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Introduction: Precocious puberty is defined as the appearance of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys. Central precocious puberty (CPP) is a rare condition that is diagnosed when premature activation of the hypothalamic-pituitary-gonadal axis is detected, in association with precocious breast development or testicular growth. Idiopathic CPP is historically considered to be the most common form, but in recent years defects in a small but growing number of genes regulating the timing of puberty have been identified in an increasing proportion of cases of CPP. Delta-like non-canonical Notch ligand 1 (DLK1) is understood to be one of the key genes involved in the etiology of CPP, although its mechanistic role is not yet fully understood. Case Presentation: We identified a novel de novo variant of DLK1 (c.835C>T; p.Gln279*) in an 8-year-old girl of Bangladeshi origin. She presented with an advanced Tanner staging of B4P4A2, significantly advanced bone age (BA, 13 years), a near-adult proportioned uterus, with a history of menarche at the age of 7.4 years. Diagnosis was confirmed by raised basal luteinizing hormone concentration. She was found to have truncal obesity associated with abnormal fasting insulin levels and mildly elevated cholesterol levels. These findings are consistent with previous literature describing an association between patients with DLK1 deficiency and an impaired metabolic profile. The patient was treated for 2 years with GnRH agonists with ongoing biochemical follow-up into adolescence. Conclusion: This case illustrates the susceptibility to metabolic derangement for patients with mutations in DLK1 and the need for ongoing monitoring after puberty. Our summary of previously identified DLK1 variants and their metabolic consequences demonstrates the frequency of obesity, lipid abnormalities, and insulin dysregulation in this patient cohort in childhood and beyond. This knowledge can guide future clinical practice for patients with CPP patients due to DLK1 deficiency. [ABSTRACT FROM AUTHOR]

Herein is reported, for the first time in the rat Harderian gland (HG), the counteractive action of melatonin (Mlt), a well‐known antioxidant radical scavenger, on the increased oxidative stress damages induced by a pro‐oxidant substance, cadmium (Cd), an environmental pollutant also considered as endocrine disruptor. HG, an infraorbital gland present in almost all terrestrial vertebrates, produces a lipid secretion to lubricate the eyeball, as well as porphyrin/Mlt as light transducers. Moreover, HG is an extra‐gonadal source of steroid sex hormones. Via ex vivo experiments lasting for 24 h, we verified the increased lipid peroxidation in Cd‐treated glands, producing morphological alteration of the glandular epithelium, as well as an increased porphyrins accumulation. Moreover, Cd also induced a decreased protein level of the steroidogenic enzymes steroidogenic acute regulatory (StAR) and 3βHSD, and an increased mast cell number. Results obtained with Mlt cotreatment demonstrated that it decreased the levels of Cd‐induced oxidative damage, with reversal of all the observed modification. Furthermore, the TUNEL assay showed that the increased number of apoptotic cells in Cd‐treated HG was counteracted by the contemporaneous Mlt administration. Results confirmed that Mlt treatment restored the levels of two autophagy markers, LC3 and p62, counteracting the autophagy Cd‐induced. Interestingly, the positive effects of Mlt alone were highlighted by the decreased rate of lipid peroxidation as compared with the control, confirming its antioxidant action. Combined data further confirmed the antioxidant action of Mlt in counteracting the degeneration provoked by reactive oxygen species (ROS) in the rat HG, a tissue extremely susceptible to oxidative stress condition. Research Highlights: –Oxidative stress Cd‐induced produces morphological and biochemical alteration in the rat Harderian gland.–Melatonin cotreatment counteracts all the damages provoked by oxidative disorders.–Melatonin alone reduces the rate of lipid peroxidation, thanks to its antioxidant properties. [ABSTRACT FROM AUTHOR]

Frog relaxin (fRLX) belongs to the relaxin/insulin gene family present in the testis of Rana esculenta and is specifically expressed by Leydig cells. Since the expression of fRLX transcript changes during the reproductive cycle and is more abundant when circulating levels of androgens are relatively high, we investigated the effect(s) of testosterone and its antagonist (cyproterone acetate, CPA) on its expression pattern, in the testis of the frog Rana esculenta. Results from in vivo and in vitro experiments demonstrate that testosterone strongly induces a significant increase of fRLX mRNA expression in frog testes and, this effect is counteracted by CPA, supporting the existence of intratesticular (autocrine/paracrine) mechanisms of action. Interestingly, in both the control and testosterone-treated testes, fRLX mRNA expression was markedly decreased 24 h post-treatment, as compared to that measured at 2 h and 8 h post-treatment, suggesting that factor(s), other than testosterone, may act(s) in controlling its expression. In addition, RT-PCR analysis and in situ hybridization performed on frog testis injected with CPA for 15 days, on alternate days, showed a strong decrease of fRLX expression, suggesting that CPA counteracts the effect of testosterone on fRLX expression. Taken together our results strongly indicate that changes in the production, by the Leydig cells, of both testosterone and fRLX may represent a marker for the study of Leydig cell activity in the testis of the frog Rana esculenta. [ABSTRACT FROM AUTHOR]

Sea urchin DNA (cytosine-5)-methyltransferase (Dnmt1) that is responsible for maintenance of DNA methylation patterns clearly shares similarity with various Dnmt1s identified in vertebrates. In this study, we determined the structure of the sea urchin Dnmt1 gene by screening a genomic library of the sea urchin Paracentrotus lividus with the complementary DNA (cDNA) as probe. Analysis of the positive clones demonstrated that the Dnmt1 gene consists of 34 exons and 33 introns spanning a distance of 35 kb. All exon-intron junction sequences agree with the GT/AG consensus with the exception of the 3′ acceptor site of intron 8 where CT replaces AG consensus.The differences in the total number of exons between sea urchin and mouse genes reside mainly in the N-terminal region of the protein (exons 5–7 of the sea urchin, exons 5–12 of the mouse) where there is very low similarity in the amino acid sequence.By reverse transcription-polymerase chain reaction using oligonucleotides spanning different regions of the cDNA we carried out a comprehensive analysis of alternative splicing of the Dnmt1 messenger RNA (mRNA) in sea urchin embryos at different stages of development. We demonstrated the presence of at least five alternative spliced mRNAs that are regulated during development and are translated in truncated or deleted proteins. [Copyright &y& Elsevier]

The biology of transposable elements (TEs) is a fascinating and complex field of investigation. TEs represent a substantial fraction of many eukaryotic genomes and can influence many aspects of DNA function that range from the evolution of genetic information to duplication, stability, and gene expression. Their ability to move inside the genome has been largely recognized as a double-edged sword, as both useful and deleterious effects can result. A fundamental role has been played by the evolution of the molecular processes needed to properly control the expression of TEs. Today, we are far removed from the original reductive vision of TEs as "junk DNA", and are more convinced that TEs represent an essential element in the regulation of gene expression. In this review, we summarize some of the more recent findings, mainly in the animal kingdom, concerning the active roles that TEs play at every level of gene expression regulation, including chromatin modification, splicing, and protein translation. [ABSTRACT FROM AUTHOR]

Insulin-like peptide 3 (INSL3) is a biomarker for Leydig cells in the testes of vertebrates, and it is principally involved in spermatogenesis through specific binding with the RXFP2 receptor. This study reports the insl3 gene transcript and the Insl3 prepropeptide expression in both non-reproductive and reproductive tissues of Danio rerio. An immunohistochemistry analysis shows that the hormone is present at a low level in the Leydig cells and germ cells at all stages of Danio rerio testis differentiation. Considering that the insl3 gene is transcribed in Leydig cells, our results highlight an autocrine and paracrine function of this hormone in the Danio rerio testis, adding new information on the Insl3 mode of action in reproduction. We also show that Insl3 and Rxfp2 belonging to Danio rerio and other vertebrate species share most of the amino acid residues involved in the ligand–receptor interaction and activation, suggesting a conserved mechanism of action during vertebrate evolution. [ABSTRACT FROM AUTHOR]

Immediate early genes play an essential role in cellular responses to different stimuli. Many of them are transcription factors that regulate the secondary response gene expression. Non-coding RNAs may also be involved in this regulatory cascade. In fact, they are emerging as key actors of gene expression regulation, and evidence suggests that their dysregulation may underly pathological states. We previously took a snapshot of both coding and long non-coding RNAs differentially expressed in neuronal cells after brain-derived neurotrophic factor stimulation. Among these, the transcription factor EGR1 (a well-known immediate early gene) and LINC00473 (a primate-specific long non-coding RNA) that has emerged as an interesting RNA candidate involved in neuronal function and in cancer. In this work, we demonstrated that LINC00473 gene expression kinetics resembled that of immediate early genes in SH-SY5Y and HEK293T cells under different cell stimulation conditions. Moreover, we showed that the expression of LINC00473 is under the control of the transcription factor EGR1, providing evidence for an interesting functional relationship in neuron function. [ABSTRACT FROM AUTHOR]

BACKGROUND: Testis-specific genes encoding for long non-coding RNA (lncRNA) have been detected in several cancers; many produce proteins with restricted or aberrant expression patterns in normal or cancer tissues. OBJECTIVE: To characterize new lncRNA involved in normal and/or pathological differentiation of testicular cells. METHODS: Using bioinformatics analysis, we found that lncRNA LOC100130460 (CAND1.11) is expressed in normal and tumor testis; its expression was assessed in several human cell lines by qRT-PCR. CAND1.11 protein, produced by a single nucleotide mutation, was studied by western blot and immunofluorescence analysis on normal, classic seminoma, and Leydig cell tumor testicular tissues. RESULTS: CAND1.11 gene is primate-specific; its expression was low in SH-SY5Y cells and increased when differentiated with retinoic acid treatment. CAND1.11 expression in PC3 cells was higher than in PNT2 cells. CAND1.11 protein is present in the human testis and overexpressed in testicular cancer tissues. CONCLUSIONS: This report is one of the few providing evidence that a lncRNA produces a protein expressed in normal human tissues and overexpressed in several testicular cancers, suggesting its involvement in regulating cell proliferation and differentiation. Although further studies are needed to validate the results, our data indicate that CAND1.11 could be a potential new prognostic biomarker to use in proliferation and cancer. [ABSTRACT FROM AUTHOR]

Purpose: Here, we report, for the first time, the temporal expression and localization of axonemal radial spoke head homolog A (RSPH6A) protein during the first wave of rat spermatogenesis and in oxidative stress conditions. Methods: For the developmental study, testes were collected from rats at different developmental stages (7, 14, 21, 28, 35, 42, and 60 postnatal days); for in vivotreatment, 24 rats were treated with cadmium and/or melatonin. From each sample, western blot (WB) and immunofluorescence (IF) analyses for RSPH6A were performed. Results: RSPH6A expression starts at 21 PND alongside the appearance of I spermatocytes (SPC) with a significant increase up to 60 PND. Data were confirmed by IF analysis, showing that RPSH6A expression is restricted to I and II SPC, spermatids, and mature sperm. In vivo experiments showed that the expression and localization of RSPH6A in the testis and epididymal spermatozoa of adult rats treated with cadmium were impaired. Interestingly, melatonin (an antioxidant), given together with Cd, can counteract its damaging effects. Conclusions: All combined data confirm that RSPH6A contributes to the onset of fertility by acting on sperm motility, raising the possibility of using RSPH6A as a marker for normal fertility in the general population. [ABSTRACT FROM AUTHOR]

The CD33 gene encodes for a member of the sialic-acid-binding immunoglobulin-type lectin (Siglec) family, and is one of the top-ranked Alzheimer's disease (AD) risk genes identified by genome-wide association studies (GWAS). Many CD33 polymorphisms are associated with an increased risk of AD, but the function and potential mechanism of many CD33 single-nucleotide polymorphisms (SNPs) in promoting AD have yet to be elucidated. We recently identified the CD33 SNP rs2455069-A>G (R69G) in a familial form of dementia. Here, we demonstrate an association between the G allele of the rs2455069 gene variant and the presence of AD in a cohort of 195 patients from southern Italy. We carried out in silico analysis of the 3D structures of CD33 carrying the identified SNP to provide insights into its functional effect. Structural models of the CD33 variant carrying the R69G amino acid change were compared to the CD33 wild type, and used for the docking analysis using sialic acid as the ligand. Our analysis demonstrated that the CD33-R69G variant may bind sialic acid at additional binding sites compared to the wild type, thus potentially increasing its affinity/specificity for this molecule. Our results led to a new hypothesis of rs2455069-A>G SNP as a risk factor for AD, suggesting that a long-term cumulative effect of the CD33-R69G variant results from the binding of sialic acid, acting as an enhancer of the CD33 inhibitory effects on amyloid plaque degradation. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we show that Prothymosin alpha (Ptma), a small, unfolded, negatively charged protein, is present in the cartilaginous fish Torpedo marmorata. The ptma gene is functional and peculiarly controlled during the male spermatogenesis of T. marmorata, as revealed by in situ hybridization and by immunocytochemistry studies. The data show that the ptma transcript is present in stage-specific germ cells, i.e. spermatocytes II and round spermatids. The Ptma protein is detectable in spermatocytes II, in round and elongated spermatids as well as in spermatozoa before their release from cysts, while it is not evident in spermatozoa located in male genital tracts. The ptma transcript and protein are also evident in some Leydig cells, located among maturing cysts containing meiotic and differentiating male cells. No expression for ptma is observed within Sertoli cells. Furthermore, immunolocalization procedures demonstrate that the protein is preferentially localized in the cytoplasm, whereas a nuclear localization is observed in round and elongated spermatids. The possibility that Ptma is involved in testis activity is discussed. [Copyright &y& Elsevier]

Simple Summary: For proper fertility, the production of good-quality spermatozoa is essential. Nowadays, many environmental pollutants affect the spermatogenetic process, at different levels. For this reason, new approaches are needed to prevent/counteract these toxic effects. Here, we showed that the excitatory amino acid D-aspartic acid (D-Asp) prevents the deadly action of ethane dimethane sulfonate (EDS) on the testosterone-secreting Leydig cells in rat testis. We found that EDS, probably via the reduced testosterone level, alters the normal histology of the seminiferous epithelium, leading to germ cells death and to the decreased protein level of two Leydig cell "markers": steroidogenic acute regulatory and prolyl endopeptidase. In addition, the same analysis performed on rats that were pre-treated with D-Asp revealed a protective role of this compound, since all the above parameters were quite normal. Moreover, we found that the protective mechanism of action involved in this scenario may be due to the ability of D-Asp to reduce the oxidative stress induced by EDS. Based on these findings, we could affirm that D-Asp may be an encouraging candidate to be used to alleviate the harmful action due to environmental pollutants exposure, in order to maintain appropriate fertility. Herein is reported the first evidence of the protective role of D-aspartic acid (D-Asp) in preventing the toxic effect exerted by the alkylating agent ethane dimethane sulfonate (EDS) in the rat testis. We confirmed that EDS treatment specifically destroyed Leydig cells (LC), resulting in the drastic decrease of the serum testosterone level and producing morphological changes in the germinal tubules, i.e., altered organization of the epithelium, loss of cell contacts and the consequent presence of empty spaces between them, and a reduce number of spermatozoa. Moreover, an increase of TUNEL-positive germ cells, other than alteration in the protein level and localization of two LC "markers", StAR and PREP, were observed. Interestingly, results obtained from rats pre-treated with D-Asp for 15 days before EDS-injection showed that all the considered parameters were quite normal. To explore the probable mechanism(s) involved in the protection exerted by D-Asp, we considered the increased oxidative stress induced by EDS and the D-Asp antioxidant effects. Thiobarbiturc acid-reactive species (TBARS) levels increased following EDS-injection, while no change was observed in the D-Asp + EDS treated rats. Our results showed that D-Asp may be used as a strategy to mitigate the toxic effects exerted by environmental pollutants, as endocrine disrupters, in order to preserve the reproductive function. [ABSTRACT FROM AUTHOR]

EGR1 is a transcription factor expressed in many cell types that regulates genes involved in different biological processes including growth, proliferation, and apoptosis. Dysregulation of EGR1 expression has been associated with many pathological conditions such as tumors and brain diseases. Known molecular mechanisms underlying the control of EGR1 function include regulation of transcription, mRNA and protein stability, and post-translational modifications. Here we describe the identification of a splicing isoform for the human EGR1 gene. The newly identified splicing transcript encodes a shorter protein compared to the canonical EGR1. This isoform lacks a region belonging to the N-terminal activation domain and although it is capable of entering the nucleus, it is unable to activate transcription fully relative to the canonical isoform. [ABSTRACT FROM AUTHOR]

Non-coding regions with dozens to several hundred base pairs of extreme conservation have been found in all metazoan genomes. The distribution of these conserved non-coding elements (CNE) within and across genomes has suggested that many of them may have roles as transcriptional regulatory elements. A combination of bioinformatics and experimental approaches can be used to identify CNEs with regulatory activity in phylogenetically distant species. Nevertheless, the high divergent rate of genomic sequences of several organisms, such as tunicates, complicates the characterization of these conserved elements and very few examples really may prove their functional activity. We used a comparative approach to facilitate the identification of CNEs among distantly related or highly divergent species and experimentally demonstrated the functional significance of these novel CNEs. We first experimentally tested, in C. robusta and D. rerio transgenic embryos, the regulatory activity of conserved elements associated to genes involved in developmental control among different chordates (Homo sapiens and Danio rerio for vertebrates, Ciona robusta and Ciona savignyi for tunicates and Branchiostoma floridae for cephalochordates). Once demonstrated the cross-species functional conservation of these CNEs, the same gene loci were used as references to locate homologous regions and possible CNEs in available tunicate genomes. Comparison of tunicate-specific and chordate-specific CNEs revealed absence of conservation of the regulatory elements in spite of conservation of regulatory patterns, likely due to evolutionary specification of the respective developmental networks. This result highlights the importance of an integrative in-silico/in-vivo approach to CNEs investigation, encompassing both bioinformatics, essential for putative CNEs identification, and laboratory experiments, pivotal for the understanding of CNEs functionality. • The Otx CNE-B retain its function in distant species as C. robusta and D. rerio. • O. dioica and B. schlosseri lack the Ipf1 gene. • Chordate-specific CNEs are not conserved in all considered tunicates. • Tunicate CNEs display expression patterns similar to the chordate-specific CNEs. [ABSTRACT FROM AUTHOR]

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a protein belonging to the formin family, which regulates, together with the small GTPase RhoA, the nucleation and the assembly of actin fibres through Wnt-Dishevelled PCP pathway. Its role has been investigated in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. In this work, we studied the expression of DAAM1 mRNA and protein by PCR and Western blot analyses and its co-localization with actin in adult mouse seminal vesicles by immunofluorescence. We show that both proteins are cytoplasmic: actin is evident at cell–cell junctions and at cell cortex; DAAM1 had a more diffused localization, but is also prominent at the apical plasmatic membrane of epithelial cells. These findings support our hypothesis of a role of DAAM1 in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular concerning actin filaments. We were also able to detect DAAM1 and actin association in the smooth muscle cells that surround the epithelium too. In this case, we could only speculate the possible involvement of this formin in muscular cells in the maintenance and the regulation of the contractile structures. The present results strongly suggest that DAAM1 could have a pivotal role in vesicle exocytosis and in the physiology of mouse seminal vesicles. [ABSTRACT FROM AUTHOR]

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a formin-family protein involved in nucleation of unbranched actin filaments and in cytoskeletal organization through Wnt-Dishevelled PCP pathway, which participates in essential biological processes, such as cell polarity, movement, and adhesion during morphogenesis and organogenesis. While its role has been investigated during development and in somatic cells, its potential association with the germinal compartment and reproduction is still unexplored. In this work, we assessed the possible association of DAAM1 with the morphogenesis of rat testis. We studied its expression and profiled its localization versus actin and tubulin, during the first wave of spermatogenesis and in the adult gonad (from 7 to 60 dpp). We show that, in mitotic phases, DAAM1 shares its localization with actin in Sertoli cells, gonocytes, and spermatogonia. Later, during meiosis, both proteins are found in spermatocytes, while only actin is detectable at the forming blood-testis barrier. DAAM1, then, follows the development of the acrosome system throughout spermiogenesis, and it is finally retained inside the cytoplasmic droplet in mature gametes, as corroborated by additional immunolocalization data on both rat and human sperm. Unlike the DAAM1, actin keeps its localization in Sertoli cells, and tubulin is associated with their protruding cytoplasm during the process. Our data support, for the first time, the hypothesis of a role for DAAM1 in cytoskeletal organization during Mammalian testis morphogenesis and gamete progression, while also hinting at its possible investigation as a morphological marker of germ cell and sperm physiology. J. Cell. Physiol. 231: 2172-2184, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to evaluate the accuracy of tridimensional endoanal ultrasound (3D-EAUS) in the diagnosis of perianal sepsis comparing the results with the surgical findings, considered as reference standard. Methods: From January 2009 to January 2013, all the patients referred for the assessment and treatment of perianal sepsis with suspected anorectal origin were enrolled in the study. All patients gave informed written consent. Prior to surgery, all the patients underwent anamnestic evaluation, clinical examination, and unenhanced and HO-enhanced 3D-EAUS. Surgery was performed by a colorectal surgeon blinded to the 3D-EAUS results. Results: A total of 212 patients with suspected perianal suppurations were assessed during the study period. In 12 patients, the HO-enhanced 3D-EAUS was not performed, and so, they were excluded from the study. Very good agreement between 3D-EAUS and examination under anesthesia (EUA) in the classification of primary fistula tracts (kappa = 0.93) and in the identification of fistula internal opening (kappa = 0.97) was found. There was a good concordance (kappa = 0.71) between 3D-EAUS and surgery in the detection of fistula secondary extensions. The overall sensitivity and specificity of 3D-EAUS in the diagnosis of perianal sepsis were 98.3 and 91.3 % respectively. Conclusion: 3D-EAUS is a safe and reliable technique in the assessment of perianal sepsis. It may assist the surgeon in delineating the fistula tract anatomy and in determining the origin of sepsis, supporting the preoperative planning of definitive and appropriate surgical therapy. [ABSTRACT FROM AUTHOR]

Objective. This study was designed to evaluate the early results of the topical application of Hydrolyzed Hibiscus esculentus Extract 3% ointment (Myoxinol 3%), a novel local product with Botox-like activity, in the conservative treatment of chronic anal fissure (CAF). Methods. Among all patients with CAF observed during the study period, 31 subjects met the inclusion criteria and underwent medical therapy with Myoxinol 3% ointment every 12 hours for 6 weeks. Two patients were lost to follow-up. Clinical and manometric follow-up was carried out eight weeks after treatment. Results. At follow-up the success rate was 72.4% (21/29); median VAS score and mean anal resting pressure were significantly lower if compared with respective baseline data. The only one adverse effect of the topical application of Myoxinol 3% ointment was perianal itch, which was reported by 3,4% (1/29) of the patients available for the analysis. However, in this case this symptom did not cause interruption of the treatment. Conclusions. The topical application of Myoxinol 3% ointment in the cure of CAF shows encouraging early results. Further researches with a larger series and a longer follow-up are needed to confirm these data. [ABSTRACT FROM AUTHOR]

In mammals, the RXFP3 is the cognate receptor of the relaxin-3 peptide ( RLN3). In teleosts, many different orthologue genes for RXFP3 are present. In particular, two paralogue genes, rxfp3-2a and rxfp3-2b, likely encode the receptors for the Rln3a peptide. The transcription of these two rxfp3 genes is differentially regulated early during zebrafish embryogenesis. Indeed, reverse transcription-polymerase chain reaction analyses show that the rxfp3-2b transcript is always present during embryo development, while the rxfp3-2a transcript is detectable only at larval stage. By in situ hybridization experiments on embryos and larvae, the rxfp3-2b transcript was revealed in the brain and in the retinal ganglion cell layer and thymus. Particularly in the brain, many territories are involved in the rxfp3-2b expression, among them the optic tectum, thalamus, preoptic area, different nerve nuclei, habenula and pineal gland. The RXFP3 spatiotemporal expression pattern appears to be conserved between Danio rerio and mammals, as also previously showed for the corresponding ligand, the RLN3. Interestingly, the brain areas expressing the rxfp3-2b receptor gene are involved in the visual system, emotional behaviors and circadian rhythm and could be functionally related to the neurotransmitter Rln3a-expressing territories. [ABSTRACT FROM AUTHOR]

We report the identification, the cDNA cloning, the temporal and spatial expression pattern analysis of the rln gene in the zebrafish Danio rerio. The deduced Rln B and A domains show different evolutionary conservation. Rln B domain shows higher similarity when compared to zebrafish and human RLN3 B domain than human RLN1 and RLN2 B domain. Differently, the zebrafish Rln A domain shows relatively low amino acid sequence similarity when compared with the same sequences. The rln gene is transcribed both during embryogenesis and in adult organism, where higher transcript level has been particularly evidenced in the brain. Moreover, we provide the first description of rln spatial expression pattern during embryonic development. In particular, we show restricted transcript localization starting at the pharyngula stage in olfactory placode, branchial arch region, and in a cell cluster near to otic vesicle. In larval stage, new transcription territories have been detected in both neural and non-neural regions. In particular, in the brain, rln expression has been revealed in telencephalic region around anterior commissure, in the preoptic area, and in restricted rombencephalic cell clusters. Expression of rln gene in extra-neural territories has been detected in the pancreatic and thyroid gland regions. Danio rerio rln expression pattern analysis reveals shared features with the mammalian RLN gene, particularly in the brain, where it might have a role in the neurophysiological processes. In addition, expression in the thyroid and pancreas region suggests a function as a paracrine and endocrine hormone. [ABSTRACT FROM AUTHOR]

Abstract: GHRELIN (GHRL) is an acylated peptide that contains 28-amino acids prevalently expressed in the stomach of several species. Specifically, it contributes to energy balance, but some new evidence highlights its role in the regulation of reproductive functions. In fact, this protein has been detected at testicular level in the tubular and interstitial compartments of several vertebrate species, and previous research has demonstrated that GHRL affects various aspects of spermatogenesis and steroidogenesis. GHRL clearly plays an inhibitory role in mammalian reproduction, in contrast GHRL stimulates reproductive functions in non mammalian vertebrate. We have focused our attention on the comparative aspect of GHRL, thus studying its expression in an amphibian seasonal breeder, Pelophylax esculentus, to verify the presence and localization, of Ghrl transcript variations during the frog reproductive cycle, in order to demonstrate that Pelophylax esculentus may represent a useful animal model to assess the role of GHRL in male fertility. [Copyright &y& Elsevier]

We report the gene characterization, the cDNA cloning and the temporal and spatial expression pattern of the relaxin receptor rxfp1 gene in the zebrafish Danio rerio. The zebrafish rxfp1 gene has the same syntenic genomic organization, and a similar exon-intron structure to the homologue human gene. Furthermore, the deduced Rxfp1 protein sequence shows a high degree of amino acid similarity when compared with the human protein and the conservation of all amino acid identity necessary for the binding with relaxin. Our results show that rxfp1 gene is active either during embryogenesis or in the adult organism, showing a wide expression pattern. Moreover, we provide the first description of rxfp1 spatial expression pattern during embryo development, showing that the transcript is already present at the early developmental stage and is distributed in all of the embryonic cells until somitogenesis. Starting at the pharyngula stage the gene expression becomes mainly restricted in the brain territories. In fact, at the larval stage, the transcript is detectable in the epiphysis, postoptic region, posterior tuberculum, hypothalamus, optic tectum, tegmentum/pons, medulla and also in the structure of a peripheral nervous system, the terminal nerve. The rxfp1 expression pattern in Danio rerio embryos is very similar to that reported in the adult mammalian brain, suggesting a pivotal role of this receptor in the neurophysiology processes already at very early developmental stages. [ABSTRACT FROM AUTHOR]

Prothymosin alpha (PTMA) is a highly acidic small polypeptide, that is, widely distributed and conserved among mammals. Its possible involvement in male gametogenesis has been mentioned but not clarified yet; in particular, it has been suggested that, in non-mammalian vertebrates, it could play a role during GC meiosis and differentiation. In the present work we investigated the possible association between PTMA and meiotic and post-meiotic phases of mammalian spermatogenesis. Three different time points during postnatal development of rat testis were analyzed, that is, 27 dpp (completed meiosis), 35 dpp (occurring spermiogenesis), and 60 dpp (first wave of spermatogenesis definitely ended). RT-PCR and Western blot analyses showed that the expression levels of both Ptma mRNA and corresponding protein decrease in total extracts from 27 to 60 dpp. The in situ hybridization localized the transcript in interstitial Leydig cells, peritubular myoid cells and, inside the tubules, in germ cells from pachytene spermatocytes to newly formed haploid spermatids. The immunohistochemistry analysis localized the protein in the same cell types at 27 dpp, while at 35 and 60 dpp the haploid cells remain the only germ cells that still express it. In particular, PTMA specific localization in the heads of spermatids and epididymal spermatozoa, associated with the acrosome system, supports for the first time the hypothesis of a direct function in male germ cells. J. Cell. Physiol. 224: 362–368, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Relaxin-3 (Rln3) is thought to function as a neurotransmitter mainly produced in the mammalian nucleus incertus and is involved in different neural processes; among them, the stress response and food intake. Here, we report the expression pattern of the duplicated zebrafish rln3b gene and compare it to the previously analyszd spatial expression pattern of the rln3a gene. Both genes, during the embryogenesis and in the adult fish, are active and show relevant differences in their expression patterns. rln3b is diffusely expressed in the brain until the pharyngula period, when, at 48 h postfertilization (hpf), the expression becomes restricted to the periaqueductal gray, where it persists also at later developmental stages. No expression was observed in the nucleus incertus cells that express the rln3a gene from 72 hpf. In the adult, both genes are expressed in brain, but only rln3b transcript is revealed in testis at the similar expression level, whereas in the other analyzed tissues the transcript levels are lower or absent. Both the putative mature protein sequences are highly conserved, this feature and their differential expression patterns might indicate a sub-functionalization during evolution with the consequent retention of the two paralogues genes. [ABSTRACT FROM AUTHOR]

The msh/Msx family is a subclass of homeobox-containing genes suggested to perform a conserved function in the patterning of the early embryo. We had already isolated a member of this gene family (Ci-msxb) in Ciona intestinalis, which has a very complex expression pattern during embryogenesis. To identify the regulatory elements controlling its tissue-specific expression, we have characterized the gene structure and the regulatory upstream region. By electroporation experiments, we demonstrated that a 3.8-kb region located upstream of the gene contains all the regulatory elements able to reproduce its spatial expression pattern. Analyzing progressively truncated fragments of this region, three discrete and separate regions driving LacZ reporter gene expression in the ventral epidermis, primordial pharynx and neural territories have been identified. We further investigated the element(s) necessary for Ci-msxb activation in the nervous system during embryonic development by in vivo and in vitro experiments. Both electroporation and gel-shift assays of overlapping wild type and mutated oligonucleotides demonstrated that a unique sequence of 30 bp is involved in Ci-msxb neural activation from neurula to larva stage. This sequence contains consensus binding sites for various ubiquitous transcription factors such as TCF11 whose possible implication in formation of the regulatory complexes is discussed. [Copyright &y& Elsevier]

Probes for the occurrence of endogenous D-aspartic acid (D-Asp) and N-methyl-D-aspartic acid (NMDA) in the neural complex and gonads of a protochordate, the ascidian Ciona intestinalis, have confirmed the presence of these two excitatory amino acids and their involvement in hormonal activity. A hormonal pathway similar to that which occurs in vertebrates has been discovered. In the cerebral ganglion D-Asp is synthesized from L-Asp by an aspartate racemase. Then, D-Asp is transferred through the blood stream into the neural gland where it gives rise to NMDA by means of an NMDA synthase. NMDA, in turn, passes from the neuronal gland into the gonads where it induces the synthesis and release of a gonadotropin-releasing hormone (GnRH). The GnRH in turn modulates the release and synthesis of testosterone and progesterone in the gonads, which are implicated in reproduction. [Copyright &y& Elsevier]

Hox genes are organized in genomic clusters. In all organisms where their role has been studied, Hox genes determine developmental fate along the antero-posterior axis. Hence, these genes represent an ideal system for the understanding of relationships between the number and expression of genes and body organization. We report in this paper that the ascidian Ciona intestinalis genome appears to contain a single Hox gene complex which shows absence of some of the members found in all chordates investigated up to now. Furthermore, the complex appears to be either unusually long or split in different subunits. We speculate that such an arrangement of Hox genes does not correspond to the chordate primordial cluster but occurred independently in the ascidian lineage. [Copyright &y& Elsevier]

Recently we demonstrated that N-methyl-d-aspartic acid (NMDA) is present as an endogenous compound in the nervous tissues and endocrine glands of the rat where it plays a role in the regulation of the luteinizing hormone, growth hormone, and prolactin (FASEB J. 14 (2000) 699; Endocrinology 141 (2000) 3861). Based on the prediction that NMDA could have future importance in neuroendocrinology, we have devised an improved method for the specific and routine determination of NMDA in biological tissue. This method is based on the detection by HPLC of methylamine (CH3NH2) which comes from the oxidation of NMDA by d-aspartate oxidase, an enzyme which specifically oxidizes NMDA, yielding CH3NH2 as one of the oxidative products of the reaction. The sensitivity of the method permits the accurate determination of NMDA in the supernatant of a tissue homogenate at levels of about 5–10 picomol/assay. However, for those tissues in which the concentration of NMDA is less than 1 nmol/g, the sample must be further purified by treatment with o-phthaldialdehyde in order to separate the NMDA from the other amino acids and amino compounds and then concentrated and analyzed by HPLC. Using this method we have conducted a comparative study in order to measure the amount of NMDA in neuroendocrine and other tissues of various animal phyla from mollusks to mammals. [Copyright &y& Elsevier]

A full-length cDNA encoding a highly conserved calmodulin was isolated from a cDNA library prepared from hatched larvae of the ascidian Ciona intestinalis. Sequence analysis has identified a 447 b.p. open reading frame, encoding a putative protein of 149 amino acid residues, with a predicted molecular weight of 16.8 kDa, showing 85-98% identity to known calmodulins. Northern blot analysis revealed a single transcript of about 0.8 kb in length, which was maternally expressed and progressively increased during development, until late tail-bud stage. Whole-mount in situ hybridizations, carried out on embryos at different stages of development, showed that starting from the neurula stage, the C. intestinaliscalmodulin ( Ci-CaM) expression became restricted to the neuroectoderm and that in larvae it was specifically detected in the nervous system. [ABSTRACT FROM AUTHOR]