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Livio CASARINI

Ricercatore t.d. art. 24 c. 3 lett. B presso: Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Medicina, Endocrinologia, Metabolismo e Geriatria


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Pubblicazioni

2019 - Abacavir, nevirapine, and ritonavir modulate intracellular calcium levels without affecting GHRH-mediated growth hormone secretion in somatotropic cells in vitro [Articolo su rivista]
Brigante, G; Riccetti, L; Lazzaretti, C; Rofrano, L; Sperduti, S; Potì, F; Diazzi, C; Prodam, F; Guaraldi, G; Lania, Ag; Rochira, V; Casarini, L
abstract

Growth Hormone (GH) deficiency is frequent in HIV-infected patients treated with antiretroviral therapy. We treated GH3 cells with antiretrovirals (nevirapine, ritonavir or abacavir sulfate; 100 pM-1 mM range), after transfection with human growth hormone releasing hormone (GHRH) receptor cDNA. Cells viability, intracellular cAMP, phosphorylation of CREB and calcium increase, GH production and secretion were evaluated both in basal condition and after GHRH, using MTT, bioluminescence resonance energy transfer, western blotting and ELISA. Antiretroviral treatment did not affect GHRH 50% effective dose (EC50) calculated for 30-min intracellular cAMP increase (Mann-Whitney's U test; p ≥ 0.05; n = 4) nor 15-min CREB phosphorylation. The kinetics of GHRH-mediated, rapid intracellular calcium increase was perturbed by pre-incubation with drugs, while GHRH failed to induce the ion increase in ritonavir pre-treated cells (ANOVA; p < 0.05; n = 3). Antiretrovirals did not impact 24-h intracellular and extracellular GH levels (ANOVA; p ≥ 0.05; n = 3). We demonstrated the association between antiretrovirals and intracellular calcium increase, without consequences on somatotrope cells viability and GH synthesis. Overall, these results suggest that antiretrovirals may not directly impact on GH axis in HIV-infected patients.


2018 - 'Spare' Luteinizing Hormone Receptors: Facts and Fiction. [Articolo su rivista]
Casarini, Livio; Santi, Daniele; Simoni, Manuela; Potì, Francesco.
abstract

It is common opinion that maximal activation of luteinizing hormone (LH)-dependent steroidogenic signal occurs at <1% of human LH/choriogonadotropin (hCG) receptor (LHCGR) occupancy. This effect would be a consequence of an excess of receptors expressed on the surface of theca cells, resulting in a pool of LHCGRs remaining unbound (spare). This concept was borrowed from historical pharmacological studies, when discrepancies between ligand-receptor binding and dose-response curves of cAMP were evaluated by treating mouse or rat Leydig cells with hCG in vitro. Recent findings demonstrated the specificity of LH- and hCG-dependent effects, receptor heterodimerization, and differing behaviors of rodent versus human gonadotropin-responsive cells, which may help to revise the 'spare' LHCGRs concept applied to human ovarian physiology and assisted reproduction.


2018 - FSH (Follicle-Stimulating Hormone) [Capitolo/Saggio]
Santi, Daniele; Casarini, Livio; R Marshall, Gary; Simoni, Manuela
abstract

Follicle-stimulating hormone (FSH) is a glycoprotein regulating development and reproduction. In both adult fertile males and females, FSH mediates spermatogenesis and folliculogenesis, acting through its G-protein coupled receptor (FSHR). Mutations and single nucleotide polymorphisms occurring within the genes encoding the hormone beta subunit and FSHR may modulate or even impair the physiological function of FSH in both males and females. Synthesis and secretion of FSH are described in the chapter, with a specific overview on the pathways activated upon FSH-FSHR interaction and the physiology of the hormone.


2018 - LH (Luteinizing Hormone) [Capitolo/Saggio]
Casarini, Livio; Santi, Daniele; R Marshall, Gary; Simoni, Manuela
abstract

Luteinizing hormone (LH) is secreted by the pituitary gland as a heterodimeric glycoprotein acting on the gonads, regulating development and reproduction. In human of fertile age, it plays a central role in follicle development and spermatogenesis stimulating the production of steroid hormones and mediating proliferative signals. LH acts on a G-protein coupled receptor (LHCGR), shared together with the pregnancy hormone choriogonadotropin (hCG), which features specific intracellular signaling and physiological function. In this chapter, the role exerted by LH during fetal life and fertile age of humans is described.


2018 - Molecular basis of androgen action on human sexual desire [Articolo su rivista]
Santi, Daniele; Spaggiari, Giorgia; Gilioli, Lisa; Potã¬, Francesco; Simoni, Manuela; Casarini, Livio
abstract

Reproduction is a fundamental process for the species maintenance and the propagation of genetic information. The energy expenditure for mating is overtaken by motivational stimuli, such as orgasm, finely regulated by steroid hormones, gonadotropins, neurotransmitters and molecules acting in the brain and peripheral organs. These functions are often investigated using animal models and translated to humans, where the androgens action is mediated by nuclear and membrane receptors converging in the regulation of both long-term genomic and rapid non-genomic signals. In both sexes, testosterone is a central player of this game and is involved in the regulation of sexual desire and arousal, and, finally, in reproduction through cognitive and peripheral physiological mechanisms which may decline with aging and circadian disruption. Finally, genetic variations impact on reproductive behaviours, resulting in sex-specific effect and different reproductive strategies. In this review, androgen actions on sexual desire are evaluated, focusing on the molecular levels of interaction.


2018 - Molecular human reproduction: advancements in clinical and basic research. [Articolo su rivista]
Casarini, Livio
abstract

EDITORIAL - NEW INSIGHTS IN MOLECULAR HUMAN REPRODUCTION


2018 - Pharmacogenetics of G-protein-coupled receptors variants: FSH receptor and infertility treatment [Articolo su rivista]
Santi, Daniele; Potì, Francesco; Simoni, Manuela; Casarini, Livio
abstract

Infertility treatment may represent a paradigmatic example of precision medicine. Follicle-stimulating hormone (FSH) has been proposed as a valuable therapeutic option both in males and in females, even if a standardized approach is far to be established. To date, several genetic mutations as well as polymorphisms have been demonstrated to significantly affect the pathophysiology of FSH-FSH receptor (FSHR) interaction, although the underlying molecular mechanisms remain unclear. This review aims to highlight possible aspects of FSH therapy that could benefit from a pharmacogenetic approach, providing an up-to-date overview of the variability of the response to FSH treatment in both sexes. Specific sections are dedicated to the clinical use of FSH in infertility and how FSHR polymorphisms may affect the therapeutic endpoints.


2018 - Probing the effect of sildenafil on Progesterone and Testosterone production by an intracellullar FRET/BRET combined approach. [Articolo su rivista]
Casarini, Livio; Riccetti, Laura; Limoncella, Silvia; Lazzaretti, Clara; Barbagallo, Federica; Pacifico, Salvatore; Guerrini, Remo; Tagliavini, Simonetta; Trenti, Tommaso; Simoni, Manuela; Sola, Marco; DI ROCCO, Giulia
abstract

Förster Resonance Energy Transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell line for studying the PDE5 inhibitor effect on hCG/LH-induced steroidogenic pathway. The sensor benefits of FRET between, a donor (D), the fluorescein-like diarsenical probe able to covalently bind a tetracysteine motif fused to the PDE5 catalytic domain and, an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant values (Kd) of 5.63.2 M and 13.70.8 M were obtained from HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS-rhodamine conjugate by sildenafil; Ki values were 3.60.3 nM (IC50= 2.3 nM) in HEK293 cells and 101.0 nM (IC50=3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by BRET allowed the exploitation of PDE5i effects on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner, thus confirming previous in vivo findings.


2018 - Response: Commentary: Efficacy of Follicle-Stimulating Hormone (FSH) Alone, FSH + luteinizing hormone, human menopausal gonadotropin or FSH + human chorionic gonadotropin on assisted reproductive technology outcomes in the "Personalized" Medicine Era: A meta-analysis [Articolo su rivista]
Santi, Daniele; Casarini, Livio; Alviggi, Carlo; Simoni, Manuela
abstract

N/A


2018 - The cAMP/PKA pathway: steroidogenesis of the antral follicular stage. [Articolo su rivista]
Riccetti, Laura; Sperduti, Samantha; Lazzaretti, Clara; Casarini, Livio; Simoni, Manuela
abstract

Pituitary gonadotropins, follicle-stimulating (FSH) and luteinizing hormone (LH) promote follicular recruitment and support antral follicle growth, maturation and selection, resulting in ovulation of the dominant follicle. FSH and LH biological functions are mediated by G protein-coupled receptors, FSHR and LHCGR, resulting in the activation of a number of signaling cascades, such as the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Some in-vitro data are consistent with the dual, proliferative and pro-apoptotic role of cAMP, leaving unanswered questions on how cAMP/PKA signaling is linked to the follicle fate. Progression of the antral stage is characterized by the presence of dynamic serum gonadotropin and estrogen levels, accompanying proliferation and steroidogenesis of growing as well as apoptosis of atretic follicles. These events are parallel to changes of FSHR and LHCGR density at the cell surface occurring throughout the antral stage, reasonably modulating the cAMP/PKA activation pattern, cell metabolism and functions. Understanding whether gonadotropins and receptor expression levels impact on the steroidogenic pathway and play a role in determining the follicular fate, may put new light on molecular mechanisms regulating human reproduction. The aim of the present review is to update the role of major players modulating the cAMP/PKA pathway and regulating the balance between proliferative, differentiating and pro-apoptotic signals.


2018 - Two hormones for one receptor: evolution, biochemistry, actions and pathophysiology of LH and hCG [Articolo su rivista]
Casarini, Livio; Santi, Daniele; Brigante, Giulia; Simoni, Manuela
abstract

Luteinizing hormone (LH) and chorionic gonadotropin (CG) are glycoproteins fundamental for sexual development and reproduction. Since they act on the same receptor (LHCGR), there is a general consensus that LH and hCG are equivalent. However, separate evolution of LHβ and hCGβ subunits occurred in primates, resulting in two molecules sharing ∼85% identity and regulating different physiological events. Pituitary, pulsatile LH production results in a ∼90 min half-life molecule targeting the gonads, to regulate gametogenesis and androgen synthesis. Trophoblast hCG, the "pregnancy hormone", exists in several isoforms and glycosylation variants with long half-lives (hours), angiogenic potential, and acts on luteinized ovarian cells as a progestational. The different molecular features of LH and hCG lead to hormone-specific LHCGR binding and intracellular signaling cascades. In ovarian cells, LH action is preferentially exerted through kinases, pERK1/2 and pAKT, resulting in irreplaceable proliferative/anti-apoptotic signals and partial agonism on progesterone production in vitro. In contrast, hCG displays notable cAMP/PKA-mediated steroidogenic and pro-apoptotic potential, which is masked by estrogen action in vivo. In vitro data are confirmed by large dataset from assisted reproduction, since the steroidogenic potential of hCG positively impacts on the number of retrieved oocytes, while LH impacts pregnancy rate (per oocyte number). Interestingly, Leydig cell in vitro exposure to hCG results in qualitatively similar cAMP/PKA and pERK1/2 activation as compared to LH, as well as testosterone. The supposed equivalence of LH and hCG is debunked by such data highlighting their sex-specific functions, thus deeming it an oversight caused by incomplete understanding of clinical data.


2017 - Central hypogonadism due to a giant, “silent” FSH-secreting, atypical pituitary adenoma: effects of adenoma dissection and short-term Leydig cell stimulation by luteinizing hormone (LH) and human chorionic gonadotropin (hCG) [Articolo su rivista]
Santi, Daniele; Spaggiari, Giorgia; Casarini, Livio; Fanelli, Flaminia; Mezzullo, Marco; Pagotto, Uberto; Granata, Antonio R. M; Carani, Cesare; Simoni, Manuela
abstract

We present a case report of an atypical giant pituitary adenoma secreting follicle-stimulating hormone (FSH). A 55-year-old patient presented for erectile dysfunction, loss of libido and fatigue. The biochemical evaluation showed very high FSH serum levels in the presence of central hypogonadism. Neither testicular enlargement nor increased sperm count was observed, thus a secretion of FSH with reduced biological activity was supposed. The histological examination after neuro-surgery showed an atypical pituitary adenoma with FSH-positive cells. Hypogonadism persisted and semen analyses impaired until azoospermia in conjunction with the reduction in FSH levels suggesting that, at least in part, this gonadotropin should be biologically active. Thus, we hypothesized a concomitant primary testicular insufficiency. The patient underwent short-term treatment trials with low doses of either recombinant luteinizing hormone (LH) or human chorionic gonadotropin (hCG) in three consecutive treatment schemes, showing an equal efficacy in stimulating testosterone (T) increase. This is the first case of atypical, giant FSH-secreting pituitary adenoma with high FSH serum levels without signs of testicular hyperstimulation, in presence of hypogonadism with plausible combined primary and secondary etiology. Hypophysectomized patients may represent a good model to assess both pharmacodynamics and effective dose of LH and hCG in the male.


2017 - Efficacy of FSH alone, FSH + LH, hMG or FSH + hCG on ART outcomes in the 'personalized' medicine era: a meta-analysis [Abstract in Rivista]
Santi, Daniele; Casarini, Livio; Alviggi, Carlo; Simoni, Manuela
abstract

Background: Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) act on the same receptor, activating different signal transduction pathways. The role of LH or hCG addition to follicle stimulating hormone (FSH) as well as menopausal gonadotropins (hMG) in controlled ovarian stimulation (COS) is debated. Aim: To compare FSH+LH, or FSH+hCG or hMG vs FSH alone on COS outcomes. Design: A meta-analysis according to PRISMA statement and Cochrane Collaboration was performed, including prospective, controlled clinical trials published until July 2016, enrolling women treated with FSH combined with other gonadotropins. Trials enrolling women with polycystic ovarian syndrome were excluded. Results: Considering 70 studies, the administration of FSH alone resulted in higher number of oocytes retrieved than FSH+LH or hMG. The MII oocytes number did not change when FSH alone was compared to FSH+LH, FSH+hCG or hMG. Embryo number and implantation rate were higher when hMG was used instead of FSH alone. Pregnancy rate was significantly higher in FSH+LH-treated group versus others. Only twelve studies reported live birth rate, not providing protocol-dependent differences. Patients’ stratification by age (median=32.5 years) and/or by GnRH agonist/antagonist identified patient subgroups benefiting from specific drug combinations. Conclusion: In COS, FSH alone results in higher oocyte number. However, hMG improves the collection of mature oocytes and embryos and increases implantation rate, although the final increased pregnancy rate is evident only in GnRH agonist protocol. On the other hand, LH addition leads to higher pregnancy rate. This study supports the concept of a different clinical action of gonadotropins in COS, reflecting previous in vitro data.


2017 - Efficacy of follicle-stimulating hormone (FSH) alone, FSH + luteinizing hormone, human menopausal gonadotropin or FSH + human chorionic gonadotropin on assisted reproductive technology outcomes in the "personalized" medicine era: A meta-analysis [Articolo su rivista]
Santi, Daniele; Casarini, Livio; Alviggi, Carlo; Simoni, Manuela
abstract

Setting: Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) act on the same receptor, activating different signal transduction pathways. The role of LH or hCG addition to follicle-stimulating hormone (FSH) as well as menopausal gonadotropins (human menopausal gonadotropin; hMG) in controlled ovarian stimulation (COS) is debated. Objective: To compare FSH + LH, or FSH + hCG or hMG vs. FSH alone on COS outcomes. Design: A meta-analysis according to PRISMA statement and Cochrane Collaboration was performed, including prospective, controlled clinical trials published until July 2016, enrolling women treated with FSH alone or combined with other gonadotropins. Trials enrolling women with polycystic ovarian syndrome were excluded (PROSPERO registration no. CRD42016048404). Results: Considering 70 studies, the administration of FSH alone resulted in higher number of oocytes retrieved than FSH + LH or hMG. The MII oocytes number did not change when FSH alone was compared to FSH + LH, FSH + hCG, or hMG. Embryo number and implantation rate were higher when hMG was used instead of FSH alone. Pregnancy rate was significantly higher in FSH + LH-treated group vs. others. Only 12 studies reported live birth rate, not providing protocol-dependent differences. Patients' stratification by GnRH agonist/antagonist identified patient subgroups benefiting from specific drug combinations. Conclusion: In COS, FSH alone results in higher oocyte number. HMG improves the collection of mature oocytes, embryos, and increases implantation rate. On the other hand, LH addition leads to higher pregnancy rate. This study supports the concept of a different clinical action of gonadotropins in COS, reflecting previous in vitro data.


2017 - Estrogen Modulates Specific Life and Death Signals Induced by LH and hCG in Human Primary Granulosa Cells In Vitro [Articolo su rivista]
Casarini, Livio; Riccetti, Laura; DE PASCALI, Francesco; Gilioli, Lisa; Marino, Marco; Vecchi, Eugenia; Morini, Daria; Nicoli, Alessia; LA SALA, Giovanni Battista; Simoni, Manuela
abstract

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are glycoprotein hormones used for assisted reproduction acting on the same receptor (LHCGR) and mediating different intracellular signaling. We evaluated the pro- and anti-apoptotic effect of 100 pM LH or hCG, in the presence or in the absence of 200 pg/mL 17β-estradiol, in long-term, serum-starved human primary granulosa cells (hGLC) and a transfected granulosa cell line overexpressing LHCGR (hGL5/LHCGR). To this purpose, phospho-extracellular-regulated kinase 1/2 (pERK1/2), protein kinase B (pAKT), cAMP-responsive element binding protein (pCREB) activation and procaspase 3 cleavage were evaluated over three days by Western blotting, along with the expression of target genes by real-time PCR and cell viability by colorimetric assay. We found that LH induced predominant pERK1/2 and pAKT activation STARD1, CCND2 and anti-apoptotic XIAP gene expression, while hCG mediated more potent CREB phosphorylation, expression of CYP19A1 and procaspase 3 cleavage than LH. Cell treatment by LH is accompanied by increased (serum-starved) cell viability, while hCG decreased the number of viable cells. The hCG-specific, pro-apoptotic effect was blocked by a physiological dose of 17β-estradiol, resulting in pAKT activation, lack of procaspase 3 cleavage and increased cell viability. These results confirm that relatively high levels of steroidogenic pathway activation are linked to pro-apoptotic signals in vitro, which may be counteracted by other factors, i.e., estrogens.


2017 - Genetics of gonadotropins and their receptors as markers of ovarian reserve and response in controlled ovarian stimulation [Articolo su rivista]
Riccetti, Laura; Pascali, Francesco De; Gilioli, Lisa; Santi, Daniele; Brigante, Giulia; Simoni, Manuela; Casarini, Livio
abstract

Several controlled ovarian stimulation (COS) protocols have been developed to increase the yield of mature oocytes retrieved in assisted reproductive techniques (ARTs). The ovarian reserve (OR) influences the COS response, and it represents the main parameter that helps clinicians in refining clinical treatments in the perspective of a "personalized" ART. This approach is even more needed in particular conditions such as poor OR or polycystic ovary syndrome. Follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotropin are currently used in COS at different combinations and with different efficacies, even if the best approach definition is controversial. Differences in individual-specific ovarian response to gonadotropin stimulation can be due to alterations of genes encoding for hormones or their receptors. In particular, FSHB c.-211G>T, FSHR p.Asn680Ser, and c.-29G>A SNP allelic combinations may be used as OR and COS response markers. The purpose of this review is to highlight the evidence-based relevance of mutations and polymorphisms in gonadotropins and their receptor genes as predictive markers of OR and COS response to achieve fine-tuned therapeutic regimens.


2017 - Gonadotropins beyond ART [Articolo su rivista]
De Vincentis, Sara; Casarini, Livio; Simoni, Manuela; Brigante, Giulia
abstract

Gonadotropins (LH, FSH and hCG) play a central role in controlling steroidogenesis and gametogenesis. For this reason, they are largely used in the treatment of infertility, especially in the setting of assisted reproductive technique. Beyond their important action in the regulation of reproduction, gona dotropins are also involved in other hormonal processes, closely interacting with other endocrine axes. Among them, the interaction between gonadal and thyroid axes is widely studied in the literature. There is evidence of an undeniable structural similarity of both hormones and receptors, maybe due to a common ancient origin. Indeed, altered levels of thyroid hormones could lead to different disorders of gonadal development and function throughout entire life, especially before and during pregnancy. Moreover, a complex interplay between insulin-like growth factors and gonadotropins has been described both at central and peripheral level. Finally, several tumors are able to produce gonadotropins or are regulated by them in their own growth. The role of gonadotropins in the regulation of cellular growth and apoptosis is evident by now, but still not fully understood.


2017 - Heterogeneous hCG and hMG commercial preparations result in different intracellular signalling but induce a similar long-term progesterone response in vitro [Articolo su rivista]
Riccetti, Laura; Klett, Danièle; Ayoub, Mohammed Akli; Boulo, Thomas; Pignatti, Elisa; Tagliavini, Simonetta; Varani, Manuela; Trenti, Tommaso; Nicoli, Alessia; Capodanno, Francesco; La Sala, Giovanni Battista; Reiter, Eric; Simoni, Manuela; Casarini, Livio
abstract

STUDY QUESTION: Are four urinary hCG/menotropin (hMG) and one recombinant preparation characterized by different molecular features and do they mediate specific intracellular signaling and steroidogenesis?SUMMARY ANSWER: hCG and hMG preparations have heterogeneous compositions and mediate preparation-specific cell signaling and early steroidogenesis, although similar progesterone plateau levels are achieved in 24 h-treated human primary granulosa cells in vitro.WHAT IS KNOWN ALREADY: hCG is the pregnancy hormone marketed as a drug for ARTs to induce final oocyte maturation and ovulation, and to support FSH action. Several hCG formulations are commercially available, differing in source, purification methods and biochemical composition.STUDY DESIGN, SIZE, DURATION: Commercial hCG preparations for ART or research purposes were compared in vitro.PARTICIPANTS/MATERIALS, SETTING, METHODS: The different preparations were quantified by immunoassay with calibration against the hCG standard (Fifth IS; NIBSC 07/364). Immunoreactivity patterns, isoelectric points and oligosaccharide contents of hCGs were evaluated using reducing and non-reducing Western blotting, capillary isoelectric-focusing immunoassay and lectin-ELISA, respectively. Functional studies were performed in order to evaluate intracellular and total cAMP, progesterone production and beta-arrestin 2 recruitment by ELISA and BRET, in both human primary granulosa lutein cells (hGLC) and luteinizing hormone (LH)/hCG receptor (LHCGR)-transfected HEK293 cells, stimulated by increasing hormone concentrations. Statistical analysis was performed using two-way ANOVA and Bonferroni post-test or Mann-Whitney's U-test as appropriate.MAIN RESULTS AND THE ROLE OF CHANCE: Heterogeneous profiles were found among preparations, revealing specific molecular weight patterns (20-75 KDa range), isoelectric points (4.0-9.0 pI range) and lectin binding (P < 0.05; n = 7-10). These drug-specific compositions were linked to different potencies on cAMP production (EC50 1.0-400.0 ng/ml range) and beta-arrestin 2 recruitment (EC50 0.03-2.0 mu g/ml) in hGLC and transfected HEK293 cells (P < 0.05; n = 3-5). In hGLC, these differences were reflected by preparation-specific 8-h progesterone production although similar plateau levels of progesterone were acheived by 24-h treatment (P >= 0.05; n = 3).LARGE SCALE DATA: N/A.LIMITATIONS, REASONS FOR CAUTION: The biological activity of commercial hCG/hMG preparations is provided in International Units (IU) by in-vivo bioassay and calibration against an International Standard, although it is an unsuitable unit of measure for in-vitro studies. The re-calibration against recombinant hCG, quantified in grams, is based on the assumption that all of the isoforms and glycosylation variants have similar immunoreactivity.WIDER IMPLICATIONS OF THE FINDINGS: hCG/hMG preparation-specific cell responses in vitro may be proposed to ART patients affected by peculiar ovarian response, such as that caused by polycystic ovary syndrome. Otherwise, all the preparations available for ART may provide a similar clinical outcome in healthy women.


2017 - Human LH and hCG stimulate differently the early signalling pathways but result in equal testosterone synthesis in mouse Leydig cells in vitro [Articolo su rivista]
Riccetti, Laura; De Pascali, Francesco; Gilioli, Lisa; Potì, Francesco; Giva, LAVINIA BEATRICE; Marino, Marco; Tagliavini, Simonetta; Trenti, Tommaso; Fanelli, Flaminia; Mezzullo, Marco; Pagotto, Uberto; Simoni, Manuela; Casarini, Livio
abstract

BACKGROUND: Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones regulating development and reproductive functions by acting on the same receptor (LHCGR). We compared the LH and hCG activity in gonadal cells from male mouse in vitro, i.e. primary Leydig cells, which is a common tool used for gonadotropin bioassay. Murine Leydig cells are naturally expressing the murine LH receptor (mLhr), which binds human LH/hCG. METHODS: Cultured Leydig cells were treated by increasing doses of recombinant LH and hCG, and cell signaling, gene expression and steroid synthesis were evaluated. RESULTS: We found that hCG is about 10-fold more potent than LH in cAMP recruitment, and slightly but significantly more potent on cAMP-dependent Erk1/2 phosphorylation. However, no significant differences occur between LH and hCG treatments, measured as activation of downstream signals, such as Creb phosphorylation, Stard1 gene expression and testosterone synthesis. CONCLUSIONS: These data demonstrate that the responses to human LH/hCG are only quantitatively and not qualitatively different in murine cells, at least in terms of cAMP and Erk1/2 activation, and equal in activating downstream steroidogenic events. This is at odds with what we previously described in human primary granulosa cells, where LHCGR mediates a different pattern of signaling cascades, depending on the natural ligand. This finding is relevant for gonadotropin quantification used in the official pharmacopoeia, which are based on murine, in vivo bioassay and rely on the evaluation of long-term, testosterone-dependent effects mediated by rodent receptor.


2017 - Human Luteinizing Hormone and Chorionic Gonadotropin Display Biased Agonism at the LH and LH/CG Receptors. [Articolo su rivista]
Riccetti, Laura; Romain, Yvinec; Danièle, Klett; Nathalie, Gallay; Yves, Combarnous; Eric, Reiter; Simoni, Manuela; Casarini, Livio; Mohammed, Akli Ayoub
abstract

Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) have been considered biologically equivalent because of their structural similarities and their binding to the same receptor; the LH/CGR. However, accumulating evidence suggest that LH/CGR differentially responds to the two hormones triggering differential intracellular signaling and steroidogenesis. The mechanistic basis of such differential responses remains mostly unknown. Here, we compared the abilities of recombinant rhLH and rhCG to elicit cAMP, β-arrestin 2 activation, and steroidogenesis in HEK293 cells and mouse Leydig tumor cells (mLTC-1). For this, BRET and FRET technologies were used allowing quantitative analyses of hormone activities in real-time and in living cells. Our data indicate that rhLH and rhCG differentially promote cell responses mediated by LH/CGR revealing interesting divergences in their potencies, efficacies and kinetics: rhCG was more potent than rhLH in both HEK293 and mLTC-1 cells. Interestingly, partial effects of rhLH were found on β-arrestin recruitment and on progesterone production compared to rhCG. Such a link was further supported by knockdown experiments. These pharmacological differences demonstrate that rhLH and rhCG act as natural biased agonists. The discovery of novel mechanisms associated with gonadotropin-specific action may ultimately help improve and personalize assisted reproduction technologies.


2017 - Primary Leydig cells naturally expressing mouse LHR do not discriminate between LH- and hCGmediated signaling in vitro [Articolo su rivista]
Riccetti, Laura; Gilioli, Lisa; Brigante, Giulia; Simoni, Manuela; Casarini, Livio
abstract

Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones fundamental for development and reproduction. These hormones were considered biologically equivalent for decades due to structural similarities and binding to the same receptor (LHCGR), although they mediate different physiological roles. Previous reports demonstrated LH- and hCGspecific intracellular signaling mediated by LHCGR in human primary granulosa cells, but few studies using rodent receptor (Lhr) are available. We investigated the Lhr-mediated activation of the cAMP/PKA-pathway, ERK1/2 and CREB phosphorylation, gene expression and steroidogenesis, in murine Leydig cells treated with LH and hCG. We found that hCG is more potent than LH in inducing cAMP production, as well as downstream the pERK1/2 activation. However, similar levels of CREB phosphorylation, Stard1 gene expression and testosterone production occurred upon LH and hCG treatment in vitro. These findings revealed that rodent Lhr mediates quantitatively, but not qualitatively, different LH- and hCG-dependent signaling, which results in similar testosterone synthesis. These data suggest that in vivo bioassay using a model expressing rodent receptor, which rely on the evaluation of testosterone-dependent endpoints, may be not suitable to quantify gonadotropins activity for clinical purpose.


2017 - Short term Leydig cell stimulation by LH and hCG in man with central hypogonadism [Abstract in Atti di Convegno]
Santi, Daniele; Spaggiari, G; Casarini, L; Fanelli, F; Mezzullo, M; Pagotto, U; Granata, Arm; Carani, C; Simoni, M
abstract

Short term Leydig cell stimulation by LH and hCG in man with central hypogonadism


2016 - Clinical Applications of Gonadotropins in the Female: Assisted Reproduction and Beyond. [Articolo su rivista]
Casarini, Livio; Simoni, Manuela; Brigante, Giulia; Santi, Daniele
abstract

Gonadotropins (LH, FSH, and hCG) act in concert in the regulation of female reproductive system. Exploiting this influence, they are part of the assisted reproductive technique protocols. In this review we analyze the effectiveness of the different available gonadotropin formulations and the consequent adverse events. Moreover, different protocols for poor-responders and polycystic ovary syndrome affected women are explored. All these clinical different approaches have specific molecular bases, covered in this review starting from evolution and population genetics, getting to in vitro studies of gonadotropins action. Beyond their application in assisted reproductive technique, gonadotropins have also been largely studied for their intertwined network of interactions with other hormones, which all together contribute to the functioning of the reproductive system and other hormonal axes. In particular, there is both clinical and molecular evidence of interaction between thyroid hormones and insulin growth factors with gonadotropins. Finally, gonadotropins are widely studied for their role in the maintenance of the proper balance between cell proliferation and differentiation, and therefore in cancer.


2016 - Follicle-stimulating hormone potentiates the steroidogenic activity of chorionic gonadotropin and the anti-apoptotic activity of luteinizing hormone in human granulosa-lutein cells in vitro [Articolo su rivista]
Casarini, Livio; Riccetti, Laura; De Pascali, Francesco; Nicoli, Alessia; Tagliavini, Simonetta; Trenti, Tommaso; La Sala, Giovanni Battista; Simoni, Manuela
abstract

Luteinizing hormone (LH) and choriogonadotropin (hCG) are glycoprotein hormones regulating ovarian function and pregnancy, respectively. Since these molecules act on the same receptor (LHCGR), they were traditionally assumed as equivalent in assisted reproduction techniques (ART), although differences between LH and hCG were demonstrated at molecular and physiological level. In this study, we demonstrated for the first time that co-treatment with a follicle-stimulating hormone (FSH) dose in the ART therapeutic range potentiates different LH- and hCG-dependent responses in vitro, measured in terms of cAMP, phospho-CREB, -ERK1/2 and -AKT activation, gene expression, progesterone and estradiol production in human granulosa-lutein cells (hGLC). We show that in the presence of FSH, hCG biopotency is about 5-fold increased, in the presence of FSH, in terms of cAMP activation. Accordingly, CREB phosphorylation and steroid production is increased under hCG and FSH co-treatment. LH effects, evaluated as steroidogenic cAMP/PKA pathway activation, do not change in the presence of FSH, which, however, increases LH-dependent ERK1/2 and AKT, but not CREB phosphorylation, resulting in antiapoptotic effects. The different modulatory activity of FSH on LH and hCG action in vitro corresponds to their different physiological functions, reflecting proliferative effects exerted by LH during the follicular phase and before trophoblast development, and the high steroidogenic potential of hCG requested to sustain pregnancy from the luteal phase onwards.


2016 - Gonadotrophin Receptors [Capitolo/Saggio]
Casarini, Livio; Huhtaniemi, Ilpo; Simoni, Manuela; Rivero Müller, Adolfo
abstract

The two gonadotrophin receptors (GnRs), luteinizing hormone receptor (LHCGR) and follicle-stimulating receptor (FSHR), belong to the glycoprotein hormone receptor subgroup of type A G protein-coupled receptors (GPCRs). LHCGR binds specifically the two structurally similar gonadotrophins, luteinizing hormone (LH) and human chorionic gonadotrophin (hCG), and FSHR binds follicle-stimulating hormone (FSH). The receptors reside on plasma membrane and transmit the gonadotrophin signal to target cells using the classical Gs/adenylyl cyclase/cyclic AMP/protein kinase A signaling cascade. Other signaling pathways (e.g., inositol phosphate, calcium) are activated at pharmacological hormone concentrations or at high receptor density. LHCGR is expressed in testicular Leydig cells and in ovarian theca, luteinizing granulosa and luteal cells. FSHR is expressed in testicular Sertoli cells and ovarian granulosa cells. LHCGR activation stimulated Leydig cell steroidogenesis, in particular testosterone production, while FSHR maintains Sertoli cell metabolism, thereby indirectly stimulating spermatogenesis. Recent basic research, using GnR, expressing cells in vitro and genetically modified mice in vivo, has elucidated novel aspects of the molecular mechanisms of gonadotrophin receptor function. The crystal structure of GnRs has also been partly resolved. Numerous inactivating and activating GnR mutations that have been discovered in patients have unraveled the molecular basis of hypogonadism and other aberrations of reproductive endocrine functions. The purpose of this chapter is to review the recent trends of GnR research and how it has elucidated the molecular mechanisms of GnR function and the role of GnR in human reproductive physiology and pathophysiology.


2016 - Is polycystic ovary syndrome a sexual conflict? A review [Articolo su rivista]
Casarini, Livio; Simoni, Manuela; Brigante, Giulia
abstract

Several studies have attempted to explain the high overall prevalence of polycystic ovary syndrome among women worldwide (about 4-10%) despite its link to subfertile phenotypes. For this reason, it is considered an evolutionary paradox. In this review, we show that several genetic loci associated with the disease differently modulate the reproductive parameters of men and women. This observation suggests that such genetic variants lead to opposite effects in the two sexes in reproductive success. Intralocus sexual conflict as a cause of the persistence polycystic ovary syndrome genotypes among humans is supported.


2016 - β-arrestins regulate gonadotropin receptor-mediated cell proliferation and apoptosis by controlling different FSHR or LHCGR intracellular signaling in the hGL5 cell line. [Articolo su rivista]
Casarini, Livio; Reiter, Eric; Simoni Manuela
abstract

Gonadotropin signaling classically involves proliferative, steroidogenic and apoptotic stimuli. In this study, we used the human granulosa cell line hGL5 to demonstrate how follicle-stimulating hormone (FSH) and luteinizing hormone (LH) differently control proliferative or apoptotic signals, revealing novel intrinsic properties of their receptors (FSHR, LHCGR). We found that, in this tumor-like cell line, the expression of endogenous FSHR and LHCGR is serum-dependent, but both receptors were unable to activate the canonical cAMP/PKA pathway upon gonadotropin stimulation, failing to produce cAMP, progesterone and G protein-coupled receptor (GPCR)-mediated apoptosis in vitro. Conversely, ligand treatment resulted in FSHR- and LHCGR-mediated ERK1/2 phosphorylation and cell proliferation due to receptor coupling to β-arrestins. The inactive cAMP/PKA pathway was unlocked by siRNA-mediated knock-down of β-arrestin 1 and 2, leading to progesterone synthesis and apoptosis. Surprisingly, FSH, but not LH treatment accelerated the cAMP/PKA-mediated apoptosis after β-arrestin silencing, an effect which could be reproduced by overexpressing the FSHR, but not the LHCGR. This work demonstrates that the expression of FSHR and LHCGR can be induced in hGL5 cells but that the FSHR-dependent cAMP/PKA pathway is constitutively silenced, possibly to protect cells from FSHR-cAMP-PKA-induced apoptosis. Also, we revealed previously unrecognized features intrinsic to the two structurally similar gonadotropin receptors, oppositely resulting in the regulation of life and death signals in vitro.


2015 - Impact of gene polymorphisms of gonadotropins and their receptors on human reproductive success [Articolo su rivista]
Casarini, Livio; Santi, Daniele; Marino, Marco
abstract

Gonadotropins and their receptors' genes carry several single-nucleotide polymorphisms resulting in endocrine genotypes modulating reproductive parameters, diseases, and lifespan leading to important implications for reproductive success and potential relevance during human evolution. Here we illustrate common genotypes of the gonadotropins and gonadotropin receptors' genes and their clinical implications in phenotypes relevant for reproduction such as ovarian cycle length, age of menopause, testosterone levels, polycystic ovary syndrome, and cancer. We then discuss their possible role in human reproduction and adaptation to the environment. Gonadotropins and their receptors' variants are differently distributed among human populations. Some hints suggest that they may be the result of natural selection that occurred in ancient times, increasing the individual chance of successful mating, pregnancy, and effective post-natal parental cares. The gender-related differences in the regulation of the reproductive endocrine systems imply that many of these genotypes may lead to sex-dependent effects, increasing the chance of mating and reproductive success in one sex at the expenses of the other sex. Also, we suggest that sexual conflicts within the FSH and LH-choriogonadotropin receptor genes contributed to maintain genotypes linked to subfertility among humans. Because the distribution of polymorphic markers results in a defined geographical pattern due to human migrations rather than natural selection, these polymorphisms may have had only a weak impact on reproductive success. On the contrary, such genotypes could acquire relevant consequences in the modern, developed societies in which parenthood attempts often occur at a later age, during a short, suboptimal reproductive window, making clinical fertility treatments necessary.


2015 - Proliferative versus apoptotic signals in granulosa cells: β-arrestins as switch between life and death signals in vitro [Abstract in Atti di Convegno]
Livio, Casarini; Manuela, Simoni
abstract

Background: The immortalized human granulosa cell line hGL5 is not responsive to FSH and LH/hCG, which fail to activate the steroidogenic cAMP/PKA pathway, CREB phosphorylation and progesterone production. Conversely, the activation of adenylyl cyclase by forskolin results in intracellular cAMP increase and steroid production, cell rounding and apoptosis, suggesting a possible FSHR and LHCGR downregulation. Curiously, in hGL5 cells the expression of some receptors (e.g. the oxytocin receptor) is absent in serum starvation conditions and increases in a serum concentration-dependent manner. Aim of the study: To investigate the mechanism of FSHR expression regulation in hGL5 cells and to evaluate whether it is associated with life/death signals in vitro. Methods. We evaluated the FSHR expression in hGL5 cells maintained under different serum concentrations (between 0 and 15%) by real time PCR and Western blotting. The response to 50 nM FSH or 100 pM LH was evaluated by measuring cAMP and progesterone production by ELISA, as well as ERK1/2 and CREB phosphorylation by Western blotting. Cell viability was assessed by proliferation assay and confocal imaging. These endpoints were evaluated in the presence and in the absence of selective inhibitors or agonists (i.e. the PKA inhibitor H-89, the phorbol ester PMA as a PKC-ERK1/2 activator, and siRNA against β-arrestin1/2). Results. The expression of FSHR and LHCGR was serum-dependent at both mRNA and protein level, being absent under starvation and increasing progressively with serum concentrations (linear regression of expression-fold values plotted against serum concentration; p<0.05; n=3). However, FSH/LH stimulation was ineffective both on cAMP and progesterone production and CREB phosphorylation (FSH/LH-stimulated cells vs controls; Mann-Whitney’s U-test; p≥0.05; n=3), suggesting uncoupling of the receptors to the Gs alpha protein. ERK1/2 phosphorylation was FSH/LH dose-dependent in the presence of serum (linear regression; p<0.05; n=3), resulting in a significant increase of cell proliferation over 4 days (FSH/LH-stimulated cells vs controls; Mann-Whitney’s U-test; p<0.05; n=3). A similar increase of cell proliferation and ERK1/2 phosphorylation was provoked by PMA (Mann-Whitney’s U-test; p<0.05; n=3). β-arrestin1/2 siRNA transfection unlocked the cAMP/PKA pathway, leading to cAMP and progesterone accumulation and CREB phosphorylation, at high basal levels (Mann-Whitney’s U-test; p<0.05; n=3). Moreover, siRNA-treated cells underwent cell rounding, pro-caspase 3 cleavage and apoptosis. The pro-apoptotic effects of cAMP/PKA pathway activation were augmented by FSH- but not LH treatment, and inhibited by selective PKA blockade by H-89. Accordingly, transfected hGL5 cells permanently overexpressing the FSHR (but not LHCGR) for 4-8 weeks showed high basal cAMP levels, cell rounding and apoptosis (Mann-Whitney’s U-test; p<0.05; n=3), revealing the dual role of the FSHR in the activation of proliferative and apoptotic signals. Conclusions. Our results suggest that β-arrestins determine the FSHR-mediated (but not LH-mediated) signaling in vitro towards proliferative- or cell death-related pathways. Our results corroborate the relationship between cAMP/PKA pathway activation and cell death in granulosa cells.


2015 - Response to letter by Azziz R., et al. [Articolo su rivista]
Casarini, Livio; Brigante, Giulia
abstract

Comment on The polycystic ovary syndrome evolutionary paradox: a genome-wide association studies-based, in silico, evolutionary explanation. [J Clin Endocrinol Metab. 2014] Letter to the editor re: Casarini and Brigante, 2014, from Azziz R., et al. [J Clin Endocrinol Metab. 2015]


2014 - FSHR polymorphism p.N680S mediates different responses to FSH in vitro [Articolo su rivista]
Casarini, Livio; Moriondo, Valeria; Marino, Marco; Adversi, Francesca; Capodanno, Francesco; Grisolia, Chiarina; La Marca, Antonio; La Sala, Giovanni Battista; Simoni, Manuela
abstract

The single nucleotide polymorphism p.N680S of the follicle-stimulating hormone (FSH) receptor (FSHR) is a discrete marker of ovarian response but previous in vitro studies failed to demonstrate differences in the response to FSH between N and S carrier cells. Here we demonstrate that p.N680S mediates different kinetics of the response to FSH in vitro. Intracellular cAMP production is faster in p.N680S N than in S homozygous human granulosa cells (45 versus 90 min to achieve the plateau, respectively; Mann-Whitney's U-test; p < 0.005; n = 4). Reflecting the cAMP kinetics, phospho-ERK1/2 and -CREB activation, AREG and STARD1 gene expressions and progesterone production were qualitatively and quantitatively different in N versus S homozygous cells. Finally, the blockade of ERK pathway by U0126 abolishes the genotype-mediated different effects on gene expression and progesterone production (Mann-Whitney's U-test; p ≥ 0.005; n = 3).


2014 - Gene polymorphisms in female reproduction [Capitolo/Saggio]
Casarini, Livio; Simoni, Manuela
abstract

This chapter presents an overview of the gene polymorphisms underlying the functions of ovarian receptors and their clinical implications in the female fecundity. A selection of genetic studies revealing significant associations between receptor polymorphisms, gene mutations, and some pathological conditions (i.e., female infertility, premature ovarian failure, polycystic ovary syndrome, endometriosis) are reviewed.


2014 - Mechanisms in endocrinology: Genetics of FSH action: a 2014-and-beyond view [Articolo su rivista]
Simoni, Manuela; Casarini, Livio
abstract

OBJECTIVE: To assess the pharmacogenetic potential of FSH for infertility treatment. DESIGN: Review of the literature and genomic databases. METHODS: Single-nucleotide polymorphism (SNP) assessed: rs6166 (c.2039A>G, p.N680S), rs6165 (c.919A>G, p.T307A), rs1394205 (c.-29G>A) in FSHR, and rs10835638 (c.-211G>T) in FSHB. Literature search via PubMed. Blast analysis of genomic information available in the NCBI nucleotide database. Comparison of allele frequency and haplotype distribution using the http://spsmart.cesga.estool. RESULTS: All these SNPs appear first in Homo, result in reduced FSH action, and are present with variable frequencies and combinations worldwide. Stringent clinical studies demonstrate that the FSHR genotype influences serum FSH levels and gonadal response in both sexes. Serum FSH levels depend on the -211G>T SNP, influencing transcriptional activity of the FSHB promoter. Genotypes reducing FSH action are overrepresented in infertile subjects. CONCLUSIONS: Although the clinical relevance of the FSHR polymorphisms alone is limited, the combination of FSHR and FSHB genotypes has a much stronger impact than either one alone in both sexes. About 20% of people are carriers of the alleles associated with lower serum FSH levels/reduced FSHR expression or activity, possibly less favorable for reproduction. Prospective studies need to investigate whether stratification of infertile patients according to their FSHR-FSHB genotypes improves clinical efficacy of FSH treatment compared with the current, naïve approach. A relative enrichment of less favorable FSHR-FSHB genotypes may be related to changes in human reproductive strategies and be a marker of some health-related advantage at the cost of reduced fertility.


2014 - The FSHR polymorphism p.N680S mediates different response kinetics to FSH in vitro [Abstract in Atti di Convegno]
Casarini, Livio; Moriondo, Valeria; Marino, Marco; Adversi, Francesca; Grisolia, Chiarina; LA MARCA, Antonio; LA SALA, Giovanni Battista; Simoni, Manuela
abstract

Introduction: FSH acts on its receptor (FSHR) resulting in signal transduction activation, gene expression and steroidogenesis. The FSHR common SNP p.N680S is a marker of gonadal response in vivo. However, in vitro dose–response experiments failed to demonstrate the molecular basis thereof so far. In this study, we systematically investigated whether p.N680S mediates different kinetics of FSH response in vitro. Design: We evaluated the activation kinetics of cAMP, phERK1/2, phCREB by ELISA and western blotting in FSHR homozygous, primary, human granulosa lutein cells (hGLC-680N, -680S) stimulated by 50 nM r-FSH for up to 2 h (short-term stimulation). Following short-term stimulation the expression of target genes was evaluated by real-time PCR after 12 h, and progesterone production kinetics over 24 h. Specific inhibitors/agonists (U0126, PMA) were used in the presence and in the absence of FSH. Results: Intracellular cAMP increased within 5–10 min in hGLC-680N, reaching the plateau in about 45 min. cAMP increase was delayed in hGLC-680S, reaching the plateau in 120 min, revealing different activation kinetics (Mann–Whitney U test; P<0.05; n=4). r-FSH-dependent cAMP stimulation kinetics resulted in different ERK1/2 and CREB phosphorylation, reaching maximal levels in 5–30 min in hGLC-680N, whereas, in hGLC-680S, these were weaker and steady over 2 h (Mann–Whitney U test; P<0.05; n=3). hGLC-680N stimulation resulted in higher expression levels of AREG and StAR (Mann–Whitney U test; P<0.05; n=4) and in subsequently different progesterone production kinetics, achieving overall higher levels in hGLC-680N vs -680S (Mann–Whitney U test; P<0.05; n=3). Interestingly, the different kinetics of progesterone production between hGLC-680N and -680S were interchanged by selective phospho-ERK1/2 blockade/activation through specific inhibitor/agonist, revealing a short-term cross-talk mediated by ERK1/2. Conclusions: This study demonstrates for the first time in vitro, how FSHR p.N680S mediates different response to FSH, resulting in different kinetics of cAMP, phERK1/2 and phCREB activation, and progesterone production.


2014 - The PCOS evolutionary paradox: a GWAS-based, in silico, evolutionary explanation [Abstract in Atti di Convegno]
Casarini, Livio; Simoni, Manuela
abstract

Introduction: PCOS is a common endocrine disorder in women exhibiting characteristics ranging from hyperandrogenic to metabolic phenotypes, more prevalent in people of African/Caucasian and Asian ancestry, respectively. Since PCOS impairs fertility without diminishing in prevalence, it was discussed as an evolutionary paradox. GWAS identified 17 SNPs with different allele frequencies, depending on ethnicity, in various susceptibility loci (FSHR, LHCGR, DENND1A, THADA, C9ORF3, YAP1, HMGA2, RAB5B/SUOX, INSR, TOX3, and SUMO1P1). The aim of this study was to analyze in silico the PCOS phenotype–genotype relationship using these SNPs for analysis of genetic clustering and distance, two measures of the degree of similarity of genetic data. Methods: HapMap and HGDP databases (hapmap.ncbi.nlm.nih.gov; www.hagsc.org/hgdp/files.html) were used as source of allele frequencies of the 17 SNPs, using data from 622 male and female individuals of various populations, grouped in Africans, Americans, European-Caucasians, Mediterranean-Middle Easterns, Central Asians, Oceanians and East Asians. Genetic clustering was calculated from SNPs data by Bayesian analysis using the STRUCTURE software (burn-in=5000/50000 MCMC reps; iterations=20; 2<K<10). The inferred ancestry of individuals was matched with PCOS phenotype data of each group, extracted from a previous meta-analysis. The measure of genetic distance was plotted against the geographic distance between the populations. Results: The 622 male and female individuals were assigned to five genetic clusters, matching with different world regions (Kruskal–Wallis/Dunn’s post-test; P<0.0001), and converging in only two main PCOS phenotypes (Anova/Bonferroni post-test; P<0.0001). The overall genetic distance, calculated using PCOS markers, increased along with the geographic distance among the populations (linear regression; r2=0.2106; P<0.0001), in a phenotype-unrelated manner. Conclusions: Phenotype–genotype correlations were demonstrated for PCOS, suggesting that its genetic gradient results from genetic drift together with intralocus sexual conflict rather than natural selection of phenotypic traits in females. Recognizing the genetic background may be important for the correct pharmacological approach to PCOS treatment.


2014 - The TRHR Gene Is Associated with Hypothalamo-Pituitary Sensitivity to Levothyroxine [Articolo su rivista]
Brigante, Giulia; Spaggiari, Giorgia; Santi, Daniele; Cioni, Katia; Gnarini, Valentina; Diazzi, Chiara; Pignatti, Elisa; Casarini, Livio; Marino, Marco; Tüttelmann, Frank; Carani, Cesare; Simoni, Manuela
abstract

Thyroidectomized patients need variable doses of levothyroxine (LT4) to obtain target thyroid-stimulating hormone (TSH) levels. Individual feedback set-points have been hypothesized and the influence of several genes in the regulation of the pituitary-thyroid axis has been demonstrated.


2014 - The polycystic ovary syndrome evolutionary paradox: a genome-wide association studies-based, in silico, evolutionary explanation. [Articolo su rivista]
Casarini, Livio; Brigante, Giulia
abstract

Objective: In this study we analyze the PCOS phenotype-genotype relationship in silico, using SNPs of representative genes for analysis of genetic clustering and distance, to evaluate the degree of genetic similarity. Data Source: 1000 Genomes, HapMap, and Human Genome Diversity Project databases were used as source of allele frequencies of the SNPs, using data from male and female individuals grouped according to their geographical ancestry. Setting and Design: Genetic clustering was calculated from SNPs data by Bayesian inference. The inferred ancestry of individuals was matched with PCOS phenotype data, extracted from a previous meta-analysis. The measure of genetic distance was plotted against the geographic distance between the populations. Results: The individuals were assigned to five genetic clusters, matching with different world regions (Kruskal-Wallis/Dunn's post test; P < .0001), and converging in two main PCOS phenotypes in different degrees of affinity. The overall genetic distance increased with the geographic distance among the populations (linear regression; R2 = 0.21; P < .0001), in a phenotype-unrelated manner. Conclusions: Phenotype-genotype correlations were demonstrated, suggesting that PCOS genetic gradient results from genetic drift due to a serial founder effect occurred during ancient human migrations. The overall prevalence of the disease supports intralocus sexual conflict as alternative to the natural selection of phenotypic traits in females.


2013 - Are pre-miR-146a and PTTG1 associated with papillary thyroid cancer? [Articolo su rivista]
M. Marino; Valentina Cirello;V. Gnarini;Carla Colombo;E. Pignatti;L. Casarini;C. Diazzi;V. Rochira;Katia Cioni;B. Madeo;C. Carani;M. Simoni;Laura Fugazzola
abstract

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, with a steadily increasing incidence in the last few decades worldwide. The predisposition to developing this carcinoma by the heterozygous state of rs2910164 within the precursor of the miR-146a has been reported, but recently not confirmed. Interestingly, on the same chromosome, almost 50 kb separate the pre-miR-146a from the pituitary tumor-transforming gene 1 (PTTG1), a proto-oncogene involved in several tumors, including thyroid cancers. In this study, we analyzed, using a case–control design, the genetic association between PTC and the genomic region encompassing pre-miR-146a rs2910164 and PTTG1 rs1862391 and rs2910202. We enrolled 307 affected patients and 206 healthy controls. The possible presence of thyroid nodules in controls was excluded by ultrasonography. All the cases were submitted to single- nucleotide polymorphism (SNP) genotyping of pre-miR-146a and PTTG1, and risk association analyses were carried out. The genotypic and allelic frequencies of pre-miR-146a rs2910164 were not statistically different in the patients and controls, and this SNP was not in linkage disequilibrium with the investigated PTTG1 SNPs. Consistently, meta-analyses, the first including all the affected cases published to date, did not confirm the previously reported association of the heterozygous CG genotype with PTC. The PTTG1 SNPs exhibited the same allelic frequency in the patients and controls and were not associated with the disease. In conclusion, in a well-selected Italian population, neither pre-miR-146a rs2910164 nor PTTG1 rs1862391 and rs2910202 were found to be associated with the risk of developing PTC.


2013 - Association of pre-miR-146a rs2910164 GG genotype with papillary thyroid cancer: a new case control study on two adjacent genes on chromosome 5, pre-miR-146a and PTTG1 [Abstract in Rivista]
Marco Marino; Valentina Cirello; Valentina Gnarini; Elisa Pignatti; Livio Casarini; Chiara Diazzi; Vincenzo Rochira; Katia Cioni; Bruno Madeo; Manuela Simoni; Laura Fugazzola
abstract

Role of the pre-miR-146a and PTTG1 on papilary thyroid cancer


2013 - Papillary thyroid cancer: a new case-control study involving pre-mir-146a and PTTG1 genes [Abstract in Rivista]
M. Marino; V. Cirello; V. Gnarini; E. Pignatti; L. Casarini; C. Diazzi; V. Rochira; K. Cioni; B. Madeo; M. Simoni; L. Fugazzola
abstract

Studio genetico dei carcinomi papillari della tiroide


2013 - Polymorphisms in gonadotropin and gonadotropin receptor genes as markers of ovarian reserve and response in in vitro fertilization [Articolo su rivista]
La Marca, Antonio; Sighinolfi, Giovanna; Argento, Cindy; Grisendi, Valentina; Casarini, Livio; Volpe, Annibale; Simoni, Manuela
abstract

Since gonadotropins are the fundamental hormones that control ovarian activity, genetic polymorphisms may alter gonadal responsiveness to glycoproteins; hence they are important regulators of hormone activity at the target level. The establishment of the pool of primordial follicles takes place during fetal life and is mainly under genetic control. Consequently, single nucleotide polymorphisms (SNPs) in gonadotropins and their receptors do not seem to be associated with any significant modification in the endowment of nongrowing follicles in the ovary. Indeed, the age at menopause, a biological characteristic strongly related to ovarian reserve, as well as markers of functional ovarian reserve such as anti-Müllerian hormone and antral follicle count, are not different in women with different genetic variants. Conversely, some polymorphisms in FSH receptor (FSHR) seem to be associated with modifications in ovarian activity. In particular, studies suggest that the Ser680 genotype for FSHR is a factor of relative resistance to FSH stimulation resulting in slightly higher FSH serum levels, thus leading to a prolonged duration of the menstrual cycle. Moreover, some FSHR gene polymorphisms show a positive association with ovarian response to exogenous gonadotropin administration, hence exhibiting some potential for a pharmacogenetic estimation of the FSH dosage in controlled ovarian stimulation. The study of SNPs of the FSHR gene is an interesting field of research that could provide us with new information about the way each woman responds to exogenous gonadotropin administration during ovulation induction.


2012 - Aromatase expression in human peripheral blood leukocytes (PBLs) and in various tissues in primates: studies in elderly humans and cynomolgus monkeys [Articolo su rivista]
E. Pignatti; L. Casarini; S. Scaltriti; J. Wistuba; S. Schlatt; A. Rossi; A. Lachhab; E. Taliani; C. Carani; M. Simoni
abstract

Background Previous analysis of aromatase gene and protein expression in PBLs, studied in children and adults, were extended to elderly subjects. In addition we assessed whether aromatase expression in PBLs could be used as a parameter of aromatase expression in other tissues, using the cynomolgus monkey as model. Methods Real-time analysis of aromatase gene expression and protein evaluation by Western blot were performed in PBLs of human elderly subjects and in various tissues from cynomolgus monkeys. Results No gender-related difference in CYP19A1 mRNA and protein expression in PBLs from human elderly women and men was found. In elderly male cynomolgus monkeys CYP19A1 mRNA and protein were expressed in all cells and tissues analysed, with the lowest levels in PBLs but no clear-cut correlation with other tissues. Conclusions Aromatase expression in PBLs in elderly human subjects is not gender-related and cannot be a surrogate of aromatase expression for other tissues.


2012 - LH and hCG Action on the Same Receptor Results in Quantitatively and Qualitatively Different Intracellular Signalling [Articolo su rivista]
L. Casarini; M. Lispi; S. Longobardi; F. Milosa; A. La Marca; D. Tagliasacchi; E. Pignatti; M. Simoni
abstract

Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED50: 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.


2012 - The TRHR gene is associated to hypothalamo-pituitary sensitivity to levothyroxine in thyroidectomized patients [Abstract in Atti di Convegno]
Brigante, G; Spaggiari, G; Cioni, K; Gnarini, V; Pignatti, E; Casarini, L; Marino, M; Tüttelmann, F; Carani, C; Simoni, M
abstract

Background: Patients thyroidectomized for thyroid cancer need variable doses of levothyroxine (LT4) to obtain TSH suppression. A predetermined thyroid function set-point for each individual has been hypothesized, suggesting a genetic influence in the regulation of pituitary-thyroid axis. We hypothesized of the TRHR gene could be associated with a different hypothalamo-pituitary sensitivity to the negative feedback of the thyroid hormones. Methods: We performed a case–control association study, enrolling 107 thyroidectomized patients, in follow-up for differentiated thyroid cancer, and 99 volunteer controls. Patients were evaluated first when TSH levels were suppressed (<0.1 mIU/l), by the lowest effective LT4 dose, and then when TSH was subsuppressed (0.1<TSH<0.5 mIU/l). We selected two SNPs of TRHR gene, rs3134105 and rs3110040, identified as informative markers, using the online database ‘HapMap’. We performed a frequency analysis of the mapped SNPs, followed by a linkage analysis using the HaploView software. Genotyping was performed using the High Resolution Melting technology. Results: The selected SNPs were in linkage disequilibrium. A significant difference between the three possible genotypes for rs3134105 was found for fT4/TSH ratio (P=0.03). Moreover, despite similar serum concentrations of fT3 and fT4 obtained by similar levothyroxine doses, carriers of at least one A allele of rs3134105 had significantly lower serum TSH levels (P=0.04) as well as higher fT3/TSH (P=0.05) and fT4/TSH ratios (P=0.02). Conclusions: We demonstrated an association between TSH and discrete alleles of the TRHR gene identified by the markers SNPs rs3134105 and rs3110040 in totally thyroidectomized patients with diagnosis of thyroid cancer under subsuppressive LT4 therapy. The TRHR gene is a determinant of hypothalamo-pituitary sensitivity to levothyroxine in such patients.


2011 - Effects of polymorphisms in gonadotropin and gonadotropin receptor genes on reproductive function. [Articolo su rivista]
L. Casarini; E. Pignatti; M. Simoni.
abstract

Gonadotropins, the action of which is mediated at the level of their gonadal receptors, play a key role in sexual development, reproductive functions and in metabolism. The involvement of the gonadotropins and their receptor genotypes on reproductive function are widely studied. A large number of gonadotropins and their receptors gene polymorphisms are known, but the only one considerable as a clear, absolute genetic marker of reproductive features or disfunctions is the FSHR Asn680Ser polymorphism, since it modulates ovarian response to FSH. The aim of these studies would to be the prediction of the genetic causes of sex-related diseases to enable a customized clinical setting based on individual response of patients undergoing gonadotropin stimulation. In this review we discuss the latest information about the effects of polymorphisms of the gonadotropins and their receptor genes on reproductive functions of both male and female, and discuss their patho-physiological implications.


2011 - Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells. [Articolo su rivista]
V. Nordhoff; B. Sonntag; D. von Tils; M. Götte; AN. Schüring; J. Gromoll; K. Redmann; L. Casarini; M. Simoni.
abstract

The study was designed to evaluate in vitro the cellular mechanisms of the single nucleotide polymorphism (SNP) p.N680S of the FSH receptor gene (FSHR) in human granulosa cells (GC) and included patients homozygous for the FSHR SNP (NN/SS) undergoing ovarian stimulation. GC were isolated during oocyte retrieval and cultured for 1–7 days. Basal oestradiol and progesterone concentrations were measured after short-term culture. The kinetics of cAMP, oestradiol and progesterone concentrations in response to various amounts of FSH were analysed in a 6–7 day culture. Basal oestradiol, but not progesterone, concentrations on day 1 of GC culture, were significantly higher in NN compared with SS (P = 0.045), but non-responsive to FSH stimulation. Immunofluorescence microscopy demonstrated the re-appearance of FSHR expression with increasing days in culture. Upon stimulation with FSH, GC cultured for 6–7 days displayed a dose-dependent increase of cAMP, oestradiol and progesterone but no difference in the EC50 values between both variants. Primary long-term GC cultures are a suitable system to study the effects of FSH in vitro. However, the experiments suggest that factors down-stream of progesterone production or external to GC might be involved in the clinically observed differences in an FSHR variant-mediated response to FSH


2011 - Two hormone for one receptors: dissecting out LH and hCG activity with an in vitro approach [Abstract in Atti di Convegno]
Casarini, Livio; LA MARCA, Antonio; Pignatti, Elisa; Simoni, Manuela
abstract

Introduction: LH and hCG act on the same receptor (LHCGR), have different half-lives and in vivo biopotency. It is not known whether they elicit the same cellular and molecular response. The aim of this study was to compare the kinetics of cAMP response to recombinant LH and hCG. Design: In COS-7 cells permanently expressing the human LHCGR (COS-7/LHCGR) we evaluated LH and hCG dose-response curves, by measuring total cAMP after 3 h of incubation. We then evaluated the time-course of intracellular cAMP production in the presence of ED50 doses of LH and hCG over 3 h. Finally we evaluated the long-term response to LH and hCG by exposing human primary granulosa lutein cells (hGLC) to ED50 doses over 12 h. All incubations were performed in the presence of IBMX. Results: In COS-7/LHCGR cells, we observed significantly different ED50 for LH (475.75±137.33 pM, mean±S.D.) and hCG (101.75±44.63 pM) (Mann–Whitney’s U-test, P=0.029; n=4). Maximal LH stimulation of intracellular cAMP, about 50 fold over control, reached a plateau in 10 min, while maximal hCG stimulation at similar levels was attained only after 1 h (Anova; P<0.05; n=3). In hGLC continuous exposure to LH and hCG resulted in a repetitive, pulsatile increase of intracellular cAMP with peaks every 3–4 h and significantly higher levels of stimulation in the presence of hCG vs LH (Anova; P<0.05; n=3). Conclusions: Equimolar concentrations of human recombinant LH and hCG result in significantly higher in vitro biopotency of hCG (about 5-fold). Equipotent concentration (ED50) of LH and hCG stimulate a faster response to LH within the first 3 h, but a quantitatively higher response to hCG over 12 h. hGLC respond to constant LH/hCG stimulation in a pulsatile fashion, suggesting a novel control of gonadotropins action at the receptor level.


2009 - Expression of the genes siamois, engrailed-2, bmp4 and myf5 during Xenopus development in presence of the marine toxins okadaic acid and palytoxin. [Articolo su rivista]
A. Franchini; L. Casarini; D. Malagoli; E. Ottaviani
abstract

The present investigation examines the effects of the marine toxins, okadaic acid (OA) and palytoxin (PTX), on some genes involved in the neural and muscular specification and patterning of Xenopus laevis. The RT-PCR analyses performed at different stages of embryonic and larval development (stages 11-47) demonstrated that both toxins induce an over-expression of the genes siamois and engrailed-2 and a different behaviour in bmp4 and myf5. Indeed, OA provoked a significant increase in bmp4 in the earliest stage (11) examined, a down-regulation from stages 12 to 17, and a renewed increase from the beginning of hatching onwards (stages 35-47). In contrast, myf5 was up-regulated in all stages up to 35. PTX induced an over-expression of both bmp4 and myf5 during the embryonic and early larval development stages. The results show that PTX induces an increase in expression levels in all tested genes, while the response to OA seems to be more stage-dependent, with the embryonic development stage more sensitive to the toxin than the larval stages.


2008 - Cytotoxic activity by the mussel Mytilus galloprovincialis and the Venus clam Chamela gallina in the Adriatic sea in 2007 [Articolo su rivista]
D. Malagoli; L. Casarini; F. Fiori; E. Ottaviani
abstract

Given the ecological and economic importance of bivalve molluscs, the evaluation of their welfare is one of the primary aims for both biologists and people working in shell fishing. After a three year-long period monitoring the cytotoxic activity exerted by the hemolymph from the mussel Mytilus galloprovincialis, we have concluded that cytotoxicity represents a useful parameter to evaluate the status of the immune activity and therefore the health of mussels in a specific period of the year. During 2007, we compared the mussel cytoxicity with that of the Venus clam Chamelea gallina from contiguous areas of the Northern Adriatic Sea. Our observations indicate that the cytotoxicity of the hemolymph of the two species follows a similar course during the year, suggesting that cytotoxic activity is primarily determined by the life/reproductive cycles.


2008 - Effects of the marine toxins okadaic acid and palytoxin on mussel phagocytosis. [Articolo su rivista]
D. Malagoli; L. Casarini; E. Ottaviani
abstract

The present study analyzes the effects of the marine toxins okadaic acid (OA) and palytoxin (PTX) on the phagocytic activity of immunocytes from the mussel Mytilus galloprovincialis.In particular, we describe how the effects of the two biotoxins are influenced by the temperature and experimental stress applied before hemolymph withdrawal. The collected data indicate that OA increases phagocytic activity only when hemolymph incubation is performed at 25 C, but not at 20 C, suggesting a certain degree of dependence of OA effects from the status of mussel immunocytes. Conversely, PTX plays an active role in immunocyte signalling transduction pathways, increases the phagocytic activity and markedly promotes the involvement of p38 mitogen-activated protein (MAP) kinase in phagocytosis. Overall, we conclude that both OA and PTX influence mussel phagocytic activity, and the toxic effects may depend on both the mollusc conditions and the activation of specific signalling pathways.


2008 - Toxicological effects of marine palytoxin evaluated by FETAX assay [Articolo su rivista]
A. Franchini; L. Casarini; E. Ottaviani
abstract

The FETAX (frog embryo teratogenesis assay Xenopus) is considered a useful bioassay to detect health hazard substances.In the study of the marine toxin palytoxin (PTX), FETAX has revealed evident impacts on embryo mortality, teratogenesisand growth at the two highest (370 and 37 nM) concentrationsused. Significant mortality rates, peaks in the number of malformed embryos and delays in growth were found, while the total sample number fell by about 80% at the end of the assay with the concentrated dose. The histological analysis to evaluatethe morpho-functional induced modifications demonstrateddamage to the nervous and muscle tissue, a general reductionin the size of the main inner visceral organs and severe injury to the heart structure in some specimens. No inflammatory response was observed.


2007 - Evaluation of the effects of the marine toxin okadaic acid by using FETAX assay [Articolo su rivista]
L. Casarini; A. Franchini; D. Malagoli; E. Ottaviani
abstract

The Frog Embryo Teratogenesis Assay Xenopus (FETAX), is a screening assay using embryos at gastrula stage of the anuran Xenopus laevis to identify substances that may pose a developmental hazard in humans. The FETAX assay evaluates three parameters, i.e. mortality, delayed growth and embryo malformation. In the present investigation, the FETAX protocol was applied to the marine toxin okadaic acid (OA) and the experiments show that OA affects the above parameters in a dose-correlated manner. The morpho-functional modifications induced in embryo organs by OA were also studied. The nervous system, tail skeletal musculature, intestine and kidney appeared particularly damaged, with the former being the most sensitive. On the whole, various advantages emerge from using the FETAX assay: different parameters can be tested simultaneously, the indication of the presence of a potentially dangerous substance is rapid and the assay is a valid alternative to mammalian systems. (c) 2007 Elsevier Ireland Ltd. All rights reserved.


2007 - Monitoring of the immune efficiency of Mytilus galloprovincialis in Adriatic sea mussel farms in 2006: regular changes of cytotoxicity during the year [Articolo su rivista]
D. Malagoli; L. Casarini; E. Ottaviani
abstract

By monitoring the course of hemolymph cytolytic activity in Mytilus galloprovincialis during 2006, we have observed important fluctuations in the percentage of cytotoxic animals over the year. The changes seem to be correlated with seasonal variations in the temperature, but observations in mussels kept in aquaria indicated that this parameter is not the main cause of the fluctuations. Data presented here suggest that normal levels of cytotoxicity can be predicted in a population for a specific period of the year, therefore confirming the value of this parameter in determining the immune efficiency of mussels at a given time.


2007 - Stress and immune response in the mussel Mytilus galloprovincialis. [Articolo su rivista]
D. Malagoli; L. Casarini; S. Sacchi; E. Ottaviani
abstract

The present study investigates the effects on immune-related parameters of various stress factors (air exposure, mechanicalstress, high temperature and extreme salinity conditions) faced by the bivalve mollusc Mytilus galloprovincialis during marketingprocedures. We observed that some stress typologies increase phagocytosis and the number of circulating immunocytes, whileothers can modify immunocyte response towards a further perturbation, i.e. the marine algal toxin yessotoxin. Our results suggest that non-lethal stress can be counteracted for sometime by increasing the level of some defence parameters. Moreover, our data indicate that fishing and transport procedures could interfere with mussel immunosurveillance.


2006 - Algal toxin yessotoxin signalling pathways involve immunocyte mussel calcium channels [Articolo su rivista]
D. Malagoli; L. Casarini; E. Ottaviani
abstract

A fragment of a putative L-type Ca2+ channel has been identified by molecular biology experiments in immunocytes from the mussel Mytilus galloprovincialis. Using the cell permeable and Ca2+-specific fluorochrome FURA 2-AM, we have demonstrated that the algal toxin yessotoxin (YTX) is able to increase intracellular Ca2+ concentration in M. galloprovincialis immunocytes. The YTX effect on Ca2+ increase is inhibited by the L-type Ca2+ channel inhibitor, verapamil, which is cAMP- and cGMP-dependent; but PKA- and nitric oxide-independent. On the basis of these observations, a possible role for YTX as a potential disturber of mussel immune efficiency is suggested. (c) 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.


2006 - Monitoring of the immune efficiency of Mytilus galloprovincialis in Adriatic sea mussel farms in 2005 [Articolo su rivista]
Malagoli, Davide; Casarini, Livio; Ottaviani, Enzo
abstract

The monthly evaluation of the cytotoxicity of hemolymph from the mussel Mytilus galloprovincialis revealed some variations in the percentage of cytotoxic animals during the year. Cytotoxicity is confirmed to be a dynamic parameter that can be used as an indicator of immune efficiency and, therefore, of the state of health of the animals.


2004 - Analysis of the expression pattern of the defensin gene in the lepidopteran Mamestra brassicae. [Articolo su rivista]
Borsatti, Federica; Casarini, Livio; Mandrioli, Mauro
abstract

Southern blotting experiments performed on M. brassicae genomic DNA after digestion with methylation-sensitive restriction enzymes indicated that defensin gene is methylated at CpG targets in the promoter region. However, defensin gene is actively transcribed despite the presence of methylation. Experiments performed by genome demethylation indicated that demethylated defensin gene resulted in altered expression after bacterial induction. In particular, if defensin gene is demethylated it has not possible to observed any increase in gene expression after induction with Gram positive bacteria. The present results are very intriguing since they indicate not only that in M. brassicae DNA methylation is not involved in gene silencing but also that cytosine methylation could be essential to assure the expression of specific genes. Finally, the above reported data apparently argue against a unifying and evolutionary conserved role of cytosine methylation from invertebrates to vertebrates. In fact, it appears that the DNA methylation/gene silencing correlation, which is typically reported in vertebrates, appears not to hold true for insects.