From Organ Damage to Effects on Inflammation, Cancer, Reproduction and Development.

Glyphosate is widely used worldwide as a potent herbicide. Due to its ubiquitous use, it is detectable in air, water and foodstuffs and can accumulate in human biological fluids and tissues representing a severe human health risk. In plants, glyphosate acts as an inhibitor of the shikimate pathway, which is absent in vertebrates. Due to this, international scientific authorities have long-considered glyphosate as a compound that has no or weak toxicity in humans. However, increasing evidence has highlighted the toxicity of glyphosate and its formulations in animals and human cells and tissues. Thus, despite the extension of the authorization of the use of glyphosate in Europe until 2022, several countries have begun to take precautionary measures to reduce its diffusion. Glyphosate has been detected in urine, blood and maternal milk and has been found to induce the generation of reactive oxygen species (ROS) and several cytotoxic and genotoxic effects in vitro and in animal models directly or indirectly through its metabolite, aminomethylphosphonic acid (AMPA). This review aims to summarize the more relevant findings on the biological effects and underlying molecular mechanisms of glyphosate, with a particular focus on glyphosate's potential to induce inflammation, DNA damage and alterations in gene expression profiles as well as adverse effects on reproduction and development.

Kisspeptin Receptor on the Sperm Surface Reflects Epididymal Maturation in the Dog.

Alongside the well-known central modulatory role, the Kisspeptin system, comprising Kiss1, its cleavage products (Kisspeptins), and Kisspeptin receptor (Kiss1R), was found to regulate gonadal functions in vertebrates; however, its functional role in the male gamete and its localization during maturation have been poorly understood. The present study analyzed Kisspeptin system in dog testis and spermatozoa recovered from different segments of the epididymis, with focus on Kiss1R on sperm surface alongside the maturation during epididymal transit, demonstrated by modification in sperm kinetic, morphology, and protamination. The proteins Kiss1 and Kiss1R were detected in dog testis. The receptor Kiss1R only was detected in total protein extracts from epididymis spermatozoa, whereas dot blot revealed Kiss1 immunoreactivity in the epidydimal fluid. An increase of the Kiss1R protein on sperm surface along the length of the epididymis, with spermatozoa in the tail showing plasma membrane integrity and Kiss1R protein (p < 0.05 vs. epididymis head and body) was observed by flow cytometry and further confirmed by epifluorescence microscopy and Western blot carried on sperm membrane preparations. In parallel, during the transit in the epididymis spermatozoa significantly modified their ability to move and the pattern of motility; a progressive increase in protaminization also occurred. In conclusion, Kisspeptin system was detected in dog testis and spermatozoa. Kiss1R trafficking toward plasma membrane along the length of the epididymis and Kiss1 in epididymal fluid suggested a new functional role of the Kisspeptin system in sperm maturation and storage.

Impact of Polyphenolic-Food on Longevity: An Elixir of Life. An Overview.

Aging and, particularly, the onset of age-related diseases are associated with tissue dysfunction and macromolecular damage, some of which can be attributed to accumulation of oxidative damage. Recently, growing interest has emerged on the beneficial effects of plant-based diets for the prevention of chronic diseases including obesity, diabetes, and cardiovascular disease. Several studies collectively suggests that the intake of polyphenols and their major food sources may exert beneficial effects on improving insulin resistance and related diabetes risk factors, such as inflammation and oxidative stress. They are the most abundant antioxidants in the diet, and their intake has been associated with a reduced aging in humans. Polyphenolic intake has been shown to be effective at ameliorating several age-related phenotypes, including oxidative stress, inflammation, impaired proteostasis, and cellular senescence, both in vitro and in vivo. In this paper, effects of these phytochemicals (either pure forms or polyphenolic-food) are reviewed and summarized according to affected cellular signaling pathways. Finally, the effectiveness of the anti-aging preventive action of nutritional interventions based on diets rich in polyphenolic food, such as the diets of the Blue zones, are discussed.

Sirt1 Activity in the Brain: Simultaneous Effects on Energy Homeostasis and Reproduction.

Diet deeply impacts brain functions like synaptic plasticity and cognitive processes, neuroendocrine functions, reproduction and behaviour, with detrimental or protective effects on neuronal physiology and therefore consequences for health. In this respect, the activity of metabolic sensors within the brain is critical for the maintenance of health status and represents a possible therapeutic target for some diseases. This review summarizes the main activity of Sirtuin1 (Sirt1), a metabolic sensor within the brain with a focus on the link between the central control of energy homeostasis and reproduction. The possible modulation of Sirt1 by natural phytochemical compounds like polyphenols is also discussed.

The Complex Interplay between Endocannabinoid System and the Estrogen System in Central Nervous System and Periphery.

The endocannabinoid system (ECS) is a lipid cell signaling system involved in the physiology and homeostasis of the brain and peripheral tissues. Synaptic plasticity, neuroendocrine functions, reproduction, and immune response among others all require the activity of functional ECS, with the onset of disease in case of ECS impairment. Estrogens, classically considered as female steroid hormones, regulate growth, differentiation, and many other functions in a broad range of target tissues and both sexes through the activation of nuclear and membrane estrogen receptors (ERs), which leads to genomic and non-genomic cell responses. Since ECS function overlaps or integrates with many other cell signaling systems, this review aims at updating the knowledge about the possible crosstalk between ECS and estrogen system (ES) at both central and peripheral level, with focuses on the central nervous system, reproduction, and cancer.

ω-3 and ω-6 Polyunsaturated Fatty Acids, Obesity and Cancer.

Recently, nutraceutical bioactive compounds in foods have been discovered for their potential health benefits regarding the prevention of chronic disorders, such as cancer, and inflammatory, cardiovascular, and metabolic diseases. Dietary omega-3 polyunsaturated fatty acids (ω-3PUFAs), including alpha-linolenic acid, docosapentaenoic acid, and eicosapentaenoic acid, are mostly attractive. They are available for the customers worldwide from commonly used foods and/or as components of commercial food supplements. The anti-inflammatory and hypotriglyceridemic effects of these fatty acids are well known, whereas pro-inflammatory properties have been recognized in their dietary counterparts, the ω-6PUFAs. Both ω-3 and ω-6PUFAs contribute to the production of lipid mediators such as endocannabinoids that are notably involved in control of food intake, energy sensing, and food-related disorders. In this review, we present ω-3 and ω-6PUFAs and their derivatives, endocannabinoids; discuss the anti-obesity effects of ω-3PUFAs; their roles in inflammation and colorectal cancer development; and how their action can be co-preventative and co-therapeutic.

Kisspeptins, new local modulators of male reproduction: A comparative overview.

Spermatogenesis is a complex process that leads to the production of male gametes within the testis through the coordination of mitotic, meiotic and differentiation events, under a deep control of endocrine, paracrine and autocrine modulators along the Hypothalamus-pituitary-gonad (HPG) axis. The kisspeptin system plays a fundamental role along the HPG axis as it is the main positive modulator upstream of the hypothalamic neurons that secrete the Gonadotropin Releasing Hormone (GnRH), the decapeptide that supports pituitary gonadotropins and the production of gonadal sex steroid. Currently, kisspeptins and their receptor, KISS1R, have a recognized activity in the central control of puberty onset, sex maturation, reproduction and sex-steroid feedback mechanisms in both animal models and human. However, kisspeptin signaling has been widely reported in peripheral tissues, particularly in the testis of mammalian and non-mammalian vertebrates, with functions related to Leydig cells physiology and steroid biosynthesis, spermatogenesis progression and spermatozoa functions, but its mandatory role within the testis is still a matter of discussion. This review provides a summary of the main intratesticular effects of kisspeptin in vertebrates, via a comparative approach. Particular emphasis was devoted to data from the anuran amphibian Pelophylax esculentus, the first animal model in which the direct intratesticular activity of kisspeptin was reported.

Bisphenol A induces DNA damage in cells exerting immune surveillance functions at peripheral and central level.

Bisphenol A (BPA) is a synthetic xenoestrogen diffused worldwide. Humans are chronically exposed to low doses of BPA from food and drinks, thus BPA accumulates in tissues posing human health risk. In this study, we investigated the effects of BPA on peripheral blood mononuclear cells (PBMC) from human healthy donors, and in glia and microglia of rat offspring at postnatal day 17 (17PND) from pregnant females who received BPA soon after coupling and during lactation and weaning. Results indicated that BPA affected Phytoemagglutinin (PHA) stimulated PBMC proliferation causing an S-phase cell cycle accumulation at nanomolar concentrations while BPA was almost ineffective in resting PBMC. Furthermore, BPA induced chromosome aberrations and the appearance of shattered cells characterized by high number of fragmented and pulverized chromosomes, suggesting that the compound could cause a massive genomic rearrangement by inducing catastrophic events. The BPA-induced DNA damage was observed mainly in TCD4+ and TCD8+ subsets of T lymphocytes and was mediated by the increase of ERK1/2 phosphorylation, p21/Waf1 and PARP1 protein expression. Intriguingly, we observed for the first time that BPA-induced effects were associated to a sex specific modulation of ERα and ERß in human PBMC. Immunofluorescence analysis of rat hippocampus corroborated in vitro findings showing that BPA induced É£H2AX phosphorylation in microglia and astrocytosis by decreasing ERα expression within the dentate gyrus. Overall these results suggest that BPA can alter immune surveillance functions at both peripheral and central level with a potential risk for cancer, neuroinflammation and neurodegeneration.

Minireview: The Epigenetic Modulation of KISS1 in Reproduction and Cancer.

Epigenetics describes how both lifestyle and environment may affect human health through the modulation of genome functions and without any change to the DNA nucleotide sequence. The discovery of several epigenetic mechanisms and the possibility to deliver epigenetic marks in cells, gametes, and biological fluids has opened up new perspectives in the prevention, diagnosis, and treatment of human diseases. In this respect, the depth of knowledge of epigenetic mechanisms is fundamental to preserving health status and to developing targeted interventions. In this minireview, we summarize the epigenetic modulation of the KISS1 gene in order to provide an example of epigenetic regulation in health and disease.

Antitumor efficacy of Kisspeptin in human malignant mesothelioma cells.

PURPOSE: Kisspeptin signaling, via its receptors GPR54, could be an essential players in the inhibition of mesothelioma progression, invasion and metastasis formation. The loss of KiSS1 by tumor cells has been associated with a metastatic phenotype but the mechanistic insights of this process are still unknown. EXPERIMENTAL DESIGN: The blockade of the metastatic process at early stage is a hot topic in cancer research. We studied the role of KiSS1 on proliferation, invasiveness, migration abilities of mesothelioma cell lines focusing on the effect on epithelial-to-mesenchymal transition (EMT). RESULTS: Treatment with the KiSS1 peptide or with a synthesis peptide with longer half-life, the FTM080, significantly inhibited cell proliferation, migration and invasion of mesothelioma cell lines; the same treatment reduced the activity of MMP-2 and MMP-9 determining consequently a marked reduction in the invasiveness of primary tumors and metastases. Thespecificexpression of EMT markers, as E-caderin, Vimentin, Slug and Snail, suggested the inhibition of EMT after treatment with KiSS1 as well as the preservation of epithelial components. CONCLUSION: Our results support anti-proliferative effect of KiSS1 in cancer cells and suggest that targeting the KiSS1/GPR54 system may represent a novel therapeutic approach for mesothelioma.

Chronic exposure to low dose of bisphenol A impacts on the first round of spermatogenesis via SIRT1 modulation.

Spermatogenesis depends on endocrine, autocrine and paracrine communications along the hypothalamus-pituitary-gonad axis. Bisphenol A (BPA), an estrogen-mimic endocrine disrupting chemical, is an environmental contaminant used to manufacture polycarbonate plastics and epoxy resins with toxic effects for male reproduction. Here we investigated whether the chronic exposure to low BPA doses affects spermatogenesis through the modulation of SIRT1, a NAD+-dependent deacetylase involved in the progression of spermatogenesis, with outcomes on apoptosis, oxidative stress, metabolism and energy homeostasis. BPA exposure via placenta first, and lactation and drinking water later, affected the body weight gain in male offspring at 45 postnatal days and the first round of spermatogenesis, with impairment of blood testis barrier, reactive oxygen species production, DNA damage and decreased expression of SIRT1. The analysis of SIRT1 downstream molecular pathways revealed the increase of acetyl-p53Lys370, γH2AX foci, the decrease of oxidative stress defenses and the higher apoptotic rate in the testis of treated animals, with partial rescue at sex maturation. In conclusion, SIRT1 pathways disruption after BPA exposure can have serious consequences on the first round of spermatogenesis.

MicroRNAs, Cancer and Diet: Facts and New Exciting Perspectives.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs able to regulate gene expression at multiple levels. They are detected in tissues, blood, and other body fluids with high stability and have a recognized role in maintaining of tissue homeostasis. Aberrant expression profile of miRNAs has been observed in several diseases, primarily cancer. As a consequence, the analysis of miRNA signature has recognized diagnostic and prognostic role in human diseases, and the development of miRNA-based therapies is currently under investigation. Recently, emerging but controversial data have revealed the possibility that diet-derived miRNAs might be transferred from food in living organisms to regulate gene expression. Thus, exogenous dietderived miRNAs might substantially contribute to the pool of circulating miRNAs to preserve tissue homeostasis and health status in recipient's organisms, opening new perspectives for diet in heath and disease. OBJECTIVE: This brief review aims at summarizing data concerning the recognized role of miRNAs as biomarkers, drugs and therapeutic targets in cancer and the detection and the activity of diet-derived miRNAs in both physiological and pathological conditions. CONCLUSION: MiRNAs have emerged as crucial molecules in anticancer therapies and diet-derived miRNAs might contribute to the pool of circulating miRNAs to preserve, maintain or restore health.

Kisspeptin regulates steroidogenesis and spermiation in anuran amphibian.

Kisspeptin (Kp) system has a recognized role in the control of gonadotropic axis, at multiple levels. Recently, a major focus of research has been to assess any direct activity of this system on testis physiology. Using the amphibian anuran, Pelophylax esculentus, as animal model, we demonstrate - for the first time in non-mammalian vertebrate - that testis expresses both Kiss-1 and Gpr54 proteins during the annual sexual cycle and that ex vivo 17B-estradiol (E2, 10-6 M) increases both proteins over control group. Since the interstitium is the main site of localization of both ligand and receptor, its possible involvement in the regulation of steroidogenesis has been evaluated by ex vivo treatment of testis pieces with increasing doses of Kp-10 (10-9-10-6 M). Treatments have been carried out in February - when a new wave of spermatogenesis occurs - and affect the expression of key enzymes of steroidogenesis inducing opposite effects on testosterone and estradiol intratesticular levels. Morphological analysis of Kp-treated testes reveals higher number of tubules with spermatozoa detached from Sertoli cells than control group and the expression of connexin 43, the main junctional protein in testis, is deeply affected by the treatment. In spite of the effects on spermatozoa observed ex vivo, in vivo administration of Kp-10 has been unable to induce sperm release in cloacal fluid. In conclusion, we demonstrate Kp-10 effects on steroidogenesis with possible involvement in the balance between testosterone and estradiol levels, and report new Kp-10 activities on spermatozoa-Sertoli cell interaction.

Kisspeptins, Estrogens and Male Fertility.

BACKGROUND: The control of male fertility requires accurate endocrine, paracrine and autocrine communications along the hypothalamus-pituitary-gonad (HPG) axis. In this respect, the possible interplay between upcoming/classical modulators of reproductive functions deserves attention in that may be a successful tool for the future exploitation of new potential therapeutic targets in the treatment of fertility disorders. METHODS: In this review we will discuss upcoming data concerning the role of kisspeptins, the products of the Kiss1 gene, and estrogens - classically considered as female hormones - as well as their possible interplay in testis. RESULTS: Kisspeptins, via the activation of kisspeptin receptor Gpr54 represent the main gatekeeper of the hypothalamic Gonadotropin Releasing Hormone (GnRH) centrally modulating the onset and maintaining reproductive functions. As a consequence, the loss of kisspeptin signalling causes hypogonadotrophic hypogonadism in humans and animal models. In spite of the well recognized functions at hypothalamic levels, recent data strongly support direct production and activity of kisspeptin in testis and its involvement in the control of Leydig cells, germ cells progression and sperm functions. Similarly, estrogens exhibit high impact on proliferative/apoptotic/differentiative events in testis, thus resulting as local key modulators for the production - but also for the release, transport and maturation - of high quality spermatozoa. CONCLUSION: This review summarizes the upcoming data from experimental models and humans concerning the testicular activity of kisspeptins and estrogens to preserve male fertility. Mutual enhancement of kisspeptin and estradiol signalling for the progression of spermatogenesis has also been discussed.

Anandamide acts via kisspeptin in the regulation of testicular activity of the frog, Pelophylax esculentus.

In the frog Pelophylax esculentus, the endocannabinoid anandamide (AEA) modulates Gonadotropin Releasing Hormone (GnRH) system in vitro and down-regulates steroidogenic enzymes in vivo. Thus, male frogs were injected with AEA ± SR141716A, a cannabinoid receptor 1 (CB1) antagonist, to evaluate possible effects on GnRH and Kiss1/Gpr54 systems, gonadotropin receptors and steroid levels. In frog diencephalons, AEA negatively affected both GnRH and Kiss1/Gpr54 systems. In testis, AEA induced the expression of gonadotropin receptors, cb1, gnrh2 and gnrhr3 meanwhile reducing gnrhr2 mRNA and Kiss1/Gpr54 proteins. Furthermore, aromatase (Cyp19) expression increased in parallel to testosterone decrease and estradiol increase. In vitro treatment of testis with AEA revealed direct effects on Cyp19 and induced the expression of the AEA-degrading enzyme Faah. Lastly, AEA effects on Faah were counteracted by the antiestrogen ICI182780, indicating estradiol mediated effect. In conclusion, for the first time we show in a vertebrate that AEA regulates testicular activity through kisspeptin system.

Kisspeptin drives germ cell progression in the anuran amphibian Pelophylax esculentus: a study carried out in ex vivo testes.

Kisspeptin, via Gpr54 receptor, regulates puberty onset in most vertebrates. Thus, the direct involvement of kisspeptin activity in testis physiology was investigated in the anuran amphibian, Pelophylax esculentus. In this vertebrate gpr54 mRNA has been localized in both interstitial compartment and spermatogonia (SPG), whereas SPG proliferation requires the cooperation between estradiol and testicular Gonadotropin releasing hormone (Gnrh). In the pre-reproductive period, dose response curve to assess the effects of Kisspeptin-10 (Kp-10) was carried out in vitro (dose range: 10(-9)-10(-6)M; incubation times: 1 and 4h); proliferative activity and germ cell progression were evaluated by expression analysis of proliferating cell nuclear antigen (pcna), estrogen receptor beta (erß), Gnrh system (gnrh1, gnrh2, gnrhr1, r2, r3) and by the count of empty, mitotic and meiotic tubules. All selected markers were up regulated at 4h Kp-10 incubation. Histological analysis also proved the increase of mitotic activity and the progression of spermatogenesis. Besides Kp-10 modulation of testicular Gnrh system, in vitro treatment with 17ß-estradiol (10(-6)M) ± the antagonist ICI182-780 (10(-5)M) revealed gnrh2 and gnrhr3 estrogen dependent expression. In the reproductive period, testes were incubated for 1 and 4h with Kp-10 (10(-7)M) or Kp-10 (10(-7)M)+kisspeptin antagonist [Kp-234 (10(-6)M)]. Results obtained in the pre-reproductive period were confirmed and Kp-234 completely counteracted Kp-10 effects. In conclusion, Kp-10 modulated the expression of pcna, erß, gnrhs and gnrhrs, inducing the progression of the spermatogenesis.