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CLARA LAZZARETTI

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Pubblicazioni

2024 - Benzo[a]pyrene disrupts LH/hCG-dependent mouse Leydig cell steroidogenesis through receptor/Gαs protein targeting [Articolo su rivista]
Lazzaretti, C.; Roy, N.; Paradiso, E.; Capponi, C.; Ferrari, T.; Reggianini, F.; Sperduti, S.; Perri, C.; Baschieri, L.; Mascolo, E.; Varani, M.; Canu, G.; Trenti, T.; Nicoli, A.; Morini, D.; Iannotti, F.; Villani, M. T.; Vicini, E.; Simoni, M.; Casarini, L.
abstract

Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzo[a]pyrene (BaP), induce reproductive toxicity. Several existing studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 μM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.

2023 - Allosteric modulation of gonadotropin receptors [Articolo su rivista]
Lazzaretti, Clara; Simoni, Manuela; Casarini, Livio; Paradiso, Elia
abstract

Gonadotropins regulate reproductive functions by binding to G protein-coupled receptors (FSHR and LHCGR) expressed in the gonads. They activate multiple, cell-specific signalling pathways, consisting of ligand-dependent intracellular events. Signalling cascades may be modulated by synthetic compounds which bind allosteric sites of FSHR and LHCGR or by membrane receptor interactions. Despite the hormone binding to the orthosteric site, allosteric ligands, and receptor heteromerizations may reshape intracellular signalling pattern. These molecules act as positive, negative, or neutral allosteric modulators, as well as non-competitive or inverse agonist ligands, providing a set of new compounds of a different nature and with unique pharmacological characteristics. Gonadotropin receptor allosteric modulation is gathering increasing interest from the scientific community and may be potentially exploited for clinical purposes. This review summarizes the current knowledge on gonadotropin receptor allosteric modulation and their potential, clinical use.

2023 - Gene expression differences between small and large follicles [Abstract in Rivista]
Baschieri, Lara; Cimadomo, Danilo; Davolio, Federica; Sperduti, Samantha; Mascolo, Elisa; Paradiso, Elia; Lazzaretti, Clara; Perri, Carmela; Roy, Neena; Innocenti, Federica; Albricci, Laura; Rienzi, Laura; Vaiarelli, Alberto; Maria, Ubaldi Filippo; Simoni, Manuela; Casarini, Livio
abstract

2023 - In vitro evaluation of the impact of two differently glycosylated recombinant FSH on signal transduction [Abstract in Rivista]
Mascolo, Elisa; Baschieri, Lara; Roy, Neena; Lazzaretti, Clara; Paradiso, Elia; Perri, Carmela; Sperduti, Samantha; Simoni, Manuela; Casarini, Livio
abstract

2023 - Intracellular cGMP increase is not involved in thyroid cancer cell death [Articolo su rivista]
Alessandro, Sara D'; Paradiso, Elia; Lazzaretti, Clara; Sperduti, Samantha; Perri, Carmela; Antoniani, Francesco; Righi, Sara; Simoni, Manuela; Brigante, Giulia; Casarini, Livio
abstract

Introduction: Type 5 phosphodiesterase (PDE5) inhibitors (PDE5i) lead to intracellular cyclic-guanosine monophosphate (cGMP) increase and are used for clinical treatment of erectile dysfunction. Studies found that cGMP may up/downregulate the growth of certain endocrine tumor cells, suggesting that PDE5i could impact cancer risk. Aim: We evaluated if PDE5i may modulate thyroid cancer cell growth in vitro. Materials and methods: We used malignant (K1) and benign (Nthy-ori 3-1) thyroid cell lines, as well as the COS7 cells as a reference model. Cells were treated 0-24 h with the PDE5i vardenafil or the cGMP analog 8-br-cGMP (nM-μM range). cGMP levels and caspase 3 cleavage were evaluated by BRET, in cGMP or caspase 3 biosensor-expressing cells. Phosphorylation of the proliferation-associated extracellularly-regulated kinases 1 and 2 (ERK1/2) was evaluated by Western blotting, while nuclear fragmentation by DAPI staining. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Both vardenafil and 8-br-cGMP effectively induced dose-dependent cGMP BRET signals (p≤0.05) in all the cell lines. However, no differences in caspase 3 activation occurred comparing PDE5i-treated vs untreated cells, at all concentrations and time-points tested (p>0.05). These results match those obtained upon cell treatment with 8-br-cGMP, which failed in inducing caspase 3 cleavage in all the cell lines (p>0.05). Moreover, they reflect the lack of nuclear fragmentation. Interestingly, the modulation of intracellular cGMP levels with vardenafil or the analog did not impact cell viability of both malignant and benign thyroid tumor cell lines, nor the phosphorylation of ERK1/2 (p>0.05). Conclusions: This study demonstrates that increased cGMP levels are not linked to cell viability or death in K1 and Nthy-ori 3-1 cell lines, suggesting that PDE5i do not impact the growth of thyroid cancer cells. Since different results were previously published, further investigations are recommended to clarify the impact of PDE5i on thyroid cancer cells.

2023 - Lack of GPER-TSHR heteromers is a hallmark of thyroid cancer [Abstract in Rivista]
Perri, Carmela; D'Alessandro, Sara; Paradiso, Elia; Lazzaretti, Clara; Mascolo, Elisa; Baschieri, Lara; Roy, Neena; Sperduti, Samantha; Simoni, Manuela; Brigante, Giulia; Casarini, Livio
abstract

2023 - LH increases the response to FSH in granulosa-lutein cells from sub/poor-responder patients in vitro [Articolo su rivista]
Sperduti, Samantha; Paradiso, Elia; Anzivino, Claudia; Lazzaretti, Clara; Limoncella, Silvia; D'Alessandro, Sara; Roy, Neena; Reggianini, Francesca; Ferrari, Tommaso; Melli, Beatrice; La Sala, Giovanni Battista; Nicoli, Alessia; Daolio, Jessica; Villani, Maria Teresa; Tagliavini, Simonetta; Trenti, Tommaso; Potì, Francesco; Sandhowe, Reinhild; Centonze, Chiara; Lispi, Monica; Simoni, Manuela; Casarini, Livio
abstract

STUDY QUESTION Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response? SUMMARY ANSWER A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women. WHAT is KNOWN ALREADY Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART. STUDY DESIGN, SIZE, DURATION hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) +/- 0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors' ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well. MAIN RESULTS AND THE ROLE of CHANCE hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P < 0.05). The addition of LH to the cell treatment significantly increased overall FSH efficacy, indicated by cAMP and progesterone levels, within all groups (P > 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders. LIMITATIONS, REASONS FOR CAUTION Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage. WIDER IMPLICATIONS of THE FINDINGS This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare.

2023 - Protein kinase B (Akt) blockade inhibits LH/hCG-mediated 17,20-lyase, but not 17[alpha]-hydroxylase activity of CYP17a1 in mouse Leydig cell steroidogenesis [Abstract in Rivista]
Paradiso, Elia; Lazzaretti, Clara; Sperduti, Samantha; Melli, Beatrice; Perri, Carmela; D'Alessandro, Sara; Baschieri, Lara; Mascolo, Elisa; Roy, Neena; Simoni, Manuela; Casarini, Livio
abstract

2023 - Protein kinase B (Akt) blockade inhibits LH/hCG-mediated 17,20-lyase, but not 17α-hydroxylase activity of Cyp17a1 in mouse Leydig cell steroidogenesis [Articolo su rivista]
Paradiso, Elia; Lazzaretti, Clara; Sperduti, Samantha; Melli, Beatrice; Trenti, Tommaso; Tagliavini, Simonetta; Roli, Laura; D'Achille, Fabio; Beltrán-Frutos, Ester; Simoni, Manuela; Casarini, Livio
abstract

: Androgens are produced by adrenal and gonadal cells thanks to the action of specific enzymes. We investigated the role of protein kinase B (Akt) in the modulation of Δ4 steroidogenic enzymes' activity, in the mouse Leydig tumor cell line mLTC1. Cells were treated for 0-24 h with the 3 × 50% effective concentration of human luteinizing hormone (LH) and choriogonadotropin (hCG), in the presence and in the absence of the specific Akt inhibitor 3CAI. Cell signaling analysis was performed by bioluminescence resonance energy transfer (BRET) and Western blotting, while the expression of key target genes was investigated by real-time PCR. The synthesis of progesterone, 17α-hydroxy (OH)-progesterone and testosterone was measured by immunoassay. Control experiments for cell viability and caspase 3 activation were performed as well. We found that both hormones activated cAMP and downstream effectors, such as extracellularly-regulated kinase 1/2 (Erk1/2) and cAMP response element-binding protein (Creb), as well as Akt, and the transcription of Stard1, Hsd3b1, Cyp17a1 and Hsd17b3 genes, boosting the Δ4 steroidogenic pathway. Interestingly, Akt blockade decreased selectively Cyp17a1 expression levels, inhibiting its 17,20-lyase, but not the 17-hydroxylase activity. This effect is consistent with lower Cyp17a1 affinity to 17α-OH-progesterone than progesterone. As a result, cell treatment with 3CAI resulted in 17α-OH-progesterone accumulation at 16-24 h and decreased testosterone levels after 24 h. In conclusion, in the mouse Leydig cell line mLTC1, we found substantial Akt dependence of the 17,20-lyase activity and testosterone synthesis. Our results indicate that different intracellular pathways modulate selectively the dual activity of Cyp17a1.

2023 - Reprogramming of reproductive signals via human luteinizing hormone/choriogonadotropin receptor (LHCGR)/G protein-coupled estrogen receptor (GPER) heteromers [Abstract in Rivista]
Lazzaretti, Clara; Paradiso, Elia; Sperduti, Samantha; Sayers, Niamh; Pelagatti, Ginevra; D'Alessandro, Sara; Perri, Carmela; Baschieri, Lara; Mascolo, Elisa; Roy, Neena; Simoni, Manuela; Hanyaloglu, Aylin; Casarini, Livio
abstract

2023 - Short-Term Exposure to Bisphenol A Does Not Impact Gonadal Cell Steroidogenesis In Vitro [Articolo su rivista]
Roy, Neena; Lazzaretti, Clara; Paradiso, Elia; Capponi, Chiara; Ferrari, Tommaso; Reggianini, Francesca; Sperduti, Samantha; Baschieri, Lara; Mascolo, Elisa; Perri, Carmela; Varani, Manuela; Canu, Giulia; Trenti, Tommaso; Nicoli, Alessia; Morini, Daria; Iannotti, Francesca; Villani, Maria Teresa; Vicini, Elena; Simoni, Manuela; Casarini, Livio
abstract

: Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

2022 - Analysis of follicle-stimulating hormone receptor (FSHR)/g-protein coupled estrogen receptor (GPER) complex internalization through early and late endosomes [Abstract in Atti di Convegno]
Lazzaretti, Clara; Casadei Garofani, Beatrice; Paradiso, Elia; D'Alessandro, Sara; Sperduti, Samantha; Roy, Neena; Mascolo, Elisa; Baschieri, Lara; Anzivino, Claudia; Simoni, Manuela; Casarini, Livio
abstract

2022 - cGMP is not involved in thyroid cancer cell death [Abstract in Atti di Convegno]
D'Alessandro, Sara; Paradiso, Elia; Lazzaretti, Clara; Sperduti, Samantha; Baschieri, Lara; Mascolo, Elisa; Roy, Neena; Anzivino, Claudia; Righi, Sara; Santi, Daniele; Brigante, Giulia; Simoni, Manuela; Casarini, Livio
abstract

2022 - Genetic signature of differentiated thyroid carcinoma susceptibility: a machine learning approach [Articolo su rivista]
Brigante, Giulia; Lazzaretti, Clara; Paradiso, Elia; Nuzzo, Federico; Sitti, Martina; Tüttelmann, Frank; Moretti, Gabriele; Silvestri, Roberto; Gemignani, Federica; Försti, Asta; Hemminki, Kari; Elisei, Rossella; Romei, Cristina; Zizzi, Eric Adriano; Deriu, Marco Agostino; Simoni, Manuela; Landi, Stefano; Casarini, Livio
abstract

To identify a peculiar genetic combination predisposing to differentiated thyroid carcinoma (DTC), we selected a set of single nucleotide polymorphisms (SNPs) associated with DTC risk, considering polygenic risk score (PRS), Bayesian statistics and a machine learning (ML) classifier to describe cases and controls in three different datasets. Dataset 1 (649 DTC, 431 controls) has been previously genotyped in a genome-wide association study (GWAS) on Italian DTC. Dataset 2 (234 DTC, 101 controls) and dataset 3 (404 DTC, 392 controls) were genotyped. Associations of 171 SNPs reported to predispose to DTC in candidate studies were extracted from the GWAS of dataset 1, followed by replication of SNPs associated with DTC risk (P < 0.05) in dataset 2. The reliability of the identified SNPs was confirmed by PRS and Bayesian statistics after merging the three datasets. SNPs were used to describe the case/control state of individuals by ML classifier. Starting from 171 SNPs associated with DTC, 15 were positive in both datasets 1 and 2. Using these markers, PRS revealed that individuals in the fifth quintile had a seven-fold increased risk of DTC than those in the first. Bayesian inference confirmed that the selected 15 SNPs differentiate cases from controls. Results were corroborated by ML, finding a maximum AUC of about 0.7. A restricted selection of only 15 DTC-associated SNPs is able to describe the inner genetic structure of Italian individuals, and ML allows a fair prediction of case or control status based solely on the individual genetic background.

2022 - Luteinizing hormone (LH)- and choriogonadotropin (hCG)-induced internalization of the receptor (LHCGR) is responsible for hormone-specific signaling [Abstract in Rivista]
Paradiso, Elia; Lazzaretti, Clara; D'Alessandro, Sara; Sperduti, Samantha; Roy, Neena; Mascolo, Elisa; Baschieri, Lara; Anzivino, Claudia; Simoni, Manuela; Casarini, Livio
abstract

2022 - Phosphodiesterase (PDE) 5 inhibitors sildenafil, tadalafil and vardenafil impact cAMP-specific PDE8 isoforms-linked second messengers and steroid production in a mouse Leydig tumor cell line [Articolo su rivista]
Limoncella, S.; Lazzaretti, C.; Paradiso, E.; D'Alessandro, S.; Barbagallo, F.; Pacifico, S.; Guerrini, R.; Tagliavini, S.; Trenti, T.; Santi, D.; Simoni, M.; Sola, M.; Di Rocco, G.; Casarini, L.
abstract

Type 5 phosphodiesterase (PDE5) blockade by inhibitors (PDE5i) results in intracellular cyclic guanosine monophosphate (cGMP) increase and smooth muscle relaxation and are used for the treatment of men erectile dysfunction. Although they have high specificity for PDE5, these inhibitors are suspected to cross-interact also with cyclic adenosine monophosphate (cAMP)-specific PDEs, inducing the intracellular accumulation of this cyclic nucleotide and related testosterone increase, positively impacting male reproductive parameters. However, the link between the use of PDE5i and the activation of cAMP-mediated steroidogenesis is still unclear. We have investigated whether three PDE5i, sildenafil, tadalafil and vardenafil, cross-interacts with the high affinity cAMP-specific enzymes type 8A and 8B PDEs (PDE8A and PDE8B), in live, transfected mouse Leydig tumor (mLTC1) and human embryonic kidney (HEK293) cell lines in vitro. The PDE5i-induced production of cAMP-dependent testosterone and its precursor progesterone was evaluated as well. We have developed PDE8A/B biosensors and modified cyclic nucleotides confirming enzyme binding to cAMP, but not to cGMP, in our cell models. cAMP binding to PDE8A/B was displaced upon cell treatment with PDE5i, revealing that sildenafil, tadalafil and vardenafil have similar effectiveness in live cells, in vitro. The cross-interaction between PDE5i and PDE8A/B supports the gonadotropin-enhanced intracellular cAMP increase, occurring together with cGMP increase, as well as steroid synthesis. Indeed, we found that Leydig cell treatment by PDE5i increases progesterone and testosterone production triggered by gonadotropins. We demonstrated that PDE5i may interact with the cAMP-specific PDE8A and PDE8B, possibly inducing intracellular cAMP and sex steroid hormone increase. These findings support clinical data suggesting that PDE5i might increase testosterone levels in men.

2022 - Regulation of antral follicular growth by an interplay between gonadotropins and their receptors [Articolo su rivista]
Casarini, L.; Paradiso, E.; Lazzaretti, C.; D'Alessandro, S.; Roy, N.; Mascolo, E.; Zareba, K.; Garcia-Gasca, A.; Simoni, M.
abstract

Knowledge of the growth and maturation of human antral follicles is based mainly on concepts and deductions from clinical observations and animal models. To date, new experimental approaches and in vitro data contributed to a deep comprehension of gonadotropin receptors’ functioning and may provide new insights into the mechanisms regulating still unclear physiological events. Among these, the production of androgen in the absence of proper LH levels, the programming of follicular atresia and dominance are some of the most intriguing. Starting from evolutionary issues at the basis of the gonadotropin receptor signal specificity, we draw a new hypothesis explaining the molecular mechanisms of the antral follicular growth, based on the modulation of endocrine signals by receptor-receptor interactions. The “heteromer hypothesis” explains how opposite death and life signals are delivered by gonadotropin receptors and other membrane partners, mediating steroidogenesis, apoptotic events, and the maturation of the dominant follicle.

2022 - The endocrine disruptor Benzo[a]Pyrene inhibits gonadotropin-mediated steroidogenesis in a mouse Leydig tumor cell line [Abstract in Atti di Convegno]
Sperduti, Samantha; Roy, Neena; Lazzaretti, Clara; Paradiso, Elia; D'Alessandro, Sara; Mascolo, Elisa; Baschieri, Lara; Santi, Daniele; Simoni, Manuela; Casarini, Livio
abstract

2021 - Endocrine Disruption of the Follicle-Stimulating Hormone Receptor Signaling During the Human Antral Follicle Growth [Articolo su rivista]
Roy, N.; Mascolo, E.; Lazzaretti, C.; Paradiso, E.; D'Alessandro, S.; Zareba, K.; Simoni, M.; Casarini, L.
abstract

An increasing number of pollutants with endocrine disrupting potential are accumulating in the environment, increasing the exposure risk for humans. Several of them are known or suspected to interfere with endocrine signals, impairing reproductive functions. Follicle-stimulating hormone (FSH) is a glycoprotein playing an essential role in supporting antral follicle maturation and may be a target of disrupting chemicals (EDs) likely impacting female fertility. EDs may interfere with FSH-mediated signals at different levels, since they may modulate the mRNA or protein levels of both the hormone and its receptor (FSHR), perturb the functioning of partner membrane molecules, modify intracellular signal transduction pathways and gene expression. In vitro studies and animal models provided results helpful to understand ED modes of action and suggest that they could effectively play a role as molecules interfering with the female reproductive system. However, most of these data are potentially subjected to experimental limitations and need to be confirmed by long-term observations in human.

2021 - Identification of key receptor residues discriminating human chorionic gonadotropin (Hcg)-and luteinizing hormone (lh)-specific signaling [Articolo su rivista]
Lazzaretti, C.; Secco, V.; Paradiso, E.; Sperduti, S.; Rutz, C.; Kreuchwig, A.; Krause, G.; Simoni, M.; Casarini, L.
abstract

(1) The human luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptor (LHCGR) discriminates its two hormone ligands and differs from the murine receptor (Lhr) in amino acid residues potentially involved in qualitative discerning of LH and hCG. The latter gon-adotropin is absent in rodents. The aim of the study is to identify LHCGR residues involved in hCG/LH discrimination. (2) Eight LHCGR cDNAs were developed, carrying “murinizing” mutations on aminoacidic residues assumed to interact specifically with LH, hCG, or both. HEK293 cells expressing a mutant or the wild type receptor were treated with LH or hCG and the kinetics of cyclic adenosine monophosphate (cAMP) and phosphorylated extracellular signal-regulated ki-nases 1/2 (pERK1/2) activation was analyzed by bioluminescence resonance energy transfer (BRET). (3) Mutations falling within the receptor leucine reach repeat 9 and 10 (LRR9 and LRR10; K225S +T226I and R247T), of the large extracellular binding domain, are linked to loss of hormone-specific induced cAMP increase, as well as hCG-specific pERK1/2 activation, leading to a Lhr-like modulation of the LHCGR-mediated intracellular signaling pattern. These results support the hypothesis that LHCGR LRR domain is the interaction site of the hormone β-L2 loop, which differs between LH and hCG, and might be fundamental for inducing gonadotropin-specific signals. (4) Taken to-gether, these data identify LHCGR key residues likely evolved in the human to discriminate LH/hCG specific binding.

2021 - L’ormone luteinizzante e la gonadotropina corionica umana: attività molecolari e cliniche mediate da un unico recettore [Articolo su rivista]
Sperduti, Samantha; Paradiso, Elia; Lazzaretti, Clara; Rochira, Vincenzo; Brigante, Giulia; Santi, Daniele; Simoni, Manuela; Casarini, Livio
abstract

2021 - Quantification of hormone membrane receptor FSHR, GPER and LHCGR transcripts in human primary granulosa lutein cells by real-time quantitative PCR and digital droplet PCR [Articolo su rivista]
Sperduti, S.; Lazzaretti, C.; Paradiso, E.; Anzivino, C.; Villani, M. T.; De Feo, G.; Simoni, M.; Casarini, L.
abstract

Background: Quantitative real time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) are methods used for gene expression analysis in several contexts, including reproductive endocrinology. Objectives: Herein, we compared qPCR and ddPCR technologies for gene expression analysis of hormone membrane receptor-encoding genes (FSHR, GPER and LHCGR), as well as the commonly used RPS7 housekeeping gene, in order to identify the most reliable method to be applied for gene expression analysis in the context of human reproduction. Methods: Total RNA was extracted from human primary granulosa lutein cells of donor patients undergoing assisted reproduction and used for gene expression analysis by qPCR and ddPCR, after finding the optimal annealing temperature. Immunostaining for protein localization in cell membranes was also performed. Results: Both techniques provided results reflecting the low number of FSHR and GPER transcripts, although ddPCR quantified the low-expressed genes with major accuracy, thanks to its higher reaction efficiency. The absolute FSHR and GPER transcript number was also determined by ddPCR, resulting in 40- to 260-fold lower amount than LHCGR transcripts. qPCR and ddPCR data are convergent with immunofluorescence analysis of membrane receptor expression in human primary granulosa lutein cells. Conclusion: These results suggest that ddPCR is the candidate technology for analysis of genes with relatively low expression levels and provides useful insights for characterizing hormone receptor expression levels in the context of reproductive endocrinology.

2021 - Sphingosine-1 phosphate induces cAMP/PKA-independent phosphorylation of the cAMP response element-binding protein (CREB) in granulosa cells [Articolo su rivista]
Paradiso, E.; Lazzaretti, C.; Sperduti, S.; Antoniani, F.; Fornari, G.; Brigante, G.; Di Rocco, G.; Tagliavini, S.; Trenti, T.; Morini, D.; Falbo, A. I.; Villani, M. T.; Nofer, J. -R.; Simoni, M.; Poti, F.; Casarini, L.
abstract

Background and aims: Sphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid. The role of the lysosphingolipid in gonads of the female is widely unclear. At nanomolar concentrations, S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P1-5, modulating different signaling pathways. S1P1 and S1P3 are highly expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. In this study, we evaluated the signaling cascade activated by S1P and its synthetic analogues in hGLC and hGL5 cells, exploring the biological relevance of S1PR-stimulation in this context. METHODS AND RESULTS. hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P1- and S1P3-specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1P-dependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P2 or S1P3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulin-encoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. However, S1P or agonists did not modulate granulosa cell viability and proliferation in our conditions. Conclusions: This study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce intracellular steroidogenic signals and progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P–S1PR axis may cooperate with gonadotropins in modulating follicle development.

2021 - The “hitchhiker’s guide to the galaxy” of endothelial dysfunction markers in human fertility [Articolo su rivista]
Santi, D.; Spaggiari, G.; Greco, C.; Lazzaretti, C.; Paradiso, E.; Casarini, L.; Poti, F.; Brigante, G.; Simoni, M.
abstract

Endothelial dysfunction is an early event in the pathogenesis of atherosclerosis and represents the first step in the pathogenesis of cardiovascular diseases. The evaluation of endothelial health is fundamental in clinical practice and several direct and indirect markers have been suggested so far to identify any alterations in endothelial homeostasis. Alongside the known endothelial role on vascular health, several pieces of evidence have demonstrated that proper endothelial functioning plays a key role in human fertility and reproduction. Therefore, this stateof‐the‐art review updates the endothelial health markers discriminating between those available for clinical practice or for research purposes and their application in human fertility. Moreover, new molecules potentially helpful to clarify the link between endothelial and reproductive health are evaluated herein.

2020 - Evolutionary, structural, and physiological differences between hCG and LH [Capitolo/Saggio]
Casarini, Livio; Lazzaretti, Clara; Paradiso, Elia; Santi, Daniele; Brigante, Giulia; Simoni, Manuela
abstract

2020 - FSH for the Treatment of Male Infertility [Articolo su rivista]
Casarini, L.; Crépieux, P.; Reiter, E.; Lazzaretti, C.; Paradiso, E.; Rochira, V.; Brigante, G.; Santi, D.; Simoni, M.
abstract

Follicle-stimulating hormone (FSH) supports spermatogenesis acting via its receptor (FSHR), which activates trophic effects in gonadal Sertoli cells. These pathways are targeted by hormonal drugs used for clinical treatment of infertile men, mainly belonging to sub-groups defined as hypogonadotropic hypogonadism or idiopathic infertility. While, in the first case, fertility may be efficiently restored by specific treatments, such as pulsatile gonadotropin releasing hormone (GnRH) or choriogonadotropin (hCG) alone or in combination with FSH, less is known about the efficacy of FSH in supporting the treatment of male idiopathic infertility. This review focuses on the role of FSH in the clinical approach to male reproduction, addressing the state-of-the-art from the little data available and discussing the pharmacological evidence. New compounds, such as allosteric ligands, dually active, chimeric gonadotropins and immunoglobulins, may represent interesting avenues for future personalized, pharmacological approaches to male infertility.

2020 - Membrane Estrogen Receptor (GPER) and Follicle-Stimulating Hormone Receptor (FSHR) Heteromeric Complexes Promote Human Ovarian Follicle Survival [Articolo su rivista]
Casarini, L.; Lazzaretti, C.; Paradiso, E.; Limoncella, S.; Riccetti, L.; Sperduti, S.; Melli, B.; Marcozzi, S.; Anzivino, C.; Sayers, N. S.; Czapinski, J.; Brigante, G.; Poti, F.; La Marca, A.; De Pascali, F.; Reiter, E.; Falbo, A.; Daolio, J.; Villani, M. T.; Lispi, M.; Orlando, G.; Klinger, F. G.; Fanelli, F.; Rivero-Muller, A.; Hanyaloglu, A. C.; Simoni, M.
abstract

Molecular Biology; Female Reproductive Endocrinology; Endocrine Regulation

2020 - Polygenic Susceptibility to Papillary Thyroid Cancer in Italian Subjects. [Abstract in Atti di Convegno]
Brigante, G.; Lazzaretti, C.; Paradiso, E.; Foersti, A.; Hemminki, K.; Elisei, R.; Romei, C.; Rochira, V.; Simoni, M.; Landi, S.; Casarini, L.
abstract

olygenic Susceptibility to Papillary Thyroid Cancer in Italian Subjects INTRODUCTION AND AIM. Thyroid cancer is the most common endocrine neoplasia, with an estimated age- standardized incidence rate of 6.7 per 100000 worldwide in 2018 [1]. This rate is rapidly increasing and papillary thy- roid carcinoma (PTC) is the main histotype. PTC suscepti- bility is the result of genetic predisposition, environmental factors and lifestyle. We studied the genetic combination that characterizes PTC affected subjects, differentiating them from healthy controls. METHODS AND RESULTS. We considered the genetic variants (SNPs) significantly associated with PTC on the PubMed database. 184 informative SNPs were selected, considering linkage disequilibrium. Then, SNPs data were extracted from the online 1000 Genomes database,comprising genome of 2504 unselected individuals col- lected worldwide. The combination of 184 SNPs associ- ated with PTC was used to group individuals in different risk-clusters according to their genetic structure, calcu- lated by Bayesian statistics, as previously performed for polycystic ovary syndrome [2]. Individuals were distrib- uted among 7 groups worldwide, indicating different de- gree of genetic predisposition to PTC. We then considered genetic data from about 1200 individuals (697 PTC versus 497 healthy controls) of Central/South Italian origin reg- istered in a GWAS, specific for PTC [3]. This first analysis was refined using the 33 SNPs reasonably most causa- tive of genetic clustering (26 with p<0.05 at trend test in GWAS and 7 with p<0.05 in the model of recessive inher- itance). At multivariate logistic regression analysis, PTC and healthy controls resulted genetically different (ODDS RATIO 188.6, 95%CI 64.35-552.8), revealing diverse pre- disposition to develop cancer. Afterwards, these results have been confirmed in an independent cohort of Italian subjects (234 PTC and 100 controls). Then, the genetic structure of each subject was indicated as a percentage of affinity to each risk-cluster and re-analyzed together with other risk factors: sex, body-mass index, area of origin and familiarity (quantified in a growing score as the degree of kinship increases). These data were analyzed together by principal component analysis and clustering of the two groups was even more pronounced. The most contributive factors to the diversity between PTC and healthy controls were genetics and familiarity. CONCLUSION. We demonstrated that PTC affected subjects are genetically different from healthy controls, and that the difference is identifiable in a peculiar combi- nation of genetic variants.

2020 - Two human menopausal gonadotrophin (hMG) preparations display different early signaling in vitro [Articolo su rivista]
Casarini, L.; Riccetti, L.; Paradiso, E.; Benevelli, R.; Lazzaretti, C.; Sperduti, S.; Melli, B.; Tagliavini, S.; Varani, M.; Trenti, T.; Morini, D.; Falbo, A.; Villani, M. T.; Jonas, K. C.; Simoni, M.
abstract

Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, β-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting β-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-β-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies.

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.

2019 - Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars [Articolo su rivista]
Riccetti, Laura; Sperduti, Samantha; Lazzaretti, Clara; Klett, Danièle; De Pascali, Francesco; Paradiso, Elia; Limoncella, Silvia; Potì, Francesco; Tagliavini, Simonetta; Trenti, Tommaso; Galano, Eugenio; Palmese, Angelo; Satwekar, Abhijeet; Daolio, Jessica; Nicoli, Alessia; Villani, Maria Teresa; Aguzzoli, Lorenzo; Reiter, Eric; Simoni, Manuela; Casarini, Livio
abstract

Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f (R))- with biosimilar preparations (Bemfola (R) and Ovaleap (R))-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f (R) or biosimilar (1 x 10(-3) -1 x 10(3) ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ increase and beta-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and beta-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 +/- 0.9-24 +/- 1.7 ng/ml; beta-arrestin 2 EC50 range = 140 +/- 14.1-313 +/- 18.7 ng/ml; Kruskal-Wallis test; p >= 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ increased upon cell treatment by 4 mu g/ml Gonal-f (R), while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC(50)s were demonstrated (Kruskal -Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.

2019 - Gnrh antagonists produce differential modulation of the signaling pathways mediated by gnrh receptors [Articolo su rivista]
Sperduti, S.; Limoncella, S.; Lazzaretti, C.; Paradiso, E.; Riccetti, L.; Turchi, S.; Ferrigno, I.; Bertacchini, J.; Palumbo, C.; Poti, F.; Longobardi, S.; Millar, R. P.; Simoni, M.; Newton, C. L.; Casarini, L.
abstract

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1–2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LβT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, β-catenin activation and mouse luteinizing-hormone β-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LβT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM–1 µM, demonstrating higher potency than Ganirelix and Teverelix,.

2019 - Inferring biallelism of two FSH receptor mutations associated with spontaneous ovarian hyperstimulation syndrome by evaluating FSH, LH and HCG cross-activity [Articolo su rivista]
Lazzaretti, C.; Riccetti, L.; Sperduti, S.; Anzivino, C.; Brigante, G.; De Pascali, F.; Poti, F.; Rovei, V.; Restagno, G.; Mari, C.; Lussiana, C.; Benedetto, C.; Revelli, A.; Casarini, L.
abstract

Research question: What is the cumulative effect of two follicle-stimulating hormone receptor (FSHR) mutations in spontaneous ovarian hyperstimulation syndrome (sOHSS) pathogenesis? Are these mutations in the mono- or biallelic state? Design: Two FSHR mutations were found in a pregnant patient affected by sOHSS with no predisposing conditions. While the p.Asn106His mutation is novel, the p.Ser128Tyr mutation has been associated with sOHSS previously. The patient's FSHR gene was analysed by Sanger sequencing, and FSHR cDNAs carrying a single or both point mutations were created by mutagenesis in vitro. cAMP activation by recombinant FSH, luteinizing hormone (LH), human chorionic gonadotropin (HCG) and thyroid-stimulating hormone (TSH) was evaluated in transfected HEK293 cells by bioluminescence resonance energy transfer. Results: All mutations decreased the 50% effective concentration of FSH calculated for cAMP (P < 0.05, n = 6), resulting in two- to 10-fold lower ligand potency. TSH failed to induce an FSHR-mediated increase in intracellular cAMP, while LH was approximately four-fold more potent than HCG in p.Ser128Tyr FSHR-expressing HEK293 cells despite lower cAMP plateau levels (P < 0.05, n = 5). The p.Ser128Tyr FSHR mutation was found to be responsible for an LH-/HCG-induced increase in cAMP when it was in the biallelic heterozygous state with p.Asn106His, but no increase in cAMP was induced in the monoallelic state. Conclusion: In-vitro data support that, in pregnant patients with sOHSS, the two FSHR mutations have an opposing effect on the pathogenesis of sOHSS and are in the biallelic heterozygous form, allowing HCG to induce a p.Ser128Tyr FSHR-mediated increase in cAMP.

2019 - Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach [Articolo su rivista]
Casarini, L.; Riccetti, L.; Limoncella, Silvia; Lazzaretti, C.; Barbagallo, F.; Pacifico, S.; Guerrini, R.; Tagliavini, S.; Trenti, T.; Simoni, M.; Sola, M.; Di Rocco, G.
abstract

Forster 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 lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can 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 (Kd) values of 5.6 ± 3.2 and 13.7 ± 0.8 μM were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS-rhodamine conjugate by sildenafil; the 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 bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.

2018 - Immunohistochemical analysis of the distribution of molecules involved in ionic and pH regulation in the lancelet Branchiostoma floridae (Hubbs, 1922) [Articolo su rivista]
Cuoghi, Ivan; Lazzaretti, Clara; Mandrioli, Mauro; Mola, Lucrezia; Pederzoli, Aurora
abstract

The aim of present work is to analyse the distribution of carbonic anhydrase II (CAII), cystic fibrosis transmembrane regulator (CFTR), vacuolar-type H+-ATPase (V-H+-ATPase), Na+/K+ ATPase, Na+/H+ exchanger (NHE) and SLC26A6 (solute carrier family 26, member 6), also known as pendrin protein, in the lancelet Branchiostoma floridae in order to go in depth in the evolution of osmoregulation and pH regulation in Chordates. In view of their phylogenetic position, lancelets may indeed provide a critical point of reference for studies on osmoregulation evolution in Chordates. The results of present work demonstrated that, except to Na+/K+ ATPase that is strongly expressed in nephridia only, all the other studied molecules are abundantly present in skin, coelomic epithelium, renal papillae and nephridia and hepatic coecum. Thus, it is possible to hypothesize that also in lancelet, as in fish, these organs are involved in pH control and ionic regulation.In the digestive tract of B. floridae, the intestine epithelium was weakly immune-reactive to all tested antibodies, while the hepatic coecum showed an intense immunoreactivity to all molecules. Since in amphioxus the hepatic coecum functions simultaneously as stomach, liver and pancreas, these immunohistochemical results proved the secretion of H+ and HCO3− ions, typical of digestive process. Colocalization studies indicated a co-expression of the studied proteins in all considered organs, excluding NHE and pendrin for renal papillae, since some renal papillae are NHE immunopositive only.

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.