Search

Evidence has been accumulated indicating that GnRH-like peptides are present in a variety of extrabrain areas of mammalian and nonmammalian vertebrates. A pioneer study carried out in the frog, Rana esculenta, demonstrated that testicular GnRH induced spermatogonial proliferation. Recently, we have shown that in proliferating spermatogonia (SPG) of frogs, a change of localization of the oncoprotein Fos, from the cytoplasm to the nucleus, occurs. This leads to the hypothesis that one or more testicular GnRH peptides may regulate SPG proliferation through Fos family proteins. Therefore, in vivo experiments in intact R. esculenta and in vitro incubations of testis fragments have been carried out using GnRH agonist (GnRHa; buserelin) and GnRH antagonist (D-pGlu(1),D-Phe(2),D-Trp(3,6)-GnRH). Cytoplasmic and nuclear Fos-like protein localization has been found by Western blot analysis in testicular extracts. Immunocytochemistry confirmed that cytoplasmic immunostaining was restricted to SPG; change of localization into the nuclear compartment was observed after GnRHa treatment. Northern blot analysis showed that treatments of testis fragments with GnRHa did not modify testicular c-fos mRNA expression. On the contrary, a Fos-like protein of 52 kDa, while not affected in vivo, disappeared from testicular cytosolic extracts after in vitro treatment with GnRHa. Contemporaneously, a 55-kDa Fos-related signal appeared in nuclear extracts. The GnRH antagonist counteracted the effects of GnRHa. Furthermore, in vivo treatments showed that GnRHa acted negatively on a 43-kDa nuclear Fos-related signal and that gonadotropins caused the decrease of 52-kDa cytoplasmic signal. In conclusion, we show, to our knowledge for the first time, that Fos is regulated by GnRHa directly (not through the pituitary) at the testicular level. The main effect appears to be related to Fos translocation from cytoplasmic to nuclear compartments of SPG.

Ethane 1,2-dimethane sulphonate (EDS) is an alkylating agent, which has a selective cytotoxic effect on Leydig cells in some mammalian species. Similarly, in the frog, Rana esculenta, Leydig cells are destroyed after a single EDS injection and regenerate after 28 days. Regeneration of Leydig cells in frogs appears to be independent of the pituitary. The present experiments in R. esculenta were carried out: a) to investigate Leydig cell responsiveness to gonadotropin stimulation during 58 days after a single EDS injection; and b) to assess whether four consecutive EDS injections induce additional effects on the testicular cell population. Our results show that androgen stimulation after gonadotropin injections is restored after 44 days from a single EDS injection. Since the interstitial compartment appears to be normal at least 28 days after EDS treatment, it is likely that new Leydig cells lack gonadotropin receptors. With respect to multiple-EDS injections, Leydig cells completely disappear in several areas and the adjacent germinal compartment is disorganised. In some cases damaged germinal compartment is still surrounded by intact Leydig cells. Surprisingly, testicular and plasma androgens strongly increase in EDS-treated animals. Therefore, Sertoli cells may produce substances inhibiting androgen production in Leydig cells. J. Exp. Zool. 287:384-393, 2000.

Michel Rauch and François Ferrière : these authors contributed equally to this work; International audience; Testicular descent corresponds to migration of the testis from the abdominal cavity to the scrotum and is essential for proper functioning of the testis. Recent advances in the characterization of estrogen receptor (ESR) subtypes and isoforms in various tissues prompted us to study ESRs within the gubernaculum testis, a structure involved in testicular descent. In the rat gubernaculum, we searched for ESR alpha (Esr1) and beta (Esr2) and for the androgen receptor (Ar), androgens being known to regulate testicular descent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that Esr1, Esr2, and Ar mRNAs were all expressed in the gubernaculum. Using PEETA (Primer extension, Electrophoresis, Elution, Tailing, and Amplification), we established that all Esr1 leader exons, previously identified in other organs, such as the uterus and pituitary, were transcribed in the gubernaculum, with the major form being O/B. The RNA protection assays, RT-PCR, and Western blot experiments revealed that isoform-specific mRNA transcripts generated by alternative splicing of the C-leader sequence on coding exons 1 and 2 of the Esr1 gene gave the 46- and 66-kDa ESR1 proteins. The ESR1 and AR proteins were found to colocalize in the parenchymal cells of the gubernaculum early in development, whereas AR also was strongly expressed in the muscular cells, both during fetal and postnatal life. The ESR2 protein was weakly expressed, principally in the muscular cells, but only once testicular descent had occurred. The levels of the 46-kDa ESR1 variant (ER46) exceeded those of the 66-kDa ESR1 form (ER66) at periods when the gubernaculum developed. Conversely, the 66-kDa form appears to predominate clearly when the gubernaculum growth was low or completed. The possible role of estrogens on the modulation of the androgen-dependent growth of the gubernaculum and, more widely, on testicular descent is discussed.

CircRNA cargo in spermatozoa (SPZ) participates in setting cell quality, in terms of morphology and motility. Cannabinoid receptor CB1 activity is correlated with a proper spermatogenesis and epididymal sperm maturation. Despite CB1 promotes endogenous skill to circularize mRNAs in SPZ, few notions are reported regarding the functional link between endocannabinoids and spermatic circRNA cargo. In CB1 knock-out male mice, we performed a complete dataset of spermatic circRNA content by microarray strategy. Differentially expressed (DE)-circRNAs, as a function of genotype, were identified. Within DE-circRNAs, we focused the attention on circLIMA1, as putative actin-cytoskeleton architecture regulator. The validation of circLIMA1 dependent-competitive endogenous RNA (ceRNA) network (ceRNET) in in vitro cell line confirmed its activity in the regulation of the cytoskeletal actin. Interestingly, a dynamic actin regulation in SPZ nuclei was found during their epididymal maturation. In this scenario, we showed for the first time an intriguing sperm nuclear actin remodeling, regulated via a ceRNET-independent pathway, consisting in the nuclear shuttling of circLIMA1-QKI interactome and downstream in Gelsolin regulation. In particular, the increased levels of circLIMA1 in CB1 knock-out SPZ, associated with an inefficient depolymerization of nuclear actin, specifically illustrate how endocannabinoids, by regulating circRNA cargo, may contribute to sperm morpho-cellular maturation.

The role of circRNA in reproduction is under investigation. CircRNAs are expressed in human testis, spermatozoa (SPZ), and seminal plasma. Their involvement in embryo development has also been suggested. Asthenozoospermia, a common cause of male infertility, is characterized by reduced or absent sperm motility in fresh ejaculate. While abnormal mitochondrial function, altered sperm tail, and genomic causes have been deeply investigated, the epigenetic signature of asthenozoospermic derived SPZ still remains unexplored. CircRNAs may take part in the repertoire of differentially expressed molecules in infertile men. Considering this background, we carried out a circRNA microarray, identifying a total of 9,138 transcripts, 22% of them novel based and 83.5% with an exonic structure. Using KEGG analysis, we evaluated the circRNA contribution in pathways related to mitochondrial function and sperm motility. In order to discriminate circRNAs with a differential expression in SPZ with differential morphological parameters, we separated sperm cells by Percoll gradient and analyzed their differential circRNA payload. A bioinformatic approach was then utilized to build a circRNA/miRNA/mRNA network. With the aim to demonstrate a dynamic contribution of circRNAs to the sperm epigenetic signature, we verified their modulation as a consequence of an oral amino acid supplementation, efficacious in improving SPZ motility.

Human mesenchymal/stromal stem cells (hMSC) are the most promising cell source for adult cell therapies in regenerative medicine. Many clinical trials have reported the use of autologous transplantation of hMSCs in several disorders, but with limited results. To exert their potential, hMSCs could exhibit efficient homing and migration toward lesion sites among other effects, but the underlying process is not clear enough. To further increase the knowledge, we studied the co-regulation between hypoxia-regulated genes and miRNAs. To this end, we investigated the miRNA expression profile of healthy hMSCs in low oxygen/nutrient conditions to mimic ischemia and compared with cells of patients suffering from critical limb ischemia (CLI). miRNAs are small, highly conserved, non-coding RNAs, skilled in the control of the target&rsquo, s expression level in a fine-tuned way. After analyzing the miRNOme in CLI-derived hMSC cells and healthy controls, and intersecting the results with the mRNA expression dataset under hypoxic conditions, we identified two miRNAs potentially relevant to the disease: miR-29b as a pathological marker of the disease and miR-638 as a therapeutic target. This study yielded a deeper understanding of stem cell biology and ischemic disorders, opening new potential treatments in the future.

Rationale: The pathogenesis of bicuspid aortic valve (BAV)–associated aortopathy is poorly understood, and no prognostic biomarker is currently available. Objective: We aimed to identify putative circulating biomarkers pathogenetically and prognostically linked to bicuspid aortopathy. Methods and Results: By reverse transcription polymerase chain reaction, we evaluated gene expression variations (versus normal aorta) of transforming growth factor-β1 (TGF-β1), connective tissue growth factor, matrix metalloproteinase-2 (MMP-2), MMP-14, endoglin (ENG), and superoxide dismutase 3 in ascending aorta samples from 50 tricuspid and 70 patients with BAV undergoing surgery for aortic stenosis (aorta diameter ≤45 mm: BAV non-dil or >45 mm: BAV dil ). Expression changes of the TGF-β1 active dimer and ENG were analyzed also by Western blot in ascending aorta samples from other 10 tricuspid aortic valve, 10 BAV non-dil , and 10 BAV dil patients. The serum concentration of study targets was assessed through ELISA and the ratio of serum TGF-β1/ENG (T/E) was evaluated. All BAV non-dil patients underwent follow-up echocardiography to assess aortic growth rate. In BAV non-dil patients, TGF-β1 and MMP-2 gene expression increased significantly, whereas MMP-14 and ENG expression decreased versus controls. Expression changes were confirmed at protein level for TGF-β1 and ENG. TGF-β1 serum concentration significantly decreased in tricuspid aortic valve and BAV non-dil patients versus healthy subjects. ENG serum concentration decreased in all patients, more markedly in BAV dil . A significant increase of the T/E ratio versus healthy subjects was unique of patients with BAV. In BAV non-dil patients, a T/E ≥9 was independently associated in multivariable analysis with higher MMP-2 and lower superoxide dismutase 3 gene expression, independent of age and aortic diameter. A significant correlation was observed between baseline T/E ratio and aortic diameter growth rate in BAV non-dil patients ( r =0.66, P Conclusions: The novel evidence of a possible value of the T/E ratio as a biomarker of BAV aortopathy was presented: further validation studies are warranted.

Human mesenchymal stromal/stem cells (hMSCs) emerged as a promising therapeutic tool for ischemic disorders, due to their ability to regenerate damaged tissues, promote angiogenesis and reduce inflammation, leading to encouraging, but still limited results. The outcomes in clinical trials exploring hMSC therapy are influenced by low cell retention and survival in affected tissues, partially influenced by lesion’s microenvironment, where low oxygen conditions (i.e. hypoxia) and inflammation coexist. Hypoxia and inflammation are pathophysiological stresses, sharing common activators, such as hypoxia-inducible factors (HIFs) and NF-κB. HIF1α and HIF2α respond essentially to hypoxia, activating pathways involved in tissue repair. Little is known about the regulation of HIF3α. Here we investigated the role of HIF3α in vitro and in vivo. Human MSCs expressed HIF3α, differentially regulated by pro-inflammatory cytokines in an oxygen-independent manner, a novel and still uncharacterized mechanism, where NF-κB is critical for its expression. We investigated if epigenetic modifications are involved in HIF3α expression by methylation-specific PCR and histone modifications. Robust hypermethylation of histone H3 was observed across HIF3A locus driven by pro-inflammatory cytokines. Experiments in a murine model of arteriotomy highlighted the activation of Hif3α expression in infiltrated inflammatory cells, suggesting a new role for Hif3α in inflammation in vivo.

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 the maintaining of 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, the transport and the 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.

Endocannabinoids are natural lipids able to bind to cannabinoid and vanilloid receptors. Their biological actions at the central and peripheral level are under the tight control of the proteins responsible for their synthesis, transport and degradation. In the last few years, several reports have pointed out these lipid mediators as critical signals, together with sex hormones and cytokines, in various aspects of animal and human reproduction. The identification of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in reproductive cells and tissues of invertebrates, vertebrates and mammals highlights the key role played by these endogenous compounds along the evolutionary axis. Here, we review the main actions of endocannabinoids on female and male reproductive events, and discuss the interplay between them, steroid hormones and cytokines in regulating fertility. In addition, we discuss the involvement of endocannabinoid signalling in ensuring a correct chromatin remodeling, and hence a good DNA quality, in sperm cells.

Purpose: Restenosis is a complex and heterogeneous pathophysiological phenomenon occurring in patients submitted to revascularization procedures. Previous studies proved the antirestenotic properties of injected allogenic mesenchymal stromal cells (MSCs) in an experimental model of rat carotid (re)stenosis induced through arteriotomy. In this study we describe some of the effects subsequent to MSC treatment of rats submitted to carotid arteriotomy and possibly responsible for their antirestenotic effect. Methods: Rat MSCs were isolated from bone marrow, expanded in vitro and characterized. Subsequently, we evaluated the effects of MSC administration via tail vein at 3 and 7 days after carotid arteriotomy both in rat serum and in injured carotids, focusing on DNA oxidative damage (8-oxo-dG detection), cell proliferation index (BrdU incorporation assay), apoptotic index (TUNEL assay), the expression of inflammation- and proliferation-related genes (RT-PCR), the release of growth factors and of inflammation-related cytokines (antibody arrays and ELISA). Results: MSC administration induced a greater cell proliferation in carotids after arteriotomy, together with an increased level of VEGF in the serum and with the higher expression of VEGF mRNA in injured carotids. Serum analysis also revealed a decreased level of the pro-inflammatory cytokines CXCL1, CXCL5, L-Selectin, ICAM-1 and LIX, and of TIMP1 and SDF-1alpha in MSC-treated rats. The MSC immunomodulatory activity was confirmed by the decreased expression of TLR2 and TLR4 in injured carotids. Conclusions: MSCs play an immunomodulatory paracrine role when injected in rats submitted to carotid arteriotomy, accompanied by the release of VEGF, possibly contributing to the accelerated repair of the injured vascular wall.

Msj-1 gene encodes a DnaJ protein highly expressed in spermatids and spermatozoa of both rodents and amphibians, possibly involved in vesicle fusion and protein quality control by means of interaction with heat shock proteins. We isolated and characterized the entire murine msj -1 gene and searched for putative msj-1 -like genes into the human genome. Furthermore, ultrastructural localization of MSJ-1 was analyzed in mouse germ cells by immunogold electron microscopy. The analysis of murine msj-1 genomic sequence reveals that it is an intron less gene. Putative promoter region was predicted within the 600 bp upstream the transcription start site. In mouse, msj-1 maps on chromosome 1, into an intronic region of UDP glucuronosyl-transferase 1 family cluster. At ultrastructural level, MSJ-1 marks the developing acrosomic vesicle and the sperm centriolar region. A blast search against the human genome database revealed two closed regions ( Ha and Hb ) on human chromosome 2 having high nucleotide identity with murine msj-1 coding region. Similarly to mouse, in human both regions map into an intronic region of UDP glycosyl-transferase 1 family polypeptide A cluster ( ugt1a @). A significant ORF encoding a putative DnaJ protein of 145 aa was predicted from Ha . Finally, expression analysis, conducted by RT-PCR in human sperm cells, demonstrated that Ha mRNA is effectively present in humans; by Western blot, a specific MSJ-1 band of approximately 30 kDa was detected in human sperm. Taken together, these data suggest that msj-1 gene might be conserved among vertebrates and might exert fundamental functions in reproduction.

mUBPy (mouse ubiquitin specific processing protease) is a de-ubiquitinating enzyme expressed in mouse testis and brain. In testis, it interacts with the DnaJ protein MSJ-1 (mouse sperm cell specific DnaJ first homologue), a molecular chaperone expressed in spermatids and spermatozoa. Since MSJ-1 is conserved among vertebrates, to demonstrate an evolutionarily conserved function of UBPy/MSJ-1 system, we assayed mUBPy presence in the anuran amphibian, the frog, Rana esculenta, during the annual sexual cycle. By Western blot we have detected a specific signal of 126 kDa in testis and isolated spermatozoa. During the annual sexual cycle, the signal gradually increases as soon as spermatogenesis resumes after the winter stasis. Using immunocytochemistry, we have localized the protein in spermatids and spermatozoa. In conclusion, UBPy/MSJ-1 system is available in R. esculenta testis suggesting a conserved fundamental function in spermatogenesis and sperm formation.

Using an anti-Fos family member antiserum, we previously described, in the testis of Rana esculenta, the presence of a nuclear 43-kDa protein that we hypothesized to be Fra-1. Using an antiserum against Fra-1, we here report on Fra-1 expression, localization, and putative activity in the R. esculenta testis during the annual reproductive cycle. Western blot analysis confirms that the nuclear 43-kDa protein is Fra-1. Immunocytochemistry demonstrates Fra-1 in peritubular myoid cells (PMC), efferent ducts, and blood vessels. We present, for the first time for a vertebrate, experimental evidence that the expression of Fra-1 in PMC is related to its activity during sperm transport from the tubular compartment to the efferent ducts.

MSJ-1 is member of the DnaJ/heat shock protein (Hsp) 40 chaperone protein family. It is present in mouse testis and spinal cord. In particular, MSJ-1 is localized in post-meiotic cells and in motoneurones of the ventral horns. To assess whether the role of this protein is evolutionarily conserved, we have investigated if msj-1 gene is expressed in the frog, Rana esculenta. Using reverse transcription-polymerase chain reaction (RT-PCR), a msj-1-like transcript was detected in testis, brain, and spinal cord. Homology ranging from 42.3 to 46.0% was found as compared with the mammalian counterparts. Muscle did not show any signal. By Western blot analysis, a signal of the predicted size of 30 kDa was evidenced in testis, brain, and spinal cord but not in ovary, heart, liver, kidney, and muscle. MSJ-1 fluctuations in the testis reveal that it appeared in concomitance with post-meiotic events during the annual sexual cycle, as shown in a previous study. The protein is localized in spermatids and is still retained in mature spermatozoa, where it has perinuclear and centriolar localization. MSJ-1 levels did not change in brain and spinal cord. Furthermore, in the brain MSJ-1 was mainly present in diencephalon and mesencephalon, while in spinal cord MSJ-1 was localized into several motoneurones of the cervical and thoracic tract. A putative role in vesicle trafficking is briefly discussed. Mol. Reprod. Dev. 68: 149–158, 2004. © 2004 Wiley-Liss, Inc.

The expression of c-fos and c-jun mRNA has been examined in the testis of a seasonal breeder (the frog, Rana esculenta) during the annual reproductive cycle, using Northern blot analysis along with measurements of plasma levels of estradiol-17 beta and androgens (testosterone + 5 alpha-dihydrotestosterone). A c-fos-like transcript of 1.9 kb was revealed using a 1.1-kb v-fos probe, while three different transcripts of 3.7, 3.4, and 2.7 kb were seen using 1.0-kb human (h)-c-jun fragment. The proto-oncogene-like mRNAs appear during the period of the year associated with the new wave of spermatogenic activity. The levels of fos-like mRNA were highest after the estradiol-17 beta peak, while low levels were concomitant with high androgen concentrations. It is concluded that there is a close correlation between c-fos- and c-jun-like expression and testicular activity in R. esculenta.

Recently, we have investigated the endocannabinoid involvement in chromatin remodeling events occurring in male spermatids. Indeed, we have demonstrated that genetic inactivation of the cannabinoid receptor type 1 (Cnr1) negatively influences chromatin remodeling mechanisms, by reducing histone displacement and indices of sperm chromatin quality (chromatin condensation and DNA integrity). Conversely, Cnr1 knock-out (Cnr1(-/-)) male mice, treated with estrogens, replaced histones and rescued chromatin condensation as well as DNA integrity. In the present study, by exploiting Cnr1(+/+), Cnr(+/-) and Cnr1(-/-) epididymal sperm samples, we show that histone retention directly correlates with low values of sperm chromatin quality indices determining sperm nuclear size elongation. Moreover, we demonstrate that estrogens, by promoting histone displacement and chromatin condensation rescue, are able to efficiently reduce the greater nuclear length observed in Cnr1(-/-) sperm. As a consequence of our results, we suggest that nucleus length may be used as a morphological parameter useful to screen out spermatozoa with low chromatin quality.

In the frog, Rana esculenta, two molecular forms of GnRH, coeluting with chicken (c) GnRH-II and salmon (s) GnRH, have been detected using HPLC and radioimmunoassay. Mammalian (m) GnRH seems to be also present. In amphibians the role of cGnRH-II seems to be primarily the involvement in the regulation of neuroendocrine processes and, while the mGnRH has been postulated to act as a neurotransmitter and/or neuromodulator, the activity of sGnRH-like material has not been investigated. Therefore, we have treated the frogs with single or multiple injections of cGnRH-II or sGnRH (6 micrograms) or both peptides (6 micrograms of each) to detect differences in the response measured as testicular or plasma androgen (testosterone plus 5 alpha-dihydrotestosterone) concentration during the annual reproductive cycle. The basal profile of testicular and plasma androgen shows that the spring peak disappeared in control animals given multiple injections and kept in short-term captivity. We show in the treatment with cGnRH-II and/or sGnRH that the effects of the peptides depend on the season, the experimental design, and the tissue in which androgen levels were measured. In particular, both peptides strongly stimulate androgen production during the autumn-winter period, the time of the greater response to the GnRHs when basal levels of steroids are highest.

Many advances have been carried out on the estrogens, GnRH and endocannabinoid system that have impact in the reproductive field. Indeed, estrogens, the generally accepted female hormones, have performed an unsuspected role in male sexual functions thanks to studies on non-mammalian vertebrates. Similarly, these animal models have provided important contributions to the identification of several GnRH ligand and receptor variants and their possible involvement in sexual behavior and gonadal function regulation. Moreover, the use of non-mammalian animal models has contributed to a better comprehension about the endocannabinoid system action in several mammalian reproductive events. We wish to highlight here how non-mammalian vertebrate animal model research contributes to advancements with implications on human health as well as providing a phylogenetic perspective on the evolution of reproductive systems in vertebrates.

BACKGROUND: The goal of our study was to molecularly dissect mesothelioma tumour pathways by mean of microarray technologies in order to identify new tumour biomarkers that could be used as early diagnostic markers and possibly as specific molecular therapeutic targets. METHODOLOGY: We performed Affymetrix HGU133A plus 2.0 microarray analysis, containing probes for about 39,000 human transcripts, comparing 9 human pleural mesotheliomas with 4 normal pleural specimens. Stringent statistical feature selection detected a set of differentially expressed genes that have been further evaluated to identify potential biomarkers to be used in early diagnostics. Selected genes were confirmed by RT-PCR. As reported by other mesothelioma profiling studies, most of genes are involved in G2/M transition. Our list contains several genes previously described as prognostic classifier. Furthermore, we found novel genes, never associated before to mesotheliom that could be involved in tumour progression. Notable is the identification of MMP-14, a member of matrix metalloproteinase family. In a cohort of 70 mesothelioma patients, we found by a multivariate Cox regression analysis, that the only parameter influencing overall survival was expression of MMP14. The calculated relative risk of death in MM patients with low MMP14 expression was significantly lower than patients with high MMp14 expression (P = 0.002). CONCLUSIONS: Based on the results provided, this molecule could be viewed as a new and effective therapeutic target to test for the cure of mesothelioma. Background: The goal of our study was to molecularly dissect mesothelioma tumour pathways by mean of microarray technologies in order to identify new tumour biomarkers that could be used as early diagnostic markers and possibly as specific molecular therapeutic targets.Methodology: We performed Affymetrix HGU133A plus 2.0 microarray analysis, containing probes for about 39,000 human transcripts, comparing 9 human pleural mesotheliomas with 4 normal pleural specimens. Stringent statistical feature selection detected a set of differentially expressed genes that have been further evaluated to identify potential biomarkers to be used in early diagnostics. Selected genes were confirmed by RT-PCR. As reported by other mesothelioma profiling studies, most of genes are involved in G2/M transition. Our list contains several genes previously described as prognostic classifier. Furthermore, we found novel genes, never associated before to mesotheliom that could be involved in tumour progression. Notable is the identification of MMP-14, a member of matrix metalloproteinase family. In a cohort of 70 mesothelioma patients, we found by a multivariate Cox regression analysis, that the only parameter influencing overall survival was expression of MMP14. The calculated relative risk of death in MM patients with low MMP14 expression was significantly lower than patients with high MMp14 expression (P = 0.002).Conclusions: Based on the results provided, this molecule could be viewed as a new and effective therapeutic target to test for the cure of mesothelioma.

The effects of cannabinoids on human health have been known since the antiquities when the extract of the plant Cannabis sativa was used because of its psychoactivity. The scientific story of the cannabinoids started in the 1960s with the isolation and characterization of the active component of the plant. After the synthesis of cannabinoid analogues, the analysis of structure-effect relationships was implemented, and this had a similar effect to a positive "Pandora's box" opening. To date, numerous roles have been ascribed to the "endocannabinoid system." Here we describe its involvement in the control of male reproduction, taking into consideration possible evolutionary speculations. Indeed, the endocannabinoid system is a very ancient signaling system, being clearly present from the divergence of the protostomian/deuterostomian.

The morphofunctional relationship between the endocannabinoid system and GnRH activity in the regulation of reproduction has poorly been investigated in vertebrates. Due to the anatomical features of lower vertebrate brain, in the present paper, we chose the frog Rana esculenta (anuran amphibian) as a suitable model to better investigate such aspects of the reproductive physiology. By using double-labeling immunofluorescence aided with a laser-scanning confocal microscope, we found a subpopulation of the frog hypothalamic GnRH neurons endowed with CB1 cannabinoid receptors. By means of semiquantitative RT-PCR assay, we have shown that, during the annual sexual cycle, GnRH-I mRNA (formerly known as mammalian GnRH) and CB1 mRNA have opposite expression profiles in the brain. In particular, this occurs in telencephalon and diencephalon, the areas mainly involved in GnRH release and control of the reproduction. Furthermore, we found that the endocannabinoid anandamide is able to inhibit GnRH-I mRNA synthesis; buserelin (a GnRH agonist), in turn, inhibits the synthesis of GnRH-I mRNA and induces an increase of CB1 transcription. Our observations point out the occurrence of a morphofunctional anatomical basis to explain a reciprocal relationship between the endocannabinoid system and GnRH neuronal activity.

Endogenous and environmental estrogens have been proved to affect male reproduction in vertebrates. Both positive and negative effects in the regulation of the reproductive tract have been described. Since it is well known that amphibians represent a useful model to study several aspects concerning reproductive activity, we have taken advantage of the frog, Rana esculenta , to study the involvement of estrogens in sperm release. We show here that pituitary hormones increased the number of peritubular myoid cells (PMCs) expressing Fra-1 and induced testicular morphological changes related to sperm release. The estrogen antagonist ICI182-780 counteracted the hypophysis driven effects. In vivo and in vitro experiments demonstrated that 17β-Estradiol acted directly on the testis to switch-on Fra-1 in PMCs. Furthermore, impairment of estrogen activity significantly reduced sperm release mainly affecting the detachment of spermatozoa from Sertoli cells (spermiation). Therefore, estrogens can be considered a new entry in the list of substances involved in spermiation.

Prevailing studies emphasize on endocannabinoid activity in brain. However, sporadic evidences hint that endocannabinoid system controls male reproduction ranging from invertebrates to vertebrates. Although N-arachidonoylethanolamine is described in rat testis, its activity is still poorly known. Type-1 cannabinoid receptor and fatty acid amide hydrolase are particularly expressed in elongating spermatids and spermatozoa suggesting that endocannabinoids affect spermiogenesis and sperm physiology. Aim of this paper is to provide an analysis of the information available in vertebrates on male germ cell progression and sperm maturation mediated by the endocannabinoid system.

The molecular hypothesis of learning and memory processes is based on changes in synaptic weights in neural networks. Aim of this study was to map neural traces of exposure to a spatial novelty were mapped by (i) the transcription factors (TFs) c-fos , c-jun and jun-B using Northern blot and immunocytochemistry (ICC), (ii) RNA synthesis by 3 H-uridine autoradiography and RNA level, (iii) NADPH-diaphorase (NADPH-d) expression by histochemistry. Thus, adult male albino rats were exposed to a Lat-maze and sacrificed at different times. Non-exposed rats served as controls. The latter showed a low constitutive expression of TF, RNA synthesis and NADPH-d across the brain. Northern blots showed a three-fold increase in TFs in exposed versus non-exposed rats in the cerebral cortex. ICC showed in exposed rats several TFs positive cells in the granular and pyramidal layers of the hippocampus and later in all layers of the somatosensory cortex, in the granular layer of the cerebellar cortex. The TF-positivity was stronger in rats exposed for the first time, and was time and NMDA-dependent. Autoradiography for RNA synthesis showed positive cells in the ependyma, hippocampus and cerebellum 6 h after testing, and in the somatosensory cortex 24 h later. In addition, exposure to novelty induced NADPH-d in the dorsal hippocampus, the caudate-putamen, all the layers of the somatosensory cortex. and the cerebellum. The positivity was absent immediately after exposure, appeared within 2 h and disappeared 24 h later. A strong neuronal discharge by the convulsant pentylenetetrazol, strongly induced TFs but not din not affect NADPH-d 2 h later. Thus, data suggest that the processing of spatial and emotional components of experience activates neural networks across different organization levels of the CNS.

Activating mutations in v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS) have recently been identified as the molecular cause underlying Costello syndrome (CS). To further investigate the phenotypic spectrum associated with germline HRAS mutations and characterize their molecular diversity, subjects with a diagnosis of CS (N = 9), Noonan syndrome (NS; N = 36), cardiofaciocutaneous syndrome (CFCS; N = 4), or with a phenotype suggestive of these conditions but without a definitive diagnosis (N = 12) were screened for the entire coding sequence of the gene. A de novo heterozygous HRAS change was detected in all the subjects diagnosed with CS, while no lesion was observed with any of the other phenotypes. While eight cases shared the recurrent c.34G>A change, a novel c.436G>A transition was observed in one individual. The latter affected residue, p.Ala146, which contributes to guanosine triphosphate (GTP)/guanosine diphosphate (GDP) binding, defining a novel class of activating HRAS lesions that perturb development. Clinical characterization indicated that p.Gly12Ser was associated with a homogeneous phenotype. By analyzing the genomic region flanking the HRAS mutations, we traced the parental origin of lesions in nine informative families and demonstrated that de novo mutations were inherited from the father in all cases. We noted an advanced age at conception in unaffected fathers transmitting the mutation.

Endocannabinoids are endogenous ligands for plasma membrane receptors (CB1 and CB2), belonging to the superfamily of G-protein-coupled receptors. They mimic some of the effects played by delta9-tetrahydrocannabinol (THC), the active principle isolated from Cannabis sativa. N-arachidonoylethanolamine (anandamide, AEA) is the main endocannabinoid described to date in the testis and in human seminal plasma. However, the activity of AEA in controlling male reproduction is still poorly understood. In this study we report on physiological activity of endocannabinoids in the male reproductive tract. Using wild type (WT) and CB1 knock out mice (CB1KO) we show that endocannabinoids act in the epididymus. Here, through CB1, they inhibit sperm motility measured as the percentage of motile spermatozoa (SPZ). In particular, while in WT mice, as expected, the percentage of motile SPZ (measured in caput and cauda of epididymus) was signiWcantly lower in the caput as compared with the cauda, in CB1KO mice a strong increase of motile SPZ in the caput was measured.

Endogenous cannabinoids and type-1 cannabinoid receptor (CB1) are widely produced and distributed in the central nervous system (CNS) and peripheral nerves in mammals. In addition, the detection of endocannabinoids and corresponding receptors in non nervous peripheral tissues indicates an involvement of the system in the control of a wide range of physiological activities, including reproduction. Recently, the existence of CB1 was also observed in lower vertebrates and in urochordate suggesting that the endocannabinoid system is phylogenetically conserved. Using RT-PCR, CB1 mRNA expression profiles were characterized in a wide range of tissues of the anuran amphibian, the frog, Rana esculenta. Besides a strong expression in the CNS, CB1 was also present in testis, kidney, liver, ovary, muscle, heart, spleen, and pituitary. The CB1 expression pattern has been characterized in both testis and CNS during the annual sexual cycle. In testis, CB1 is poorly expressed during the winter stasis of the spermatogenesis rising during the breeding season and resumption period. An expression profile mismatching to that observed in testis was detected in whole-brain preparations during the sexual cycle; in particular in the diencephalon, the encephalic area mainly involved in the control of reproductive functions. Furthermore, fluctuations inside isolated encephalic areas and spinal cord were observed all over the reproductive cycle. In conclusion, CB1 receptor is expressed in R. esculenta CNS and testis. As far as the gonad it concerns, our results suggest the involvement of the endocannabinoids in the control of reproductive function. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc.

The presence of c-jun like mRNA was assessed in the brain of the frog, Rana esculenta, during the annual sexual cycle. In parallel, Jun protein and GnRH molecular form (mammalian and chicken II also indicated as GnRH1 and GnRH2, respectively) activity was studied in order to establish possible relationships. Northern blot analysis of total RNA reveals the presence of a 2.7 kb c-jun-like mRNA. Western blots, carried out on cytoplasmic and nuclear protein extracts, show the presence of Jun immunoreactive band of 39 kDa in brain and pituitary. Fluctuations of c-jun-like mRNA and Jun immunoreactive protein (cytoplasmic and nuclear) levels in brains during the year indicate relationships among transcription, translation, and nuclear activity. In particular, mRNA levels increase gradually from September until November when Jun protein concentration peaks in cytosolic extracts. Conversely, the nuclear protein reaches highest concentration in July when the cytosolic level shows low values. Immunocytochemical studies confirm the presence of Jun immunoreactivity in both cytoplasmic and nuclear compartments of several brain areas, including those primarily involved in gonadotropin discharge (e.g., anterior preoptic area and preoptic nucleus). GnRH molecular forms and Jun are colocalized in anterior preoptic area and preoptic nucleus. Moreover, during the period characterized by GnRH release, Jun levels strongly decrease in nuclei. Finally, we show that treatments with a GnRH analog (buserelin, Hoechst, Frankfurt) increase Jun levels in brain nuclear extracts.

The mechanisms underlying the complexity of spermatogenesis and spermiogenesis have deeply been studied in recent years. Transgenic animals, gene-targeting techniques, and lower vertebrate animal models have led to the discovery of some of the intratesticular signals involved in germ cell progression. This review wish to give the state of the art about it with particular emphasis on the comparative approach.

Testicular morphology of vertebrate testis indicates requirement of local control. In urodeles, the testis is organized in lobes of increasing maturity throughout the cephalocaudal axis. The anuran testis is organized in tubules. Spermatogenesis occurs in cysts composed by Sertoli cells enveloping germ cells at synchronous stages. Moreover, in numerous species germ cell progression lasts a year which defines the sexual cycle. Due to the above quoted features, research on factors regulating germ cell progression in amphibians may reach greater insight as compared with mammalian animal models. In particular, studies on endocrine and paracrine/autocrine factors involved in the regulation of germ cell functions reveal that fos activation and a J protein, previously specifically found in mouse testis, exert an important role in spermatogonial proliferation and maturation of post-meiotic stages, respectively.

The role of Fos proteins in the regulation of germ cell progression during spermatogenesis has been studied in the frog, Rana esculenta. A peculiarity of this animal model is the finding of Fos in cytoplasmic compartment of primary spermatogonia during the resting period of the annual reproductive cycle. Interestingly, Fos is localized in the nuclear compartment when spermatogenesis resumes. Using Western blot analysis, we show that a 52-kDa Fos protein occurs in testicular cytosolic preparations, whereas two different Fos signals of 43 and 68 kDa are typical of the nuclear compartment. The 68-kDa Fos immunoreactive protein increases in nuclear extracts in concomitance with spermatogonia (SPG) proliferation either during the annual sexual cycle or in experimental animal groups where SPG proliferation was induced by thermal stimulus (24 C). Indeed, an increase in proliferating cell nuclear antigen was detectable after thermal induction of mitotic activity. A decrease in the 52-kDa signal and a concomitant increase in the 68-kDa signal is observed in testes of 24 C treated groups. The use of alkaline phosphatase and alkaline phosphatase inhibitors indicates that the 68-kDa protein is a phosphorylated form. Estrogens, which are able to induce SPG proliferation, are responsible for the appearance of the 43-kDa Fos form in nuclear testicular extracts. In conclusion, our results show, for the first time in a vertebrate species, that storage in the cytoplasm, on the one hand, and appearance as well as phosphorylation of Fos proteins in the nucleus of germ cells, on the other hand, regulate spermatogenesis progression during the seasonal breeding. Moreover, the phosphorylated 68-kDa Fos form may be involved in mechanisms underlying SPG proliferation. (Endocrinology 143: 163–170, 2002)

C-fos activity was determined in the brain of the frog, Rana esculenta, during the annual sexual cycle. The localization of GnRH molecular forms (mammalian- and chicken-GnRHII) was also carried out to determine whether or not the proto-oncogene and the peptides showed a functional relationship. Northern blot analysis of total RNA revealed the presence of a single strong signal of c-fos like mRNA of 1.9 Kb during February and April. This was followed by expression of c-Fos protein (Fos) in several brain areas during March and July shown by immunocytochemistry. In particular, the olfactory region, the lateral and medial pallium, the nucleus lateralis septi, the ventral striatum, the caudal region of the anterior preoptic area, the suprachiasmatic nucleus, the ventral thalamus, tori semicircularis and ependymal layers of the tectum were immunostained. There was no overlap between Fos immunoreactive perikarya and GnRH immunoreactive perikarya (e.g. gonadotrophin-releasing hormone (GnRH) in the rostral part and Fos in the caudal region of the anterior preoptic area). Interestingly, a cytoplasmic localization of Fos was also observed by immunocytochemistry and gel retardation experiments supported this observation. Cytoplasmic extracts from September-October animals bound the AP1 oligonucleotide. The complex was not available in the nuclear extracts from the same preparation, suggesting that, besides Fos, Jun products were also present. Conversely, nuclear but not cytosolic binding was detected in the brain of animals collected in July. In conclusion, we show that Fos and GnRH activity does not correlate in the frog brain and, for the first time in a vertebrate species, we give evidence of a cytoplasmic AP1 complex in neuronal cells.

Proto-oncogenes are said to influence the regulation of cellular growth and differentiation. Myc, Fos, Jun, and Mos protein localization has been studied by immunocytochemistry in the testis of the frog, Rana esculenta, during the annual reproductive cycle. Oncoproteins have been localized in the primary and secondary (I and II) spermatogonia (SPG). Myc and Mos also appear in I and II spermatocytes (SPC) while Jun appears in II SPC. Myc, Fos, and Jun in SPG translocate in the nucleus during the periods of active spermatogenesis. Myc, Fos, and Jun are also localized in Sertoli cells. Fos is present in interstitial cells during the period characterized by the androgen peak which precedes the sharp increase of estradiol. It is suggested that proto-oncogene activity exerts a regulatory role in steroidogenesis and spermatogenesis.

Estradiol-17beta (E2) is suspected to exert a role in the regulation of testicular activity. Using a nonmammalian vertebrate model (the frog, Rana esculenta), we have investigated whether c-fos activity is detectable in the testis during the annual sexual cycle and whether E2 exerts a regulatory role on spermatogenesis through fos activity. FOS protein is available in testicular nuclear extracts (about 60 kDa) and, surprisingly, also in cytosolic extracts (about 60, 80, and 100 kDa). Estradiol induces primary spermatogonia (ISPG) proliferation [this effect is counteracted by antiestrogens (Tamoxifen and ICI 182-780)] and FOS appearance in testicular cytosolic extracts as well as c-fos transcription. Also, this effect is counteracted by ICI 182-780. Interestingly, the number of FOS immunopositive nuclei of ISPG strongly increases after E2 treatment, whereas a great increase of immunopositivity in the cytoplasm of ISPG is observed with the contemporaneous treatment with antiestrogens. In conclusion, our results demonstrate that E2 induces ISPG multiplication in the frog, R. esculenta, and, for the first time in a vertebrate species, that it triggers c-fos activity in the testis. Moreover, E2 may be involved in mechanisms related to FOS transport in the nucleus of ISPG to induce the mitotic activity.

Msj-1 (mouse sperm cell-specific DnaJ first homologue) is a gene specifically expressed in germ cells at haploid stages. The protein first appears in round spermatids, accumulates in the periacrosomal region of elongating spermatids, and is maintained in spermatozoa. The msj-1 expression pattern is consistent with a role for this DnaJ protein in the spermiogenesis process. In this study, we used two experimental models, the anuran amphibian Rana esculenta and the wobbler mutant mouse, to explore the role of MSJ-1 during spermatogenesis, with a focus on spermiogenesis. Mice homozygous for the recessive mutation wobbler (wr/wr), a mutation of unknown identity, produce sperm cells characterized by a missing acrosome. In Rana esculenta testis, detection of high levels of MSJ-1 protein coincided with the appearance of postmeiotic germ cells during the annual sexual cycle. Conversely, elimination of the meiotic and postmeiotic stages, through gonadotropin administration at low temperature, abolished the MSJ-1 immunoreactive signal. In 20-day-old mice, when postmeiotic germ cells appeared for the first time, MSJ-1 mRNA and protein were observed in +/+ testis but were barely detectable in wr/wr testis. In adult testis, reduced MSJ-1 protein levels were observed in both +/wr and wr/wr testis, as compared with +/+ controls. Similarly, numbers of spermatids that stained by immunofluorescence for MSJ-1 appeared to be progressively reduced in adult +/+, +/wr, and wr/wr mouse testes, respectively. Characterization of the endocrine status of wobbler testis revealed reduced transcript levels of estrogen receptor alpha and reduced intratesticular androgen levels. However, androgen treatment did not affect MSJ-1 protein levels in either frogs or mice. In conclusion, our data in Rana esculenta and the wobbler mouse demonstrate a tight correlation between MSJ-1 protein expression and postmeiotic stages. In particular, the findings in the wobbler testis suggest a role for this protein in acrosomogenesis.