Costanzo Moretti

Expression of functional leptin receptors in rodent Leydig cells.

Several studies indicate that the size of body fat stores and the circulating levels of the adipocyte-derived hormone leptin are able to influence the activity of the hypothalamic-pituitary-gonadal axis. The leptin-hypothalamic-pituitary-gonadal interactions have been mainly studied at the level of the central nervous system. In this study, we investigated the possibility that leptin may have direct effects on the rodent Leydig cell function. To probe this hypothesis, we first analyzed the expression of leptin receptors (OB-R) in rodent Leydig cells in culture. RT-PCR studies showed that rat Leydig cells express both the long (OB-Rb) and short isoform (OB-Ra) of leptin receptor, whereas MLTC-1 cells (a murine Leydig tumor cell line) express only the long isoform. Short-term (30 ‐90 min) incubation of rat Leydig cells with increasing concentrations of leptin (2‐500 ng/ml) led to a significant and dose-dependent inhibition of human (h)CGstimulated testosterone (T) production (;60% reduction, IC50 5 20 ng/ml) but no change in basal androgen release. Also, leptin (150 ng/ml) amplified hCG-induced intracellular cAMP formation (1- to 2-fold) without modifying basal cAMP levels. Subsequent experiments showed that leptin inhibited 8Br-cAMP-stimulated T production, indicating that leptin’s effect is exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T secretion was accompanied by a significant reduction of androstenedione and a concomitant rise of the precursor metabolites pregnenolone, progesterone, and 17-OHprogesterone, conceivable with a leptin-induced lesion of 17,20 lyase activity. Separate experiments performed with the MLTC-1 cells (not expressing cytochrome P450 ‐17a) showed that leptin, though amplifying hCG-stimulated cAMP production, did not modify hCG-stimulated pregnenolone and progesterone release. These results further indicate that leptin action on steroidogenesis occurs downstream of progesterone synthesis. Northern Blot experiments showed no acute effect of leptin on cytochrome P450 ‐17a messenger RNA accumulation in rat Leydig cells in basal and hCG-stimulated conditions, excluding that the rapid changes observed were caused by messenger RNA degradation. In conclusion, these findings, for the first time, show that leptin has direct, receptor-mediated actions on rodent Leydig cells in culture, at concentrations within the range of obese men. (Endocrinology 140: 4939 ‐ 4947, 1999)

Sam68 regulates translation of target mRNAs in male germ cells, necessary for mouse spermatogenesis

Sam68 is a KH-type RNA-binding protein involved in several steps of RNA metabolism with potential implications in cell differentiation and cancer. However, its physiological roles are still poorly understood. Herein, we show that Sam68−/− male mice are infertile and display several defects in spermatogenesis, demonstrating an essential role for Sam68 in male fertility. Sam68−/− mice produce few spermatozoa, which display dramatic motility defects and are unable to fertilize eggs. Expression of a subset of messenger mRNAs (mRNAs) is affected in the testis of knockout mice. Interestingly, Sam68 is associated with polyadenylated mRNAs in the cytoplasm during the meiotic divisions and in round spermatids, when it interacts with the translational machinery. We show that Sam68 is required for polysomal recruitment of specific mRNAs and for accumulation of the corresponding proteins in germ cells and in a heterologous system. These observations demonstrate a novel role for Sam68 in mRNA translation and highlight its essential requirement for the development of a functional male gamete.

Expression of PACAP and its type-I receptor isoforms in the rat ovary

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the secretin/glucagon/vasoactive intestinal peptide (VIP)/growth hormone releasing hormone (GHRH) family of neuropeptides, several of which stimulate steroidogenesis in ovarian granulosa cells. PACAP receptors are of two major subtypes; the type I receptor (PACAP-I-R) has much higher affinity for PACAP than VIP, and the type II receptor (PACAP-II-R) has similar affinity for both peptides. In the rat ovary, expression of the PACAP gene was demonstrated by amplification of ovarian RNA by the reverse transcription/polymerase chain reaction (RT-PCR). In addition, hybridization of Northern blots of rat ovarian poly(A)+ RNA with a 706-nt rat hypothalamic PACAP-I-R cDNA probe revealed the presence of a 7.0 kb PACAP receptor transcript, similar to that detected in brain and hypothalamus. RT-PCR using specific primers for the PACAP-I-R gene yielded products of the expected size with RNA obtained from ovarian tissue, brain, and hypothalamus. The authenticity of the PCR products was confirmed by Southern blotting and nested PCR, which revealed at least three splice variants of the PACAP-I-R in the rat ovary. These findings demonstrate that both PACAP and PACAP-I-R isoforms are expressed in the rat ovary, where they could exert autocrine or paracrine actions on granulosa cell function.

Molecular basis of thyrotropin and thyroid hormone action during implantation and early development

BACKGROUND Implantation and early embryo development are finely regulated processes in which several molecules are involved. Evidence that thyroid hormones (TH: T4 and T3) might be part of this machinery is emerging. An increased demand for TH occurs during gestation, and any alteration in maternal thyroid physiology has significant implications for both maternal and fetal health. Not only overt but also subclinical hypothyroidism is associated with infertility as well as with obstetric complications, including disruptions and disorders of pregnancy, labor, delivery, and troubles in early neonatal life. METHODS We searched the PubMed and Google Scholar databases for articles related to TH action on ovary, endometrium, trophoblast maturation and embryo implantation. In addition, articles on the regulation of TH activity at cellular level have been reviewed. The findings are hereby summarized and critically discussed. RESULTS TH have been shown to influence endometrial, ovarian and placental physiology. TH receptors (TR) and thyrotropin (thyroid-stimulating hormone: TSH) receptors (TSHR) are widely expressed in the feto-maternal unit during implantation, and both the endometrium and the trophoblast might be influenced by TH either directly or through TH effects on the synthesis and activity of implantation-mediating molecules. Interestingly, due to the multiplicity of mechanisms involved in TH action (e.g. differential expression of TR isoforms, heterodimeric receptor partners, interacting cellular proteins, and regulating enzymes), the TH concentration in blood is not always predictive of their cellular availability and activity at both genomic and nongenomic level. CONCLUSIONS In addition to the known role of TH on the hormonal milieu of the ovarian follicle cycle, which is essential for a womans fertility, evidence is emerging on the importance of TH signaling during implantation and early pregnancy. Based on recent observations, a local action of TH on female reproductive organs and the embryo during implantation appears to be crucial for a successful pregnancy. Furthermore, an imbalance in the spatio-temporal expression of factors involved in TH activity might induce early arrest of pregnancy in women considered as euthyroid, based on their hormonal blood concentration. In conclusion, alterations of the highly regulated local activity of TH may play a crucial, previously underestimated, role in early pregnancy and pregnancy loss. Further studies elucidating this topic should be encouraged.

Association of epicardial fat thickness with the severity of obstructive sleep apnea in obese patients

BACKGROUND The correlation between obesity and severity of obstructive sleep apnea (OSA) is controversial. Although fat excess is a predisposing factor for the development of OSA, it has not been determined whether fat distribution rather than obesity per se is associated with OSA severity. Epicardial fat thickness (EFT) is an independent index of visceral adiposity and cardiometabolic risk. We investigated the relation between fat distribution and cardiometabolic risk factors, including EFT and common carotid intima-media thickness (cIMT), with the severity of OSA in obese patients. METHODS One hundred and fifteen obese patients (56 males, 59 females) with polysomnographic evidence of OSA (≥ 5 apnea/hypopnea events per hour) of various degrees, without significant differences in grade of obesity as defined by body mass index (BMI), were evaluated. The following parameters were measured: BMI, body composition by dual energy X-ray absorptiometry, EFT, right ventricular end-diastolic diameter (RVEDD) and cIMT by ultrasound, and parameters of metabolic syndrome (waist circumference, arterial blood pressure, fasting glucose, HDL-cholesterol and triglycerides). RESULTS EFT, RVEDD, cIMT and trunk/leg fat mass ratio showed a positive correlation with OSA severity in univariate analysis (r=0.536, p<0.001; r=0.480, p<0.001; r=0.345, p<0.001; r=0.330, p<0.001, respectively). However, multiple linear regression analysis showed that EFT was the most significant independent correlate of the severity of OSA (R(2)=0.376, p=0.022). CONCLUSIONS The present study suggests that, in obese patients, EFT may be included among the clinical parameters associating with OSA severity. The association of EFT with OSA, both cardiovascular risk factors, is independent of obesity as defined by classical measures.

Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT

BackgroundThe Aurora kinase family members, Aurora-A, -B and -C, are involved in the regulation of mitosis, and alterations in their expression are associated with cell malignant transformation. To date no information on the expression of these proteins in medullary thyroid carcinoma (MTC) are available. We here investigated the expression of the Aurora kinases in human MTC tissues and their potential use as therapeutic targets.MethodsThe expression of the Aurora kinases in 26 MTC tissues at different TNM stages was analyzed at the mRNA level by quantitative RT-PCR. We then evaluated the effects of the Aurora kinase inhibitor MK-0457 on the MTC derived TT cell line proliferation, apoptosis, soft agar colony formation, cell cycle and ploidy.ResultsThe results showed the absence of correlation between tumor tissue levels of any Aurora kinase and tumor stage indicating the lack of prognostic value for these proteins. Treatment with MK-0457 inhibited TT cell proliferation in a time- and dose-dependent manner with IC50 = 49.8 ± 6.6 nM, as well as Aurora kinases phosphorylation of substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it. Cytofluorimetric analysis confirmed that MK-0457 induced accumulation of cells with ≥ 4N DNA content without inducing apoptosis. Finally, MK-0457 prevented the capability of the TT cells to form colonies in soft agar.ConclusionsWe demonstrate that Aurora kinases inhibition hampered growth and tumorigenicity of TT cells, suggesting its potential therapeutic value for MTC treatment.

Obstructive sleep apnea and bone mineral density in obese patients

Context Obesity and its co-morbidities may adversely affect bone mineral density (BMD). Obstructive sleep apnea (OSA) is a major complication of obesity. To date, the effects of OSA on BMD in obese patients have been poorly studied. Objective To examine whether the severity of OSA independently correlates with BMD in obese patients. Methods One hundred and fifteen obese subjects with OSA (Apnea/Hypopnea Index [AHI] ≥5 events per hour) were included in the study. BMD was measured at lumbar spine, total hip, and femoral neck by dual energy X-ray absorptiometry. Body mass index, lean mass, and representative measures of metabolic syndrome (waist circumference, fasting plasma glucose, blood pressure, HDL-cholesterol, triglycerides) and inflammation (ESR, CRP, fibrinogen) were also evaluated. Results BMD did not differ among obese individuals regardless of OSA severity. Correlation coefficient analysis for all the covariates showed a lack of association between AHI and BMD that was strongly influenced by age and weight. Conclusion Our study does not support an independent association between AHI and BMD in obese patients. Controlled studies involving a greater number of patients are warranted.

Insulin-Sensitizers, Polycystic Ovary Syndrome and Gynaecological Cancer Risk

Preclinical, early phase clinical trials and epidemiological evidence support the potential role of insulin-sensitizers in cancer prevention and treatment. Insulin-sensitizers improve the metabolic and hormonal profile in PCOS patients and may also act as anticancer agents, especially in cancers associated with hyperinsulinemia and oestrogen dependent cancers. Several lines of evidence support the protection against cancer exerted by dietary inositol, in particular inositol hexaphosphate. Metformin, thiazolidinediones, and myoinositol postreceptor signaling may exhibit direct inhibitory effects on cancer cell growth. AMPK, the main molecular target of metformin, is emerging as a target for cancer prevention and treatment. PCOS may be correlated to an increased risk for developing ovarian and endometrial cancer (up to threefold). Several studies have demonstrated an increase in mortality rate from ovarian cancer among overweight/obese PCOS women compared with normal weight women. Long-term use of metformin has been associated with lower rates of ovarian cancer. Considering the evidence supporting a higher risk of gynaecological cancer in PCOS women, we discuss the potential use of insulin-sensitizers as a potential tool for chemoprevention, hypothesizing a possible rationale through which insulin-sensitizers may inhibit tumourigenesis.

A Small Supernumerary Marker Derived from the Pericentromeric Region of Chromosome 5: Case Report and Delineation of Partial Trisomy 5p Phenotype

A 17-year-old girl presented with a distinct phenotype mainly featuring craniofacial dysmorphism, including a disproportioned large, round, elongated face; hypertelorism; deep-set eyes with short palpebral fissures; obesity (BMI 37), and a neuropsychiatric disorder with high-functioning autism. Postnatal conventional cytogenetic analyses from peripheral blood revealed a mosaic small supernumerary marker chromosome (sSMC) with a mos 47,XX,+mar[7]/46,XX[43] karyotype. By cenM-FISH technique, the sSMC was identified as a ring derivative of chromosome 5. Metaphase FISH analysis with a set of dedicated probes defined its origin from the pericentromeric region of chromosome 5, including the NIPBL gene at 5p13.2. Such sSMCs, exceedingly rare in the literature, underlie proximal trisomy 5p. In order to delineate a core phenotype of proximal trisomy 5p, we compared our patients features with those of 6 patients found in the literature with similar der(5) chromosomes. Furthermore, a dozen individuals with 5p13 (micro)duplication syndrome was compared and discussed. We identified highly distinctive craniofacial dysmorphism, obesity, and intellectual disability and/or autism spectrum disorder as typical features of proximal 5p trisomy. In the critical region (band 5p13), the NIPBL gene is likely to be a major determinant of the neurobehavioral phenotype, and its presence at the sSMC level may be relevant to predict clinical outcome.

Lack of Influence of the Androgen Receptor Gene CAG-Repeat Polymorphism on Clinical and Electrocardiographic Manifestations of the Brugada Syndrome in Man.

Background Clinical studies suggest that testosterone (T) plays an important role in the male predominance of the clinical manifestations of the Brugada syndrome (BS). However, no statistically significant correlations have been observed between T levels and electrocardiogram (ECG) parameters in the BS patients. We investigated whether the hormonal pattern and the variation within CAG repeat polymorphism in exon 1 of the androgen receptor (AR) gene, affecting androgen sensitivity, are associated with the Brugada ECG phenotype in males. Methods and Results 16 male patients with BS (mean age 45.06 ± 11.3 years) were studied. 12-lead ECG was recorded. Blood levels of follicle-stimulating hormone, luteinizing hormone, prolactin, testosterone, free-T, dihydrotestosterone, 17-β-estradiol, estrone, 3-alpha-androstanediol-glucuronide, delta-4-androstenedione, dehydroepiandrosterone sulphate, progesterone, 17-hydroxyprogesterone, and sex hormone binding globulin were assayed. Genotyping of CAG repeats on DNA extracted from leukocytes was carried out. No relationship was found between hormone values and ECG parameters of BS. BS patients showed the CAG length normally recognized in the human polymorphism range and the number of CAG repeats did not correlate with the ECG pattern of BS. Conclusions The AR CAG repeat length does not correlate with the ECG features of the patients affected by BS. The search for genes downstream AR activation as possibly responsible for the increased risk of spontaneous arrhythmias in BS males after puberty is warranted.