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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. 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 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.

ObjectiveTo review systematically community-based primary care interventions for epilepsy in low- and middle-income countries to rationalize approaches and outcome measures in relation to epilepsy care in these countries.MethodsA systematic search of PubMed, EMBASE, Global Index Medicus, CINAHL, and Web of Science was undertaken to identify trials and implementation of provision of antiseizure medications, adherence reinforcement, and/or health care provider or community education in community-based samples of epilepsy. Data on populations addressed, interventions, and outcomes were extracted from eligible articles.ResultsThe 24 reports identified comprise mostly care programs addressing active convulsive epilepsy. Phenobarbital has been used most frequently, although other conventional antiseizure medications (ASMs) have also been used, but none of the newer. Tolerability rates in these studies are high, but overall attrition is considerable. Other approaches include updating primary health care providers, reinforcing treatment adherence in clinics, and raising community awareness. In these programs, the coverage of existing treatment gap in the community, epilepsy-related mortality, and comorbidity burden are only fleetingly addressed. None, however, explicitly describe sustainability plans.ConclusionsCost-free provision, mostly of phenobarbital, has resulted in short-term seizure freedom in roughly half of the people with epilepsy in low- and middle-income countries. Future programs should include a range of ASMs. These should cover apart from seizure control and treatment adherence, primary health care provider education, community awareness, and referral protocols for specialist care. Programs should incorporate impact assessment at the local level. Sustainability in the long term as much as resilience and scalability should be addressed in future initiatives.

La neurofibromatosi tipo 1 (NF1) e una malattia multisistemica, clinicamente eterogenea, che puo coinvolgere anche il sistema endocrino. Il feocromocitoma/paraganglioma associato alla NF1 ha frequenza maggiore rispetto alla popolazione generale e presenta un moderato rischio di malignita. Altre manifestazioni endocrinologiche sono la bassa statura, la puberta precoce, alterazioni del metabolismo osseo, alterazioni tiroidee e metaboliche. L’endocrinologo dovrebbe pertanto essere parte integrante del team multidisciplinare preposto alla gestione clinica di questi pazienti.

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 &ldquo, markers&rdquo, 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.

Background. Anti-reflux surgery is an effective treatment for gastroesophageal reflux disease (GERD). Nevertheless, surgery is still indicated with great caution in relation to the risk of complications, and in particular to postoperative dysphagia (PD). Objective. To compare the clinical outcomes, with particular focus on the incidence and severity of PD, of laparoscopic Nissen–Rossetti fundoplication (NRF) and floppy Nissen fundoplication (FNF) with complete fundus mobilization, in the surgical treatment of GERD. Methods. Ninety patients with GERD were enrolled. Forty-four patients (21[47.7%] men, 23[52.2%] women; mean age 42.4 ± 14.3 years) underwent NRF (Group A), and 46 patients (23[50%] men, 23[50%] women; mean age 43.3 ± 15.4 years) received laparoscopic FNF with complete fundus mobilization (Group B). Clinical assessment was performed using a structured questionnaire and SF-36 quality of life (QoL) score. PD was assessed using a validated classification, and an overall outcome was also determined by asking the patient to score it. Results. At 24-month follow-up, 38 (88.3%) patients in Group A vs 39 (86.6%) in Group B reported to be completely satisfied with reflux relief and free of protonic pump inhibitors (PPIs), while 3 (6.9%) in Group A vs 2(4.4%) in Group B reported occasional PPI intake and 2(4.6%) in Group A vs 4(8.8%) in Group B needed regular PPI use. Persistent PD was observed in 8(18.6%) patients in Group A and in 2(4.4%) in Group B ( P = .03). No significant differences were found in the QoL score and in the overall outcome perceived by the patients. Conclusion. FNF, with complete fundus mobilization, appears to be associated with a lower rate of postoperative persistent dysphagia.

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.

SummaryIn this paper, with the aim to find new genes involved in mammalian spermatogenesis, we isolated, for the first time in the rat testis, a partial cDNA clone that encoded EH domain binding protein 1-like 1 (Ehbp1l1), a protein that has a single calponin homology domain (CH). Bioinformatic analysis showed that EHBP1l1 contains three domains: the N-terminal C2-like, the CH and the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domains, which are evolutionarily conserved in vertebrates. We found that Ehbp1l1 mRNA was expressed in several rat tissues, including the liver, intestine, kidney and also in the testis during its development, with a higher level in testis from 12-month-old animals. Interestingly,in situhybridization experiments revealed that Ehbp1l1 is specifically expressed by types I and II spermatocytes, this result was validated by RT-PCR performed on total RNA obtained from enriched fractions of different testicular cell types. As EHBP1l1 has been described as linked to vesicular transport to the actin cytoskeleton and as an effector of the small GTPase Rab8, we hypothesized that it could participate both in cytoskeletal remodelling and in the regulation of vesicle sorting from thetrans-Golgi network to the apical plasma membrane. Our findings provide a better understand of the molecular mechanisms of the differentiation process of spermatogenesis; Ehbp1l1 may also be used as a new marker of testicular activity.

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.

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.

Prolyl endopeptidase (PREP) is a post-proline cleaving enzyme. It is involved in the regulation of multiple inositol polyphosphate phosphatase activity implicated in the pathway of inositol 1,4,5 trisphosphate, resulting in the modulation of cytosolic Ca2+ levels. Besides its peptidase activity, PREP was identified as binding partner of tubulin, suggesting that it may participate in microtubule-associate processes. In this paper, we evaluated the expression of PREP mRNA and protein by PCR and Western blot analyses and its colocalization with tubulin by immunofluorescence in adult mouse seminal vesicles. We show that both proteins are cytoplasmic: tubulin is localized at the apical half part of the cell; while PREP has a more diffuse localization, showing a prominent distribution at the apical cytoplasm. These findings support our hypothesis of a specific role of PREP in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular its association with tubulin filaments. Moreover, it may regulate the Ca2+ levels, which promotes the final step of the vesicular exocytosis, namely the fusion of the vesicles with the plasma membrane. These results strongly suggest for a pivotal role of PREP in vesicle exocytosis as well as in the physiology of mouse seminal vesicles. Summary Prolyl endopeptidase (PREP) is a post-proline cleaving enzyme. It is involved in the regulation of multiple inositol polyphosphate phosphatase activity implicated in the pathway of inositol 1,4,5-trisphosphate, resulting in the modulation of cytosolic Ca2+ levels. Besides its peptidase activity, PREP was identified as a binding partner of tubulin, suggesting that it may participate in microtubule-associate processes. In this paper, we evaluated the expression of PREP mRNA and protein by polymerase chain reaction and western blot analyses and its co-localization with tubulin by immunofluorescence in adult mouse seminal vesicles. We showed that both proteins are cytoplasmic: tubulin is localized at the apical half part of the cell, while PREP has a more diffuse localization, showing a prominent distribution at the apical cytoplasm. These findings support our hypothesis of a specific role for PREP in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular its association with tubulin filaments. Moreover, it may regulate Ca2+ levels, and promote the final step of vesicular exocytosis, namely the fusion of the vesicles with the plasma membrane. These results strongly suggest that there is a pivotal role for PREP in vesicle exocytosis, as well as in the physiology of mouse seminal vesicles.

Maternal stress during pregnancy adversely affects developmental fetal programming. Glucocorticoid excess is one of those conditions that underlie the prenatal stress and can lead to many pathological disorders later in life. Beyond the obvious use of mammalian model organisms to uncover the different mechanisms at the basis of prenatal stress effects, zebrafish represents a complementary fruitful model for this research field. Here we demonstrated that the application of an experimental paradigm, which simulates prenatal stress by exposing embryos to cortisol excess, produced an alteration of gene expression pattern. In particular, the transcript level of hsd11b2, a gene involved in the cortisol catabolism, was affected in prenatally stressed larvae, even after many hours from the removal of cortisol excess. Interestingly, the expression pattern of c-fos, a marker gene of neural activity, was affected in prenatally stressed larvae even in response to a swirling and osmotic stress challenge. Our data corroborate the idea of zebrafish as a useful model organism to study prenatal stress effects on vertebrate development.

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.

The aim of the present article was to investigate the oogenic cycle of Mytilus galloprovincialis sampled in the Bay of Naples, and to immunolocalize 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and P450 aromatase, enzymes involved in the synthesis of two sex hormones: testosterone and 17β-estradiol. We demonstrate that the oogenic cycle starts in late summer-early fall and continues in early winter when the first event of spawning occurs; other spawning events take place until June, when the ovary is spent and contains a few empty ovarian follicles and numerous somatic cells, that is, adipogranular cells and vesicular connective tissue cells. During the oogenic cycle, apoptotic events occur at the level of oogonia, previtellogenic oocytes, as well as follicle cells; by contrast, necrosis events probably take place in vitellogenic oocytes, which, once degenerated, transfer their content to healthy oocytes. Finally, the present data demonstrate that 3β-HSD, 17β-HSD, and P450 aromatase are present in the ovary both during the reproductive and nonreproductive phases. The possible role of these enzymes during the Mytilus galloprovincialis reproductive cycle is discussed. Anat Rec, 302:1039-1049, 2019. © 2019 Wiley Periodicals, Inc.

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.

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.

RXFP2 is one of the 4 receptors for relaxin insulin-like peptides, in particular it binds with high affinity the INSL3 peptide. INSL3/RXFP2 pair is essential for testicular descent during placental mammalian development. The evolutionary history of this ligand/receptor pair has received much attention, since its function in vertebrate species lacking testicular descent, such as the fishes, remains elusive. Herein, we analyzed the expression pattern of three rxfp2 homologue genes in zebrafish embryonic development. For all the three rxfp2 genes (rxfp2a, rxfp2b, and rxfp2-like) we showed the presence of maternally derived transcripts. Later in the development, rxfp2a is only expressed at larval stage, whereas rxfp2b is expressed in all the analyzed stage with highest level in the larvae. The rxfp2-like gene is expressed in all the analyzed stage with a transcript level that increased starting at early pharyngula stage. The spatial localization analysis of rxfp2-like gene showed that it is expressed in many cell clusters in the developing brain. In addition, other rxfp2-like-expressing cells were identified in the retina and oral epithelium. This analysis provides new insights to elucidate the evolution of rxfp2 genes in vertebrate lineage and lays the foundations to study their role in vertebrate embryonic development.

The bivalve Mytilus galloprovincialis has a broad geographic distribution, represent an important species for the ecology of coastal waters, also constituting a major aquaculture species. In the present work, molecular and tissue biomarkers were examined in mussel populations (M. galloprovincialis) located in four different areas of the coastal water of the Campania Region. During an annual life cycle, we analyzed the expression patterns of several genes commonly used to estimate cellular stress response and damage, namely p53, p63, HSP70, MT-10, and MT-20, related tissue lesions (pathogens, inflammations, digestive tubules damage), oxidative stress indicators (H 2 O 2 , SOD specific activity) and associated environmental data. The computed Principal Component Analysis showed that the areas were discernible based on the environmental data and biomarker results. About animal health status, mussels from Gulf of Pozzuoli and Naples's harbor did show a thinnest epithelial cell of digestive tubules compared to mussels sampled from other sampling sites; moreover, high prevalence of cases of intersex in three of the examinated areas were observed. The presence of a potential zoonotic pathogen (Nocardia crassostreae) was identified, appearing as an important possible emerging disease. We also reported the OIE notifiable protozoa Marteilia refringens in three areas out of four. The likely impact of both observed pathogens on the mussel health and shellfish aquaculture needs to be urgently addressed. Results are discussed considering animal histopathological health parameters and biological effects.

Mytilus galloprovincialis female specimens were collected from two mussel farms located in two sites next to Castel dell’Ovo, a historical complex located in the Naples Bay. Such sites were named, respectively, A‐area and B‐area for the different microbiological parameters so that mussels from A‐area can be sold without purification, whereas mussels from B‐area must be purified before sale. The mussels were collected during the nonreproductive (summer 2009) and reproductive periods (autumn 2009). Gonadosomatic index, structural organization of the ovary, presence of apoptosis, estrogen receptors expression, as well as the bisphenol A (BPA) content in the ovaries, were evaluated. Ovaries from specimens collected in area B showed a different and significant distribution of the investigated biomarkers as well as of BPA content in respect to those measured in the A‐area specimens, confirming that mussels are valid sentinel organisms to biomonitor in the Naples bay too.

Cells respond to neural stimuli through the rapid and transient induction of a set of genes called immediate early genes (IEGs). Among these genes, EGR1 (early growth response gene 1) encodes for a transcription factor known to be involved in different biological functions such as cell growth, differentiation and apoptosis. In the neuronal development, EGR1 has been hypothesized to have a role in differentiation, neuronal plasticity, and in the development of learning and memory. The scope of our study is to evaluate the involvement of EGR1 in the neuronal cell proliferation, survival and death. To this aim, we used SH-SY5Y neuroblastoma cell line and CRISPR-Cas9 technology to generate an EGR1 knock-out (KO) neuronal cell model. EGR1-KO cells show clear morphological differences, and higher proliferation and migration rate compared to WT cells. Under different stress conditions, such as serum withdrawal and H2O2-induced oxidative stress, KO cells have higher survival rate than WT cells. A preliminary molecular analysis demonstrated that KO cells react to stress-induced conditions by triggering autophagy process in contrast to WT cells. Therefore, EGR1 knockout cells may represent an interesting model for studying the molecular mechanisms regulated by EGR1 for the proper development, differentiation and survival of neurons.

The transcription factor EGR1 is an immediate-early gene often used as an indirect marker to measure neuronal activity. It has been hypothesized to have a role in many biological processes in neuronal cells, such as neuronal plasticity and development of learning and memory. The expression of EGR1 is often altered in neurodegenerative diseases and psychiatric disorders including Alzheimer’s disease and schizophrenia. However, the role of EGR1 and the transcriptional programs under its control in neuronal development are not fully understood. In this regard, we established a neuronal cell model with a defective EGR1 gene. We generated a knockout cell (KO) line for EGR1 gene using one of the most popular neuronal cell line, the SH-SY5Y, that can be differentiated to a more mature neuron-like phenotype using retinoic acid (RA). EGR1-KO cells show clear morphological differences compared to WT cells, particularly evident for neurite arborization. In addition, EGR1-KO cells show higher proliferation and migration rate compared to WT cells. Under different stress conditions, such as serum withdrawal and H2O2-induced oxidative stress, KO cells have higher survival rate than WT cells. A preliminary molecular analysis demonstrated that KO cells react to stress-induced conditions likely involving autophagy process in contrast to WT cells. Surprisingly, EGR1-KO cells undergo cell death after RA treatment in contrast to WT cells that undergo neuronal differentiation. Molecular analyses of the EGR1-KO model show a dysregulation of many differentiation markers, including those related to neurite maturation and synaptogenesis. In fact, the western blotting analysis shows a higher level of GAP43 and SYT1 proteins in KO cells compared to WT. Therefore, EGR1 knockout cell model obtained by CRISPR/Cas9 method may represent an interesting model for studying the molecular mechanisms regulated by EGR1 for the proper development, differentiation, and survival of neurons.

Relaxin (RLN) and insulin (INSL)-like peptides are member of the INSL/RLN superfamily, which are encoded by seven genes in humans and can activate the G-protein coupled receptor RXFP 1–4. These peptides evolved from a common ancestor, RLN3-like gene. Two rounds of whole genome duplication (WGD) in early vertebrate evolution, together with an additional WGD in the teleost lineage, caused an expansion of RLN genes set in the genome of Danio rerio. In particular, six RLN genes are present: a single copy of rln and insl3 genes, and two paralogs for the rln3 gene (rln3a and rln3b), and the insl5 gene (insl5a and insl5b). We have already reported the presence of rln3a and rln3b genes in the developing zebrafish brain, as well as the expression of rln gene in the developing zebrafish brain and extraneural territories, such as thyroid gland and pancreas. Here, we report for the first time the expression of the two parologs genes for insl5, insl5a, and insl5b in D. rerio embryonic development. The corresponding transcripts of both the paralogs are present in all embryonic stages analyzed by RT-qPCR. In situ hybridization analyses showed a restricted signal in intestinal cells and the pancreatic region at 72 hpf for insl5a, while at 96 hpf both genes are expressed in specific intestinal cells. Furthermore, in adult zebrafish intestine tissue, in situ hybridation experiments showed that insl5a transcript is specifically localized in the goblet cells, while insl5b transcript is in enteroendocrine cells. These data revealed a high degree of gene expression pattern conservation for such genes in vertebrate evolution.

Relaxin peptides exert different functions in reproduction and neuroendocrine processes via interaction with two evolutionarily unrelated groups of receptors: RXFP1 and RXFP2 on one hand, RXFP3 and RXFP4 on the other hand. Evolution of receptor genes after splitting of tetrapods and teleost lineage led to a different retention rate between mammals and fish, with the latter having more gene copies compared to the former. In order to improve our knowledge on the evolution of the relaxin ligands/receptors system and have insights on their function in early stages of life, in the present paper we analyzed the expression pattern of five zebrafish RXFP3 homologue genes during embryonic development. In our analysis, we show that only two of the five genes are expressed during embryogenesis and that their transcripts are present in all the developmental stages. Spatial localization analysis of these transcripts revealed that the gene expression is restricted in specific territories starting from early pharyngula stage. Both genes are expressed in the brain but in different cell clusters and in extra-neural territories, one gene in the interrenal gland and the other in the pancreas. These two genes share expression territories with the homologue mammalian counterpart, highlighting a general conservation of gene expression regulatory processes and their putative function during evolution that are established early in vertebrate embryogenesis.

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. © 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

In vertebrate species, the MDM2 and MDM4 gene paralogs negatively regulate the activity of p53 family members and are involved in the development of a number of tumor types. Comparative genomic analyses have shown the presence of a single mdm homolog gene in invertebrates; its interaction with p53 and its involvement in tumor pathogenesis was demonstrated only in the mussel Mytilus trossulus. In addition to p53-related activity, a pro-inflammatory role for MDM2 in mammals has recently been described. In the present paper, we report the Real-time RT-PCR expression analysis of the mdm homolog gene in digestive gland tissue of Mytilus galloprovincialis collected from four different sites in the Campania region (Italy) during a single year. Our results revealed a positive correlation between the expression level of the mdm homolog and the percentage of chronic inflammatory lesions, both of which are increased during the summer period, suggesting a previously unidentified involvement of mdm in inflammatory processes in invertebrate species. Results obtained pointed out the potential interest of the use of mdm gene expression in marine food and seawater quality evaluation monitoring programs.

We hypothesized that pathological perineal descent may be responsible for the failure of operations for obstructed defecation syndrome and that correcting excessive perineal descent may improve the outcome in this group of patients.The purpose of this study was to report the short-term preliminary results of a novel surgical procedure, transverse perineal support, for the correction of pathological perineal descent.This was a prospective, uncontrolled, open-label study.The study was conducted in a hospital and a university center.Among 25 patients observed with failure of previous surgery for obstructed defecation syndrome, 12 with pathological perineal descent underwent transverse perineal support, were followed-up at 6 months, and constituted the object of analysis.The surgical procedure was performed positioning a porcine dermal implant just above the perineum superficial fascia sutured to the periosteum membrane of ischiatic tuberosities at the insertion of the superficial transverse perineal muscle.The main outcome measures were obstructed defecation syndrome score and x-ray and magnetic resonance defecographic imaging evaluation of perineal descent and anorectal manometric parameters.The postoperative median obstructed defecation syndrome score was 7.0 (range, 3-8), showing a statistically significant difference if compared with the preoperative score of 13.5 (range, 9-18; p = 0.0005). The mean postoperative maximum intrarectal pressure was 69.4 ± 11.1 mm Hg, significantly higher than the preoperative pressure of 45.9 ± 12.8 mm Hg (p0.0001). At postoperative x-ray and magnetic resonance imaging defecography, the mean fixed and dynamic perineal descents were significantly lower than the preoperative descents (p = 0.02 for fixed perineal descent and p = 0.0004 for dynamic perineal descent). Of the 4 patients (33.3%) with preoperative pathological dynamic perineal descent, only 1 showed a persistent pathological dynamic perineal descent. No early or late complication was observed.The study was limited by its small size and short follow-up time.Transverse perineal support appears to be a promising, safe, and effective procedure in the treatment of obstructed defecation syndrome associated with pathological perineal descent (see Video, Supplemental Digital Content 1, http://links.lww.com/DCR/A225).

The use of a dedicated score represents an essential tool for the clinical staging of obstructed defecation syndrome (ODS), for subsequent diagnostic and therapeutic options, and for the evaluation of the results. This study was designed to develop and validate a new, simple, and disease-specific scoring system for ODS. The questionnaire consists of 5 items: excessive straining, incomplete rectal evacuation, use of enemas and/or laxatives, vaginal-anal-perineal digitations, and abdominal discomfort and/or pain. Each item was graded from 0 to 5 with a score ranging from 0 ( no symptoms) to 20 ( very severe symptoms). A specific statistical analysis identifies the new score as a valuable and concise instrument, which demonstrates, overall, excellent concurrent validity, reproducibility, internal consistency, and discriminant validity for the diagnosis and grading of ODS. The use of this questionnaire may improve uniformity in clinical research and may allow a more precise evaluation of symptom severity and treatment effectiveness in ODS.

Purpose. This multicenter randomized study was designed to compare the clinical and functional results of stapled transanal rectal resection (STARR) performed with 2 staplers (PPH-01 vs. PPH-03) in the treatment of hemorrhoidal disease associated with a large internal rectal prolapse. Methods. From a total of 937 patients, referred for hemorrhoidal disease in the 20 centers involved in the study, 425 (45.3%) with prolapsed hemorrhoids associated with a large internal rectal prolapse were randomized to undergo STARR with PPH-01 or PPH-03. Postoperative evaluation was made at 3, 6, and 12 months. Results. The incidence of bleeding at the stapled line was significantly lower in the PPH-03 group than in the PPH-01 group (58/207 [28.0%] vs. 145/201 [72.1%]; P < .0001); the mean number of hemostatic stitches was significantly higher in the PPH-01 than in the PPH-03 group (3.2 ± 0.1 vs. 1.8 ± 0.8; P < .0001). The mean operative time was 25.1 ± 11.5 minutes in the PPH-03 group and 38.1 ± 15.7 minutes in the PPH-01 group ( P < .0001). No major complications occurred in either of the groups. At 12-month follow-up, the success rate in the 2 groups was 94.5% in the PPH01 group and 94.2% in the PPH03 group. Conclusion. STARR performed for the treatment of hemorrhoidal disease associated with a large rectal prolapse is a safe and effective procedure. The use of the PPH-03 stapler instead of the PPH-01 guarantees a statistically significant reduction of intraoperative bleeding and a significant decrease of the operative time.

Stapled transanal rectal resection has become the primary surgical procedure for surgical treatment of obstructed defecation syndrome caused by rectocele or rectal intussusception. The procedure is generally performed with 2 circular staplers. Recently, a dedicated contour-curved stapler was developed.This study was designed to compare the effects of these stapler types on relief of symptoms.A randomized controlled trial was conducted at a regional referral center in Naples, Italy.Patients with obstructed defecation syndrome and rectocele or rectal intussusception, treated from November 2005 through September 2007.Participants were randomly assigned to undergo stapled transanal rectal resection with 2 circular staplers or the contour-curved stapler.The primary end point was success rate at 24 months, defined by symptom improvement on an obstructed defecation syndrome scale. Secondary end points included success rate at 12 months, Agachan-Wexner constipation score, and rates of early and late complications at 12 and 24 months.Of 198 patients evaluated, 63 patients (31.8%) satisfied criteria. Follow-up data were available for 61 patients: 30 patients (28 women) in the circular stapler group (mean age, 53; range, 41-75 years) and 31 (29 women) in the contour-curved stapler group (mean age, 55; range, 38-69 years). At 24-month follow-up, success was achieved in 21 patients (70.0%) with the circular staplers and in 27 (87.0%) with the contour-curved stapler (P = .10). Symptom scores improved significantly in both groups from baseline to 12 months (P.0001). Improvement was maintained in the contour-curved stapler group: mean score, 5.0 (SD, 1.6) at 12 months and 5.5 (1.5) at 24 months (P = .20). In the circular stapler group, symptom scores worsened from 4.5 (1.5) at 12 months to 9.0 (1.3) at 24 months (P.0001). At 24 months, the groups differed significantly in symptom scores (P.0001) and constipation scores (P = .03). No significant differences were seen in duration of postoperative hospital stay or rate of early or late complications.Stapled transanal rectal resection with either circular or contour-curved staplers can achieve relief of symptoms in patients with obstructed defecation syndrome. The contour-curved stapler appears to result in more stable clinical results over time.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles., Highlights • The Uncx4.1 and Uncx genes derive from the teleost-specific whole-genome duplication. • Uncx genes are expressed during embryogenesis in unique and overlapping domains. • Uncx gene expression during somite differentiation is regulated by FGF signaling. • Synteny and expression profiles correlate Uncx genes with axon guidance.

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.

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.

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.

Prothymosin alpha (PTMA) is a small acidic protein abundantly and ubiquitously expressed in mammals and involved in different biological activities. Until now, its specific function in spermatogenesis has never been properly investigated. Recently, the isolation of a cDNA encoding for PTMA from the testis of the frog Rana esculenta has been reported: ptma transcript is highly expressed throughout the frog reproductive cycle, peaking in September/October, in concomitance with the germ cell maturation; it is specifically localized in the cytoplasm of primary and secondary spermatocytes and, at a lower level, in the interstitial compartment, in Leydig cells.In this article we support the involvement of PTMA in the meiotic phases of frog spermatogenesis. The expression of ptma mRNA increases in the testis of frogs treated with the antiandrogen cyproterone acetate, which blocks the II meiotic division and induces an increase in SPC cysts; on the contrary, it highly decreases in the testis of animals kept at 4 degrees C and treated with human corionic gonadotropin, in concomitance with the induced block of spermatogenesis and the disappearance of meiotic cells in the tubules. Furthermore, for the first time we have also evidenced by immunohistochemistry the expression of PTMA in the nuclei of secondary spermatocytes, spermatids, and spermatozoa, as well as in the cytoplasm of interstitial Leydig cells. Taken together our data suggest for an important role of PTMA in germ cell maturation and/or differentiation during R. esculenta spermatogenesis.

We examined the spatial expression of the relaxin-3 gene in the developing zebrafish brain, one of the vertebrate model systems in which this gene has been identified. Until the pharyngula stage, the gene is expressed diffusely in the brain, where, starting at about 40 hpf, the transcripts appear restricted in a midbrain cell cluster of the periaqueductal gray. Later, at 72 hpf, the transcripts are still evident in that cluster and distributed in a larger cell number; at this stage, the gene is also expressed posteriorly, in a smaller cell group that, as we report for the first time, could be homologous to mammalian nucleus incertus. The gene expression persists in both cell clusters at 96 hpf. This pattern indicates both conserved and divergent expression features of the relaxin-3 gene among developing zebrafish and rat brains, where only scattered cells express the gene in the periaqueductal gray. Developmental Dynamics 237:3864–3869, 2008. © 2008 Wiley-Liss, Inc.

We show that ptma, a single copy gene found in all organisms investigated so far, is duplicated in zebrafish. The two genes, ptmaa and ptmab, are individually controlled as indicated by their different expression patterns during embryonic development. Only the ptmab transcript is observed at 4 and 8 hpf of development in all embryonic cells, whereas both genes are expressed at later stages as revealed by in situ hybridization studies. In most cases, the two genes are expressed in the same territories, but only the ptmaa transcript was found in the trigeminal ganglion and in endodermal pouches. In the eye, at 72 hpf, the ptmaa and ptmab transcripts were found in amacrine cells, whereas only the ptmab transcript appeared in horizontal cells. The existence of two prothymosin genes indicates that their function in cell proliferation and differentiation is more complex in fishes than in mammals. Developmental Dynamics 237:1112–1118, 2008. © 2008 Wiley-Liss, Inc.

In P. lividus sea urchin the H3.3 histone variant is coded by an mRNA characterized by a long 3′UTR containing ARE (AU-Rich element) motifs. RNA stability assays performed in rabbit reticulocyte lysate showed that such 3′UTR affects the degradation rate of the transcripts. In fact, chimeric molecules containing the 3′UTR of H3.3 transcript, ligated to the coding region of the rabbit β-globin transcript, were unstable whereas chimeric molecules containing mainly the coding region of the H3.3 transcript were stable as the wild-type globin mRNA. Three proteins (45kDa, 32kDa and 25kDa) that bind specifically the 3′UTR have been revealed in the whole protein extracts of embryos at different stages of development. PLAUF, a P. lividus RNA-binding protein similar to human and rodent AUF1 proteins, was identified as the 32kDa factor using anti-PLAUF antibody in Western blot and supershift mobility assays. Moreover the recombinant GST–PLAUF protein specifically binds part of the H3.3 3′UTR and in vitro affects the half-life of the transcript. In addition in situ hybridization experiments demonstrated that PLAUF and H3.3 histone mRNAs co-localize in embryos at different stages of development. In conclusion all the reported results suggest that PLAUF can bind in vivo the 3′UTR of the H3.3 histone mRNA and plays some role in the stability of the mRNA.

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. 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 H2O2-enhanced 3D-EAUS. Surgery was performed by a colorectal surgeon blinded to the 3D-EAUS results. A total of 212 patients with suspected perianal suppurations were assessed during the study period. In 12 patients, the H2O2-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. 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.

Preliminary results have shown that various proteins bind long 3′UTR of the transcript for Paracentrotus lividus sea urchin H3.3 histone variant and are probably implicated in mRNA instability. In order to identify these RNA-binding proteins, we screened a λ-ZAPII cDNA expression library prepared from poly(A) mRNA extracted from sea urchin embryos at blastula stage. We isolated a cDNA that codes for a novel RNA-binding protein homologous to rat and human AUF1 family proteins and we refer to it as PLAUF. Proteins present in the whole lysate of the phages expressing PLAUF bound specifically in vitro the 3′UTR of the H3.3 histone transcript. Northern blot analysis revealed three PLAUF transcripts that are already present in unfertilized eggs; during development their amount increased starting from 4-blastomere embryos and reached the plateau at blastula stage. While the transcription start point was unique, longer 3′UTRs were revealed by 3′RACE approach and further cDNA library screening. Moreover RT-PCR showed the presence of at least one alternative spliced mRNA that codes for a protein with different COOH terminus. The structure of the PLAUF gene was determined by screening a P. lividus sea urchin genomic library with the PLAUF cDNA as probe. Analysis of the positive clones showed that the PLAUF gene is split in 10 exons and 9 introns spanning a distance of about 10 kb. Moreover we demonstrated that the exon 9 was alternative spliced during mRNA processing.

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.

Summary Prothymosin (PTMA) is a highly acidic, intrinsically disordered protein, which is widely expressed and conserved throughout evolution; its uncommon features are reflected by its involvement in a variety of processes, including chromatin remodelling, transcriptional regulation, cell proliferation and death, immunity. PTMA has also been implicated in spermatogenesis: during vertebrate germ cell progression in the testis the protein is expressed in meiotic and post-meiotic stages, and it is associated with the acrosome system of the differentiating spermatids in mammals. Then, it finally localizes on the inner acrosomal membrane of the mature spermatozoa, suggesting its possible role in both the maturation and function of the gametes. In the present work we studied PTMA expression during the spermatogenesis of the adult zebrafish, a species in which two paralogs have been described. Our data show that ptma transcripts are expressed in the testis, and localize in meiotic and post-meiotic germ cells, namely spermatocytes and spermatids. Consistently, the protein is expressed in spermatocytes, spermatids, and spermatozoa: its initial perinuclear distribution is extended to the chromatin region during cell division and, in haploid phases, to the cytoplasm of the developing and final gametes. The nuclear localization in the acrosome-lacking spermatozoa suggests a role for PTMA in chromatin remodelling during gamete differentiation. These data further provide a compelling starting point for the study of PTMA functions during vertebrate fertilization. Prothymosin α (PTMA) is a highly acidic, intrinsically disordered protein, which is widely expressed and conserved throughout evolution; its uncommon features are reflected by its involvement in a variety of processes, including chromatin remodelling, transcriptional regulation, cell proliferation and death, immunity. PTMA has also been implicated in spermatogenesis: during vertebrate germ cell progression in the testis the protein is expressed in meiotic and post-meiotic stages, and it is associated with the acrosome system of the differentiating spermatids in mammals. Then, it finally localizes on the inner acrosomal membrane of the mature spermatozoa, suggesting its possible role in both the maturation and function of the gametes. In the present work we studied PTMA expression during the spermatogenesis of the adult zebrafish, a species in which two paralogs have been described. Our data show that ptma transcripts are expressed in the testis, and localize in meiotic and post-meiotic germ cells, namely spermatocytes and spermatids. Consistently, the protein is expressed in spermatocytes, spermatids, and spermatozoa: its initial perinuclear distribution is extended to the chromatin region during cell division and, in haploid phases, to the cytoplasm of the developing and final gametes. The nuclear localization in the acrosome-lacking spermatozoa suggests a role for PTMA in chromatin remodelling during gamete differentiation. These data further provide a compelling starting point for the study of PTMA functions during vertebrate fertilization.

Analysis of the genome structure of the Paracentrotus lividus (sea urchin) DNA methyltransferase (DNA MTase) gene showed the presence of an open reading frame, named METEX, in intron 7 of the gene. METEX expression is developmentally regulated, showing no correlation with DNA MTase expression. In fact, DNA MTase transcripts are present at high concentrations in the early developmental stages, while METEX is expressed at late stages of development. Two METEX cDNA clones (Met1 and Met2) that are different in the 3′ end have been isolated in a cDNA library screening. The putative translated protein from Met2 cDNA clone showed similarity with Escherichia coli endonuclease III on the basis of sequence and predictive three-dimensional structure. The protein, overexpressed in E. coli and purified, had functional properties similar to the endonuclease specific for apurinic/apyrimidinic (AP) sites on the basis of the lyase activity. Therefore the open reading frame, present in intron 7 of the P. lividus DNA MTase gene, codes for a functional AP endonuclease designated SuAP1.

A cDNA clone encoding for a Prothymosin alpha (Prot-alpha) has been isolated and characterized from the testis of the frog Rana esculenta. Frog Prothymosin alpha (fProt-alpha) predicted a 109 amino acid protein with a high homology to the mammalian Prot-alpha. fProt-alpha contains 28 aspartic and 25 glutamic acid residues and presents the typical basic KKQK amino acid sequence in the close carboxyl terminal region. Northern blot analysis revealed that fProt-alpha is highly expressed in the testis. A different expression of fProt-alpha transcript was found during the frog reproductive cycle with a peak in September/October in concomitance with germ cell maturation, strongly suggesting a role for this protein in the testicular activity. In situ hybridization evidenced that the only germ cells expressing fProt-alpha are the primary and secondary spermatocytes; in addition, the hybridization signal was stronger in the October testis. Taken together, our findings indicate that fProt-alpha might contribute to the efficiency of frog spermatogenesis with a role during the meiosis. This study is the first report on the isolation and characterization of a Prot-alpha in a non-mammalian vertebrate. In addition, our results indicate that the testis of the frog R. esculenta may be a useful model to increase the knowledge concerning the physiological role of Prot-alpha in vertebrates.

RXFP2 is one of the 4 receptors for relaxin insulin-like peptides, in particular it binds with high affinity the INSL3 peptide. INSL3/RXFP2 pair is essential for testicular descent during placental mammalian development. The evolutionary history of this ligand/receptor pair has received much attention, since its function in vertebrate species lacking testicular descent, such as the fishes, remains elusive. Herein, we analyzed the expression pattern of three rxfp2 homologue genes in zebrafish embryonic development. For all the three rxfp2 genes (rxfp2a, rxfp2b, and rxfp2-like) we showed the presence of maternally derived transcripts. Later in the development, rxfp2a is only expressed at larval stage, whereas rxfp2b is expressed in all the analyzed stage with highest level in the larvae. The rxfp2-like gene is expressed in all the analyzed stage with a transcript level that increased starting at early pharyngula stage. The spatial localization analysis of rxfp2-like gene showed that it is expressed in many cell clusters in the developing brain. In addition, other rxfp2-like-expressing cells were identified in the retina and oral epithelium. This analysis provides new insights to elucidate the evolution of rxfp2 genes in vertebrate lineage and lays the foundations to study their role in vertebrate embryonic development.