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Human mesenchymal stem cells (hMSCs) are attractive for clinical and experimental purposes due to their capability of self-renewal and of differentiating into several cell types. Autologous hMSCs transplantation has been proven to induce therapeutic angiogenesis in ischemic disorders. However, the molecular mechanisms underlying these effects remain unclear. A recent report has connected MSCs multipotency to sirtuin families, showing that SIRT1 can regulate MSCs function. Furthermore, SIRT1 is a critical modulator of endothelial angiogenic functions. Here, we described the generation of an immortalized human mesenchymal bone marrow-derived cell line and we investigated the angiogenic phenotype of our cellular model by inhibiting SIRT1 by both the genetic and pharmacological level. We first assessed the expression of SIRT1 in hMSCs under basal and hypoxic conditions at both RNA and protein level. Inhibition of SIRT1 by sirtinol, a cell-permeable inhibitor, or by specific sh-RNA resulted in an increase of premature-senescence phenotype, a reduction of proliferation rate with increased apoptosis. Furthermore, we observed a consistent reduction of tubule-like formation and migration and we found that SIRT1 inhibition reduced the hypoxia induced accumulation of HIF-1α protein and its transcriptional activity in hMSCs. Our findings identify SIRT1 as regulator of hypoxia-induced response in hMSCs and may contribute to the development of new therapeutic strategies to improve regenerative properties of mesenchymal stem cells in ischemic disorders through SIRT1 modulation.

Kisspeptins are involved in the regulation of hypothalamic-pituitary-gonadal axis, Leydig cell functions, and testosterone secretion, acting as endogenous ligands of the KISS1 receptor. ANKRD31 protein participates in male fertility, regulating meiotic progression, and epididymal sperm maturation. Here, we show that in Leydig cells, KISS1 receptor and ANKRD31 proteins physically interact; the formation of this protein complex is enhanced by Kisspeptin-10 that also modulates F-actin synthesis, favoring histone acetylation in chromatin and gene expression via the cytoskeletal–nucleoskeletal pathway. Kp/KISS1R system deregulation, expression impairment of cytoskeletal–nucleoskeletal mediators, Leydig gene targets, and the decreased testosterone secretion in Ankrd31−/− testis strongly supported our hypothesis. Furthermore, cytochalasin D treatment subverted the gene expression induction dependent on Kisspeptin-10 action. In conclusion, the current work highlights a novel role for the Kisspeptin-10 in the induction of the cytoskeletal–nucleoskeletal route, downstream a physical interaction between KISS1 receptor and ANKRD31, with gene expression activation as final effect, in Leydig cells.

3D embryonic stem cell (ESC) aggregates self-organize into embryo-like structures named gastruloids that recapitulate the axial organization of post-implantation embryos. Crucial in this process is the symmetry-breaking event that leads to the emergence of asymmetry and spatially ordered structures from homogeneous cell aggregates. Here, we show that budesonide, a glucocorticoid drug widely used to treat asthma, prevents ESC aggregates to break symmetry. Mechanistically, the effect of budesonide is glucocorticoid receptor independent. RNA sequencing and lineage fate analysis reveal that budesonide counteracts exit from pluripotency and modifies the expression of a large set of genes associated with cell migration, A-P axis formation, and WNT signaling. This correlates with reduced phenotypic and molecular cell heterogeneity, persistence of E-CADHERIN at the cell-cell interface, and cell aggregate compaction. Our findings reveal that cell-cell adhesion properties control symmetry breaking and cell fate transition in 3D gastruloids and suggest a potential adverse effect of budesonide on embryo development.

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.

Spermatozoa (SPZ) are motile cells, characterized by a cargo of epigenetic information including histone post-translational modifications (histone PTMs) and non-coding RNAs. Specific histone PTMs are present in developing germ cells, with a key role in spermatogenic events such as self-renewal and commitment of spermatogonia (SPG), meiotic recombination, nuclear condensation in spermatids (SPT). Nuclear condensation is related to chromatin remodeling events and requires a massive histone-to-protamine exchange. After this event a small percentage of chromatin is condensed by histones and SPZ contain nucleoprotamines and a small fraction of nucleohistone chromatin carrying a landascape of histone PTMs. Circular RNAs (circRNAs), a new class of non-coding RNAs, characterized by a nonlinear back-spliced junction, able to play as microRNA (miRNA) sponges, protein scaffolds and translation templates, have been recently characterized in both human and mouse SPZ. Since their abundance in eukaryote tissues, it is challenging to deepen their biological function, especially in the field of reproduction. Here we review the critical role of histone PTMs in male germ cells and the profile of circRNAs in mouse and human SPZ. Furthermore, we discuss their suggested role as novel epigenetic biomarkers to assess sperm quality and improve artificial insemination procedure.

The involvement of sirtuins (SIRTs) in modulating metabolic and stress response pathways is attracting growing scientific interest. Some SIRT family members are located in mitochondria, dynamic organelles that perform several crucial functions essential for eukaryotic life. Mitochondrial dysfunction has emerged as having a key role in a number of human diseases, including cancer. Here, we investigated mitochondrial damage resulting from treatment with a recently characterized pan-SIRT inhibitor, MC2494. MC2494 was able to block mitochondrial biogenesis and function in terms of ATP synthesis and energy metabolism, suggesting that it might orchestrate cell response to metabolic stress and thereby interfere with cancer promotion and progression. Targeting mitochondrial function could thus be considered a potential anticancer strategy for use in clinical therapy.

Fourier-Transform Infrared micro-spectroscopy (µFT-IR) was used for an in vitro investigation on spermatozoa (SPZ) samples separately collected from caput and cauda of mouse epididymis. SPZ are characterized by deep biochemical changes during the transit along the epididymis and they can constitute ideal candidates for a µFT-IR investigation, thanks to the ability of this technique in analyzing cells at a molecular level. Appreciable differences were reported in the infrared spectra from caput and cauda SPZ, and biochemical changes in protein, nucleic acid, lipid, and carbohydrate content of cells were evidenced. The present investigation indicates that µFT-IR can constitute a valuable tool for monitoring, in an easy and fast way, the changes suffered by SPZ during the transit along the epididymis.

The progressive and physiological decline in ovarian function depends on the rate of follicular loss by atresia, contributing to the reduction in ovarian reserve. Genetics and environmental factors play important roles in ovarian senescence and in the onset of ovarian dysfunctions such as diminished ovarian reserve. A better understanding of the mechanisms underlying ovarian aging and their regulation by genetic and environmental factors is needed to evaluate ovarian reserve and to predict fertility potential by identification of more accurate and less invasive markers. We report transcriptomic data (i) implicating novel (e.g. EIF2 signalling) and well-known pathways (e.g. TGFβ signalling), and (ii) defining a unique set of non-coding RNA (ncRNA), both associated with ovarian function. The latter includes miRNAs (e.g. Mir143 and Mir145), snoRNAs (e.g. Snord16a and Snora34), and one lncRNA (Gas5), which are differentially expressed in middle-aged ovaries (12 months) vs young-aged (3 months) from CD1 mice. Experimental analysis confirms that ovary lifespan varies across genetic backgrounds in mice and, genetics influences the response to environmental perturbations such as diet. Moreover, the identified ncRNAs were verified in a model of reproductive dysfunction promoted by the environmental toxicant ethylenthiourea. We also report the increase of miRNA143 and miRNA145 in follicular fluid of women with diminished ovarian reserve. Their levels inversely correlate with the hormonal profile and with the number of the oocytes recruited upon hormonal stimulation. Overall, we report a transcriptomic signature for ovarian dysfunction in vivo that provides a valuable resource for translational research in human reproductive aging.

Endocannabinoids are lipid mediators, enzymatically synthesized and hydrolyzed, that bind cannabinoid receptors. Together with their receptors and metabolic enzymes, they form the “endocannabinoid system”. Anandamide and 2-arachidonoylglycerol are the main endocannabinoids studied in testis. In the present study, using the first wave of spermatogenesis as an in vivo model to verify the progressive appearance of germ cells in seminiferous tubules (i.e. spermatogonia, spermatocytes and spermatids), we analyzed the expression of the main enzymes and receptors of endocannabinoid system in rat testis. In particular, the expression profile of the main enzymes metabolizing anandamide and 2-arachidonoylglycerol as well as the expression of cannabinoid receptors, CB1 and CB2, and specific markers of mitotic, meiotic and post-meiotic germ cell appearance or activities have been analyzed by RT-PCR and appropriately correlated. Our aim was to envisage a relationship between expression of endocannabinoid system components and temporal profile of germ cell appearance or activity as well as among ECS components. Results show that expression of endocannabinoid system components is related to germ cell progression. In particular, CB2 and 2-arachidonoylglycerol appear to be related to mitotic/meiotic stages while CB1 and anandamide appear to be related to spermatogonia stem cells activity and spermatids appearance, respectively. Our data also suggest that a functional interaction among endocannabinoid system components occurs in the testis. Indeed in vitro-incubated testis show that AEA-CB2 activity affects negatively MAGL levels via up-regulation of CB1 suggesting a CB1/CB2 mediated-relationship between AEA and 2-AG. Finally, we provide the first evidence that CB1 is present in fetal gonocytes, during mitotic arrest.

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.

Aims Mammalian cardiomyogenesis occurs through a multistep process that requires a complex network of tightly regulated extracellular signals, which integrate with the genetic and epigenetic machinery to maintain, expand, and regulate the differentiation of cardiac progenitor cells. Pluripotent embryonic stem cells (ESCs) recapitulate many aspects of development, and have provided an excellent opportunity to dissect the molecular mechanisms underlying cardiomyogenesis, which is still incompletely defined. Methods and results We provide new in vivo evidence that the G-protein-coupled receptor angiotensin receptor-like 1 ( Apj ) is expressed in the mesodermal cells of the second heart field, a population of cardiac progenitors that give rise to a major part of the definitive heart. By combining loss-and-gain of function studies in mouse ESCs, we show that Apj (i) controls the balance between proliferation and cardiovascular differentiation, (ii) regulates the Nodal/Bone Morphogenetic Protein antagonist Cerberus and the Baf60c/Smarcd3 subunit of the Brg1/Brm-associated factors (BAF) chromatin-remodelling complex. Conclusion We propose a model in which Apj controls a regulatory Cerberus–Baf60c pathway in pluripotent stem cell cardiomyogenesis, and speculate that this regulatory circuit may regulate cardiac progenitor cell behaviour.

Bisphenol A (BPA) is one of the commonest Endocrine Disruptor Compounds worldwide. It interferes with vertebrate reproduction, possibly by inducing deregulation of epigenetic mechanisms. To determine its effects on female reproductive physiology and investigate whether changes in the expression levels of genes related to reproduction are caused by histone modifications, BPA concentrations consistent with environmental exposure were administered to zebrafish for three weeks. Effects on oocyte growth and maturation, autophagy and apoptosis processes, histone modifications and DNA methylation were assessed by Real-Time PCR (qPCR), histology and chromatin immunoprecipitation combined with qPCR analysis (ChIP-qPCR). The results showed that 5 μg/L BPA down-regulated oocyte maturation-promoting signals, likely through changes in the chromatin structure mediated by histone modifications and promoted apoptosis in mature follicles. These data indicate that the negative effects of BPA on the female reproductive system may be due to its upstream ability to deregulate epigenetic mechanism.

Pluripotent stem cells have the ability to undergo self-renewal and to give rise to all cells of the tissues of the body. However, this definition has been recently complicated by the existence of distinct cellular states that display these features. Here, we provide a detailed overview of the family of pluripotent cell lines derived from early mouse and human embryos and compare them with induced pluripotent stem cells. Shared and distinct features of these cells are reported as additional hallmark of pluripotency, offering a comprehensive scenario of pluripotent stem cells.

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.

The HSA21 encoded Single-minded 2 (SIM2) transcription factor has key neurological functions and is a good candidate to be involved in the cognitive impairment of Down syndrome. We aimed to explore the functional capacity of SIM2 by mapping its DNA binding sites in mouse embryonic stem cells. ChIP-sequencing revealed 1229 high-confidence SIM2-binding sites. Analysis of the SIM2 target genes confirmed the importance of SIM2 in developmental and neuronal processes and indicated that SIM2 may be a master transcription regulator. Indeed, SIM2 DNA binding sites share sequence specificity and overlapping domains of occupancy with master transcription factors such as SOX2, OCT4 (Pou5f1), NANOG or KLF4. The association between SIM2 and these pioneer factors is supported by co-immunoprecipitation of SIM2 with SOX2, OCT4, NANOG or KLF4. Furthermore, the binding of SIM2 marks a particular sub-category of enhancers known as super-enhancers. These regions are characterized by typical DNA modifications and Mediator co-occupancy (MED1 and MED12). Altogether, we provide evidence that SIM2 binds a specific set of enhancer elements thus explaining how SIM2 can regulate its gene network in neuronal features.

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.

Endocannabinoids are natural lipids regulating a large array of physiological functions and behaviors in vertebrates. The endocannabinoid system is highly conserved in evolution and comprises several specific receptors (type-1 and type-2 cannabinoid receptors), their endogenous ligands (e.g. anandamide and 2-arachidonoylglycerol), and a number of biosynthetic and degradative enzymes. In the last few years, endocannabinoids have been described as critical signals in the control of male and female reproduction at multiple levels: centrally, by targeting hypothalamic Gonadotropin-Releasing-Hormone secreting neurons and pituitary, and locally, with direct effects on the gonads. These functions are supported by the extensive localization of cannabinoid receptors and endocannabinoid metabolic enzymes at different levels of the hypothalamic-pituitary-gonadal axis in mammals, as well as bonyfish and amphibians. In vivo and in vitro studies indicate that endocannabinoids centrally regulate gonadal functions by modulating the Gonadotropin Releasing Hormone-gonadotropin-steroid network through direct and indirect mechanisms. Several proofs of local endocannabinoid regulation have been found in the testis and male genital tracts, since endocannabinoids control Sertoli and Leydig cells activity, germ cell progression, as well as the acquisition of sperm functions. A comparative approach usually is a key step in the study of physiological events leading to the building of a general model. Thus, in this review we summarize the action of endocannabinoids at different levels of the male reproductive axis, with special emphasis, where appropriate, on data from non-mammalian vertebrates.

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.

Sik1 (salt inducible kinase 1) is a serine/threonine kinase that belongs to the stress- and energy-sensing AMP-activated protein kinase family. During murine embryogenesis, sik1 marks the monolayer of future myocardial cells that will populate first the primitive ventricle, and later the primitive atrium suggesting its involvement in cardiac cell differentiation and/or heart development. Despite that observation, the involvement of sik1 in cardiac differentiation is still unknown. We examined the sik1 function during cardiomyocyte differentiation using the ES-derived embryoid bodies. We produced a null embryonic stem cell using a gene-trap cell line carrying an insertion in the sik1 locus. In absence of the sik1 protein, the temporal appearance of cardiomyocytes is delayed. Expression profile analysis revealed sik1 as part of a genetic network that controls the cell cycle, where the cyclin-dependent kinase inhibitor p57(Kip2) is directly involved. Collectively, we provided evidence that sik1-mediated effects are specific for cardiomyogenesis regulating cardiomyoblast cell cycle exit toward terminal differentiation.

Endocannabinoids, via cannabinoid receptors (CB1 and CB2), affect reproductive functions at both local and central level. Due to the high complexity of the endocannabinoid system, to the widespread distribution outside the nervous system and to the high degree of evolutionary conservation, a deep CB1 molecular characterization among species may be useful to elucidate the activity of endocannabinoids at multiple levels. In this review we report CB1 characterization in non-mammalian animal models and, in particular, in the anuran amphibian, the frog, Rana esculenta; we also describe its expression during the annual sexual cycle. Moreover, since reproductive functions are under control of gonadotropin releasing hormone (GnRH), cb1 mRNA and protein expression profile in the forebrain has been compared to those of GnRH-I, the mammalian form primarily involved in gonadotropin release.

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.

Sulfatases are involved in several biological functions such as degradation of macromolecules in the lysosomes. In patients with multiple sulfatase deficiency, mutations in theSUMF1gene cause a reduction of sulfatase activities because of a posttranslational modification defect. We have generated a mouse line carrying a null mutation in theSumf1gene. Sulfatase activities are completely absent inSumf1−/−mice, indicating that Sumf1 is indispensable for sulfatase activation and that mammals, differently from bacteria, have a single sulfatase modification system. Similarly to multiple sulfatase deficiency patients,Sumf1−/−mice display frequent early mortality, congenital growth retardation, skeletal abnormalities, and neurological defects. All examined tissues showed progressive cell vacuolization and significant lysosomal storage of glycosaminoglycans.Sumf1−/−mice showed a generalized inflammatory process characterized by a massive presence of highly vacuolated macrophages, which are the main site of lysosomal storage. Activated microglia were detected in the cerebellum and brain cortex associated with remarkable astroglyosis and neuronal cell loss. Between 4 and 6 months of age, we detected a strong increase in the expression levels of inflammatory cytokines and of apoptotic markers in both the CNS and liver, demonstrating that inflammation and apoptosis occur at the late stage of disease and suggesting that they play an important role in both the systemic and CNS phenotypes observed in lysosomal disorders. This mouse model, in which the function of an entire protein family has been silenced, offers a unique opportunity to study sulfatase function and the mechanisms underlying lysosomal storage diseases.

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.

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.

Organogenesis is a complex event, often dependent on inductive tissue interactions, that ultimately promote expression and activation of a combinatorial sequence of transcription factors which are involved in controlling migration, growth and differentiation. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

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.