DAAM1 and PREP are involved in human spermatogenesis.

During differentiation of the male gamete, there is a massive remodelling in the shape and architecture of all the cells in the seminiferous epithelium. The cytoskeleton, as well as many associated proteins, plays a pivotal role in this process. To better characterise the factors involved, we analysed two proteins: the formin, dishevelled-associated activator of morphogenesis 1 (DAAM1), which participates in the regulation of actin polymerisation, and the protease, prolyl endopeptidase (PREP), engaged in microtubule-associated processes. In our previous studies we demonstrated their involvement in cytoskeletal dynamics necessary for correct postnatal development of the rat testis. Here, we used samples of testicular tissue obtained from infertile men by testicular sperm extraction and the spermatozoa of asthenoteratozoospermic patients. By western blot and immunofluorescent analysis, we found that DAAM1 and PREP expression and localisation were impaired in both the testis and spermatozoa, and in particular in the midpiece as well as in the principal and end-pieces of the flagella, as compared with spermatozoa of normospermic men. Our results provide new knowledge of the dynamics of spermatogenesis, raising the possibility of using DAAM1 and PREP as new markers of normal fertility.

Childhood trauma and cortisol awakening response in symptomatic patients with anorexia nervosa and bulimia nervosa.

OBJECTIVE: Exposure to trauma during childhood is a risk factor for eating disorders (EDs) in adulthood. The biological mechanisms underlying such increased risk seem to involve the endogenous stress response system (i.e., the hypothalamic-pituitary-adrenal [HPA] axis), which undergoes trauma-induced functional changes that may persist later in life. In the present study, we examined the effects of childhood trauma experiences on HPA-axis activity, comparing saliva cortisol awakening response (CAR) in adult patients with anorexia nervosa (AN) or bulimia nervosa (BN) with CAR in adult healthy controls. METHOD: Twenty-three patients with symptomatic AN, 21 patients with symptomatic BN, and 29 healthy women collected saliva samples at awakening and again after 15, 30, and 60 min. Participants also completed the Childhood Trauma Questionnaire and eating-related psychopathological rating scales. RESULTS: According to the Childhood Trauma Questionnaire, 13 individuals with AN and 12 individuals with BN, but none of the healthy women, reported childhood maltreatment. Compared with the control group, the non-maltreated AN patient group exhibited an enhanced CAR, whereas the group of non-maltreated BN patients showed a normal CAR. Moreover, both AN and BN patient groups with childhood maltreatment exhibited statistically significant blunting of CAR compared with non-maltreated groups. DISCUSSION: The present findings add to the evidence supporting the concept that there is a dysregulation of HPA-axis activity in symptomatic patients with EDs and suggest that childhood trauma exposure may contribute to such dysregulation.

First evidence of prothymosin α localization in the acrosome of mammalian male gametes.

Prothymosin α (PTMA) is a highly acidic intrinsically unstructured protein. Its expression in male gonads is evolutionary conserved; in rat testis it is specifically localized in the cytoplasm of post-meiotic germ cells, in association with the developing acrosome system. In the present paper we investigated on PTMA localization inside the head of mammalian spermatozoa (SPZ). We chose a confocal approach to ascertain whether PTMA is expressed in the acrosome or in the perinuclear theca, two regions that are tightly linked and partially overlapped in the mature haploid cells. The obtained results showed that PTMA is specifically localized in the acrosome of rat epididymal SPZ; the same experimental approach evidenced, for the first time, PTMA presence in human ejaculated SPZ. A Western blot analysis on protein extracts from human sperm head fractions confirmed the confocal data and demonstrated that the peptide is specifically associated with the inner acrosomal membrane fraction. Finally, when the acrosome reaction was induced in vitro by progesterone treatment on both rat and human sperm, PTMA signal was retained in the apical region of reacted SPZ. In conclusion, this study confirms the conservation of PTMA distribution in vertebrate male gametes and strongly supports a role for this polypeptide in their physiology.

Antioxidant Capacity of Carotenoid Extracts from the Haloarchaeon Halorhabdus utahensis.

Herein, we report on the production, characterization, and antioxidant power assessment of carotenoids from the haloarchaeon Halorhabdus utahensis. It was grown at 37 °C and 180 rpm agitation in halobacteria medium supplemented with glucose, fructose, and xylose, each at concentrations of 0.2%, 1%, and 2%, and the carotenoid yield and composition were investigated. The microorganism produced the carotenoids under all the conditions tested, and their amount followed the order glucose < xylose < fructose. The highest yield was achieved in 2% fructose growth medium with 550.60 ± 7.91 µg/g dry cell and 2428.15 ± 49.33 µg/L. Separation and identification of the carotenoids were performed by RP-HPLC and HPLC/APCI-ITMSn. Bacterioruberin was the main carotenoid detected and accounted for 60.6%, 56.4%, and 58.9% in 2% glucose, 1% xylose, and 2% fructose extracts, respectively. Several geometric isomers of bacterioruberin were distinguished, and representatives of monoanhydrobacterioruberin, and bisanhydrobacterioruberin were also detected. The assignment to cis-isomers was attempted through analysis of the UV/Vis spectra, intensity of cis peaks, and spectral fine structures. The extracts exhibited superoxide scavenging activity higher than butylhydroxytoluene, ascorbic acid, and Trolox, selected as antioxidant references. The anti-hyaluronidase capacity was investigated, and the 2% fructose extract showed the highest activity reaching 90% enzyme inhibition with 1.5 µg. The overall data confirm that Hrd. utahensis can be regarded as an interesting source of antioxidants that can find applications in the food and cosmetic sectors.

The acute salivary ghrelin response to a psychosocial stress is enhanced in symptomatic patients with bulimia nervosa: a pilot study.

BACKGROUND: Stress is a precipitating factor for both binge eating and bulimia nervosa (BN); however, the biological mechanisms through which it may trigger binge eating are poorly understood. There is evidence that the adrenal hormone cortisol and the gastric peptide ghrelin might be involved in stress-induced food ingestion. We hypothesized that symptomatic patients with BN might disclose deranged responses of ghrelin and/or cortisol to stressors and that this could be related to their binge-eating behaviour. METHODS: Here we investigated salivary cortisol and ghrelin responses to the Trier Social Stress Test (TSST) in 10 women with acute BN and 10 age-matched healthy females. Eating-related psychopathology and behaviours were assessed by self-report measures. RESULTS: No significant differences emerged between bulimic patients and healthy controls in the pre-stress salivary levels of both cortisol and ghrelin. The BN patients displayed normal cortisol but enhanced ghrelin responses to TSST. No significant correlations emerged between stress-induced salivary hormone changes and self-report measures of binge eating. CONCLUSION: To our knowledge, this is the first study showing deranged salivary ghrelin reactivity to a psychosocial stressor in symptomatic patients with BN. The extent to which this could contribute to the binge-eating behaviour of BN subjects awaits clarification.

Application of Vibrational Spectroscopies in the Qualitative Analysis of Gingival Crevicular Fluid and Periodontal Ligament during Orthodontic Tooth Movement.

Optical vibrational techniques show a high potentiality in many biomedical fields for their characteristics of high sensitivity in revealing detailed information on composition, structure, and molecular interaction with reduced analysis time. In the last years, we have used these techniques for investigating gingival crevicular fluid (GCF) and periodontal ligament (PDL) during orthodontic tooth treatment. The analysis with Raman and infrared signals of GCF and PDL samples highlighted that different days of orthodontic force application causes modifications in the molecular secondary structure at specific wavenumbers related to the Amide I, Amide III, CH deformation, and CH3/CH2. In the present review, we report the most relevant results and a brief description of the experimental techniques and data analysis procedure in order to evidence that the vibrational spectroscopies could be a potential useful tool for an immediate monitoring of the individual patient's response to the orthodontic tooth movement, aiming to more personalized treatment reducing any side effects.

Expression of melatonin (MT1, MT2) and melatonin-related receptors in the adult rat testes and during development.

It is well known that melatonin provokes reproductive alterations in response to changes in hours of daylight in seasonally breeding mammals, exerting a regulatory role at different levels of the hypothalamic-pituitary-gonadal axis. Although it has also been demonstrated that melatonin may affect testicular activity in vertebrates, until now, very few data support the hypothesis of a local action of melatonin in the male gonads. The aim of this study was to investigate whether MT1, MT2 melatonin receptors and the H9 melatonin-related receptor, are expressed in the adult rat testes and during development. A semi-quantitative RT-PCR method was used to analyse the expression of MT1, MT2 and H9 receptors mRNAs in several rat tissues, mainly focusing on testes during development and adult life. Our results provide molecular evidences of the presence of both MT1 and, for the first time, MT2 melatonin receptors as well as of the H9 melatonin-related receptor in the examined tissues, including adult testes. During development MT1 and MT2 transcripts are expressed at lower levels in testes of rats from 1 day to 1 week of age, lightly increased at 2 weeks of age and remained permanently expressed throughout development until 6 months. These data strongly support the hypothesis that melatonin acts directly in male vertebrate gonads suggesting that rat testes may be a suitable model to verify the role of indolamine in vertebrate testicular activity.

Association between A218C polymorphism of the tryptophan-hydroxylase-1 gene, harm avoidance and binge eating behavior in bulimia nervosa.

Genes involved in serotonin transmission are likely involved in the biological predisposition to bulimia nervosa. We investigated whether the A218C polymorphism of the tryptophan-hydroxylase-1 gene was associated to bulimia nervosa and/or to some phenotypic aspects of the disorder. One hundred eighty Caucasian women (91 patients with bulimia nervosa and 89 healthy controls) were enrolled into the study. They underwent a blood sample collection for A218C polymorphism of the tryptophan-hydroxylase-1 genotyping and a clinical evaluation assessing comorbidity for Axis I and II psychiatric disorders, harm avoidance personality dimension and bulimic symptoms. The distribution of both tryptophan-hydroxylase-1 A218C genotypes and alleles did not significantly differ between patients and controls. Bulimic women with the AA genotype exhibited a more severe binge eating behavior and higher harm avoidance scores than those with CC genotype. These findings support the idea that tryptophan-hydroxylase-1 A218C polymorphism does not play a part in the genetic susceptibility to bulimia nervosa, but it seems to be involved in predisposing bulimic patients to a more disturbed eating behavior and higher harm avoidance.

Underweight subjects with anorexia nervosa have an enhanced salivary cortisol response not seen in weight restored subjects with anorexia nervosa.

The cortisol response to awakening (CAR) has been reported to be enhanced in symptomatic patients with anorexia nervosa (AN). However, it has been not established whether the dysregulation of CAR was a primary phenomenon or a change secondary to malnutrition. Therefore, we aimed to explore the salivary CAR in both underweight and weigh-restored women with AN. Fifty-nine women volunteered for the study. They were 18 underweight AN women, 15 weight-restored AN women and 26 normal-weight healthy women. Saliva samples were collected in the morning, immediately on awakening and after 15, 30 and 60min to measure saliva levels of cortisol. Participants' anxiety levels in the morning of sampling were measured by the State-Trait Anxiety Inventory. As compared to control women, underweight AN patients showed an enhanced CAR whereas weight-restored patients had a normal CAR. These results could be not explained by group differences in body mass index or levels of anxiety. These findings show, for the first time, that the enhanced CAR occurring in the acute phase of AN is not seen in weight-recovered patients, suggesting that the dysregulated activity of the hypothalamus-pituitary-adrenal axis of symptomatic AN patients is a state-dependent phenomenon.

First evidence of a cDNA encoding for a melatonin receptor (mel 1b) in brain, retina, and testis of Pelophylax esculentus.

Melatonin, nocturnally secreted by the pineal gland, regulates a variety of physiological functions, including reproduction. Here, we investigated the evidence of melatonin binding sites in frog tissue (brain, retina, and testis) through saturation and competition binding experiments. In the frog, Pelophylax esculentus, our results confirm the presence of a single class of melatonin-specific binding sites in the brain and retina, but not in the testis. Further experiments have been done using biomolecular approaches (PCR analysis). Here, we report the isolation of a cDNA encoding for a melatonin receptor type (mel 1b) from brain, retina, and testis of the P. esculentus. PCR analysis revealed that melatonin expression is higher in the brain and retina, whereas it is lower in the testis. The presence of a melatonin receptor transcript in the frog testis corroborates our previous results obtained in in vitro experiments that suggest that melatonin might act directly in male vertebrate gonads, and indicates that the frog testis may be a suitable model to verify the role of indolamine in testicular activity.

Abnormal diurnal patterns of salivary α-amylase and cortisol secretion in acute patients with anorexia nervosa.

OBJECTIVES: The evidence that the activity of the sympathetic nervous system (SNS) is decreased in acute anorexia nervosa (AN) is not consistent. Therefore, we aimed to assess the SNS basal activity in malnourished AN patients through the measurement of diurnal salivary levels of α-amylase, whose secretion is regulated by the SNS. As secondary aim, we measured also salivary cortisol. METHODS: Eight symptomatic female patients with restrictive AN and eight age-matched healthy women underwent saliva sample collection at awakening and over the day. α-amylase and cortisol were assayed by ELISA method. RESULTS: In both patients and controls, saliva α-amylase levels significantly decreased during 60 min after awakening and then progressively rose towards the afternoon/evening. AN patients exhibited significantly reduced levels of the salivary enzyme with a significant decrease in its overall diurnal secretion and a dysregulated secretory pattern. As compared to control women, AN patients exhibited significantly enhanced levels of salivary cortisol at awakening, an enhanced and advanced cortisol secretion after awakening but no significant change in the overall diurnal secretion of the salivary hormone. CONCLUSIONS: These results suggest that the activity of the SNS, evaluated through the assessment of the diurnal secretion of salivary α-amylase, is impaired in the acute phase of AN whereas the cortisol awakening response is enhanced.

Inhibition of the increased 17beta-estradiol-induced mast cell number by melatonin in the testis of the frog Rana esculenta, in vivo and in vitro.

In the present study, we have utilized 17beta-estradiol to induce the increase of mast cell number in order to verify the melatonin effect on mast cell accumulation in the frog testicular interstitium. Data obtained from in vivo experiments confirm that 17beta-estradiol increases the mast cell number and indicate a melatonin-inhibitory role in their accumulation in the frog testis. In addition, melatonin interferes with the effects of estradiol on the increase of mast cell number in short-term cultured testes, and this result has also been obtained in a dose-response experiment at physiological concentration. The data suggest that melatonin acts on mast cell number directly via its local action in the frog gonads. In conclusion, our study shows, for the first time, that melatonin may interfere, probably via estrogen receptors, with the differentiation and/or proliferation of mast cells induced by estradiol treatment either in vivo or in vitro in the testis of the frog Rana esculenta.

Effects of melatonin treatment on Leydig cell activity in the testis of the frog Rana esculenta.

This study was conducted to verify the effect(s) of melatonin treatment on frog Leydig cells. Morphological observation after melatonin treatment indicates that many frog Leydig cells show degenerative changes (i.e. heterochromatic nuclei, loss of cellular adhesion) while in adjacent germinal tubules several Sertoli cells show heterochromatic nuclei, confirming the presence of a paracrine effect between interstitial and germinal compartments. The effect of melatonin on frog Leydig cell steroidogenesis was investigated in in vitro experiments; after 6 h of incubation melatonin severely inhibits both control and GnRH-induced testosterone secretion. In addition, in order to verify the effect of indolamine on frog Leydig cell activity, we investigated, by in situ hybridization, the presence of frog relaxin (fRLX, a transcript specifically expressed by these cells) in the testes of melatonin-injected animals after 48 h. fRLX signal completely disappeared from the testis of melatonin- injected frogs. The results of the present study indicate that melatonin treatment provokes Leydig cell morphological changes, blocks GnRH-antagonist-induced testosterone secretion and decreases fRLX expression. Taken together these results strongly indicate that melatonin acts on Leydig cells in the testis of the frog Rana esculenta.